115-09-3BABMCXWQNSQAOC-UHFFFAOYSA-MBABMCXWQNSQAOC-UHFFFAOYSA-M
Methylmercuric(II) chlorideMethyl mercuric(II) chloride (Methyl mercuric chloride) (MeHg)
Mercury, chloromethyl-
Chlormethylquecksilber
chloromethylmercure
chloromethylmercury
clorometilmercurio
Mercury methyl chloride
Methylmercuric chloride
Methylmercury chloride
Methylmercury monochloride
METHYL-QUECKSILBER-CHLORID
Monomethyl mercury chloride
NSC 19998
DTXSID50208137487-94-7LWJROJCJINYWOX-UHFFFAOYSA-LLWJROJCJINYWOX-UHFFFAOYSA-L
Mercuric chlorideMercury(II) chloride
Bichloride of mercury
Calochlor
Corrosive sublimate
Dichloromercury
Dichlorure de mercure
dicloruro de mercurio
MERCURIC BICHLORIDE
MERCURY (II) CHLORIDES
Mercury bichloride
Mercury chloromercurate(II)
mercury dichloride
Mercury perchloride
Mercury(2+) chloride
MERCURY, BICHLORIDE, GRANULAR
NSC 353255
QUECKSILBER(II)-CHLORID
Quecksilberdichlorid
Sublimate
UN 1624
DTXSID502081179-06-1HRPVXLWXLXDGHG-UHFFFAOYSA-NHRPVXLWXLXDGHG-UHFFFAOYSA-N
Acrylamide2-Propenamide
2-Propene amide
acrilamida
Acrylamid
Acrylamide monomer
Acrylic acid amide
Acrylic amide
Bio-Acrylamide 50
Ethylenecarboxamide
NSC 7785
Propenamide
UN 2074
UN3426
Vinyl amide
DTXSID502002751312-24-4Mercury chlorideDTXSID4085872460-35-5DLFVBJFMPXGRIB-UHFFFAOYSA-NDLFVBJFMPXGRIB-UHFFFAOYSA-N
AcetamideAcetamid
acetamida
Acetic acid amide
Acetimidic acid
Ethanamide
Ethanimidic acid
Methanecarboxamide
NSC 25945
DTXSID7020005103-90-2RZVAJINKPMORJF-UHFFFAOYSA-NRZVAJINKPMORJF-UHFFFAOYSA-N
Acetaminophen4-Acetamidophenol
APAP
Paracetamol
4-hydroxyacetanilide
Acetamide, N-(4-hydroxyphenyl)-
4-(Acetylamino)phenol
4-(N-Acetylamino)phenol
4-Acetaminophenol
4'-Hydroxyacetanilide
Abensanil
Acetagesic
Acetalgin
ACETAMIDE, N-(4-HYDROXYPHENYL)
Acetaminofen
Acetanilide, 4'-hydroxy-
ACETANILIDE, 4-HYDROXY-
Algotropyl
Alvedon
Anaflon
Apamide
Banesin
Ben-u-ron
Bickie-mol
Biocetamol
Cetadol
Citramon P
Claratal
Clixodyne
Dafalgan
Daphalgan
Dial-a-gesic
Disprol
Doliprane
Dolprone
Dymadon
Efferalgan
Endophy
Febrilex
Febrilix
Febro-Gesic
Febrolin
Fepanil
Finimal
Gattaphen T
Gelocatil
Gutte Enteric
Homoolan
Jin Gang
Lestemp
Liquagesic
Lonarid
Lyteca Syrup
Minoset
Momentum
N-(4-Hydroxyphenyl)acetamide
N-Acetyl-4-aminophenol
N-Acetyl-4-hydroxyaniline
N-Acetyl-p-aminophenol
Napafen
Naprinol
Nobedon
NSC 109028
NSC 3991
Ortensan
p-(Acetylamino)phenol
p-Aceaminophenol
Pacemol
p-Acetamidophenol
p-Acetoaminophen
P-ACETYLAMINOPHENOL
Paldesic
panadeine
Panadol
Panadol Actifast
Panadol Extend
Panaleve
Panasorb
Panodil
Paracetamol DC
Paracetamole
Parageniol
Paramol
Paraspen
Parelan
Pasolind N
Perfalgan
Phenaphen
Phendon
p-Hydroxyacetanilide
Prodafalgan
Puerxitong
Pyrinazine
Resfenol
Resprin
Rhodapop NCR
Salzone
Tabalgin
Tachipirina
Tempanal
Tralgon
Tylenol
TylolHot
Valadol
Valgesic
Vermidon
Vick Pyrena
DTXSID2020006968-81-0VGZSUPCWNCWDAN-UHFFFAOYSA-NVGZSUPCWNCWDAN-UHFFFAOYSA-N
AcetohexamideBenzenesulfonamide, 4-acetyl-N-[(cyclohexylamino)carbonyl]-
1-(p-Acetylbenzenesulfonyl)-3-cyclohexylurea
1-[(p-Acetylphenyl)sulfonyl]-3-cyclohexylurea
Acetohexamid
acetohexamida
Dimelin
Dimelor
Dymelor
Gamadiabet
Hypoglicil
Metaglucina
Minoral
N-(p-Acetylphenylsulfonyl)-N'-cyclohexylurea
Ordimel
Tsiklamid
Urea, 1-[(p-acetylphenyl)sulfonyl]-3-cyclohexyl-
DTXSID702000767-66-3HEDRZPFGACZZDS-UHFFFAOYSA-NHEDRZPFGACZZDS-UHFFFAOYSA-N
ChloroformTrichloromethane
Methane, trichloro-
CARBON TRICHLORIDE
Chloroforme
cloroformo
Formyl trichloride
Methane trichloride
Methane,trichloro-
NSC 77361
Trichloroform
UN 1888
DTXSID1020306110-00-9YLQBMQCUIZJEEH-UHFFFAOYSA-NYLQBMQCUIZJEEH-UHFFFAOYSA-N
FuranDivinylene oxide
furanne
Furfuran
Oxacyclopentadiene
Tetrole
UN 2389
DTXSID60206467429-90-5XAGFODPZIPBFFR-UHFFFAOYSA-NAZDRQVAHHNSJOQ-UHFFFAOYSA-N
AluminumAisin Metal Fiber
Al 050P-H24
ALC Fine
Alcan XI 1391
Almi-Paste SSP 303AR
Aloxal 3010
Alpaste 00-0506
Alpaste 0100M
Alpaste 0100MA
Alpaste 0100M-C
Alpaste 0200M
Alpaste 0200T
Alpaste 0230M
Alpaste 0230T
Alpaste 0241M
Alpaste 0300M
Alpaste 0500M
Alpaste 0539X
Alpaste 0620MS
Alpaste 0625TS
Alpaste 0638-70C
Alpaste 0700M
Alpaste 0780M
Alpaste 0900M
Alpaste 100M
Alpaste 100MS
Alpaste 100MSR
Alpaste 1100M
Alpaste 1100MA
Alpaste 1100N
Alpaste 1100NA
Alpaste 1109MA
Alpaste 1109MC
Alpaste 1200M
Alpaste 1200T
Alpaste 1260MS
Alpaste 1500MA
Alpaste 1700NL
Alpaste 1810YL
Alpaste 1830YL
Alpaste 1900M
Alpaste 1900XS
Alpaste 1950M
Alpaste 1950N
Alpaste 210N
Alpaste 2172EA
Alpaste 2173
Alpaste 240T
Alpaste 241M
Alpaste 417
Alpaste 46-046
Alpaste 4-621
Alpaste 4919
Alpaste 50-63
Alpaste 50-635
Alpaste 51-148B
Alpaste 51-231
Alpaste 5205N
Alpaste 5207N
Alpaste 52-509
Alpaste 52-568
Alpaste 5301N
Alpaste 5302N
Alpaste 53-119
Alpaste 5422NS
Alpaste 54-452
Alpaste 54-497
Alpaste 54-542
Alpaste 55-516
Alpaste 55-519
Alpaste 55-574
Alpaste 5620NS
Alpaste 5630NS
Alpaste 5640NS
Alpaste 56-501
Alpaste 5650NS
Alpaste 5653NS
Alpaste 5654NS
Alpaste 5680N
Alpaste 5680NS
Alpaste 60-600
Alpaste 60-760
Alpaste 60-768
Alpaste 62-356
Alpaste 6340NS
Alpaste 6370NS
Alpaste 6390NS
Alpaste 640NS
Alpaste 65-388
Alpaste 66NLB
Alpaste 710N
Alpaste 7130N
Alpaste 7160N
Alpaste 7160NS
Alpaste 725N
Alpaste 740NS
Alpaste 7430NS
Alpaste 7580NS
Alpaste 7620NS
Alpaste 7640NS
Alpaste 7670M
Alpaste 7670NS
Alpaste 7675NS
Alpaste 7679NS
Alpaste 7680N
Alpaste 7680NS
Alpaste 76840NS
Alpaste 7730N
Alpaste 7770N
Alpaste 7830N
Alpaste 8004
Alpaste 8080N
Alpaste 8260NAR
Alpaste 891K
Alpaste 91-0562
Alpaste 92-0592
Alpaste 93-0595
Alpaste 93-0647
Alpaste 94-2315
Alpaste 95-0570
Alpaste 96-0635
Alpaste 96-2104
Alpaste 97-0510
Alpaste 97-0534
Alpaste AW 520B
Alpaste AW 612
Alpaste AW 9800
Alpaste F 795
Alpaste FM 7680K
Alpaste FX 440
Alpaste FX 910
Alpaste FZ 0534
Alpaste FZU 40C
Alpaste G
Alpaste HR 8801
Alpaste HS 2
Alpaste J
Alpaste K 9800
Alpaste MC 666
Alpaste MC 707
Alpaste MF 20
Alpaste MG 01
Alpaste MG 1000
Alpaste MG 1300
Alpaste MG 500
Alpaste MG 600
Alpaste MH 6601
Alpaste MH 8801
Alpaste MH 9901
Alpaste MR 7000
Alpaste MR 9000
Alpaste MS 630
Alpaste N 1700NL
Alpaste NS 7670
Alpaste O 100N
Alpaste O 2130
Alpaste O 300M
Alpaste P 0100
Alpaste P 1950
Alpaste S
Alpaste SAP 110
Alpaste SAP 414P
Alpaste SAP 550N
Alpaste SCR 5070
Alpaste TCR 2020
Alpaste TCR 2060
Alpaste TCR 2070
Alpaste TCR 3010
Alpaste TCR 3030
Alpaste TCR 3040
Alpaste TCR 3130
Alpaste TD 200T
Alpaste UF 500
Alpaste WB 0230
Alpaste WD 500
Alpaste WJP-U 75C
Alpaste WX 0630
Alpaste WX 7830
Alpaste WXA 7640
Alpaste WXM 0630
Alpaste WXM 0650
Alpaste WXM 0660
Alpaste WXM 1415
Alpaste WXM 1440
Alpaste WXM 5422
Alpaste WXM 760b
Alpaste WXM 7640
Alpaste WXM 7675
Alpaste WXM-T 60B
Alpaste WXM-U 75
Alpaste WXM-U 75C
Altop X
Aluchrome Ultrafin Super
Alumat 1600
Alumet H 30
aluminio
Aluminium
Aluminium Flake
Aluminum 27
Aluminum atom
Aluminum element
Aluminum Flake PCF 7620
Aluminum granules
ALUMINUM METAL/GRANULE
ALUMINUM PASTE
ALUMINUM PIGMENT
ALUMINUM TURNINGS
Alumi-paste 640NS
Alumipaste 91-0562
Alumipaste 98-1822T
Alumipaste AW 620
Alumipaste CR 300
Alumipaste GX 180A
Alumipaste GX 201A
Alumipaste HR 7000
Alumipaste HR 850
Alumipaste MG 11
Alumipaste MH 8801
Aquamet NPW 2900
Aquapaste 205-5
Aquasilver LPW
Astroflake 40
Astroflake Black N 020
Astroflake Black N 070
Astroflake LG 40
Astroflake LG 70
Astroflake Silver N 040
Astroshine NJ 1600
Astroshine T 8990
Atomizalumi VA 200
C.I. PIGMENT METAL 1
Chromal IV
Chromal X
Decomet 1001/10
Decomet 2018/10
Decomet High Gloss Al 1002/10
Ecka AS 081
Eckart 9155
Eterna Brite 301-1
Eterna Brite 601-1
Eterna Brite 651-1
Eterna Brite EBP 251PA
Eterna Brite Primier 251PA
Ferro FX 53-038
Friend Color F 500GR-W
Friend Color F 500WT
Friend Color F 700RE-W
Friend Color F 701RE-W
Hi Print 60T
High Print 60T
Hisparkle HS 2
Hydro Paste 8726
Hydrolac WHH 2153
Hydrolan 3560
Hydrolux Reflexal 100
Hydroshine WS 1001
JISA 51010P
Kryal Z
Lansford 243
LE Sheet 800
Leafing Alpaste
LG-H Silver 25
Lunar Al-V 95
Metallux 161
Metallux 2154
Metallux 2192
Metalure
Metalure 55350
Metalure L 55350
Metalure L 59510
Metalure W 2001
Metapor
Metasheen 1800
Metasheen HR 0800
Metasheen KM 100
Metasheen KM 1000
Metasheen Slurry 1807
Metasheen Slurry 1811
Metasheen Slurry KM 100
Metax G
Metax S
Mirror Glow 1000
Mirror Glow 600
Mirrorsheen
Noral Aluminium
Noral Ink Grade Aluminium
Obron 10890
Offset FM 4500
Puratronic
Reflexal 145
Reynolds 400
Reynolds 4-301
Reynolds 4-591
Reynolds 667
SAP 260PW-HS
SAP-FM 4010
SBC 516-20Z
Scotchcal 7755SE
Serumekku
Setanium 50MIS-H8
Siberline ET 2025
Siberline ST 21030E1
Silvar A
Silver VT 522
Silverline SSP 353
Silvex 793-20C
Sparkle Silver 3141ST
Sparkle Silver 3500
Sparkle Silver 3641
Sparkle Silver 5000AR
Sparkle Silver 516AR
Sparkle Silver 5242AR
Sparkle Silver 5245AR
Sparkle Silver 5271AR
Sparkle Silver 5500
Sparkle Silver 5745
Sparkle Silver 7000AR
Sparkle Silver 7005AR
Sparkle Silver 7500
Sparkle Silver 960-25E1
Sparkle Silver E 1745AR
Sparkle Silver L 1526AR
Sparkle Silver Premier 751
Sparkle Silver SS 3130
Sparkle Silver SS 5242AR
Sparkle Silver SS 5588
Sparkle Silver SSP 132AR
Special PCR 507
Splendal 6001BG
Spota Mobil 801
SSP 760-20C
Stapa Aloxal PM 2010
Stapa Aloxal PM 3010
Stapa Aloxal PM 4010
Stapa Hydrolac BG 8n.1
Stapa Hydrolac BGH Chromal X
Stapa Hydrolac PM Chromal VIII
Stapa Hydrolac W 60NL
Stapa Hydrolac WH 16
Stapa Hydrolac WH 66NL
Stapa Hydrolux 2192
Stapa Hydrolux 8154
Stapa IL Hydrolan 2192-55900G
Stapa Metallic R 607
Stapa Metallux 1050
Stapa Metallux 211
Stapa Metallux 212
Stapa Metallux 2196
Stapa Metallux 274
Stapa Mobilux 181
Stapa Offset 3000
Stapa PV 10
Stapa VP 46432G
Starbrite 2100
Super Fine 18000
Super Fine 22000
Supramex 2022
Toyo Aluminum 02-0005
Toyo Aluminum 93-3040
Transmet K 102HE
Tufflake 3645
Tufflake 5843
UN 1396
US Aluminum 809
Valimet H 2
Valimet H 3
White Silver 7080N
White Silver 7130N
DTXSID30402737440-43-9BDOSMKKIYDKNTQ-UHFFFAOYSA-NBDOSMKKIYDKNTQ-UHFFFAOYSA-N
CadmiumCadimium
CADMIUM BLUE
CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER
DTXSID10239407439-97-6QSHDDOUJBYECFT-UHFFFAOYSA-NQSHDDOUJBYECFT-UHFFFAOYSA-N
MercuryLiquid silver
Mercure
MERCURIC METAL TRIPLE DISTILLED
mercurio
Mercury element
Quecksilber
Quicksilver
UN 2024
UN 2809
DTXSID10241727440-61-1JFALSRSLKYAFGM-UHFFFAOYSA-NJFALSRSLKYAFGM-UHFFFAOYSA-N
UraniumUranium, isotope of mass 238
238U Element
UN 2979 (DOT)
Uranium I
DTXSID10425227440-38-2RQNWIZPPADIBDY-UHFFFAOYSA-NRQNWIZPPADIBDY-UHFFFAOYSA-N
ArsenicAs
Arsenic black
ARSENIC METAL
arsenico
Grey arsenic
UN 1558
DTXSID40238867440-22-4BQCADISMDOOEFD-UHFFFAOYSA-NBQCADISMDOOEFD-UHFFFAOYSA-N
SilverAg Nanopaste NPS-J 90
Ag Sphere 2
Ag-C-GS
Algaedyn
Arctic Silver 3
Argentum
Astroflake 5
Carey Lea silver
Colloidal silver
Dotite XA 208
Du Pont 4943
ECM 100AF4810
Enlight 600
Enlight silver plate 600
Epinall
Finesphere SVND 102
Fordel DC
FP 5369-502
Jelcon SH 1
Jungindai Takasago 300
KS (metal)
LCP 1-19SFS
Metz 3000-1
Nanomelt AGC-A
Nanomelt Ag-XA 301
Nanomelt Ag-XF 301
Nanomelt Ag-XF 301H
Nanopaste NPS-J 90
Perfect Silver
Puff Silver X 1200
RT 1710S-C1
SD (metal)
Shell Silver
Silbest E 20
Silbest F 20
Silbest J 18
Silbest TC 12
Silbest TC 20E
Silbest TC 25A
Silbest TCG 1
Silbest TCG 7
Silcoat AgC 103
Silcoat AgC 2011
Silcoat AgC 209
Silcoat AgC 2190
Silcoat AgC 222
Silcoat AgC 2411
Silcoat AgC 74T
Silcoat AgC-A
Silcoat AgC-AO
Silcoat AgC-B
Silcoat AgC-BO
Silcoat AgC-D
Silcoat AgC-G
Silcoat AgC-GS
Silcoat AgC-L
Silcoat AgC-O
Silcoat GS
Silcoat RF 200
Silflake 135
Silsphere 514
Silver atom
Silver element
Silver Flake 1
Silver Flake 25
Silver Flake 52
Silver Flake 7A
SILVER FLAKES
Silver metal
Silvest TCG 11N
Technic 299
Technic 450
Techno Alpha 175
DTXSID40243057439-96-5PWHULOQIROXLJO-UHFFFAOYSA-NPWHULOQIROXLJO-UHFFFAOYSA-N
ManganeseColloidal manganese
Cutaval
Manganese element
Manganese fulleride
Manganese metal alloy
Manganese-55
manganeso
DTXSID20241697440-02-0PXHVJJICTQNCMI-UHFFFAOYSA-NPXHVJJICTQNCMI-UHFFFAOYSA-N
NickelCarbonyl 255
Carbonyl Ni 123
Carbonyl Ni 283
Carbonyl Nickel 123
Carbonyl Nickel 283
Carbonyl Nickel 287
Cerac N 2003
CNS 10 Micron
Exmet 4 Ni X-4/0
Fibrex P
Incofoam
Nickel element
NICKEL ROUND ANODES
Nicrobraz LM:BNi 2
Ni-Flake 95
Novamet 123
Novamet 4SP
Novamet 4SP10
Novamet 525
Novamet CNS 400
Novamet HCA 1
Novamet NI 255
Raney nickel
Raney nickel 2800
UN 1325
UN 2881
DTXSID20209257440-66-6HCHKCACWOHOZIP-UHFFFAOYSA-NHCHKCACWOHOZIP-UHFFFAOYSA-N
ZincZn
Asarco L 15
C.I. Pigment Black 16
Merrillite
NC-Zinc
Rheinzink
Stapa TE Zinc AT
UF (metal)
UN 1436
Zinc dust
Zinc Dust 3
Zinc Dust 500 mesh
Zinc Dust LS 2
Zinc Dust MCS
Zinc Flakes GTT
ZINC METAL
ZINC MOSSY
ZINC STRIP
ZINC, MOSSY
Zincsalt GTT
DTXSID7035012CL:0000129microglial cellCL:0000127astrocyteMP:0003674oxidative stressGO:0008219cell deathMP:0001847brain inflammationGO:0099536synaptic signalingGO:0007612learningGO:0007613memoryGO:0002263cell activation involved in immune responseGO:0002526acute inflammatory response1increased11pathological2decreasedMethylmercuric(II) chloride2016-11-29T18:42:202016-11-29T18:42:20Mercuric chloride2016-11-29T18:42:192016-11-29T18:42:19Acrylamide2017-11-08T11:15:192017-11-08T11:15:19Mercury chloride2018-10-08T04:33:292018-10-08T04:33:29Acetaminophen2016-11-29T18:42:262016-11-29T18:42:26Chloroform2016-11-29T18:42:272016-11-29T18:42:27furan2020-05-01T14:35:222020-05-01T14:35:22Platinum2022-02-04T14:36:542022-02-04T14:36:54Aluminum2022-02-04T14:42:112022-02-04T14:42:11Cadmium2017-10-25T08:33:122017-10-25T08:33:12Mercury2016-11-29T18:42:192016-11-29T18:42:19Uranium2021-08-05T14:28:502021-08-05T14:28:50Arsenic2021-04-27T00:15:212021-04-27T00:15:21Silver 2022-02-03T11:20:112022-02-03T11:20:11Manganese2022-02-04T14:47:232022-02-04T14:47:23Nickel2022-02-04T14:47:592022-02-04T14:47:59Zinc2022-02-04T15:05:002022-02-04T15:05:00nanoparticles2016-12-21T09:40:062016-12-21T09:40:06SARS-CoV2020-03-01T10:42:462020-03-01T10:42:46Sars-CoV-2<p>Virus from the coronaviridae family related to SARS-CoV, 229E, NL63, OC43, HKU1 and MERS.</p>
<p>Transmitted by aerosols</p>
2021-02-23T04:50:402022-09-09T05:09:36Chemical2017-02-07T13:22:422017-02-07T13:22:42Virus2018-05-29T07:10:012018-05-29T07:10:01bacteria2021-02-23T05:15:412021-02-23T05:15:4110116rat10090mouse7955zebra fishWCS_9606humanWCS_9031Gallus gallusWikiUser_26rodents9606Homo sapiensWikiUser_25human and other cells in culture10116Rattus norvegicusWCS_7955zebrafish9541Macaca fascicularisWCS_9606humans10095mice9685catWCS_7227fruit flyWCS_160004gastropodsWikiUser_28VertebratesWikiUser_14MonkeyWCS_90988fathead minnow36500salmonid fishBinding, Thiol/seleno-proteins involved in protection against oxidative stressBinding, SH/SeH proteins involved in protection against oxidative stressMolecular<p style="text-align:justify">In the brain, thiol (SH)- and seleno-containing proteins involved in protection against oxidative stress are mainly located in mitochondria and in the cytoplasm of the different neural cell types (Comini, 2016; Hoppe et al. 2008; Barbosa et al. 2017; Zhu et al. 2017). The main SH-containing peptide involved in protection against oxidative stress is Glutathione (GSH), a tripeptide acting as a cofactor for the enzyme peroxidase and thus serving as an indirect antioxidant donating the electrons necessary for its decomposition of H<sub>2</sub>O<sub>2</sub>. The seleno-containing proteins of interest are: (i) the Glutathione Peroxidase (GPx) family, involved in detoxification of hydroperoxides; (ii) the Thioredoxin Reductase (TrxR) family, involved in the regeneration of reduced thioredoxin (Pillai et al., 2014; ), and the less studied SelH, K, S, R, W, and P selenoproteins <!--[endif]---->(Pisoschi and Pop, 2015, Reeves and Hoffmann, 2009). Binding of chemicals to these proteins induces either their inactivation or favor their degradation (Farina et al. 2009; Zemolin et al. 2012). Of particular importance, the GSH/GPx and thioredoxin (Trx)/TrxR systems are the two main redox regulators of mammalian cells and the disruption of their activities can compromise cell viability (Ren et al. 2016).<!--![endif]----></p>
<ul>
<li>Binding of Hg to thiol groups was analyzed by multiple collector inductively coupled plasma mass spectometry (Wiederhold et al., 2010).</li>
<li>The binding affinity of methylmercury by various selenium-containing lingands was investigated by proton magnetic resonance spectometry (Sugiura et al., 1978; Arnold et al., 1986).</li>
<li>A methylene blue-mediated enzyme biosensor was developed for the detection of mercury-glutathione complex. The biosensor was the enzyme horseradish peroxidase. The binding site of HgCl<sub>2</sub> with the enzyme was a cysteine residue-SH (Han et al., 2001).</li>
<li style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:7pt"><span style="font-size:11.0pt">A photometric method to quantify GSH loss after reactio with organic electrophiles has also been reported (Böhme et al., 2009).</span></span></span></li>
<li>Binding of mercuric chloride to GSH was measured by high performance liquid chromatography (HPLC)-ultraviolet (UV) detection, HPLC-inductively coupled mass spectometry and HPLC-electrospray ionization mass spectometry (Qiao et al., 2017).</li>
<li> Carvalho et al. (2011) determined the binding of MeHg or Hg2+ with purified Thioredoxin Reductase using mass spectrometry. The liquid chromatography was not applied because they have used a pure chemical system, i.e, without living cells.</li>
<li>Mass spectra analysis allowed to measure the binding of mercury chloride and methylmercury to proteins of the mamallian thioredixin system, thioredoxin reductase (Trx) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx) (Carvahlo et al., 2008)</li>
<li>The methodology to detect acrylamide-cysteine adducts has been performed by liquid chromatography coupled to tandem mass spectrometry (Martyniuk et al. 2013). In this paper the authors dected by using a shotgun proteomic approach a total of 15,243 peptides in ACR-exposed N27 cells. And from those 15,243 peptides, 103 peptides (from 100 different proteins) contained acrylamide-cysteine adducts.</li>
</ul>
<p style="text-align:justify">Due to the ubiquitous distribution of the SH-/ and seleno-proteins involved in protection against oxidative stress and inview of the strong affinity of MeHg and Hg<sup>2+</sup> for thiolate and selenolate groups the binding of MeHg and Hg<sup>2+</sup> to thiol and selenol groups is expected to occur in the living cells of all taxonomic groups found in the biosphere.The conservation of these effects across different vertebrate species indicates that thiol- and selenol-containing proteins (particularly, TrxR and GPx) can also be important targets of electrophilic forms of Hg(EpHg<sup>+</sup> or MeHg and Hg<sup>2+</sup>) toxicity in fish and birds (Heinz, 1979; Carvalho et al. 2008b; Heinz et al. 2009; Xu et al.2012, 2016). The disruption of the Trx and GSH systems by MeHg and Hg<sup>2+</sup>have been demonstrated in zebra-sea breams (Branco et al. 2011; 2012a,b) and salmon (<em>Salmo salar, </em>Bernstssen et al. 2003). MeHg can also interfere with the Trx and GSH systems in zebrafish (Yang et al. 2007; Cambier et al. 2012).</p>
UBERON:0000955brainHighFemaleHighMaleHighDuring brain developmentNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot Specified<p style="text-align:justify">Arnold, A.P.,K.-S. Tan, D.L. Rabenstein (1986), Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. 23. Complexation of Methylmercury by Selenohydryl-Containing Amino Acids and Related Molecules. <em>Inorganic Chemistry</em>, 25 (14), pp. 2433-2437.</p>
<p style="text-align:justify">Barbosa, N.V., et al. (2017), Organoselenium compounds as mimics of selenoproteins and thiol modifier agents (2017) Metallomics, 9 (12), pp. 1703-1734.</p>
<p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Boehme, A. et al. (2009), Kinetic gluthathione chemoassay to quantify thiol reactivity of organic electrophiles – Application to a, b-unsaturated ketones, acrylates, and propiolates. <em>Chem. Res. Toxicol</em>. 22(4): 742-50. doi: 10.1021/tx800492x.</span></span></p>
<p style="text-align:justify">Berntssen, M.H, A. Aatland, R.D. Handy (2003), Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr. <em>Aquatic Toxicology</em>. 65, pp.55-72.</p>
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<p> </p>
2017-11-09T04:01:322022-07-15T09:18:22Oxidative Stress Oxidative Stress Molecular<p style="text-align:justify">Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.</p>
<p style="text-align:justify">In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).</p>
<p><span style="font-size:16px"><span style="background-color:white"><span style="color:#2f5597">ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, <span style="background-color:white">catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol</span></span></span><span style="color:#2f5597">, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O<sub>2</sub></span><span style="background-color:white"><span style="color:#2f5597"><span style="background-color:white">. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (</span></span></span></span><span style="color:#2f5597">Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017)<span style="font-size:16px"><span style="background-color:white"><span style="background-color:white">.</span></span></span></span></p>
<p><span style="color:#27ae60"><span style="font-size:18px"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="background-color:white">However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). </span></span></span></span></span></span></p>
<p style="text-align:justify">Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Sources of ROS Production</span></span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Direct Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO<sub>2</sub>*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and more O<sub>2</sub> (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021). </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Indirect Sources:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008). As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O<sub>2</sub>, and inorganic phosphate (P<sub>i</sub>) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria have its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS are also produced through nicotinamide adenine dinucleotide phosphate oxidase (NOX) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A<sub>2</sub> (PLA<sub>2</sub>), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).</span></span></span></span></p>
<p><strong>Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage.</strong><span style="color:#27ae60"> Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed</span></p>
<ul>
<li>Detection of ROS by chemiluminescence <span style="font-size:12px">(<span style="font-family:arial,helvetica,sans-serif">https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)</span></span></li>
<li>Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.</li>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). </li>
<li>TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. </li>
<li>8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015).</li>
</ul>
<p><strong>Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:</strong></p>
<ul>
<li>Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus</li>
<li>Western blot for increased Nrf2 protein levels</li>
<li>Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus</li>
<li>qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)</li>
<li>Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014)</li>
<li>OECD TG422D describes an ARE-Nrf2 Luciferase test method</li>
<li>In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation</li>
</ul>
<p> </p>
<table border="1" cellpadding="1" cellspacing="1">
<tbody>
<tr>
<td><strong>Assay Type & Measured Content</strong></td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics </strong><strong>(Length / Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>ROS Formation in the Mitochondria assay</strong> (Shaki et al., 2012)</p>
</td>
<td>“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”</td>
<td>0, 50, 100 and 200 μM of Uranyl Acetate</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Mitochondrial Antioxidant Content Assay</strong> Measuring GSH content</p>
(Shaki et al., 2012)</td>
<td>“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein.”</td>
<td>
<p>0, 50, 100, or 200 <em>μ</em>M Uranyl Acetate</p>
</td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>H<sub>2</sub>O<sub>2</sub> Production Assay</strong> Measuring H<sub>2</sub>O<sub>2</sub> Production in isolated mitochondria</p>
(Heyno et al., 2008)</td>
<td>“Effect of CdCl<sub>2</sub> and antimycin A (AA) on H<sub>2</sub>O<sub>2</sub> production in isolated mitochondria from potato. H<sub>2</sub>O<sub>2</sub> production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” (</td>
<td>
<p>0, 10, 30  <em>μ</em>M Cd<sup>2+</sup></p>
2  <em>μ</em>M<br />
antimycin A</td>
<td> </td>
</tr>
<tr>
<td>
<p><strong>Flow Cytometry ROS & Cell Viability</strong></p>
(Kruiderig et al., 1997)</td>
<td>“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At <em>t </em>5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”</td>
<td> </td>
<td>
<p>Strong/easy</p>
medium</td>
</tr>
<tr>
<td>
<p><strong>DCFH-DA Assay</strong> Detection of hydrogen peroxide production (Yuan et al., 2016)</p>
</td>
<td>
<p>Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H<sub>2</sub>O<sub>2 </sub>to form fluorescent production. </p>
</td>
<td>0-400 µM</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td>
<p><strong>H2-DCF-DA Assay</strong> Detection of superoxide production (Thiebault et al., 2007)</p>
</td>
<td>This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.</td>
<td>0–600 µM</td>
<td>
<p>Long/ Easy</p>
<p>High accuracy</p>
</td>
</tr>
<tr>
<td><strong>CM-H2DCFDA Assay</strong></td>
<td>**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**</td>
<td> </td>
<td> </td>
</tr>
</tbody>
</table>
<p>Direct Methods of Measurement</p>
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<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Method of Measurement</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">References</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Description</span></span></strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">OECD-Approved Assay</span></span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Chemiluminescence </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Lu, C. et al., 2006; </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as uminol and lucigenin are commonly used to amplify the signal. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
<p> </p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Spectrophotometry </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Direct or Spin Trapping-Based Electron Paramagnetic Resonance (EPR) Spectroscopy </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The unpaired electrons (free radicals) found in ROS can be detected with EPR, and is known as electron paramagnetic resonance. A variety of spin traps can be used. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Nitroblue Tetrazolium Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The Nitroblue Tetrazolium assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis of DHE is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Amplex Red Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescence analysis to measure extramitochondrial or extracellular H<sub>2</sub>O<sub>2</sub> levels. In the presence of horseradish peroxidase and H<sub>2</sub>O<sub>2</sub>, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Dichlorodihydrofluorescein Diacetate (DCFH-DA) </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">An indirect fluorescence analysis to measure intracellular H<sub>2</sub>O<sub>2</sub> levels. H<sub>2</sub>O<sub>2</sub> interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">HyPer Probe </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Fluorescent measurement of intracellular H<sub>2</sub>O<sub>2</sub> levels. HyPer is a genetically encoded fluorescent sensor that can be used for <em>in vivo</em> and<em> in situ </em>imaging. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Cytochrome c Reduction Assay </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The cytochrome c reduction assay is used to measure O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> levels. O</span></span><sub><span style="font-size:12.0pt"><span style="color:#2f5597">2</span></span></sub><span style="background-color:white"><span style="color:#2f5597">•</span></span><sup><span style="font-size:12.0pt"><span style="color:#2f5597">–</span></span></sup><span style="font-size:12.0pt"><span style="color:#2f5597"> is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm. </span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Proton-electron double-resonance imagine (PEDRI)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule. </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Glutathione (GSH) depletion </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Biesemann, N. et al., 2018) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., </span></span><span style="color:#2f5597"><a href="http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html"><span style="font-size:12.0pt"><span style="color:#2f5597">http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html</span></span></a></span><span style="font-size:12.0pt"><span style="color:#2f5597">). </span></span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Thiobarbituric acid reactive substances (TBARS) </span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Griendling, K. K., et al., 2016)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit. </span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Protein oxidation (carbonylation)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Azimzadeh et al., 2017; Azimzadeh etal., 2015; Ping et al., 2020)</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:141px"><span style="color:#27ae60">Seahorse XFp Analyzer </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:151px"><span style="color:#27ae60">Leung et al. 2018 </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:255px"><span style="color:#27ae60">The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR). </span></td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"><span style="color:#27ae60">No </span></td>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Molecular Biology:</span></span></strong><span style="font-size:12.0pt"><span style="color:#2f5597"> Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: </span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Method of Measurement</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">References</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">Description</span></span></strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-size:12.0pt"><span style="color:#2f5597">OECD-Approved Assay</span></span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Immunohistochemistry </span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Amsen, D., de Visser, K. E., and Town, T., 2009)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus </span></span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px">
<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Quantitative polymerase chain reaction (qPCR) </span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:139px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Forlenza et al., 2012)</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences) </span></span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px">
<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis</span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">(Jackson, A. F. et al., 2014)</span></span></span></span></span></p>
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<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway</span></span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; vertical-align:top; width:75px">
<p style="text-align:center"><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#2f5597">No</span></span></span></span></span></p>
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<p><span style="color:#27ae60"><strong>Taxonomic applicability: </strong>Occurrence of oxidative stress is not species specific. </span></p>
<p><span style="color:#27ae60"><strong>Life stage applicability:</strong> Occurrence of oxidative stress is not life stage specific. </span></p>
<p><span style="color:#27ae60"><strong>Sex applicability: </strong>Occurrence of oxidative stress is not sex specific. </span></p>
<p><span style="color:#27ae60"><strong>Evidence for perturbation by prototypic stressor:</strong> There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009). </span></p>
HighMixedHighAll life stagesHighHigh<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ahmad, S. et al. (2021), “60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression”, <em>Journal Insect Science,</em> Vol. 21/5, Oxford University Press, Oxford, </span><a href="https://doi.org/10.1093/jisesa/ieab080" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1093/jisesa/ieab080</a></span></span></p>
<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif">Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, <em>Antioxidants & Redox Signaling</em>, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, <a href="https://doi.org/10.1089/ars.2010.3400" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1089/ars.2010.3400</a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in <em>Inflammation and Cancer</em>, Humana Press, Totowa, </span></span><a href="https://doi.org/10.1007/978-1-59745-447-6_5" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#1155cc">https://doi.org/10.1007/978-1-59745-447-6_5</span></span></span></a> </span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, <em>Journal of Proteome Research</em>, Vol. 14/2, American Chemical Society, Washington, </span></span></span><a href="https://doi.org/10.1021/pr501141b" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1021/pr501141b</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, <em>International Journal of Radiation Biology</em>, Vol. 93/9, Informa, London, </span></span></span><a href="https://doi.org/10.1080/09553002.2017.1339332" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1080/09553002.2017.1339332</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="color:#27ae60">Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, <em>American Journal of Physiology - Cell Physiology</em>, Vol. 318/5, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/ajpcell.00520.2019." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/ajpcell.00520.2019.</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, <em>Journal of ocular pharmacology and therapeutics</em>, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, <a href="https://doi.org/10.1089/jop.2000.16.285.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1089/jop.2000.16.285.</a> </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Biesemann, N. et al., (2018), “High Throughput Screening of Mitochondrial Bioenergetics in Human Differentiated Myotubes Identifies Novel Enhancers of Muscle Performance in Aged Mice”, <em>Scientific Reports, </em>Vol. 8/1,</span></span> <span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Nature Portfolio, London, </span></span><a href="https://doi.org/10.1038/s41598-018-27614-8" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s41598-018-27614-8</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">. </span></span></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in <em>The Pathophysiologic Basis of Nuclear Medicine</em>, Springer, New York, pp. 540-548</span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Fletcher, A. E (2010), “Free radicals, antioxidants and eye diseases: evidence from epidemiological studies on cataract and age-related macular degeneration”, <em>Ophthalmic Research</em>, Vol. 44, Karger International, Basel, <a href="https://doi.org/10.1159/000316476.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1159/000316476.</a> </span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in <em>Cytokine Protocols, </em>Springer, New York, </span></span><a href="https://doi.org/10.1007/978-1-61779-439-1_2" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1007/978-1-61779-439-1_2</span></span></a><strong> </strong></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Forrester, S.J. et al. </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">(2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, <em>Physiological Reviews, </em>Vol. 98/3<strong>,</strong> American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/physrev.00038.201" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/physrev.00038.201</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="color:#27ae60"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814 </span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Times New Roman,Times,serif">Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, <em>Current eye research</em>, Vol. 31/1, Informa, London, <a href="https://doi.org/10.1080/02713680500477347.%22%20/t%20%22_blank" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/02713680500477347.</a> </span></p>
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<p style="margin-left:48px; text-align:left"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Nguyen Dinh Cat, A. et al. (2013), “Angiotensin II, NADPH oxidase, and redox signaling in the vasculature”, <em>Antioxidants & redox signaling,</em> Vol. 19/10<strong>,</strong> </span><span style="background-color:white"><span style="color:black">Mary Ann Liebert, Larchmont, </span></span><a href="https://doi.org/10.1089/ars.2012.4641" style="color:#0563c1; text-decoration:underline"><span style="background-color:white">https://doi.org/10.1089/ars.2012.4641</span></a></span></span></p>
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<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Powers, S.K. and M.J. Jackson. (2008), “Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production”, <em>Physiological Reviews,</em> Vol. 88/4<strong>,</strong> American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/physrev.00031.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/physrev.00031.2007</span></span></a></span></span></p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Raimondi, V., F. Ciccarese and V. Ciminale. (2020), “Oncogenic pathways and the electron transport chain: a dangeROS liason”, <em>British Journal of Cancer, </em>Vol. 122/2, Nature Portfolio, London, </span></span></span><a href="https://doi.org/10.1038/s41416-019-0651-y" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1038/s41416-019-0651-y</span></span></a></span></span></p>
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<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Ungvari, Z. et al. (2013), “Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity”, <em>The Journals of Gerontology Series A: Biological Sciences and Medical Sciences</em>, Vol. 68/12, Oxford University Press, Oxford, </span></span></span><a href="https://doi.org/10.1093/gerona/glt057." style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1093/gerona/glt057.</span></span></a> </span></span></p>
<p style="margin-left:48px"> </p>
<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Vargas-Mendoza, N. et al. (2021), “Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition”, <em>Life, </em>Vol. 11/11, Multidisciplinary Digital Publishing Institute, Basel, </span></span></span><a href="https://doi.org/10.3390/life11111269" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.3390/life11111269</span></span></a></span></span></p>
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<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, <em>Journal of applied physiology</em>, Vol. 106/1, American Physiological Society, Rockville, </span></span></span><a href="https://doi.org/10.1152/japplphysiol.01278.2007" style="color:#0563c1; text-decoration:underline"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">https://doi.org/10.1152/japplphysiol.01278.2007</span></span></a><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"><span style="color:#212121">.</span></span></span></span></span></p>
<p style="margin-left:48px"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="color:black">Zhao, R. Z. et al. (2019), “Mitochondrial electron transport chain, ROS generation and uncoupling”, <em>International journal of molecular medicine</em>, Vol. 44/1, </span><span style="color:black">Spandidos</span><span style="background-color:white"><span style="color:black"> Publishing Ltd</span></span><span style="color:black">., Athens, </span><a href="https://doi.org/10.3892/ijmm.2019.4188" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3892/ijmm.2019.4188</a></span></span></p>
2017-05-30T13:58:172023-03-21T15:16:10Glutamate dyshomeostasisGlutamate dyshomeostasisCellular<p>Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system (CNS), where it plays major roles in multiple aspects, such as development, learning, memory and response to injury (Featherstone, 2010). However, it is well recognized that Glu at high concentrations at the synaptic cleft acts as a toxin, inducing neuronal injury and death (Meldrum, 2000; Ozawa et al., 1998) secondary to activation of glumatergic <em>N</em>-methyl D-aspartate (NMDA) receptors and Ca<sup>2+</sup> influx. Glu dyshomeostasis is a consequence of perturbation of astrocyte/neuron interactions and the transport of this amino acid, as will be discussed below.</p>
<p>Astrocytes are critically involved in neuronal function and survival, as they produce neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), as well as express two main glutamate transporters responsible for the removal of excessive Glu from the synaptic clefts (Chai et al., 2013; Sheldon et al., 2007). Glutamate is the major excitatory neurotransmitter in the CNS, playing a major role in memory and cognitive function (Platt, 1997), and Glu transporters as such prevent the overstimulation of post-synaptic glutamate receptors that lead to excitotoxic neuronal injury (Sattler et al., 2001; Dobble, 1999). Among the five subtypes of Glu transporters identified, glutamate aspartate transporter (GLAST) and Glu transporter-1 (GLT-1) [excitatory amino acid transporter (EAAT) 1 and 2 in humans, respectively], are predominantly expressed in astrocytes. They are responsible for the uptake of excess glutamate from the extracellular space (Furuta et al., 1997; Lehre et al., 1995; Tanaka, 2000), supported by the fact that knockdown of either GLT-1 or GLAST in mice increases extracellular glutamate levels, leading to excitotoxicity related neurodegeneration and progressive paralysis (Bristol and Rothstein, 1996). In the adult brain, EAAT2 accounts for >90% of extracellular glutamate clearance (Danbolt, 2001; Kim et al., 2011; Rothstein et al., 1995), and genetic deletion of both alleles of GLT-1 in mice leads to the development of lethal seizures (Rothstein et al., 1996). On the other hand, EAAT1-3 play a major role during human brain development, in particular in corticogenesis, where they are expressed in proliferative zones and in radial glia, and alterations of Glu transporters contributes to disorganized cortex seen in migration disorders (Furuta et al., 2005;Regan et al., 2007). Indeed, disruption of <a href="http://www.sciencedirect.com/topics/neuroscience/glutamic-acid" title="Learn more about Glutamic acid">glutamate</a> signaling is thought to be part of the etiology underlying some neurodevelopmental disorders such as autism and schizophrenia (<a name="bb0075"></a><a href="http://www.sciencedirect.com/science/article/pii/S0889159116300587?via%3Dihub#b0075">Chiocchetti et al., 2014; Schwartz et al., 2012</a>). Genes involved in glutamatergic pathways, affecting receptor signalling, metabolism and transport, were enriched in genetic variants associated with autism spectrum disorders (Chiocchetti et al, 2014).</p>
<p>Extracellular Glu released by neurons is taken up by astrocytes, which is converted into glutamine (Gln) by glutamine synthetase (GS), a thiol-containing enzyme (cf MIE, Binding to SH-/seleno containing proteins). Intercellular compartmentation of Gln and Glu, the so-called Gln/Glu-GABA cycle (GGC), is critical for optimal CNS function.13C NMR studies have demonstrated that the ratio of Gln/Glu is extremely high and increases with brain activity (Shen et al., 1999). Thus the GGC gives rise to the amino acid neurotransmitters Glu and GABA via dynamic astrocyte neuron interactions. Glu released at synaptic terminals is taken up by surrounding astrocytes via GLT-1 and GLAST (Rothstein et al., 1994; 1996). A small proportion of the astrocytic formed Gln via a reaction mediated by GS is transported into the extracellular space by Gln carriers, with a predominant role for System N/A transporter (SNAT3), which belongs to the bidirectional transporter System N (Chaudhry et al., 2002).</p>
<p>In addition to System N, release of Gln from astrocytes is mediated by other transport systems, including Systems L (LAT2) and ASC (ASCT2). Extracellular Gln is taken up into GABAergic and Glu-ergic neurons by the unidirectional System A transporters SNAT1 (Melone et al., 2004) and SNAT2 (Grewal et al., 2009). Once in neurons, Gln is converted to Glu by the mitochondrial enzyme phosphate-activated glutaminase (Kvamme et al., 2001). Additionally, Glu is packaged into synaptic vesicles by the vesicular VGLUT transporter (Bellocchio et al., 1998), released into the extracellular space and taken up by astrocytes where it is converted back to Gln by GS, thus completing the GGC (<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633698/figure/F1/">Fig. 1</a><u>).</u></p>
<p><img alt="" src="https://aopwiki.org/system/dragonfly/production/2018/02/01/4vdwamei14_Diapositive1.jpg" /></p>
<p>Figure 1: Schematic representation of Glu and Gln transport systems related to the GGC. From Sidorik-Wegrzynowicz and Aschner, 2013)</p>
<p><u> L-Glu transporter activities can be quantified by direct or indirect methods:</u></p>
<ul>
<li>Direct quantification, L-Glu transporter activities are determined by the amount of <sup>3</sup>H-labeled ligand (L-Glu or D-aspartate) taken up by the cells (Primary mixed astrocyte and neuron cultures [Perez-Dominguez et al., 2014]; primary astrocyte cultures [Matos et al., 2008; Li et al., 2006; Hazell et al., 2003]; Xenopus laevis oocyte overexpressing the L-Glu transporter subtype of interest [Sogaard et al., 2013; Trotti et al., 2001]; transfected HeLa cells [Zhang and Qu, 2012]) or tissues (ex. Hippocampal tissue [Selkirk et al., 2005])</li>
<li>Indirect quantification, L-Glu transporter activities are determined by the L-Glu residue in the medium or buffer after incubation with cells expressing the different L-Glu transporters (Brison et al., 2014; Xin et al., 2019; Jin et al., 2015; Gu et al., 2014; Abe et alt.2000], .).</li>
<li>The transport activity of the different L-Glu transporter subtypes should be determined in the presence of the appropriate inhibitors as shown in the table 1. Ex: The glutamate uptake activity via EAAT1 can be determined in the presence of dihydrokainic acid (DHK), a specific inhibitor for GLT-1, as described in Mutkus et al. (2005). Expression level of L-Glu transporter subtypes should be confirmed using Western blotting or immunocytochemistry. It is interesting to note that pure astrocyte culture express only GLAST (EAAT1) (Danbolt et al., 2016); whereas In mixed astrocyte and neuron cultures, GLAST (EAAT1) and GLT-1(EAAT2) are expressed (Danbolt et al., 2016). The expression of GLT1 (EAAT2) is suggested to be induced by soluble factors (Gegelashvili et al., 1997, 2000; Plachez et al., 2000; Martinez-Lozada et al., 2016). </li>
<li>The L-Glu concentrations in medium or in incubation buffer can be quantified by commercially-available kits quantifying the final products of the redox reaction in which L-Glu is a substrate. </li>
</ul>
<p style="margin-left:9.75pt"><u>The kits using colorimetric final products (OD=450 nm):</u></p>
<ul>
<li>Glutamate Assay Kit from Abcam (ab83389)</li>
<li>Glutamate Colorimetric Assay Kit from BioVision (K629)</li>
<li>Glutamate Assay Kit from Merck (MAK004) </li>
</ul>
<p style="margin-left:9.75pt"><u>The kit using the bioluminescent metabolite:</u></p>
<ul>
<li>Glutamate-Glo™ Assay from Promega (J7021)</li>
</ul>
<p><strong>Table 1:</strong> Summary based on the reviews of Murphy-Royal et al., 2017 and Pajarillo et al., 2019, with some modification. Concerning the physiological functions of each EAAT subtype, see the review by Danbolt (Danbolt, 2001).</p>
<table border="1" bordercolor="#ccc" cellpadding="5" cellspacing="0" class="Table" style="border-collapse:collapse">
<tbody>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Human</span></span></strong></span></span></p>
</td>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Rodent</span></span></strong></span></span></p>
</td>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Distribution</span></span></strong></span></span></p>
</td>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Non-specific inhibitors</span></span></strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:27px; vertical-align:top; width:214px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Specific inhibitors</span></span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:71px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT1</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:72px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">GLAST</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:97px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">High expression in astrocytes at developmental stage</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">DL-threo-b-benzyloxyaspartate</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">(TBOA) and its variants (e.g. PMBTBOA and TFB-TBOA)</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">(Bridges et al.,1999; Lebrun et al., 1997; Shimamoto et al., 1998; Shimamoto, 2008)</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">UCPH101 (Erichsen et al., 2010)</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT2</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">GLT-1</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Astrocytes (>90% adult CNS L-Glu uptake)</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Neuronal terminals (hippocampus, cortex, still controversial)</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">WAY213613 (Dunlop et al., 2005)</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">DHK (Arriza et al., 1994; Bridges et al., 1999</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT3</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAC1</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Neurons. Especially high in hippocampal neurons.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Also function as Cys transporter (Watts et al., 2014)</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT4</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT4</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Purkinje cells in the cerebellum</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT5</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">EAAT5</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Retina.</span></span></span></span><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"> Very weak in the CNS</span></span></span></span></p>
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<p> </p>
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<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shimamoto K, Lebrun B, Yasudakamatani Y., Sakaitani M, Shigeri Y, Yumoto N, Nakajima T. DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. 1998; Mol. Pharmacol. 53, 195e201.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shimamoto K. Glutamate transporter blockers for elucidation of the function of excitatory neurotransmission systems, 2008; Chem Rec. 8, 182e199.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Sidoryk-Wegrzynowicz%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23360507">Sidoryk-Wegrzynowicz M</a><sup>1</sup>, <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Aschner%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23360507">Aschner M</a>. Manganese toxicity in the central nervous system: the glutamine/glutamate-γ-aminobutyric acid cycle. <a href="https://www.ncbi.nlm.nih.gov/pubmed/23360507" title="Journal of internal medicine.">J Intern Med.</a> 2013 May;273(5):466-77. doi: 10.1111/joim.12040.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sogaard R, Borre L, Braunstein TH, Madsen KL, MacAulay N. Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1. J Biol Chem. 2013 Jul 12;288(28):20195-207.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Tanaka K. Functions of glutamate transporters in the brain. Neuroscience research. 2000;37:15–19.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Trotti D, Peng JB, Dunlop J, Hediger MA. Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters. Brain Res. 2001 Sep 28;914(1-2):196-203.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Watts SD, Torres-Salazar D, Divito CB, Amara SG, Cysteine transport</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">through excitatory amino acid transporter 3 (EAAT3). PLoS One 2014; 9 e109245.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Xin W, Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase. Cell Rep., May 2019; 31116973.</span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhang X, Qu S. The Accessibility in the External Part of the TM5 of the Glutamate Transporter EAAT1 Is Conformationally Sensitive during the Transport Cycle. PLoS One. 2012; 7(1): e30961.</span></span></p>
<p> </p>
2017-11-09T04:04:172022-07-15T09:45:16Cell injury/deathCell injury/deathCellular<p style="text-align:justify">Two types of cell death can be distinguished by morphological features, although it is likely that these are two ends of a spectrum with possible intermediate forms. Apoptosis involves shrinkage, nuclear disassembly, and fragmentation of the cell into discrete bodies with intact plasma membranes. These are rapidly phagocytosed by neighbouring cells. An important feature of apoptosis is the requirement for adenosine triphosphate (ATP) to initiate the execution phase. In contrast, necrotic cell death is characterized by cell swelling and lysis. This is usually a consequence of profound loss of mitochondrial function and resultant ATP depletion, leading to loss of ion homeostasis, including volume regulation, and increased intracellular Ca2+. The latter activates a number of nonspecific hydrolases (i.e., proteases, nucleases, and phospholipases) as well as calcium dependent kinases. Activation of calpain I, the Ca2+-dependent cysteine protease cleaves the death-promoting Bcl-2 family members Bid and Bax which translocate to mitochondrial membranes, resulting in release of truncated apoptosis-inducing factor (tAIF), cytochrome c and endonuclease in the case of Bid and cytocrome c in the case of Bax. tAIF translocates to cell nuclei, and together with cyclophilin A and phosphorylated histone H2AX (γH2AX) is responsible for DNA cleavage, a feature of programmed necrosis. Activated calpain I has also been shown to cleave the plasma membrane Na+–Ca2+ exchanger, which leads to build-up of intracellular Ca2+, which is the source of additional increased intracellular Ca2+. Cytochrome c in cellular apoptosis is a component of the apoptosome.</p>
<p style="text-align:justify">DNA damage activates nuclear poly(ADP-ribose) polymerase-1(PARP-1), a DNA repair enzyme. PARP-1 forms poly(ADP-ribose) polymers, to repair DNA, but when DNA damage is extensive, PAR accumulates, exits cell nuclei and travels to mitochondrial membranes, where it, like calpain I, is involved in AIF release from mitochondria. A fundamental distinction between necrosis and apoptosis is the loss of plasma membrane integrity; this is integral to the former but not the latter. As a consequence, lytic release of cellular constituents promotes a local inflammatory reaction, whereas the rapid removal of apoptotic bodies minimizes such a reaction. The distinction between the two modes of death is easily accomplished in vitro but not in vivo. Thus, although claims that certain drugs induce apoptosis have been made, these are relatively unconvincing. DNA fragmentation can occur in necrosis, leading to positive TUNEL staining <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">(<span style="font-size:16px">see explanation below</span>)</span></span>. Conversely, when apoptosis is massive, it can exceed the capacity for rapid phagocytosis, resulting in the eventual appearance of secondary necrosis.</p>
<p style="text-align:justify">Two alternative pathways - either extrinsic (receptor-mediated) or intrinsic (mitochondria-mediated) - lead to apoptotic cell death. The initiation of cell death begins either at the plasma membrane with the binding of TNF or FasL to their cognate receptors or within the cell. The latter is due to the occurrence of intracellular stress in the form of biochemical events such as oxidative stress, redox changes, covalent binding, lipid peroxidation, and consequent functional effects on mitochondria, endoplasmic reticulum, microtubules, cytoskeleton, or DNA. The intrinsic mitochondrial pathway involves the initiator, caspase-9, which, when activated, forms an “apoptosome” in the cytosol, together with cytochrome c, which translocates from mitochondria, Apaf-1 and dATP. The apoptosome activates caspase-3, the central effector caspase, which in turn activates downstream factors that are responsible for the apoptotic death of a cell (Fujikawa, 2015). Intracellular stress either directly affects mitochondria or can lead to effects on other organelles, which then send signals to the mitochondria to recruit participation in the death process (Fujikawa, 2015; Malhi et al., 2010).<sup> </sup>Constitutively expressed nitric oxide synthase (nNOS) is a Ca2+-dependent cytosolic enzyme that forms nitric oxide (NO) from L-arginine, and NO reacts with the free radical such as superoxide (O2−) to form the very toxic free radical peroxynitrite (ONOO−). Free radicals such as ONOO−, O2 − and hydroxyl radical (OH−) damage cellular membranes and intracellular proteins, enzymes and DNA (Fujikawa, 2015; Malhi et al., 2010; Kaplowitz, 2002; Kroemer et al., 2009). </p>
<p> </p>
<p><strong>Necrosis:</strong></p>
<p style="text-align:justify">Lactate dehydrogenase (LDH) is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. To detect the leakage of LDH into cell culture medium, a tetrazolium salt is used in this assay. In the first step, LDH produces reduced nicotinamide adenine dinucleotide (NADH) when it catalyzes the oxidation of lactate to pyruvate. In the second step, a tetrazolium salt is converted to a colored formazan product using newly synthesized NADH in the presence of an electron acceptor. The amount of formazan product can be colorimetrically quantified by standard spectroscopy. Because of the linearity of the assay, it can be used to enumerate the percentage of necrotic cells in a sample (Chan et al., 2013). </p>
<p style="text-align:justify">The MTT assay is a colorimetric assay for assessing cell viability. NAD(P)H-dependent cellular oxidoreductase enzymes may reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes include XTT, MTS and the WSTs. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light (Berridgeet al.,2005).</p>
<p style="text-align:justify">Propidium iodide (PI) is an intercalating agent and a fluorescent molecule used to stain necrotic cells. It is cell membrane impermeant so it stains only those cells where the cell membrane is destroyed. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm (Moore et al.,1998)</p>
<p style="text-align:justify">Alamar Blue (resazurin) is a fluorescent dye. The oxidized blue non fluorescent Alamar blue is reduced to a pink fluorescent dye in the medium by cell activity (O'Brien et al., 2000) (12).</p>
<p style="text-align:justify">Neutral red uptake, which is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in lysosomes (Repetto et al., 2008)(13). <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Moreover, quantification of ATP, signaling the presence of metabolically active cells, can be performed (CellTiter-Glo; Promega).</span></span></p>
<p style="text-align:justify">ATP assay: Quantification of ATP, signaling the presence of metabolically active cells (CellTiter-Glo; Promega).</p>
<p style="text-align:justify"><br />
<strong>Apoptosis:</strong></p>
<p style="text-align:justify">TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.</p>
<p style="text-align:justify">Caspase activity assays measured by fluorescence. During apoptosis, mainly caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. Antibodies to the 85 kDa fragment of cleaved PARP or to caspase-3 both serve as markers for apoptotic cells that can be monitored using immunofluorescence (Li, Peng et al., 2004).</p>
<p style="text-align:justify">Hoechst 33342 staining: Hoechst dyes are cell-permeable and bind to DNA in live or fixed cells. Therefore, these stains are often called supravital, which means that cells survive a treatment with these compounds. The stained, condensed or fragmented DNA is a marker of apoptosis (Loo, 2002; Kubbies and Rabinovitch, 1983). </p>
<p style="text-align:justify">Acridine Orange/Ethidium Bromide staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis.</p>
<p>Cell death is an universal event occurring in cells of any species (Fink and Cookson,2005).<sup> </sup></p>
CL:0000255eukaryotic cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHighHigh<ul>
<li>Fujikawa, D.G. (2015), The role of excitotoxic programmed necrosis in acute brain injury, Comput Struct Biotechnol J, vol. 13, pp. 212-221.</li>
<li>Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</li>
<li>Kaplowitz, N. (2002), Biochemical and Cellular Mechanisms of Toxic Liver Injury, Semin Liver Dis, vol. 22, no. 2,<span style="color:#000000"> </span><a class="external free" href="http://www.medscape.com/viewarticle/433631" rel="nofollow" target="_blank"><span style="color:#000000">http://www.medscape.com/viewarticle/433631</span></a><span style="color:#000000"> </span>(accessed on 20 January 2016).</li>
<li>Kroemer, G. et al., (2009), Classification of cell death: recommendations of the Nomenclature Committee on Cell Death, Cell Death Differ, vol. 16, no. 1, pp. 3-11.</li>
<li>Chan, F.K., K. Moriwaki and M.J. De Rosa (2013), Detection of necrosis by release of lactate dehydrogenase (LDH) activity, Methods Mol Biol, vol. 979, pp. 65–70.</li>
<li>Berridge, M.V., P.M. Herst and A.S. Tan (2005), Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, vol. 11, pp 127-152.</li>
<li>Moore, A, et al.(1998), Simultaneous measurement of cell cycle and apoptotic cell death,Methods Cell Biol, vol. 57, pp. 265–278.</li>
<li>Li, Peng et al. (2004), Mitochondrial activation of apoptosis, Cell, vol. 116, no. 2 Suppl,pp. S57-59, 2 p following S59.</li>
<li>Loo, D.T. (2002), TUNEL Assay an overview of techniques, Methods in Molecular Biology, vol. 203: In Situ Detection of DNA Damage, chapter 2, Didenko VV (ed.), Humana Press Inc.</li>
<li>Kubbies, M. and P.S. Rabinovitch (1983), Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes, Cytometry, vol. 3, no. 4, pp. 276–281.</li>
<li>Fink, S.L. and B.T. Cookson (2005), Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells, Infect Immun, vol. 73, no. 4, pp.1907-1916.</li>
<li>O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267(17): 5421-5426.</li>
<li>Repetto G, del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols 3(7): 1125-1131.</li>
</ul>
2016-11-29T18:41:222022-07-15T09:46:25NeuroinflammationNeuroinflammationTissue<p>Neuroinflammation or brain inflammation differs from peripheral inflammation in that the vascular response and the role of peripheral bone marrow-derived cells are less conspicuous. The most easily detectable feature of neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signalling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001), as well as in the production of reactive oxygen (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signalling and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004).</p>
<p>Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006 ; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells scan the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defence), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005; Moehle and West, 2015): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been descibed recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1alpha), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.</p>
<p> </p>
<p><strong>Neuroinflammation and Brain development</strong></p>
<p>During brain development, microglia are known to play a critical role as shapers of neural circuits, by providing trophic factors and by remodeling and pruning synapses (Rajendran and Paolicelli, 2018). In addition to playing a role in synaptic management, microglia are important for the pruning of dying neurons and in the clearance of debris (<a href="#_ENREF_43" title="Harry, 2013 #5042">Harry, 2013</a>). Microglia seem to affect also processes associated with neuronal proliferation and differentiation (Harry and Kraft, 2012). Similarly to microglia, astrocytes have instructive roles in neurogenesis, gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis, and synaptic pruning (Reemst et al., 2016).</p>
<p>The development-dependent reactivity of microglial cells and astrocytes is not well known. Ischemic insult appears to elicit similar microglial reactivity both during brain development and in adulthood (<a href="#_ENREF_3" title="Baburamani, 2014 #6737">Baburamani et al, 2014</a>; <a href="#_ENREF_54" title="Leonardo, 2009 #6879">Leonardo & Pennypacker, 2009</a>). In contrast, treatment with lead acetate was previously shown to result in a more pronounced microglial and astrocyte reactivity in immature 3D rat brain cell cultures as compared to mature ones (<a href="#_ENREF_101" title="Zurich, 2002 #3368">Zurich et al, 2002</a>). Astrocyte reactivity was also more pronounced in immature 3D rat brain cell cultures following paraquat exposure, whereas development-dependent differences in the phenotype of reactive microglia were observed (Sandström et al., 2017). This suggests that neuroinflammation is occurring during brain development and may express a different phenotype than in adulthood, and that dysfunction of microglia and astrocyte during brain development could contribute to neurodevelopmental disorders and potentially to late-onset neuropathology (Reemst et al., 2016).</p>
<p> </p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Neuroinflammation in relation to COVID19</strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 patients with moderate and severe COVID-19 presented an elevated plasma levels of glial fibrillary acidic protein (GFAP), which is known as biochemical indicator of glial activation (Kanberg et al., 2020).</span></span></p>
<p>Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:</p>
<ul>
<li>Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.</li>
<li>The most frequently used astrocyte marker is GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflammatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for staining of astrocytes (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.</li>
<li>All immunocytochemical methods can also be applied to cell culture models.</li>
<li>In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).</li>
<li>Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of activation markers. This can for instance be done by PCR quantification of inflammatory factors, by measurement of the respective mediators, e.g. by ELISA-related immuno-quantification. Such markers include:</li>
<li>Pro- and anti-inflammatory cytokine expression (IL-1β; TNF-α, Il-6, IL-4); or expression of immunostimmulatory proteins (e.g. MHC-II)</li>
<li>Itgam, CD86 expression as markers of M1 microglial phenotype</li>
<li>Arg1, MRC1, as markers of M2 microglial phenotype</li>
</ul>
<p>For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</p>
<p> </p>
<p><strong>Regulatory example using the KE</strong></p>
<p>Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67). It has been used on rare occasions for other toxicant evaluations.</p>
<p>Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure, <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">or SARS-CoV-2 and other coronavirus infection. </span>Some references (non-exhaustive list) are given below for illustration:</span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif">Human: Vennetti et al., 2006</span></p>
<p>Monkey (Macaca fascicularis): Charleston et al., 1994, 1996</p>
<p>Rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002</p>
<p>Mouse: Liu et al., 2012</p>
<p>Zebrafish: Xu et al., 2014.</p>
UBERON:0000955brainHighMixedHighDuring brain development, adulthood and agingHighHighModerateLowModerate<p><span style="font-size:12px">Aguzzi, A., Barres, B.A., Bennett, M.L., 2013. Microglia: scapegoat, saboteur, or something else? Science 339(6116), 156-161.</span></p>
<p><span style="font-size:12px">Aloisi, F., 2001. Immune function of microglia. Glia 36, 165-179.</span></p>
<p><span style="font-size:12px">Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287</span></p>
<p><span style="font-size:12px">Banati, R. B. (2002). "Visualising microglial activation <em>in vivo</em>." Glia 40: 206-217. </span></p>
<p><span style="font-size:12px">Baburamani AA, Supramaniam VG, Hagberg H, Mallard C (2014) Microglia toxicity in preterm brain injury. <em>Reprod Toxicol</em> <strong>48:</strong> 106-112</span></p>
<p><span style="font-size:12px">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:12px">Carson, M.J., Thrash, J.C., Walter, B., 2006. The cellular response in neuroinflammation: The role of leukocytes, microglia and astrocytes in neuronal death and survival. Clin Neurosci Res 6(5), 237-245.</span></p>
<p><span style="font-size:12px">Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.</span></p>
<p><span style="font-size:12px">Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.</span></p>
<p><span style="font-size:12px">Claycomb, K.I., Johnson, K.M., Winokur, P.N., Sacino, A.V., Crocker, S.J., 2013. Astrocyte regulation of CNS inflammation and remyelination. Brain Sci 3(3), 1109-1127.</span></p>
<p><span style="font-size:12px">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></p>
<p><span style="font-size:12px">Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451</span></p>
<p><span style="font-size:12px">Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</span></p>
<p><span style="font-size:12px">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93.</span></p>
<p><span style="font-size:12px">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.</span></p>
<p><span style="font-size:12px">Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</span></p>
<p><span style="font-size:12px">Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.</span></p>
<p><span style="font-size:12px">Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35</span></p>
<p><span style="font-size:12px">Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5</span></p>
<p><span style="font-size:12px">Harry GJ and Kraft AD (2012) Microglia in the developing brain: apotential target with lifetime effects. <a href="https://www.ncbi.nlm.nih.gov/pubmed/22322212" title="Neurotoxicology.">Neurotoxicology.</a> 33(2):191-206.</span></p>
<p><span style="font-size:12px">Harry GJ (2013) Microglia during development and aging. <em>Pharmacology & therapeutics</em> <strong>139:</strong> 313-326</span></p>
<p><span style="font-size:12px">Kanberg N, et al. Neurochemical evidence of astrocytic and neuronal injury commonly found in COVID-19. Neurology. 2020 Sep 22;95(12):e1754-e1759</span></p>
<p><span style="font-size:12px">Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444</span></p>
<p><span style="font-size:12px">Kraft AD, Harry GJ., Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International Journal of Environmental research and Public Health., 2011, 8(7): 2980-3018.</span></p>
<p><span style="font-size:12px">Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360</span></p>
<p><span style="font-size:12px">Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318</span></p>
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<p><span style="font-size:12px">Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969</span></p>
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2016-11-29T18:41:232022-07-15T09:54:27Decrease of neuronal network functionNeuronal network function, DecreasedOrgan<p><strong>Biological state:</strong> There are striking differences in neuronal network formation and function among the developing and mature brain. The developing brain shows a slow maturation and a transient passage from spontaneous, long-duration action potentials to synaptically-triggered, short-duration action potentials.</p>
<p>Furthermore, at this precise developmental stage the neuronal network is characterised by "hyperexcitability”, which is related to the increased number of local circuit recurrent excitatory synapses and the lack of γ-amino-butyric acid A (GABAA)-mediated inhibitory function that appears much later. This “hyperexcitability” disappears with maturation when pairing of the pre- and postsynaptic partners occurs and synapses are formed generating population of postsynaptic potentials and population of spikes followed by developmental GABA switch. Glutamatergic neurotransmission is dominant at early stages of development and NMDA receptor-mediated synaptic currents are far more times longer than those in maturation, allowing more calcium to enter the neurons. The processes that are involved in increased calcium influx and the subsequent intracellular events seem to play a critical role in establishment of wiring of neural circuits and strengthening of synaptic connections during development (reviewed in Erecinska et al., 2004). Neurons that do not receive glutaminergic stimulation are undergoing developmental apoptosis.</p>
<p>During the neonatal period, the brain is subject to profound alterations in neuronal circuitry due to high levels of synaptogenesis and gliogenesis. For example, in neuroendocrine regions such as the preoptic area-anterior hypothalamus (POA-AH), the site of gonadotropin-releasing hormone (GnRH) system is developmentally regulated by glutamatergic neurons. The changes in the expression of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B system begin early in postnatal development, before the onset of puberty, thereby playing a role in establishing the appropriate environment for the subsequent maturation of GnRH neurons (Adams et al., 1999).</p>
<p><strong>Biological compartments:</strong> Neural network formation and function happen in all brain regions but it appears to onset at different time points of development (reviewed in Erecinska et al., 2004). Glutamatergic neurotransmission in hippocampus is poorly developed at birth. Initially, NMDA receptors play important role but the vast majority of these premature glutamatergic synapses are “silent” possibly due to delayed development of hippocampal AMPA receptors. In contrast, in the cerebral cortex the maturation of excitatory glutamatergic neurotransmission happens much earlier. The “silent” synapses disappear by PND 7-8 in both brain regions mentioned above.</p>
<p>There is strong evidence suggesting that NMDA receptor subunit composition controls synaptogenesis and synapse stabilization (Gambrill and Barria, 2011). It is established fact that during early postnatal development in the rat hippocampus, synaptogenesis occurs in parallel with a developmental switch in the subunit composition of NMDA receptors from NR2B to NR2A. It is suggested that early expression of NR2A in organotypic hippocampal slices reduces the number of synapses and the volume and dynamics of spines. In contrast, overexpression of NR2B does not affect the normal number and growth of synapses. However, it does increase spine motility, adding and retracting spines at a higher rate. The C terminus of NR2B, and specifically its ability to bind CaMKII, is sufficient to allow proper synapse formation and maturation. Conversely, the C terminus of NR2A was sufficient to stop the development of synapse number and spine growth. These results indicate that the ratio of synaptic NR2B over NR2A controls spine motility and synaptogenesis, and suggest a structural role for the intracellular C terminus of NR2 in recruiting the signalling and scaffolding molecules necessary for proper synaptogenesis. Interestingly, it was found that genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent, the deletion of NR3A accelerates the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 sugesting that glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (Henson et al., 2012).</p>
<p><strong>General role in biology:</strong> The development of neuronal networks can be distinguished into two phases: an early ‘establishment’ phase of neuronal connections, where activity-dependent and independent mechanisms could operate, and a later ‘maintenance’ phase, which appears to be controlled by neuronal activity (Yuste and Sur, 1999). These neuronal networks facilitate information flow that is necessary to produce complex behaviors, including learning and memory (Mayford et al., 2012).</p>
<p><em>Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? </em></p>
<p><strong>In vivo:</strong> The recording of brain activity by using electroencephalography (EEG), electrocorticography (ECoG) and local field potentials (LFP) assists towards the collection of signals generated by multiple neuronal cell networks. Advances in computer technology have allowed quantification of the EEG and expansion of quantitative EEG (qEEG) analysis providing a sensitive tool for time-course studies of different compounds acting on neuronal networks' function (Binienda et al., 2011). The number of excitatory or inhibitory synapses can be functionally studied at an electrophysiological level by examining the contribution of glutamatergic and GABAergic synaptic inputs. The number of them can be determined by variably clamping the membrane potential and recording excitatory and inhibitory postsynaptic currents (EPSCs or IPSCs) (Liu, 2004).</p>
<p><strong>In vitro:</strong> Microelectrode array (MEA) recordings are also used to measure electrical activity in cultured neurons (Keefer et al., 2001, Gramowski et al., 2000; Gopal, 2003; Johnstone et al., 2010). MEAs can be applied in high throughput platforms to facilitate screening of numerous chemical compounds (McConnell et al., 2012). Using selective agonists and antagonists of different classes of receptors their response can be evaluated in a quantitative manner (Novellino et al., 2011; Hogberg et al., 2011).</p>
<p>Patch clamping technique can also be used to measure neuronal network activity.In some cases, if required, planar patch clamping technique can also be used to measure neuronal networks activity (e.g., Bosca et al., 2014).</p>
<p>In vitro studies in brain slices applying electrophysiological techniques showed significant variability among species (immature rats, rabbits and kittens) related to synaptic latency, duration, amplitude and efficacy in spike initiation (reviewed in Erecinska et al., 2004).</p>
UBERON:0000955brainHighMixedHighDuring brain developmentHighHighHighHigh<p>Adams MM, Flagg RA, Gore AC., Perinatal changes in hypothalamic N-methyl-D-aspartate receptors and their relationship to gonadotropin-releasing hormone neurons. Endocrinology. 1999 May;140(5):2288-96.</p>
<p>Binienda ZK, Beaudoin MA, Thorn BT, Ali SF. (2011) Analysis of electrical brain waves in neurotoxicology: γ-hydroxybutyrate. Curr Neuropharmacol. 9: 236-239.</p>
<p>Bosca, A., M. Martina, and C. Py (2014) Planar patch clamp for neuronal networks--considerations and future perspectives. Methods Mol Biol, 2014. 1183: p. 93-113.</p>
<p>Erecinska M, Cherian S, Silver IA. (2004) Energy metabolism in mammalian brain during development. Prog Neurobiol. 73: 397-445.</p>
<p>Gambrill AC, Barria A. NMDA receptor subunit composition controls synaptogenesis and synapse stabilization. Proc Natl Acad Sci U S A. 2011:108(14):5855-60.</p>
<p>Gopal K. (2003) Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis. Neurotoxicol Teratol. 25: 69-76.</p>
<p>Gramowski A, Schiffmann D, Gross GW. (2000) Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultures on microelectrode arrays. Neurotoxicology 21: 331-342.</p>
<p>Henson MA, Larsen RS, Lawson SN, Pérez-Otaño I, Nakanishi N, Lipton SA, Philpot BD. (2012) Genetic deletion of NR3A accelerates glutamatergic synapse maturation. PLoS One. 7(8).</p>
<p>Hogberg HT, Sobanski T, Novellino A, Whelan M, Weiss DG, Bal-Price AK. (2011) Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons. Neurotoxicology 32: 158-168.</p>
<p>Johnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ. (2010) Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology 31: 331-350.</p>
<p>Keefer E, Norton S, Boyle N, Talesa V, Gross G. (2001) Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays. Neurotoxicology 22: 3-12.</p>
<p>Liu G. (2004) Local structural balance and functional interaction of excitatory and inhibitory synapses in hippocampal dendrites. Nat Neurosci. 7: 373-379.</p>
<p>Mayford M, Siegelbaum SA, Kandel ER. (2012) Synapses and memory storage. Cold Spring Harb Perspect Biol. 4. pii: a005751.</p>
<p>McConnell ER, McClain MA, Ross J, LeFew WR, Shafer TJ. (2012) Evaluation of multi-well microelectrode arrays for neurotoxicity screening using a chemical training set. Neurotoxicology 33: 1048-1057.</p>
<p>Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. (2011) Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals. Front Neuroeng. 4: 4.</p>
<p>Yuste R, Peinado A, Katz LC. (1992) Neuronal domains in developing neocortex. Science 257: 665-669.</p>
2016-11-29T18:41:242018-05-28T11:36:00Impairment, Learning and memoryImpairment, Learning and memoryIndividual<p> </p>
<p>Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.</p>
<p>The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).</p>
<p>Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).</p>
<p>For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.</p>
<p><strong>In laboratory animals:</strong> in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, <span style="color:#3498db">Hebb-Williams maze</span>, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows.</p>
<p>1) RAM, Barnes, MWM, <span style="color:#3498db">Hebb-Williams maze </span>are examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The <span style="color:#3498db">Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004).</span></p>
<p>2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).</p>
<p>3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).</p>
<p>4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001).</p>
<p><span style="color:#3498db">5) Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </span></p>
<p><strong>In humans:</strong> A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include:</p>
<p>1) Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).</p>
<p>2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).</p>
<p>3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).</p>
<p>4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).</p>
<p>5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).</p>
<p>6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).</p>
<p><span style="color:#3498db">7) Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015).</span></p>
<p><strong>In Honey Bees:</strong> For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)."</p>
<p>Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).</p>
<p><span style="color:#3498db"><strong>Life stage applicability: </strong>This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </span></p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong> This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). </span></p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor: </strong>Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). </span></p>
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<p> </p>
2016-11-29T18:41:242023-03-22T16:30:39Tissue resident cell activationTissue resident cell activationCellular<p>Tissue resident cell activation is considered as a hallmark of inflammation irrespective of the tissue type. Strategically placed cells within tissues respond to noxious stimuli, thus regulating the recruitment of neutrophil and the initiation and resolution of inflammation (Kim and Luster, 2015). Examples for these cells are resident immune cells, parenchymal cells, vascular cells, stromal cells, or smooth muscle cells. These cells may be specific for a certain tissue, but they have a common tissue-independent role.</p>
<p>Under healthy conditions there is a homeostatic state, characterized as a generally quiescent cellular milieu. Various danger signals or alarmins that are involved in induction of inflammation like pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs) activate these resident cells in affected tissues. </p>
<p>Examples of well-characterized DAMPs (danger signals or alarmins) (Saïd-Sadier and Ojcius, 2012)</p>
<table border="1" cellpadding="0" cellspacing="3">
<thead>
<tr>
<th>
<p>DAMPs</p>
</th>
<th style="width:193px">
<p>Receptors</p>
</th>
<th style="width:299px">
<p>Outcome of receptor ligation</p>
</th>
</tr>
</thead>
<tbody>
<tr>
<td>
<p>Extracellular nucleotides<br />
(ATP, ADP, adenosine)</p>
</td>
<td style="width:193px">
<p>PI, P2X and P2Y receptors (ATP, ADP); Al, A2A, A2B and A3 receptors (adenosine)</p>
</td>
<td style="width:299px">
<p>Dendritic cell (DC) maturation, chemotaxis, secretion of cytokines (IL-1β, IL-18), inflammation</p>
</td>
</tr>
<tr>
<td>
<p>Extracellular heat shock<br />
proteins</p>
</td>
<td style="width:193px">
<p>CD14, CD91, scavenger<br />
receptors, TLR4, TLR2, CD40</p>
</td>
<td style="width:299px">
<p>DC maturation, cytokine induction, DC, migration to lymph nodes</p>
</td>
</tr>
<tr>
<td>
<p>Extracellular HMGB1</p>
</td>
<td style="width:193px">
<p>RAGE, TLR2, TLR4</p>
</td>
<td style="width:299px">
<p>Chemotaxis, cytokine induction, DC activation, neutrophil recruitment, inflammation, activation of immune cells</p>
</td>
</tr>
<tr>
<td>
<p>Uric acid crystals</p>
</td>
<td style="width:193px">
<p>CD14, TLR2, TLR4</p>
</td>
<td style="width:299px">
<p>DC activation, cytokine induction, neutrophil recruitment, gout induction</p>
</td>
</tr>
<tr>
<td>
<p>Oxidative stress</p>
</td>
<td style="width:193px">
<p>Intracellular redox-sensitive proteins</p>
</td>
<td style="width:299px">
<p>Cell death, release of endogenous DAMPs, inflammation</p>
</td>
</tr>
<tr>
<td>
<p>Laminin</p>
</td>
<td style="width:193px">
<p>Integrins</p>
</td>
<td style="width:299px">
<p>Neutrophil recruitment, chemotaxis</p>
</td>
</tr>
<tr>
<td>
<p>S100 proteins or<br />
calgranulins</p>
</td>
<td style="width:193px">
<p>RAGE</p>
</td>
<td style="width:299px">
<p>Neutrophil recruitment, chemotaxis, cytokine secretion, apoptosis</p>
</td>
</tr>
<tr>
<td>
<p>Hyaluronan</p>
</td>
<td style="width:193px">
<p>TLR2, TLR4, CD44</p>
</td>
<td style="width:299px">
<p>DC maturation, cytokine production, adjuvant activity</p>
</td>
</tr>
</tbody>
</table>
<p>Activation refers to a phenotypic modification of the resident cells that includes alterations in their secretions, activation of biosynthetic pathways, production of pro-inflammatory proteins and lipids, and morphological changes. While these represent a pleiotropic range of responses that can vary with the tissue, there are a number of common markers or signs of activation that are measurable.</p>
<p>Examples of Common markers are</p>
<ul>
<li><span style="color:#3498db">CD11b</span></li>
<li><span style="color:#3498db">Iba1</span></li>
<li><span style="color:#3498db">GFAP</span></li>
<li><span style="color:#3498db">CD68</span></li>
<li><span style="color:#3498db">CD86</span></li>
<li><span style="color:#3498db">Mac-1</span></li>
<li>NF-kB</li>
<li>AP-1</li>
<li>Jnk</li>
<li>P38/mapk</li>
</ul>
<p>These described commonalities allow the use of this KE as a hub KE in the AOP network. However, despite the similarities in the inflammatory process, the type of reactive cells and the molecules triggering their reactivity may be tissue-specific. Therefore, for practical reasons, a tissue specific description of the reactive cells and of the triggering factors is necessary in order to specify in a tissue-specific manner, which cell should be considered and what should be measured.</p>
<p><strong>BRAIN </strong></p>
<p>The most easily detectable feature of brain inflammation or neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines, chemokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signaling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001) (cf KE: pro-inflammatory mediators, increased), as well as in the production of reactive oxygen species (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signaling, and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004). Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells survey the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defense), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been described recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1a), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.</p>
<p><strong>Regulatory examples using the KE</strong></p>
<p>Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67). It has been used on rare occasions for other toxicant evaluations.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Kupffer cells (KCs) are a specialized population of macrophages that reside in the liver; they were first described by Carl Wilhelm von Kupffer (1829–1902) [Haubrich 2004]. KCs constitute 80%-90% of the tissue macrophages in the reticuloendothelial system and account for approximately 15% of the total liver cell population [Bouwens et al., 1986]. They play an important role in normal physiology and homeostasis as well as participating in the acute and chronic responses of the liver to toxic compounds. Activation of KCs results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species. This activation appears to modulate acute hepatocyte injury as well as chronic liver responses including hepatic cancer. Understanding the role KCs play in these diverse responses is key to understanding mechanisms of liver injury [Roberts et al.,2007]. Besides the release of inflammatory mediators including cytokines, chemokines, lysosomal and proteolytic enzymes KCs are a main source of TGF-β1 (transforming growth factor-beta 1, the most potent profibrogenic cytokine). In addition latent TGF-β1 can be activated by KC-secreted matrix metalloproteinase 9 (MMP-9)[Winwood and Arthur, 1993; Luckey and Peeterson, 2001] through the release of biologically active substances that promote the pathogenic process. Activated KCs also release ROS like superoxide generated by NOX (NADPH oxidase), thus contributing to oxidative stress. Oxidative stress also activates a variety of transcription factors like NF-κB, PPAR-γ leading to an increased gene expression for the production of growth factors, inflammatory cytokines and chemokines. KCs express TNF-α (Tumor Necrosis Factor-alpha), IL-1 (Interleukin-1) and MCP-1 (monocyte-chemoattractant protein-1), all being mitogens and chemoattractants for hepatic stellate cells (HSCs) and induce the expression of PDGF receptors on HSCs which enhances cell proliferation. Expressed TNF-α, TRAIL (TNF-related apoptosis-inducing ligand), and FasL (Fas Ligand) are not only pro-inflammatory active but also capable of inducing death receptor-mediated apoptosis in hepatocytes [Guo and Friedman, 2007; Friedman 2002; Roberts et al., 2007]. Under conditions of oxidative stress macrophages are further activated which leads to a more enhanced inflammatory response that again further activates KCs though cytokines (Interferon gamma (IFNγ), granulocyte macrophage colony-stimulating factor (GM-CSF), TNF-α), bacterial lipopolysaccharides, extracellular matrix proteins, and other chemical mediators [Kolios et al., 2006; Kershenobich Stalnikowitz and Weissbrod 2003].</p>
<p>Besides KCs, the resident hepatic macrophages, infiltrating bone marrow-derived macrophages, originating from circulating monocytes are recruited to the injured liver via chemokine signals. KCs appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C+ monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. The profibrotic functions of KCs (HSC activation via paracrine mechanisms) during chronic hepatic injury remain functionally relevant, even if the infiltration of additional inflammatory monocytes is blocked via pharmacological inhibition of the chemokine CCL2 [Baeck et al., 2012; Tacke and Zimmermann, 2014].</p>
<p>KC activation and macrophage recruitment are two separate events and both are necessary for fibrogenesis, but as they occur in parallel, they can be summarised as one KE.</p>
<p>Probably there is a threshold of KC activation and release above which liver damage is induced. Pre-treatment with gadolinium chloride (GdCl), which inhibits KC function, reduced both hepatocyte and sinusoidal epithelial cell injury, as well as decreased the numbers of macrophages appearing in hepatic lesions and inhibited TGF-β1 mRNA expression in macrophages. Experimental inhibition of KC function or depletion of KCs appeared to protect against chemical-induced liver injury [Ide et al.,2005]. </p>
<p><strong><span style="font-size:18px">In General:</span></strong></p>
<p>Measurement <u>targets</u> are cell surface and intracellular markers; the specific markers may be cell and species-specific. </p>
<p>Available <u>methods</u> include cytometry, immunohistochemistry, gene expression sequencing; western blotting, ELISA, and functional assays.</p>
<h2><strong><span style="font-size:18px">BRAIN </span></strong></h2>
<p>Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:</p>
<ol>
<li>Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.</li>
<li>The most frequently used astrocyte marker is glial fibrillary acidic protein, GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflamatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for astrocyte staining (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.</li>
<li>All immunocytochemical methods can also be applied to cell culture models.</li>
<li>In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).</li>
<li>Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of M1/M2 phenotype markers. This can for instance be done by PCR quantification, immunocytochemistry, immunoblotting.</li>
</ol>
<ul>
<li>Itgam, CD86 expression as markers of M1 microglial phenotype</li>
<li>Arg1, MRC1, as markers of M2 microglial phenotype</li>
</ul>
<p style="margin-left:21.3pt">(for descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014)</p>
<p style="margin-left:21.3pt"><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p style="margin-left:21.3pt">Kupffer cell activation can be measured by means of expressed cytokines, e.g. tissue levels of TNF-a [Vajdova et al,2004], IL-6 expression, measured by immunoassays or Elisa (offered by various companies), soluble CD163 [Grønbaek etal., 2012; Møller etal.,2012] or increase in expression of Kupffer cell marker genes such as Lyz, Gzmb, and Il1b, (Genome U34A Array, Affymetrix); [Takahara et al.,2006]</p>
<p>Extend to at least invertebrates</p>
<p>Not to plants and not to single-celled organisms</p>
<p><span style="font-size:14px"><strong>BRAIN:</strong></span></p>
<p>Tissue resident activation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:</p>
<p>Human: Vennetti et al., 2006</p>
<p>Monkey (Macaca fascicularis): Charleston et al., 1994, 1996</p>
<p>Rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002</p>
<p>Mouse: Liu et al., 2012</p>
<p>Zebrafish: Xu et al., 2014.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human: Su et al., 2002; Kegel et al., 2015; Boltjes et al.,2014</p>
<p>Rat: Luckey and Peterson,2001</p>
<p>Mouse: Dalton t al., 2009</p>
<p><span style="color:#3498db"><strong>Life stage applicability:</strong> This key event is mainly applicable to all life stages most evidence is derived from adult models (Betlazar et al., 2016; Paladini et al., 2021). </span></p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong> This key event is not sex specific (Betlazar et al., 2016; Paladini et al., 2021). </span></p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor:</strong> Current literature provides ample evidence of tissue resident cell activation being induced by ionizing radiation (Allen et al., 2020; Krukowski et al., 2018; Parihar et al., 2020; Parihar et al., 2018; Parihar et al., 2016; Poulose et al., 2011; Raber et al., 2019; Sumam et al., 2013). </span></p>
Not SpecifiedAll life stagesNot SpecifiedNot SpecifiedNot SpecifiedNot SpecifiedNot Specified<p><span style="color:#3498db">Allen, B. D. et al. (2020), "Mitigation of helium irradiation-induced brain injury by microglia depletion", Journal of Neuroinflammation, Vol. 17/1, Nature, https://doi.org/10.1186/s12974-020-01790-9. </span></p>
<p><span style="color:#3498db">Betlazar, C. et al. (2016), "The impact of high and low dose ionising radiation on the central nervous system", Redox Biology, Vol. 9, Elsevier, Amsterdam, https://doi.org/10.1016/j.redox.2016.08.002. </span></p>
<p>Chan JK, Roth J, Oppenheim JJ, Tracey KJ, Vogl T, Feldmann M, Horwood N, Nanchahal J., Alarmins: awaiting a clinical response. J Clin Invest. 2012 Aug;122(8):2711-9.</p>
<p>Davies LC, Jenkins SJ, Allen JE, Taylor PR, Tissue-resident macrophages, Nat Immunol. 2013 Oct;14(10):986-95. </p>
<p>Escamilla-Tilch M, Filio-Rodríguez G, García-Rocha R, Mancilla-Herrera I, Mitchison NA, Ruiz-Pacheco JA, Sánchez-García FJ, Sandoval-Borrego D, Vázquez-Sánchez EA, The interplay between pathogen-associated and danger-associated molecular patterns: an inflammatory code in cancer? Immunol Cell Biol. 2013 Nov-Dec;91(10):601-10.</p>
<p>Hussell T, Bell TJ, Alveolar macrophages: plasticity in a tissue-specific context, Nat Rev Immunol. 2014 Feb;14(2):81-93.</p>
<p>Kim ND, Luster AD. The role of tissue resident cells in neutrophil recruitment ,Trends Immunol. 2015 Sep;36(9):547-55.</p>
<p><span style="color:#3498db">Krukowski, K. et al. (2018), "Female mice are protected from space radiation-induced maladaptive responses", Brain, Behavior, and Immunity, Vol. 74, Academic Press Inc., https://doi.org/10.1016/j.bbi.2018.08.008. </span></p>
<p><span style="color:#3498db">Paladini, M. S. et al. (2021), "Microglia depletion and cognitive functions after brain injury: From trauma to galactic cosmic ray", Neuroscience Letters, Vol. 741, Elsevier, Amsterdam, https://doi.org/10.1016/j.neulet.2020.135462. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2016), "Cosmic radiation exposure and persistent cognitive dysfunction", Scientific Reports, Vol. 6/June, Nature Publishing Group, https://doi.org/10.1038/srep34774. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2018), "Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice", Experimental Neurology, Vol. 305, Academic Press Inc., https://doi.org/10.1016/j.expneurol.2018.03.009. </span></p>
<p><span style="color:#3498db">Parihar, V. K. et al. (2020), "Sex-Specific Cognitive Deficits Following Space Radiation Exposure", Frontiers in behavioral neuroscience, Vol. 14, Frontiers, https://doi.org/10.3389/fnbeh.2020.535885. </span></p>
<p><span style="color:#3498db">Poulose, S. M. et al. (2011), "Exposure to 16O-particle radiation causes aging-like decrements in rats through increased oxidative stress, inflammation and loss of autophagy", Radiation Research, Vol. 176/6, BioOne, https://doi.org/10.1667/RR2605.1. </span></p>
<p><span style="color:#3498db">Raber, J. et al. (2019), "Combined Effects of Three High-Energy Charged Particle Beams Important for Space Flight on Brain, Behavioral and Cognitive Endpoints in B6D2F1 Female and Male Mice", Frontiers in physiology, Vol. 10, Frontiers, https://doi.org/10.3389/fphys.2019.00179. </span></p>
<p>Saïd-Sadier N, Ojcius DM., Alarmins, inflammasomes and immunity. Biomed J. 2012 Nov-Dec;35(6):437-49.</p>
<p>Schaefer L, Complexity of danger: the diverse nature of damage-associated molecular patterns, J Biol Chem. 2014 Dec 19;289(51):35237-45.</p>
<p><span style="color:#3498db">Suman, S. et al. (2013), "Therapeutic and space radiation exposure of mouse brain causes impaired dna repair response and premature senescence by chronic oxidant production", Aging, Vol. 5/8, https://doi.org/10.18632/aging.100587. </span></p>
<p><span style="font-size:14px"><strong>BRAIN:</strong></span></p>
<p>Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287</p>
<p>Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.</p>
<p>Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.</p>
<p>Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.</p>
<p>Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</p>
<p>Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93.</p>
<p>Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.</p>
<p>Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</p>
<p>Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.</p>
<p>Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35</p>
<p>Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5</p>
<p>Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444</p>
<p>Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</p>
<p>Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360</p>
<p>Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318</p>
<p>Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.</p>
<p>Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.</p>
<p>Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.</p>
<p>Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.</p>
<p>Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148</p>
<p>Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346</p>
<p>Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19. </p>
<p>Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969</p>
<p>Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.</p>
<p>Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174</p>
<p>Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721</p>
<p>Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389</p>
<p>Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.</p>
<p>Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</p>
<p>Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162</p>
<p>von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.</p>
<p>Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.</p>
<p>Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.</p>
<p>Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p><span style="color:#000000">Baeck, C. et al. (2012), Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury, Gut, vol. 61, no. 3, pp.416–426.</span></p>
<p><span style="color:#000000">Boltjes, A. et al. (2014), The role of Kupffer cells in hepatitis B and hepatitis C virus infections, J Hepatol, vol. 61, no. 3, pp. 660-671.</span></p>
<p><span style="color:#000000">Bouwens, L. et al. (1986), Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver, Hepatology, vol. 6, no. 6, pp. 718-722.</span></p>
<p><span style="color:#000000">Dalton, S.R. et al. (2009), Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice, Biochem Pharmacol, vol. 77, no. 7, pp. 1283-1290.</span></p>
<p><span style="color:#000000">Friedman, S.L. (2002), Hepatic Fibrosis-Role of Hepatic Stellate Cell Activation, MedGenMed, vol. 4, no. 3, pp. 27.</span></p>
<p><span style="color:#000000">Grønbaek, H. et al. (2012), Soluble CD163, a marker of Kupffer cell activation, is related to portal hypertension in patients with liver cirrhosis, Aliment Pharmacol Ther, vol 36, no. 2, pp. 173-180.</span></p>
<p><span style="color:#000000">Guo, J. and S.L. Friedman (2007), Hepatic Fibrogenesis, Semin Liver Dis, vol. 27, no. 4, pp. 413-426.</span></p>
<p><span style="color:#000000">Haubrich, W.S. (2004), Kupffer of Kupffer cells, Gastroenterology, vol. 127, no. 1, p. 16</span></p>
<p><span style="color:#000000">Ide, M. et al. (2005), Effects of gadolinium chloride (GdCl(3)) on the appearance of macrophage populations and fibrogenesis in thioacetamide-induced rat hepatic lesions, J. Comp. Path, vol. 133, no. 2-3, pp. 92–102.</span></p>
<p><span style="color:#000000">Kegel, V. et al. (2015), Subtoxic concentrations of hepatotoxic drugs lead to Kupffer cell activation in a human in vitro liver model: an approach to study DILI, Mediators Inflamm, 2015:640631, </span><a href="http://doi.org/10.1155/2015/640631"><span style="color:#000000">http://doi.org/10.1155/2015/640631</span></a><span style="color:#000000">.</span></p>
<p><span style="color:#000000">Kershenobich Stalnikowitz, D. and A.B. Weissbrod (2003), Liver Fibrosis and Inflammation. A Review, Annals of Hepatology, vol. 2, no. 4, pp.159-163.</span></p>
<p><span style="color:#000000">Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer Cells in the Pathogenesis of Liver Disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</span></p>
<p><span style="color:#000000">Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</span></p>
<p><span style="color:#000000">Møller, H.J. (2012), Soluble CD163.Scand J Clin Lab Invest, vol. 72, no. 1, pp. 1-13.</span></p>
<p><span style="color:#000000">Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.</span></p>
<p><span style="color:#000000">Su, G.L. et al. (2002), Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14, Am J Physiol Gastrointest Liver Physiol, vol. 283, no. 3, pp. G640-645.</span></p>
<p><span style="color:#000000">Tacke, F. and H.W. Zimmermann (2014), Macrophage heterogeneity in liver injury and fibrosis, J Hepatol, vol. 60, no. 5, pp. 1090-1096.</span></p>
<p><span style="color:#000000">Takahara, T et al. (2006), Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis, World J Gastroenterol, vol. 12, no. 40, pp. 6473-6499.</span></p>
<p><span style="color:#000000">Vajdova, K. et al. (2004), Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury, Liver Transpl, vol. 10, no. 4, pp. 520–528</span></p>
<p><span style="color:#000000">Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</span></p>
<p> </p>
2017-11-28T08:59:272023-03-22T16:03:39Increased Pro-inflammatory mediatorsIncreased pro-inflammatory mediatorsTissue<p>Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators.</p>
<p>Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators.</p>
<p>Table1: a non-exhaustive list of examples for pro-inflammatory mediators</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width:253px">
<p><strong>Classes of inflammatory mediators</strong></p>
</td>
<td style="width:361px">
<p><strong>Examples</strong></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Pro-inflammatory cytokines</p>
</td>
<td style="width:361px">
<p>TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Prostaglandins</p>
</td>
<td style="width:361px">
<p>PGE2</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Bradykinin</p>
</td>
<td style="width:361px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Vasoactive amines</p>
</td>
<td style="width:361px">
<p>histamine, serotonin</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive oxygen species (ROS)</p>
</td>
<td style="width:361px">
<p>O<sup>2-</sup>, H<sub>2</sub>O<sub>2</sub></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive nitrogen species (RNS)</p>
</td>
<td style="width:361px">
<p>NO, iNOS</p>
</td>
</tr>
</tbody>
</table>
<p>The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). <span style="color:#2980b9">Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6.</span> In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015).</p>
<p>Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE.</p>
<p style="margin-right:13px; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:7pt"><span style="font-size:11.0pt">Regulatory examples using the KE</span></span></strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:7pt"><span style="font-size:11.0pt">CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7. </span></span></span></p>
<p> </p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail.</p>
<p>Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and</p>
<p>inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules,</p>
<p>and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific</p>
<p>TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006].</p>
<p>TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack</p>
<p>suppressive function and also promote tissue inflammation [Dardalhon et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured <em>in vitro</em>, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system.</p>
<p>The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects.</p>
<p>TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999]</p>
<p>TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998]</p>
<p>In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006]</p>
<p>TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006]</p>
<p>TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]</p>
<p> </p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:11pt">T<span style="font-size:14px">he specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts as used within different AOP descriptions</span></span><span style="font-size:14px">.</span></span></p>
<p><span style="font-size:14px">In general, quantification of inflammatory markers can be done by:</span></p>
<ul>
<li><span style="font-size:14px">qRT-PCR (mRNA expression)</span></li>
<li><span style="font-size:14px">ELISA</span></li>
<li><span style="font-size:14px">Immunocytochemistry</span></li>
<li><span style="font-size:14px">Immunoblotting</span></li>
</ul>
<p><span style="font-size:14px">For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span><br />
</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There are several assays for TGB-β1 measurement available.</p>
<p>e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000]</p>
<p><span style="color:#2980b9">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="color:#2980b9">Assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">Reference </span></p>
</td>
<td>
<p><span style="color:#2980b9">Description </span></p>
</td>
<td>
<p><span style="color:#2980b9">OECD Approved Assay </span></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>
<p><span style="color:#2980b9">RT-qPCR </span></p>
</li>
<li>
<p><span style="color:#2980b9">Q-PCR </span></p>
</li>
</ul>
</td>
<td>
<p><span style="color:#2980b9">(Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Measures mRNA expression of cytokines, chemokines and inflammatory markers </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoblotting (western blotting) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Lee et al., 2008) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Whole blood stimulation assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Thurm & Halsey, 2005) </span></p>
</td>
<td>
<p><span style="color:#2980b9"> Detects inflammatory cytokines in blood </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Imaging tests </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Rollins & Miskolci, 2014) </span></p>
</td>
<td>
<p><span style="color:#2980b9">A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions. </span></p>
<p><span style="color:#2980b9">Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Flow-cytometry </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Karanikas et al., 2000) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Detects the intracellular cytokines with stimulation. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Plate based assay technique using antibodies to detect presence of a protein in a liquid sample. </span></p>
<p><span style="color:#2980b9">Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Inflammatory cytokine arrays </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009) </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunohistochemistry (IHC) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Coons et al., 1942) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human [Santibañez et al., 2011]</p>
<p>Rat [Luckey and Petersen, 2001]</p>
<p>Mouse [Nan et al., 2013]</p>
<p><strong>BRAIN:</strong></p>
<p><span style="font-size:14px">Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span></p>
<p> </p>
<p><span style="color:#2980b9"><strong>Taxonomic applicability</strong>: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020).</span></p>
<p><span style="color:#2980b9"><strong>Life stage applicability</strong>: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016). </span></p>
<p><span style="color:#2980b9"><strong>Sex applicability</strong>: Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020). </span></p>
<p><span style="color:#2980b9"><strong>Evidence for perturbation by a prototypic stressor</strong>: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).</span></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot SpecifiedNot Specified<p> <span style="color:windowtext">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Hamadi N, Sheikh A, Madjid N, Lubbad L, Amir N, Shehab SA, Khelifi-Touhami F, Adem A: Increased pro-inflammatory cytokines, glial activation and oxidative stress in the hippocampus after short-term bilateral adrenalectomy. BMC Neurosci 2016, <strong>17:</strong>61.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Taetzsch T, Levesque S, McGraw C, Brookins S, Luqa R, Bonini MG, Mason RP, Oh U, Block ML (2015) Redox regulation of NF-kappaB p50 and M1 polarization in microglia. Glia 5, <strong>63:</strong>423-440.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Vesce S, Rossi D, Brambilla L, Volterra A (2007) Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation. Int Rev Neurobiol. 82 :57-71.</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext"> <strong>LIVER:</strong></span></span></p>
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</ul>
<p><span style="font-size:14px"><span style="color:windowtext"> </span></span> </p>
<p><span style="font-size:14px"><span style="color:#2980b9">Abdel-Magied, N., S. M., Shedid and Ahmed, A. G. (2019), “Mitigating effect of biotin against irradiation-induced cerebral cortical and hippocampal damage in the rat brain tissue”, Environmental Science and Pollution Research, Vol. 26/13, Springer, London, </span><a href="https://doi.org/10.1007/S11356-019-04806-X" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1007/S11356-019-04806-X</span></a><span style="color:#2980b9">. </span></span></p>
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<p><span style="color:#2980b9"><span style="font-size:14px">Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel, https://doi.org/10.3390/ijms19113669. </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Cho, H. J. et al. (2017), “Role of NADPH Oxidase in Radiation-induced Pro-oxidative and Pro-inflammatory Pathways in Mouse Brain”, International Journal of Radiation Biology, Vol. 93/11, Informa, London, </span><a href="https://doi.org/10.1080/09553002.2017.1377360" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.1080/09553002.2017.1377360</span></a><span style="color:#2980b9">. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Coons, A. H. et al. (1942), “The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody”, The Journal of Immunology, Vol. 45/3, American Association of Immunologists, Minneapolis, pp. 159-169 </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, The Journal of Immunology, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135 </span></span></p>
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<p><span style="color:#2980b9"><span style="font-size:14px">Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2 </span></span></p>
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<p><span style="color:#2980b9"><span style="font-size:14px">Goldsmith, S. J. (1975), "Radioimmunoassay: Review of basic principles", Seminars in Nuclear Medicine, Vol. 5/2, https://doi.org/10.1016/S0001-2998(75)80028-6. </span></span></p>
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<p><span style="color:#2980b9"><span style="font-size:14px">Kalm, M., K. Roughton and K. Blomgren. (2013), "Lipopolysaccharide sensitized male and female juvenile brains to ionizing radiation", Cell Death & Disease, Vol. 4/12, Nature Publishing Group, Berlin, https://doi.org/10.1038/cddis.2013.482. </span></span></p>
<p><span style="color:#2980b9"><span style="font-size:14px">Karanikas, V. et al. (2000), “Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy”, Clinical Cancer Research, Vol. 6/3, American Association for Cancer Research, Philadelphia, pp. 829–837 </span></span></p>
<p><span style="font-size:14px"><span style="color:#2980b9">Kim, S. H. et al. (2002), “Expression of TNF-alpha and TGF-beta 1 in the rat brain after a single high-dose irradiation”, Journal of Korean Medical Science, Vol. 17/2, Korean Medical Association, Seoul, </span><a href="https://doi.org/10.3346/JKMS.2002.17.2.242" rel="noreferrer noopener" target="_blank"><span style="color:#2980b9">https://doi.org/10.3346/JKMS.2002.17.2.242</span></a><span style="color:#2980b9">. </span></span></p>
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<p><span style="color:#2980b9"><span style="font-size:14px">Lee, W. H. et al. (2010), “Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain”, International Journal of Radiation Biology, Vol. 86/2, Informa, London, https://doi.org/10.3109/09553000903419346. </span></span></p>
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2017-11-28T09:00:542023-03-21T15:50:49Decreased protection against oxidative stressProtection against oxidative stress, decreasedCellular<p style="text-align:justify">High levels reactive oxygen species (ROS) can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS, such as glutathione and selenoenzymes.</p>
<p style="text-align:justify"> </p>
<p style="text-align:justify">Glutathione (GSH) is the most abundant low molecular mass thiol compound synthesized in cells, reaching intracellular concentrations of 1–10 mM, and is the major antioxidant and redox buffer in human cells. In fact, GSH serves as a reducing agent for ROS and other unstable molecules generated by catalytic systems, including glutathione peroxidase (GPx)(Forman, 2009).</p>
<p> </p>
<p>Selenium plays a crucial role in antioxidant defense, as one Se atom is absolutely required at the active site of all selenoenzymes, such as GPx and thioredoxin reductase (TrxR), in the form of selenocystein (Rayman, 2000). GPx is an antioxidant enzyme that, in the presence of tripeptide GSH, adds two electrons to reduce H<sub>2</sub>O<sub>2</sub> and lipid peroxides to water and lipid alcohols, respectively, while simultaneously oxidizing GSH to glutathione disulfide. The GPx/GSH system is thought to be a major defense in low-level oxidative stress, and decreased GPx activity or GSH levels may lead to the absence of adequate H<sub>2</sub>O<sub>2</sub> and lipid peroxides detoxification, which may be converted to OH-radicals and lipid peroxyl radicals, respectively, by transition metals (Fe<sup>2+</sup>) (Brigelius-Flohe, 2013). Thioredoxin reductase (TrxR) is essential for maintaining intracellular redox status. The expression of this small (12 kDa) ubiquitous thiol-active protein is induced by ROS and an elevated serum level may indicate a state of oxidative stress. In this regard, TrxR, a NADPH-dependent lipid hydroperoxide reductase, uses NADPH to maintain the levels of reduced Trx via a mechanism similar to that used by GR to maintain GSH levels, contributing to the maintenance of thiol redox homeostasis in proteins. Importantly, the inhibition of TrxR impairs the cyclical regeneration of Trx activity, as Trx remains in the oxidized state <!--[endif]---->(Bjornstedt, 1995, Zhong, 2002). Other, less studied selenoproteins, such as SelP, H, K, S, R, and W selenoproteins, play a role in antioxidant defense <!--[endif]---->(Pisoschi, 2015, Reeves, 2009) <!--![endif]----><!--![endif]----></p>
<ul>
<li>Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html)</li>
<li>Reduction of GPx activity. The activity of GPx can be measured by a colorimetric assay, using a commercially available kit (e.g., Abcam ab102530)</li>
<li>Reduction of TrxR activity. The activity of TrxR can be measured by a colorimetric assay, using a commercially available kit (e.g., Abcam ab83463)</li>
<li>Reduction of Selenoprotein R activity. The methionine sulfoxide reductase activity of SelR can be measured by HPLC (Chen, 2013)</li>
<li>Selenoprotein P depletion. The depletion in SelP can be measured using an ELISA (e.g., MyBiosource #MBS9301054)</li>
<li>Selenoprotein W depletion. The depletion in SelW can be measured using an ELISA (e.g., MyBiosource #MBS9312544)</li>
<li>Selenoprotein S depletion. The depletion in SelS can be measured using an ELISA (e.g., MyBiosource #MBS9306607)</li>
<li>Selenoprotein H and K depletion. The depletion in SelH and K can be measured by western blotting <!--[endif]---->(Lee, 2015, Novoselov, 2007)<!--![endif]----></li>
</ul>
<p>Glutathione, GPx and TrxR are present in bacteria, archea, algae, and in the majority of animals, including humans.</p>
UBERON:0000955brainNot SpecifiedMaleNot SpecifiedFemaleNot SpecifiedAll life stagesNot SpecifiedNot SpecifiedNot Specified<p><span style="font-size:12px">B<span style="font-family:Arial,Helvetica,sans-serif">jornstedt, M., Hamberg, M., Kumar, S., Xue, J., Holmgre, A. (1995) Human thioredoxin reductase directly reduces lipid hydroperoxides by nadph and selenocysteine strongly stimulates the reaction via catalytically generated selenols. <em>J Biol Chem</em> <strong>270</strong>, 11761-11764.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Brigelius-Flohe, R., Maiorino, M. (2013) Glutathione peroxidases. <em>Biochim Biophys Acta</em> <strong>1830</strong>, 3289-3303.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Chen, P.<em> et al.</em> (2013) Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer's Disease. <em>PLoS One</em> <strong>8</strong>, e66384.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Forman, H.J., Zhang, H., Rinna, A. (2009) Glutathione: overview of its protective roles, measurement, and biosynthesis. <em>Mol Aspects Med</em> <strong>30</strong>, 1-12.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Lee, J.H.<em> et al.</em> (2015) Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation. <em>J Biol Chem</em> <strong>290</strong>, 29941-29952.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Novoselov, S.V.<em> et al.</em> (2007) Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern. <em>J Biol Chem</em> <strong>282</strong>, 11960-11968.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Pisoschi, A.M., Pop, A. (2015) The role of antioxidants in the chemistry of oxidative stress: A review. <em>Eur J Med Chem</em> <strong>97</strong>, 55-74.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Rayman, M.P. (2000) The importance of selenium to human health. <em>Lancet</em> <strong>356</strong>, 233-241.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Reeves, M.A., Hoffmann, P.R. (2009) The human selenoproteome: recent insights into functions and regulation. <em>Cell Mol Life Sci</em> <strong>66</strong>, 2457-2478.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Zhong, L., Holmgren, A. (2002) Mammalian thioredoxin reductases as hydroperoxide reductases. <em>Methods Enzymol</em> <strong>347</strong>, 236-243.</span></span></p>
2018-09-13T04:24:062022-07-15T09:28:06abf46be0-eca6-460e-a8a1-3a74fc278d9c8a6d35bc-34cd-4eb7-9c4e-9769d6de41ea<p>Thiol (SH) and selenol (SeH) compounds exhibit reactivity toward electrophiles and oxidants and have high binding affinities for metals <!--[endif]---->(Higdon, 2012; Nagy, 2013; Winterbourn, 2008; Winther, 2014). Glutathione is a thiol-containing tripeptide acting as a cofactor for the enzyme peroxidase and thus serving as an indirect antioxidant donating the electrons necessary for its decomposition of H<sub>2</sub>O<sub>2</sub>, and is also involved in many other cellular functions (Kohen, 2002). Selenoproteins contain selenocysteine amino acid residues. The selenoprotein family is composed of proteins exerting diverse functions, among them several are oxidoreductases classified as antioxidant enzymes <!--[endif]---->(Labunskyy, 2014; Reeves, 2009). Relevant for this KER there are two well-studied selenoprotein families which are described to be expressed in the brain; (i) the Glutathione Peroxidase (GPx) family, involved in detoxification of hydroperoxides; (ii) the Thioredoxin Reductase (TrxR) family, involved in the regeneration of reduced thioredoxin (Pillai, 2014), but also the less studied SelH, K, S, R, W, and P selenoproteins <!--[endif]---->(Pisoschi, 2015; Reeves, 2009).<!--![endif]----><!--![endif]----><!--![endif]----></p>
<p><!--[endif]----><!--[endif]----><!--[endif]----></p>
<p>As summarized in the table 1, binding to the thiol/selenol groups of the selenoproteins cited above can result in structural modifications of these proteins, which in turn inhibits their catalytic activity and thereby reduces or blocks their metabolic capacity to neutralize reactive oxygen species <!--[endif]---->(Fernandes, 1996; Rajanna, 1995). Similarly, binding to the thiol group of glutathione will decrease its anti-oxidant capacity.<!--![endif]----></p>
<!--[endif]---->
<p><img alt="" 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" /></p>
<p> </p>
<p>Figure (Poole, 2015) Structures of cysteinyl and selenocysteinyl residues within proteins. The aminoacyl groups are shown to the left, with dotted lines representing peptide bonds to the next residue on either side. Both protonated (left) and deprotonated (right) forms of these amino acids are depicted with average pKa values.</p>
<!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]---->
<p> </p>
<p> </p>
<p><!--![endif]----><!--![endif]----><!--![endif]----></p>
<p> </p>
<p> </p>
<p><!--[endif]----></p>
<p><br />
<em>GPx family</em></p>
<p><!--[endif]----><!--![endif]----><!--![endif]----></p>
<p>GPxs are tetrameric enzymes. Their thiol groups can either act directly as a reductant, or catalyze reduction of hydrogen peroxide and/or phospholipid hydroperoxides through glutathione co-factors <!--[endif]---->(Hanschmann, 2013, Labunskyy, 2014).<!--![endif]----></p>
<p><!--[endif]----></p>
<p><em>TrxR family</em></p>
<p>TxRs are homodimeric flavoenzymes, which mediate the reduction of oxidized Txn at the expense of NADPH (Birben, 2012). Inhibition of TrxR enzymes has been shown to lead to oxidative stress (Branco, 2017).</p>
<p><em>SelP</em></p>
<p>Downregulation of intracellular SelP by use of small interfering RNA (siRNA) impaired the viability of human astrocytes and made them more susceptible to hydroperoxide-induced oxidative stress, pointing to a direct contribution of SeP to ROS clearance <!--[endif]---->(Steinbrenner, 2006).<!--![endif]----></p>
<p><strong>Table 1</strong></p>
<table border="1" cellpadding="0" cellspacing="0" class="Tabellenraster1" style="border-collapse:collapse; border:none; mso-border-alt:solid windowtext .5pt; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-yfti-tbllook:1184">
<tbody>
<tr>
<td style="width:77.5pt">
<p><strong><span style="color:#1C1C1C">Selenoprotein family</span></strong></p>
</td>
<td style="width:48.05pt">
<p><strong><span style="color:#1C1C1C">Protein name</span></strong></p>
</td>
<td style="width:128.15pt">
<p><strong><span style="color:#1C1C1C">Normal brain function</span></strong></p>
</td>
<td style="width:121.2pt">
<p><strong><span style="color:#1C1C1C">Disruption leading to oxidative stress</span></strong></p>
</td>
<td style="width:89.2pt">
<p><strong><span style="color:#1C1C1C">Reference</span></strong></p>
</td>
</tr>
<tr>
<td style="width:77.5pt">
<p><span style="color:#1C1C1C">Glutathione</span></p>
</td>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">GSH</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">GSH is a major endogenous antioxidant functioning directly in neutralization of free radicals and reactive oxygen compounds. GSH is the reduced form of glutathione and its SH group of cysteine is able to reduce and/or maintain reduced form of other molecules.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Disruptions leads to increased oxidative stress and apoptosis.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">Hall, 1999</span></p>
<p><span style="color:#1C1C1C">Dringen, 2000</span></p>
<p><span style="color:#1C1C1C"> </span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="width:77.5pt">
<p><span style="color:#1C1C1C">Glutathione Peroxidase (GPx) Family</span></p>
</td>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">GPx1</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Peroxide/ROS reduction</span></p>
<p><span style="color:#1C1C1C">(Promotes neuroprotection in response to oxidative challenge).</span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C">Brain expression levels are highest in microglia and lower levels detected in neurons.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Brains of GPx1−/− mice are more vulnerable to mitochondrial toxin treatment, ischemia/ reperfusion, and cold-induced brain injury.</span></p>
<p><span style="color:#1C1C1C">Cultured neurons from GPx1−/− mice were reported to be more susceptible to Aβ-induced oxidative stress, and addition of ebselen reversed this.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">Lindenau, 1998</span></p>
<p><span style="color:#1C1C1C">Klivenyi, 2000</span></p>
<p><span style="color:#1C1C1C">Flentjar, 2002 </span></p>
<p><span style="color:#1C1C1C">Crack, 2001 and 2006</span></p>
<p><span style="color:#1C1C1C"> </span></p>
</td>
</tr>
<tr>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">GPx4</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Reduction of phospholipid</span></p>
<p><span style="color:#1C1C1C">Hydroperoxides.</span></p>
<p><span style="color:#1C1C1C">Only in neurons during normal conditions.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Brains of GPx4+/− mice were shown to have increased lipid peroxidation (a sign of oxidative stress).</span></p>
<p><span style="color:#1C1C1C">Injury-induced GPx4 expression in astrocytes.</span></p>
<p><span style="color:#1C1C1C">In vivo over expression of GPx4 protects against oxidative stress-induced apoptosis.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">Ran, 2004</span></p>
<p><span style="color:#1C1C1C">Borchert, 2006 </span></p>
<p><span style="color:#1C1C1C">Savaskan, 2007 </span></p>
<p><span style="color:#1C1C1C">Chen, 2008</span></p>
<p><span style="color:#1C1C1C"> </span></p>
</td>
</tr>
<tr>
<td style="height:216.1pt; width:77.5pt">
<p><span style="color:#1C1C1C">Thioredoxin Reductase (TrxR) Family</span></p>
</td>
<td style="height:216.1pt; width:48.05pt">
<p><span style="color:#1C1C1C">TrxR1</span></p>
<p><span style="color:#1C1C1C">TrxR2</span></p>
</td>
<td style="height:216.1pt; width:128.15pt">
<p><span style="color:#1C1C1C">Cytocsolic, mitochondrial, nuclear localization. Contribute to the reduction of hydrogen peroxide and oxidative stress, and regulates redox-sensitive</span></p>
<p><span style="color:#1C1C1C">transcription factors that</span></p>
<p><span style="color:#1C1C1C">control cellular transcription</span></p>
<p><span style="color:#1C1C1C">mechanisms.</span></p>
<p><span style="color:#1C1C1C">Regulate the induction of the antioxidant enzyme heme oxygenase 1 (HO-1).</span></p>
</td>
<td style="height:216.1pt; width:121.2pt">
<p><span style="color:#1C1C1C">Overexpression of human Trx1 and Trx2 protects retinal ganglion cells against oxidative stress-induced neurodegeneration.</span></p>
<p><span style="color:#1C1C1C">Exogenously administered human rTrx ameliorates neuronal damage after transient middle cerebral artery occlusion in mice, reduces oxidative/nitrative stress and neuronal apoptosis after cerebral ischemia/reperfusion injury in mice</span></p>
</td>
<td style="height:216.1pt; width:89.2pt">
<p><span style="color:#1C1C1C">Gladyshev, 1996</span></p>
<p><span style="color:#1C1C1C">Zhong, 2000</span></p>
<p><span style="color:#1C1C1C">Hattori, 2004</span></p>
<p><span style="color:#1C1C1C">Trigona, 2006</span></p>
<p><span style="color:#1C1C1C">Papp, 2007</span></p>
<p><span style="color:#1C1C1C">Munemasa, 2008</span></p>
<p><span style="color:#1C1C1C">Arbogast, 2010</span></p>
<p><span style="color:#1C1C1C">Ma, 2012</span></p>
<p><span style="color:#1C1C1C">Burk, 2013</span></p>
<p><span style="color:#1C1C1C">Pitts, 2014</span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C"> </span></p>
</td>
</tr>
<tr>
<td rowspan="6" style="width:77.5pt">
<p><span style="color:#1C1C1C">Other relevant seleno- proteins</span></p>
</td>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">SelH</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Nuclear localization. Redox sensing.</span></p>
</td>
<td style="width:121.2pt">
<p>Hypersensitivity of SelH shRNA HeLa cells to paraquat- and H2O2-induced oxidative stress.</p>
<p><span style="color:#1C1C1C"> </span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">(Panee, 2007)(Novoselov, 2007)</span></p>
<p><span style="color:#1C1C1C">(Wu, 2014)</span></p>
</td>
</tr>
<tr>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">SelK</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Transmembrane protein</span></p>
<p><span style="color:#1C1C1C">localized to the ER membrane.</span></p>
<p><span style="color:#1C1C1C">ER homeostasis and oxidative stress response.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Protects HepG2 cells from ER stress agent-induced apoptosis.</span></p>
<p><span style="color:#1C1C1C">Overexpression of SelK attenuated the intracellular reactive oxygen species level and protected cells from oxidative stress-induced toxicity in cardiomyocytes</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">(Shchedrina, 2011)</span></p>
<p><span style="color:#1C1C1C">(Du, 2010)</span></p>
<p><span style="color:#1C1C1C">(Lu, 2006)</span></p>
</td>
</tr>
<tr>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">SelS</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Transmembrane protein</span></p>
<p><span style="color:#1C1C1C">localized to the ER membrane.</span> <span style="color:#1C1C1C">Catalyze the reduction of disulfide bonds and peroxides.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">SelS overexpression increased astrocyte resistance to ER-stress and inflammatory stimuli, and suppression of SelS compromised astrocyte viability.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">(Liu, 2013)</span></p>
<p><span style="color:#1C1C1C">(Fradejas, 2011)</span></p>
<p><span style="color:#1C1C1C">(Fradejas, 2008)</span></p>
<p><span style="color:#1C1C1C"> (Gao, 2007)</span></p>
</td>
</tr>
<tr>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">MSRB1, SelR, SelX</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Function in reduction of oxidized methionine residues, and actin polymerization.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Induce expression of MSRB1 protects neurons from amyloid β-protein insults in vitro and in vivo.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">(Lee, 2013)</span></p>
<p><span style="color:#1C1C1C">(Moskovitz, 2011)(Pillai, 2014)</span></p>
</td>
</tr>
<tr>
<td style="width:48.05pt">
<p><span style="color:#1C1C1C">SelW</span></p>
</td>
<td style="width:128.15pt">
<p><span style="color:#1C1C1C">Expressed in synapses. Plays an antioxidant role in cells.</span></p>
</td>
<td style="width:121.2pt">
<p><span style="color:#1C1C1C">Rat in vivo overexpression of SelW was shown to protect glial cells against oxidative stress caused by heavy metals and 2,20-Azobis.</span></p>
<p><span style="color:#1C1C1C">Silencing of SelW made neurons more sensitive to oxidative stress.</span></p>
</td>
<td style="width:89.2pt">
<p><span style="color:#1C1C1C">(Reeves, 2009)</span></p>
<p><span style="color:#1C1C1C">(Sun, 2001)</span></p>
<p><span style="color:#1C1C1C">(Loflin, 2006)</span></p>
<p><span style="color:#1C1C1C">(Raman, 2013)</span></p>
<p><span style="color:#1C1C1C">(Chung, 2009)</span></p>
</td>
</tr>
<tr>
<td style="height:10.4pt; width:48.05pt">
<p><span style="color:#1C1C1C">SelP</span></p>
</td>
<td style="height:10.4pt; width:128.15pt">
<p><span style="color:#1C1C1C">Is important for selenium transport, distribution and retention within the brain.</span></p>
<p><span style="color:#1C1C1C">Acts as a ROS-detoxifying enzyme. </span></p>
<p><span style="color:#1C1C1C">Protects human astrocytes from induced oxidative.</span></p>
</td>
<td style="height:10.4pt; width:121.2pt">
<p><span style="color:#1C1C1C">SelP-/- mice show neurological dysfunction and that Se content and GPx activity were reduced within brain, Se supplementation to diet attenuated. neurological dysfunctions.</span></p>
<p><span style="color:#1C1C1C">SelP-/- mice have reported deficits in PV-interneurons due to diminished antioxidant defense capabilities. Decreased neuronal selenoprotein synthesis may be a functional outcome of SelP </span></p>
<p><span style="color:#1C1C1C">Colocalization of Sel P with amyloid plaques</span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C">SelP can function as an antioxidant enzyme against reactive lipid intermediates</span></p>
</td>
<td style="height:10.4pt; width:89.2pt">
<p><span style="color:#1C1C1C">(Steinbrenner, 2009)(Arbogast, 2010)(Zhang, 2008)</span></p>
<p><span style="color:#1C1C1C">(Hill, 2003;Hill, 2004)</span></p>
<p><span style="color:#1C1C1C">(Cabungcal, 2006)</span></p>
<p><span style="color:#1C1C1C">(Pitts, 2012)</span></p>
<p><span style="color:#1C1C1C">(Byrns, 2014)</span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C">(Schomburg, 2003)</span></p>
<p><span style="color:#1C1C1C"> </span></p>
<p><span style="color:#1C1C1C">(Rock, 2010)</span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>Mercury</strong></p>
<p>Thiol- and selenol containing proteins have a high affinity for binding metals which contributes to the target site – brain – distribution of such toxicants (Farina, 2011).</p>
<p>The selenol group (-SeH) of selenocysteines is generally more reactive than thiols (-SH) towards mercury (Sugiura 1976, Khan, 2009). Methyl mercury (MeHg) can target both the GPx and TrxR proteins thereby decreasing protection against oxidative stress and therefore causing increased oxidative stress and neurotoxicity <!--[endif]---->(Branco, 2017, Carvalho, 2008, Farina, 2011).</p>
<p>Note: The binding of HgCl<sub>2</sub> and MeHg is always studied in vitro on the isolated protein, whereas the effects on the activity of the proteins involved in protection against oxidative stress is mostly studied in isolated cells, mitochondrial fractions or in animals. Therefore the concentrations cannot be compared. Binding of Hg to thiol groups and to various selenium-containing proteins: Glutathione, thioredoxin reductase, thioredoxin, glutaredoxin, glutathione reductase was measured using purified proteins (Carvahlo et al., 2008, 2011; Wiederhold et al., 2010; Sugiura et al., 1978; Arnold et al., 1986; Han et al., 2001; Qiao et al., 2017).<!--![endif]----></p>
<p> </p>
<p><!--[endif]----></p>
<p> </p>
<p><strong>Table 2</strong></p>
<table border="1" cellpadding="0" cellspacing="0" style="width:669px">
<tbody>
<tr>
<td style="width:98px">
<p><strong>KE<sub>up</sub></strong></p>
<p><strong>Binding, Thiol/seleno-proteins involved in protection against oxidative stress</strong></p>
</td>
<td style="width:121px">
<p><strong>KE<sub>down</sub></strong></p>
<p><strong>Decreased protection against oxidative stress </strong></p>
<p> </p>
</td>
<td style="width:96px">
<p><strong>Species; in vivo / in vitro</strong></p>
<p> </p>
<p> </p>
</td>
<td style="width:111px">
<p><strong>Stressor</strong></p>
<p> </p>
<p> </p>
</td>
<td style="width:64px">
<p><strong>Dose/ conc. +</strong></p>
<p><strong>Duration of exp.</strong></p>
</td>
<td style="width:108px">
<p><strong>Protective/ aggravating evidence</strong></p>
<p> </p>
</td>
<td style="width:70px">
<p><strong>Reference</strong></p>
<p> </p>
<p> </p>
</td>
</tr>
<tr>
<td style="width:98px">
<p>Binding of 2.5 mol of Hg<sup>2+</sup> /mol of TrX1</p>
<p>(Carvahlo et al., 2008)</p>
</td>
<td style="width:121px">
<p>Inhibition of TrX</p>
<p>Inhibition of TrXR</p>
</td>
<td style="width:96px">
<p>Recombinant rat TrX</p>
<p>HeLa and HEK293 cells</p>
</td>
<td style="width:111px">
<p>HgCl<sub>2</sub></p>
</td>
<td style="width:64px">
<p>IC<sub>50</sub> 7.2 nM</p>
</td>
<td style="width:108px">
<p>Selenite (5 mM)</p>
</td>
<td style="width:70px">
<p>(Carvahlo et al., 2008, 2011)</p>
</td>
</tr>
<tr>
<td style="width:98px">
<p>Binding of 5 mol of Hg<sup>2+</sup> /mol of TrX1</p>
<p>(Carvahlo et al., 2008)</p>
</td>
<td style="width:121px">
<p>Inhibition of TrX</p>
<p>Inhibition of TrXR</p>
</td>
<td style="width:96px">
<p>Recombinant rat TrX</p>
<p>HeLa and HEK293 cells</p>
</td>
<td style="width:111px">
<p>MeHg</p>
</td>
<td style="width:64px">
<p>IC<sub>50</sub> 19.7 nM</p>
</td>
<td style="width:108px">
<p>Selenite (5 mM)</p>
</td>
<td style="width:70px">
<p>(Carvahlo et al., 2008, 2011)</p>
</td>
</tr>
<tr>
<td style="width:98px">
<p>Binding to GR and GrX (Carvahlo et al., 2008)</p>
</td>
<td style="width:121px">
<p>Total inhibition</p>
</td>
<td style="width:96px">
<p>Purified proteins</p>
</td>
<td style="width:111px">
<p>Hg2+</p>
</td>
<td style="width:64px">
<p>10 nM</p>
</td>
<td style="width:108px">
<p> </p>
</td>
<td style="width:70px">
<p>(Carvahlo et al., 2008)</p>
</td>
</tr>
<tr>
<td style="width:98px">
<p>Binding to GR and GrX (Carvahlo et al., 2008)</p>
</td>
<td style="width:121px">
<p>50% of inhibition</p>
</td>
<td style="width:96px">
<p>Purified proteins</p>
</td>
<td style="width:111px">
<p>MeHg</p>
</td>
<td style="width:64px">
<p>80 nM</p>
</td>
<td style="width:108px">
<p> </p>
</td>
<td style="width:70px">
<p>(Carvahlo et al. 2008)</p>
</td>
</tr>
<tr>
<td style="width:98px">
<p> </p>
</td>
<td style="width:121px">
<p>Inhibition of TrxR and GSH activities.</p>
<p>TrxR activity <em>– cytosolic: 0.7 fold; mitochondrial: 0.4 fold)</em></p>
<p> </p>
<p> </p>
<p><em>TrxR1&2 expression – slight decrease, not quantified</em></p>
<p><em>GSH – 0.7-fold</em></p>
</td>
<td style="width:96px">
<p>Human neuroblastoma cells (SH-SY5Y)</p>
</td>
<td style="width:111px">
<p>MeHg</p>
</td>
<td style="width:64px">
<p>1 µM</p>
<p> </p>
</td>
<td style="width:108px"> </td>
<td style="width:70px">
<p>(Branco, 2017)</p>
</td>
</tr>
<tr>
<td style="width:98px"> </td>
<td style="width:121px">
<p>Depletion of GSH levels.</p>
<p><em>GSH-activity:</em></p>
<p><em>10µM – 0.75-fold</em></p>
<p><em>30µM – 0.6-fold</em></p>
<p><em>100µM – 0,5-fold</em></p>
</td>
<td style="width:96px">
<p>Mouse brain mito-chondrial-enriched</p>
<p>fractions</p>
</td>
<td style="width:111px">
<p>MeHg</p>
</td>
<td style="width:64px">
<p>10, 30, and 100 μM</p>
<p> </p>
<p>30 minutes</p>
</td>
<td style="width:108px">
<p>The co-incubation with diphenyl diselenide (100 μM)completely prevented the disruption of mitochondrial activity.</p>
</td>
<td style="width:70px">
<p>(Meinerz, 2011)</p>
</td>
</tr>
<tr>
<td style="width:98px"> </td>
<td style="width:121px">
<p>Depleted GSH levels.</p>
</td>
<td style="width:96px">
<p>Adult male Wistar rats</p>
</td>
<td style="width:111px">
<p>mercuricchloride</p>
</td>
<td style="width:64px">
<p>30ppm in drinking water</p>
</td>
<td style="width:108px"> </td>
<td style="width:70px">
<p>(Agrawal, 2015)</p>
</td>
</tr>
</tbody>
</table>
<p><!--[endif]----><!--[endif]----><!--[endif]----><!--[endif]----></p>
<p> </p>
<p><strong>Acrylamide</strong> (acrylamide is a common food contaminant generated by heat processing)</p>
<p><em>No literature supporting the link “SH/SeH binding leads to decreased protection against oxidative stress” for <strong>acrylamide as stressor</strong> in brain/neural tissue can be found.</em></p>
<p><!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----></p>
<p>Another important group of thiol-containing proteins are the metal-binging detoxifying metallothioneins. This protein family bind mercury and lead, and this binding thus serves as a protective mechanism and also protects against metal toxicity and oxidative stress (Aschner, 2006).</p>
<p>Lactational exposure to methylmercury (10 mg/L in drinking water) significantly increased cerebellar GSH level and GR activity. Possibly a compensatory response to mercury-induced oxidative stress <!--[endif]---->(Franco, 2006).<!--![endif]----></p>
<p>MeHg was shown to inhibit cerebral thioredoxin reductase activity in vitro but not in brain of mice (Wagner et al., 2010). However, it has to be noted that the exposure of mice to MeHg was only 24h.</p>
<p>Inhibition og GR and GrX by Hg2+ and MeHg was observed on the puried protein, but not in HeLa cells incubated with the same concentrations for 24h (Carvahlo et al., 2008).</p>
<p>See Table 2</p>
Not SpecifiedMaleNot SpecifiedFemaleHighAll life stagesNot SpecifiedNot SpecifiedNot Specified<p>Experimental evidences has been observed in rat, mice and human cells (Agrawal, 2015; Meinerz, 2011; Branco, 2017)</p>
<p style="margin-left:21.3pt"><!--[if supportFields]><span
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minor-latin;color:black;mso-themecolor:text1'><span style='mso-element:field-begin'></span><span
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2018-09-13T04:29:322020-02-10T09:40:038a6d35bc-34cd-4eb7-9c4e-9769d6de41ea027ac0d2-2c92-4699-8e71-c493895739f9<p>High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS, including reducing agents, glutathione peroxidases, thioredoxin reductases. Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. Ensuing from this definition, a decrease in cellular antioxidant protection will lead to the increase of oxidative stress.</p>
<p>The cell has important defense mechanisms to protect itself from oxidative stress. The cellular defense mechanisms are numerous and include repair mechanisms, prevention mechanisms, physical defenses, as well as antioxidant defense such as antioxidant enzymes, low-molecular-weight antioxidants and chelating agents (Kohen, 2002). Whenever one or many of these mechanisms are decreased, the balance will tilt towards the production of ROS, and thus generate oxidative stress. In this KER we focus on the decreased protection due to interference with the antioxidant defense system.</p>
<table border="1" cellpadding="0" cellspacing="0" style="width:694px">
<tbody>
<tr>
<td style="width:105px">
<p><a name="_GoBack"></a><strong>KE<sub>up</sub></strong></p>
<p> </p>
<p><strong>Decreased protection against oxidative stress</strong></p>
</td>
<td style="width:121px">
<p><strong>KE<sub>down</sub></strong></p>
<p> </p>
<p><strong>Oxidative stress </strong></p>
</td>
<td style="width:96px">
<p><strong>species; in vivo / in vitro</strong></p>
</td>
<td style="width:116px">
<p><strong>Stressor</strong></p>
</td>
<td style="width:69px">
<p><strong>Dose/ conc. +</strong></p>
<p><strong>Duration of exp.</strong></p>
</td>
<td style="width:110px">
<p><strong>Protective/ aggravating evidence</strong></p>
</td>
<td style="width:76px">
<p><strong>Reference</strong></p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Post-transcriptional effects on GPx1 and TrxR1 expression and activity</p>
<p> </p>
<p><em>TrxR1 – 2-fold</em></p>
<p><em>GPx1 – 0.6-fold</em></p>
</td>
<td style="width:121px">
<p>Disturbance of redox-response and induction of oxidative stress</p>
<p> </p>
<p> </p>
<p><em>SOD – 2-fold</em></p>
<p><em>ROS – increased</em></p>
</td>
<td style="width:96px">
<p>Mouse myoblast C2C12,</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>0.4 µM</p>
<p> </p>
<p>9 h</p>
</td>
<td style="width:110px">
<p>Treatment with ebselen suppressed MeHg-induced oxidative stress</p>
</td>
<td style="width:76px">
<p>(Usuki, 2011)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Inhibition of TrxR and GSH activities</p>
<p> </p>
<p> </p>
<p><em>TrxR1&2 – 0.6-fold</em></p>
<p><em>GSH – 0.7-fold</em></p>
</td>
<td style="width:121px">
<p>Oxidative stress shown by shift in GSSG/GSH ratio</p>
<p> </p>
<p><em>GSSG/GSH – 1.5-fold</em></p>
</td>
<td style="width:96px">
<p>Human neuroblastoma cells (SH-SY5Y)</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>1 µM</p>
</td>
<td style="width:110px">
<p>Se supplementation gave some extent of oxidative stress protection</p>
</td>
<td style="width:76px">
<p>(Branco, 2017)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Inhibition of GPx activity</p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GPx – 0.4-fold</em></p>
</td>
<td style="width:121px">
<p>Increased ROS formation and lipid peroxidation</p>
<p> </p>
<p> </p>
<p><em>ROS – 1.75-fold</em></p>
<p><em>Total peroxidase – 4.5-fold</em></p>
<p><em>Lipid perox. – 3-fold</em></p>
</td>
<td style="width:96px">
<p>Mouse brain</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>40 mg/L in drinking</p>
<p>water</p>
<p> </p>
<p>21-days</p>
</td>
<td style="width:110px">
<p>Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation</p>
</td>
<td style="width:76px">
<p>(Franco, 2009)</p>
</td>
</tr>
<tr>
<td style="height:66px; width:105px">
<p>Inhibition of GPx activity</p>
<p> </p>
<p> </p>
<p><em>GPx – 0.7-fold</em></p>
</td>
<td style="height:66px; width:121px">
<p>Increased ROS formation and lipid peroxidation</p>
<p> </p>
<p><em>Total H<sub>2</sub>O<sub>2</sub> – 1.5-fold</em></p>
</td>
<td style="height:66px; width:96px">
<p>Human neuro-blastoma SH-SY5Y cells.</p>
</td>
<td style="height:66px; width:116px">
<p>MeHg</p>
</td>
<td style="height:66px; width:69px">
<p>1 µM (nominal)</p>
</td>
<td style="height:66px; width:110px">
<p>Inhibition of GPx substantially enhanced MeHg toxicity</p>
</td>
<td style="height:66px; width:76px">
<p>(Franco, 2009)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Decreased GPx1, activity in cerebral cortex and hippocampus</p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GPx1 – 0.5-fold</em></p>
</td>
<td style="width:121px">
<p>Induction of oxidative stress (oxidative damage product from the reaction of ROS and deoxy-thymidine in DNA)</p>
</td>
<td style="width:96px">
<p>Male C57BL/6NJcl mice</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>1.5 mg kg<sup>−1</sup> day<sup>−1</sup></p>
<p> </p>
<p>6-weeks</p>
</td>
<td style="width:110px"> </td>
<td style="width:76px">
<p>(Fujimura, 2017)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Depletion of GSH levels</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GSH-activity:</em></p>
<p><em>10µM – 0.75-fold</em></p>
<p><em>30µM – 0.6-fold</em></p>
<p><em>100µM – 0,5-fold</em></p>
</td>
<td style="width:121px">
<p>Increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS).</p>
<p> </p>
<p><em>Mitochondrial viability</em>:</p>
<p><em>10µM – 0.75-fold</em></p>
<p><em>30µM – 0.6-fold</em></p>
<p><em>100µM – 0,5-fold</em></p>
<p><em>Total hydroperoxidases:</em></p>
<p><em>10µM – 1.0-fold</em></p>
<p><em>30µM – 1.2-fold</em></p>
<p><em>100µM – 1.75-fold</em></p>
</td>
<td style="width:96px">
<p>Mouse brain mitochondrial-enriched fractions</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>10, 30, and 100 μM</p>
<p> </p>
<p>30 minutes</p>
</td>
<td style="width:110px">
<p>The co-incubation with diphenyl diselenide (100 μM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels. thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress</p>
</td>
<td style="width:76px">
<p>(Meinerz, 2011)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Depletion of mono- and disulfide</p>
<p>glutathione in neuronal, glial and mixed cultures</p>
<p> </p>
<p><em>GSH activity – 0.83-fold</em></p>
</td>
<td style="width:121px">
<p>increased reactive oxygen species (ROS) formation measured by dichlorodihydro-fluorescein</p>
<p>(DCF) fluorescence</p>
<p> </p>
<p><em>DCF – 1.2-1.5-fold</em></p>
</td>
<td style="width:96px">
<p>Mouse primary cortical cultures</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>5 µM</p>
<p> </p>
<p>24h</p>
</td>
<td style="width:110px">
<p>glutathione monoethyl ester (GSHME : 100 µM) protects against oxidative stress formation</p>
</td>
<td style="width:76px">
<p>(Rush, 2012)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Reduced glutathione (GSH) content decreased in liver, kidney and brain.</p>
</td>
<td style="width:121px">
<p>Increased lipid peroxidation and generation of reactive oxygen species</p>
</td>
<td style="width:96px">
<p>Adult male albino Sprague-Dawley rat</p>
</td>
<td style="width:116px">
<p>Dimethylmercury (DMM)</p>
</td>
<td style="width:69px">
<p>10 mg/kg bw</p>
<p> </p>
<p>3-days</p>
</td>
<td style="width:110px">
<p>Supplementation with Se (2 mmol/kg and 0.5 mg/kg partially protected against DMM-induced tissue damage.</p>
</td>
<td style="width:76px">
<p>(Deepmala, 2013)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Reduced glutathione (GSH) level and acetyl cholinesterase activity, as well as reduced antioxidant enzyme glutathione peroxidase (GPx)</p>
</td>
<td style="width:121px">
<p>Increased lipid peroxidation level and DNA damage.</p>
</td>
<td style="width:96px">
<p>Adult male Sprague-Dawley rats</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>1 mg kg<sup>-1</sup> orally</p>
<p> </p>
<p>6 months</p>
</td>
<td style="width:110px"> </td>
<td style="width:76px">
<p>(Joshi, 2014)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Depleted GSH levels</p>
</td>
<td style="width:121px">
<p>Antioxidant imbalance and lipid peroxidation.</p>
</td>
<td style="width:96px">
<p>Adult male Wistar rats</p>
</td>
<td style="width:116px">
<p>mercuricchloride</p>
</td>
<td style="width:69px">
<p>30 ppm in drinking water</p>
</td>
<td style="width:110px"> </td>
<td style="width:76px">
<p>(Agrawal, 2015)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>GPx1 significantly decreased prior to neurotoxic effects being visible</p>
<p> </p>
<p><em>GPx1 – 0.7-fold</em></p>
</td>
<td style="width:121px">
<p>Increased lipid peroxidation and later neuronal cell death.</p>
<p> </p>
<p><em>Lipid peroxidation - 1.75-fold</em></p>
</td>
<td style="width:96px">
<p>Primary cultured mouse cerebellar granule cells</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>300 nM nominal</p>
<p> </p>
<p>24h</p>
</td>
<td style="width:110px">
<p>Overexpression of GPx-1 prevented MeHg-induced neuronal death</p>
</td>
<td style="width:76px">
<p>(Farina, 2009)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Reduction of GPx activity and increased glutathione reductase activity</p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GPx – 0.7-fold</em></p>
</td>
<td style="width:121px">
<p>Increased oxidative stress – shown by increased TBA-RS and 8-OHdG content, as well as reduction of complexes I, II, and IV activities</p>
<p> </p>
<p><em>H<sub>2</sub>O<sub>2</sub> – 1.6-fold</em></p>
</td>
<td style="width:96px">
<p>Adult male Swiss albino mice</p>
</td>
<td style="width:116px">
<p>MeHg</p>
</td>
<td style="width:69px">
<p>3–5 µg/g brain tissue</p>
<p> </p>
<p>21-days</p>
</td>
<td style="width:110px">
<p>Treatment with diphenyl diselenide (PhSe)<sub>2</sub> (5 µmol/kg) reversed MeHg’s inhibitory effect on mitochondrial activities, as well as the increased oxidative stress parameters</p>
</td>
<td style="width:76px">
<p>(Glaser, 2013)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Decreased level of GSH in blood, liver, heart, brain, lung and testis</p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GSH – 0.4-0.7 fold</em></p>
</td>
<td style="width:121px">
<p>Lipid peroxidation (increase in malondialdehyde levels in blood, liver, heart, brain, lung and testis)</p>
<p> </p>
<p><em>Lipid peroxidation – 1.4-2.0 fold</em></p>
</td>
<td style="width:96px">
<p>Rats</p>
</td>
<td style="width:116px">
<p>Acrylamide</p>
</td>
<td style="width:69px">
<p>15 mg kg<sup>-1</sup> day<sup>-1</sup></p>
<p>60 days</p>
<p> </p>
<p>gastric gavage</p>
</td>
<td style="width:110px">
<p>All effects prevented by co-treatment with boron</p>
</td>
<td style="width:76px">
<p>(Acaroz, 2018)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Decreased level of GSH in liver, kidney, brain, lung and testis</p>
<p> </p>
<p> </p>
<p><em>GSH – 0.4-0.6 fold</em></p>
</td>
<td style="width:121px">
<p>Lipid peroxidation (increase in malondialdehyde levels liver, kidney, brain, lung and testis)</p>
<p><em>Lipid peroxidation – 1.6-2.0 fold</em></p>
</td>
<td style="width:96px">
<p>Rats</p>
</td>
<td style="width:116px">
<p>Acrylamide</p>
</td>
<td style="width:69px">
<p>40 mg kg<sup>-1</sup> day<sup>-1</sup></p>
<p>10 days</p>
<p>i.p.</p>
</td>
<td style="width:110px">
<p>All effects prevented by co-treatment with resveratrol</p>
</td>
<td style="width:76px">
<p>(Alturfan, 2012)</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Decreased level of GSH and decreased activity of GPx and SOD in cerebellum</p>
<p> </p>
<p> </p>
<p> </p>
<p><em>GSH – 0.5 fold</em></p>
<p><em>GPx</em> <em>– 0.6 fold</em></p>
<p><em>SOD – 0.7 fold</em></p>
<p> </p>
</td>
<td style="width:121px">
<p>Increased lipid peroxidation (MDA) and DNA fragmentation (comet assay) in cerebellum</p>
<p> </p>
<p><em>Lipid peroxidation –1.9 fold</em></p>
</td>
<td style="width:96px">
<p>Rats</p>
</td>
<td style="width:116px">
<p>Acrylamide</p>
</td>
<td style="width:69px">
<p>40 mg kg<sup>-1</sup> day<sup>-1</sup></p>
<p>12 days</p>
<p>gavage</p>
</td>
<td style="width:110px">
<p>All effects prevented by melatonin</p>
</td>
<td style="width:76px">
<p>Pan et al., 2015</p>
</td>
</tr>
<tr>
<td style="width:105px">
<p>Decreased level of GSH/GSSG ratio</p>
<p> </p>
<p><em>GSH/GSSG ratio – 0.85 to 0.70 after 0.5-1 mM, 24h and 0.85 to 0.45 fold after 01-1 mM, 96h</em></p>
</td>
<td style="width:121px">
<p>Increased ROS production, increased lipid peroxidation (4-HNE), increased oxidative DNA damage (8-OHdG)</p>
<p> </p>
<p><em>ROS – 1.5 to 3.2 fold after after 0.5-1 mM, 24h</em></p>
<p><em>4-HNE: 1.6 fold after 1mM, 96h</em></p>
<p><em>8-OHdG: 1.7 and 2.1 fold after 0.1 and 1 mM, 96h</em></p>
<p> </p>
</td>
<td style="width:96px">
<p>Primary cultured mouse astrocytes and microglia</p>
</td>
<td style="width:116px">
<p>Acrylamide</p>
</td>
<td style="width:69px">
<p>0-1 mM</p>
<p>24-96 h</p>
</td>
<td style="width:110px">
<p> </p>
</td>
<td style="width:76px">
<p>Zhao et al., 2017</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>No uncertainties, since a decrease in protection against oxidative stress leads, by definition, to an increase in oxidative stress</p>
<p>Cf table above on Empirical Evidence</p>
HighMaleHighFemaleHighAll life stagesHighHighNot SpecifiedNot Specified<p>The link between decrease in antioxidant protection and induction of oxidative stress can be found in Zebrafish, rodents (mouse and rat) and in man, but may not be restricted to these species.</p>
<p style="margin-left:21.3pt">Acaroz, U.<em> et al.</em> (2018) The ameliorative effects of boron against acrylamide-induced oxidative stress, inflammatory response, and metabolic changes in rats. <em>Food Chem Toxicol</em> <strong>118</strong>, 745-752.</p>
<p style="margin-left:21.3pt">Agrawal, S.<em> et al.</em> (2015) Changes in tissue oxidative stress, brain biogenic amines and acetylcholinesterase following co-exposure to lead, arsenic and mercury in rats. <em>Food Chem Toxicol</em> <strong>86</strong>, 208-216.</p>
<p style="margin-left:21.3pt">Alturfan, A.A.<em> et al.</em> (2012) Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats. <em>Mol Biol Rep</em> <strong>39</strong>, 4589-4596.</p>
<p style="margin-left:21.3pt">Branco, V.<em> et al.</em> (2017) Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury. <em>Redox Biol</em> <strong>13</strong>, 278-287.</p>
<p style="margin-left:21.3pt">Deepmala, J.<em> et al.</em> (2013) Protective effect of combined therapy with dithiothreitol, zinc and selenium protects acute mercury induced oxidative injury in rats. <em>J Trace Elem Med Biol</em> <strong>27</strong>, 249-256.</p>
<p style="margin-left:21.3pt">Farina, M.<em> et al.</em> (2009) Probucol increases glutathione peroxidase-1 activity and displays long-lasting protection against methylmercury toxicity in cerebellar granule cells. <em>Toxicol Sci</em> <strong>112</strong>, 416-426.</p>
<p style="margin-left:21.3pt">Franco, J.L.<em> et al.</em> (2009) Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase. <em>Free Radic Biol Med</em> <strong>47</strong>, 449-457.</p>
<p style="margin-left:21.3pt">Fujimura, M., Usuki, F. (2017) In situ different antioxidative systems contribute to the site-specific methylmercury neurotoxicity in mice. <em>Toxicology</em> <strong>392</strong>, 55-63.</p>
<p style="margin-left:21.3pt">Glaser, V.<em> et al.</em> (2013) Protective effects of diphenyl diselenide in a mouse model of brain toxicity. <em>Chem Biol Interact</em> <strong>206</strong>, 18-26.</p>
<p style="margin-left:21.3pt">Joshi, D.<em> et al.</em> (2014) Reversal of methylmercury-induced oxidative stress, lipid peroxidation, and DNA damage by the treatment of N-acetyl cysteine: a protective approach. <em>J Environ Pathol Toxicol Oncol</em> <strong>33</strong>, 167-182.</p>
<p style="margin-left:21.3pt">Kohen, R., Nyska, A. (2002) Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. <em>Toxicol Pathol</em> <strong>30</strong>, 620-650.</p>
<p style="margin-left:21.3pt">Meinerz, D.F.<em> et al.</em> (2011) Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions. <em>Braz J Med Biol Res</em> <strong>44</strong>, 1156-1163.</p>
<p style="margin-left:21.3pt">Pan, X., et al. (2015) Melatonin attenuates oxidative damage induced by acrylamide invitro and in vivo. <em>Ox. Med. Cell Longevity</em> <strong>Vol 2015</strong>, Article ID 703709.</p>
<p style="margin-left:21.3pt">Rush, T.<em> et al.</em> (2012) Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1. <em>Neurotoxicology</em> <strong>33</strong>, 476-481.</p>
<p style="margin-left:21.3pt">Usuki, F.<em> et al.</em> (2011) Post-transcriptional defects of antioxidant selenoenzymes cause oxidative stress under methylmercury exposure. <em>J Biol Chem</em> <strong>286</strong>, 6641-6649.</p>
<p style="margin-left:21.3pt">Zhao, M et al. (2017) Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model. <em>Toxicol in Vitro</em> <strong>39</strong>, 119-125.</p>
2018-09-13T04:34:342020-02-07T04:27:38027ac0d2-2c92-4699-8e71-c493895739f96587456e-da6c-4c65-8e14-4c2dd61bb9ef<p>In the central nervous system (CNS), glutamate (Glu) is rapidly taken up at the synaptic cleft to mitigate potential excitotoxicity (Meldrum, 2000). Reuptake is carried out by the electrochemical gradient of Glu across the plasma membrane and is accomplished by Glu transporter proteins, referred to as excitatory amino acid transporters (EAATs). These transporter proteins are predominantly expressed in astrocytes, but they are also be found in other neural cells, such as oligodendrocyte, neuron, and microglia membranes (Danbolt, 2001). Functional Glu transporters are located on cell surface membranes. The activities of these transporters are regulated by a redistribution of these proteins to or from the plasma membrane (Robinson 2002), under the control of several signaling pathways. Five different families of EAATs have been recognized (EAAT1–EAAT5). They vary in Na<sup>+</sup> and/or K<sup>+</sup> coupling abilities. Their names differ based on the presence of the transporter in human or in other mammals (see <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343888/table/ijms-18-00353-t004/">Table 1</a>).</p>
<p> </p>
<table border="1" cellpadding="0" cellspacing="0">
<thead>
<tr>
<th>
<p><strong>Transporter (Human)</strong></p>
</th>
<th>
<p><strong>Transporter (Mammals)</strong></p>
</th>
<th>
<p><strong>Occurrence (Cell)</strong></p>
</th>
</tr>
</thead>
<tbody>
<tr>
<td>
<p>EAAT1</p>
</td>
<td>
<p>GLAST</p>
</td>
<td>
<p>Astrocyte, oligodendrocyte, microglia</p>
</td>
</tr>
<tr>
<td>
<p>EAAT2</p>
</td>
<td>
<p>GLT-1</p>
</td>
<td>
<p>Astrocyte, oligodendrocyte</p>
</td>
</tr>
<tr>
<td>
<p>EAAT3</p>
</td>
<td>
<p>EAAC1</p>
</td>
<td>
<p>Neuron (somatodendritic), astrocyte (low)</p>
</td>
</tr>
<tr>
<td>
<p>EAAT4</p>
</td>
<td>
<p>EAAT4</p>
</td>
<td>
<p>Purkinje cell, astrocyte</p>
</td>
</tr>
<tr>
<td>
<p>EAAT5</p>
</td>
<td>
<p>EAAT5</p>
</td>
<td>
<p>Müller cell (retina)</p>
</td>
</tr>
</tbody>
</table>
<p>Table 1: Glu transporters in human and mammals and their occurrence in CNS cells. From Rajda et al., 2017</p>
<p> </p>
<p>These transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various 'energy-generating' systems, including the Na(+)/K(+)-ATPase, the Na<sup>+</sup>/Ca<sup>2</sup><sup>+</sup> exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the 'fuel' to support glutamate uptake (Robinson and Jackson, 2016). The Na<sup>+ </sup>gradient, which depends on Na/K ATPase pump and consequently of ATP production and intracellular levels, provides the energy to move Glu from the outside into the cells, accompanied by two Na<sup>+</sup> and an H<sup>+</sup> ; at the same time, K<sup>+</sup> moves in the opposite direction (Boron and Boulpaep, 2003). Mitochondrial dysfunction leads to a decrease in ATP synthesis, impaired Ca<sup>2+</sup> content, and concomitant increase in the levels of ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species) (Beal, 2005). Free radicals, which are electrically unstable, have a central role in several physiological and pathological processes. Both ROS and RNS originate from endogenous and exogenous sources. Mitochondria, endoplasmic reticulum, peroxisomes, phagocytic cells, and others serve as endogenous sources, and environmental factors, such as alcohol, tobacco, pollution, industrial solvents, pesticides, heavy metals, specified medicines, etc. make up the prepondarance of exogenous factors. Significant amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS,) are formed during oxidative phosphorylation, when the greatest amount of ATP is produced. Cellular antioxidants production serves as a countermeasure against this process (Su et al., 2013; Szalardi et al., 2015). Most cells, including astrocytes, have protective mechanisms against ROS, predominantly in the form of the tripeptide thiol, glutathione (GSH) (Hsie et al., 1996). This process stays in a highly sensitive balance. In the specific case when ROS and RNS synthesis exceeds antioxidant synthesis it results in oxidative stress (Reddy, 2006; Ghafouribar et al., 2008; Su et al., 2013; Szalardi et al., 2015; Valko et al., 2007; Yankovskaya et al., 2003; Senoo-Matsuda et al., 2003; Schon and Manfredi, 2003).</p>
<p>Due to the tight coupling of Glu transporters with energy production, and to the important role of Glu transporters in Glu homeostasis, perturbations of energy metabolism such as mitochondrial dysfunction and increased production of ROS lead to Glu dyshomeostasis (Boron and Boulpaep, 2003). In particular, it was shown that ROS inhibit glutamate uptake by astrocytes (Sorg et al., 1997), and that <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697293/"><span style="color:#000000"> glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels</span></a><span style="color:#000000"> </span>(Liu et al., 2009).</p>
<p><strong>Porciuncula et al., (2003).</strong> Methylmercury (2-10 µM) inhibits glutamate uptake in synaptic vesicles isolated from rat brain in a concentration-dependent manner (with LD<sub>50</sub> at 50 µM). It also inhibits the H<sup>+</sup>-ATPase activity in a concentration-dependent manner with similar LD<sub>50</sub>. This suggests that the vesicular glutamate uptake is impaired by methylmercury and that this effect involves the H<sup>+</sup>-ATPase.</p>
<p><strong>Roos et al., (2009).</strong> Methylmercury induced ROS production in rat brain cortical slices after 2h exposure at 100 and 200 µM and after 5h exposure at 50 µM. Guanosine (0.5 - 5 µM), ebselen (1-5 µM) and diphenyl diselenide (1-5 µM) blocked the methylmercury-induced ROS production. The inhibitor of NMDA receptors, MK801 (50 µM) equally blocked the methylmercury-induced ROS production by two potential mechanisms of action: (i) mercury by affecting mitochondria iincreased ROS formation, which decrease glutamate uptake and consequently increased extracellular glutamate acting on NMDA receptors; (ii) The ROS formation is secondary to overstimulation of NMDA receptors, due to mercury-induced decrease in glutamate uptake.</p>
<p><strong>Roos et al., (2011).</strong> Experiments performed in isolated mitochondria from rat liver slices showed that methylmercury (25 µM) increased ROS production (measured by dichlorofluoroscein). Methionine treatment (50-250 µM) was effective in reducing ROS formation.</p>
<p><strong>Juarez et al., (2002)</strong> Microdialysis probe in adult Wistar rats showed that acute exposure to methylmercury (10, 100 µM) induced an increase release of extracellular glutamate (9.8 fold at 10 µM and 2.4 fold at 100 µM). This extracellular glutamate level remained elevated at least 90 min following methylmercury exposure.</p>
<p><strong>Allen et al., (2001).</strong> Cerebral cortical astrocytes were treated with methylmercury (1 µM for 24h or 10 µM for 30 min) and loaded with [U-<sup>13</sup>C] glutamate. In the methylmercruy-treated group, a decrease of [U-<sup>13</sup>C] lactate was observed. This lactate can only be derived from mitochondrial metabolism, via the tricarboxylic acid, showing a link between mitochondrial dysfunction and glutamate metabolism. In addition, the decreased lactate production might be detrimental to surrounding cells, since lactate has been shown to be an important substrate for neurons.</p>
<p>The relationship between oxidative stress associated to mitochondrial dysfunction and glutamate dyshomeostasis is complex and may be bidirectional. Glutamate dysfunction, due to decreased glutamate uptake, can secondarly induce increased ROS production and consequently oxidative stress.</p>
<p>The astrocytic enzyme glutamine synthetase (GS), transforming glutamate in glutamine, which is taken up by neurons, is also a SH-containing protein, which is inhibited by mercury binding (Kwon and Park, 2003). This participate to glutamate dyshomeostasis linking this KE directly to the MIE.</p>
<p>According to <strong>Porciuncula et al., (2003), </strong>a decrease of 50% of H<sup>+</sup>-ATP activity was associated to a decrease of 50% of glutamate uptake following exposure of synaptic vesicles with 5 uM of methylmercury.</p>
<p><strong>Xu et al. (2012)</strong> and <strong>Feng et al. (2014)</strong> observed that in rats treated with 12 umoles/kg for 4 weeks a 4-fold increase in ROS level in cerebral cortex, and a 2-fold increase in protein and DNA peroxidation were associated with about 20% increase of glutamate and 30% decrease of glutamine.</p>
HighMaleHighFemaleNot SpecifiedAll life stagesHighHigh<p>Experimental evidences has been observed mainly in rodent, but due to occurrence of oxidative stress and the presence of glutamate in different taxa, it may be much broader, as suggested by similar observations in C. elegans (Wu et al., 2015).</p>
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<p>Xu, B., Xu, Z.F., Deng, Y., Liu, W., Yang, H.B., Wei, Y.G., 2012. Protective effects of MK-801 on methylmercury-induced neuronal injury in rat cerebral cortex: involvement of oxidative stress and glutamate metabolism dysfunction. Toxicology 300, 112-120.</p>
<p>Yankovskaya V., Horsefield R., Törnroth S., Luna-Chavez C., Miyoshi H., Léger C., Byrne B., Cecchini G., Iwata S. 2003. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science.299:700–704. doi: 10.1126/science.1079605.</p>
2017-11-09T04:10:292020-02-06T11:57:386587456e-da6c-4c65-8e14-4c2dd61bb9efeb624ffb-7315-4087-82d2-94e1a4f9d480<p>Glutamate is the major excitatory neurotransmitter in the mammalian CNS, where it plays key roles in development, learning, memory and response to injury. However, glutamate at high concentrations at the synaptic cleft acts as a toxin, inducing neuronal injury and death <!--[endif]---->(Meldrum, 2000; Ozawa et al., 1998). Glutamate-mediated neurotoxicity has been dubbed as “excitotoxicity”, referring to the consequence of the overactivation of the <em>N</em>-methyl D-aspartate (NMDA)–type glutamate receptors (cf AOP 48), leading to increased Na<sup>+</sup> and Ca<sup>2+</sup> influx into neurons <!--[endif]---->(Choi, 1992; Pivovarova and Andrews, 2010). Increased intracellular Ca<sup>2+</sup> levels are associated with the generation of oxidative stress and neurotoxicity <!--[endif]---->(Lafon-Cazal et al., 1993). Accordingly, the control of extracellular levels of glutamate dictates its physiological/pathological actions and this equilibrium is maintained primarily by the action of several glutamate transporters (such as GLAST, GLT1, and EAAC1) located on astrocytic cell membranes, which remove the excitatory neurotransmitter from the synaptic cleft, keeping its extracellular concentrations below toxic levels <!--[endif]---->(Anderson and Swanson, 2000; Maragakis and Rothstein, 2001; Szydlowska and Tymianski, 2010).<!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----></p>
<p><!--[endif]----><!--[endif]----><!--[endif]----><!--[endif]----></p>
<p> </p>
<p>In addition to synaptic transmission, physiological stimulation of glutamate receptors can mediate trophic effects and promote neuronal plasticity. During development, NMDA receptors initiate a cascade of signal transduction events and gene expression changes primarily involving Ca<sup>2+</sup>-mediated signaling, induced by activation of either Ca<sup>2+</sup>- permeable receptor channels or voltage-sensitive Ca<sup>2+</sup> channels. The consecutive activation of major protein kinase signaling pathways, such as Ras-MAPK/ERK and PI3-K-Akt, contributes to regulation of gene expression through the activation of key transcription factors, such as CREB, SRF, MEF-2, NF-kappaB. Metabotropic glutamate receptors can also engage these signaling pathways, in part by transactivating receptor tyrosine kinases. Indirect effects of glutamate receptor stimulation are due to the release of neurotrophic factors, such as brain derived neurotrophic factor through glutamate-induced release of trophic factors from glia. The trophic effect of glutamate receptor activation is developmental stage-dependent and may play an important role in determining the selective survival of neurons that made proper connections. During this sensitive developmental period, interference with glutamate receptor function may lead to widespread neuronal loss (Balazs, 2006).</p>
<p><!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----></p>
<p>Glutamate dyshomeostasis and in particular excess of glutamate in the synaptic cleft will lead to overactivation of ionotropic glutamate receptors and cause cell injury/death, as described in AOP 48. The excess of glutamate can result from decreased uptake in astrocytes <!--[endif]---->(Aschner et al., 2000; Brookes and Kristt, 1989), or neurons <!--[endif]---->(Moretto et al., 2005; Porciuncula et al., 2003). But also from the increased release (Reynolds and Racz, 1987). This neurotoxic cascade involves calcium overload and ROS production leading to oxidative stress <!--[endif]---->(Ceccatelli et al., 2010; Lafon-Cazal, 1993; Meldrum, 2000; Ozawa, 1998). Chemicals binding to sulfhydryl (SH)-/seleno-proteins cause a direct oxidative stress by perturbing mitochondrial respiratory chain proteins and by decreasing anti-oxidant defense mechanism (see KER : MIE to KEdown oxidative stress) and an indirect oxidative stress via perturbation of glutamate homeostasis/excitotoxicity. Thus, there may be some redundancy in the empirical support between this KER and the KER linking KEup oxidative stress and KEdown cell injury/death.<!--![endif]----><!--![endif]----><!--![endif]----></p>
<p><!--[endif]----><!--[endif]----><!--[endif]----></p>
<p>Glutamate has been shown to regulate BDNF production <!--[endif]---->(Tao et al., 2002). Accordingly, glutamate may also indirectly contribute to cell injury/death by inducing modifications in the brain levels of trophic factors, since it is known that changes in trophic support can lead to cell injury/death, as well as to perturbation in the physiological establishment of neuronal network <!--[endif]---->(Zhao et al., 2017).<!--![endif]----><!--![endif]----></p>
<p><!--[endif]----><!--[endif]----><!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----><!--![endif]----></p>
<table border="1" cellpadding="0" cellspacing="0" style="width:671px">
<tbody>
<tr>
<td style="width:108px">
<p><a name="_GoBack"></a><strong>KE<sub>up</sub></strong></p>
<p><strong>Glutamate dyshomeostasis</strong></p>
</td>
<td style="width:109px">
<p><strong>KE<sub>down</sub></strong></p>
<p><strong>Cell injury/death</strong></p>
</td>
<td style="width:116px">
<p><strong>species; developmental stage of exposure to stressor</strong></p>
</td>
<td style="width:78px">
<p><strong>Stressor</strong></p>
</td>
<td style="width:84px">
<p><strong>Dose or conc.</strong></p>
<p><strong>Duration</strong></p>
</td>
<td style="width:107px">
<p><strong>Protective/ aggravating evidence</strong></p>
</td>
<td style="width:70px">
<p><strong>Reference</strong></p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Dose-dependent increase in glutamate content in cerebral cortex (+ 60% at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:109px">
<p>Increased apoptosis rate measured by flow cytometry</p>
<p>(+ 1100% at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:116px">
<p>Rat</p>
<p>adult exposure</p>
</td>
<td style="width:78px">
<p>MeHgCl</p>
</td>
<td style="width:84px">
<p>4 µmol kg-<sup>1</sup></p>
<p>12 µmol kg-<sup>1</sup></p>
<p>i.p injection</p>
<p>5 injections per week during 4 weeks</p>
</td>
<td style="width:107px">
<p>Pretreatment with dextro-methorphan (low-affinity, noncompetitive NMDAR antagonist)</p>
<p>partially decreased Glu content and apoptosis induced by MeHgCl</p>
</td>
<td style="width:70px">
<p>(Feng et al., 2014)</p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Dose-dependent increase in glutamate content in cerebral cortex (+ 22% at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:109px">
<p>Increased apoptosis rate measured by flow cytometry</p>
<p>(+ 850% at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:116px">
<p>Rat</p>
<p>adult exposure</p>
</td>
<td style="width:78px">
<p>MeHgCl</p>
</td>
<td style="width:84px">
<p>4 µmol kg-<sup>1</sup></p>
<p>12 µmol kg-<sup>1</sup></p>
<p>i.p injection</p>
<p>5 injections per week during 4 weeks</p>
</td>
<td style="width:107px">
<p>Pretreatment with memantine (low-affinity, noncompetitive NMDAR antagonist)</p>
<p>partially decreased Glu content and apoptosis induced by MeHgCl</p>
</td>
<td style="width:70px">
<p>(Liu et al., 2013)</p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Increase in glutamate content in cerebral cortex (1.12 fold at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:109px">
<p>Increased apoptosis rate measured by flow cytometry</p>
<p>(+ 630% at 12 µmol kg-<sup>1</sup>)</p>
</td>
<td style="width:116px">
<p>Rat</p>
<p>adult exposure</p>
</td>
<td style="width:78px">
<p>MeHgCl</p>
</td>
<td style="width:84px">
<p>4 µmol kg-<sup>1</sup></p>
<p>12 µmol kg-<sup>1</sup></p>
<p>i.p injection</p>
<p>5 injections per week during 4 weeks</p>
</td>
<td style="width:107px">
<p>Pretreatment with MK801 (noncompetitive NMDAR antagonist)</p>
<p>partially decreased apoptosis induced by MeHgCl</p>
</td>
<td style="width:70px">
<p>(Xu et al., 2012)</p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Decreased glutamine uptake</p>
</td>
<td style="width:109px">
<p>Reduction in inner mitochondrial membrane potential</p>
</td>
<td style="width:116px">
<p>Rat</p>
<p>astrocyte cultures</p>
</td>
<td style="width:78px">
<p>MeHg</p>
</td>
<td style="width:84px">
<p>1, 5, 10 µM</p>
<p>1 and 5 min</p>
</td>
<td style="width:107px"> </td>
<td style="width:70px">
<p>(Yin et al., 2007)</p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Changes in intracellular glutamate concentration</p>
</td>
<td style="width:109px">
<p>Cell death measured by MTT reduction and LDH release</p>
</td>
<td style="width:116px">
<p>Mouse</p>
<p>astrocytes, neurons in mono- or co-cultures</p>
</td>
<td style="width:78px">
<p>MeHg</p>
</td>
<td style="width:84px">
<p>1-50 µM</p>
<p>24h</p>
</td>
<td style="width:107px"> </td>
<td style="width:70px">
<p>(Morken et al., 2005)</p>
</td>
</tr>
<tr>
<td style="height:141px; width:108px">
<p>Concentration-dependent inhibition of glutamate uptake and stimulation of glutamate release</p>
</td>
<td style="height:141px; width:109px">
<p>Cell death measured by MTT reduction</p>
</td>
<td style="height:141px; width:116px">
<p>Mouse</p>
<p>cerebellar granule cells in culture</p>
</td>
<td style="height:141px; width:78px">
<p>HgCl<sub>2</sub></p>
<p>MeHgCl</p>
</td>
<td style="height:141px; width:84px">
<p>10<sup>-7</sup>-10<sup>-4</sup> M</p>
<p>10 min</p>
</td>
<td style="height:141px; width:107px"> </td>
<td style="height:141px; width:70px">
<p>(Fonfria et al., 2005)</p>
</td>
</tr>
<tr>
<td style="width:108px">
<p>Dose-dependent decreased cortical glutamate concentration</p>
</td>
<td style="width:109px">
<p>Dose-dependent abnormal neuronal morphology</p>
</td>
<td style="width:116px">
<p>Rat</p>
<p>Young (3-4 weeks)</p>
</td>
<td style="width:78px">
<p>Acrylamide</p>
</td>
<td style="width:84px">
<p>5, 15, 30 mg</p>
<p>kg<sup>-1</sup></p>
<p>5 injections per week during 4 weeks</p>
<p>gavage</p>
</td>
<td style="width:107px"> </td>
<td style="width:70px">
<p>(Tian et al., 2015)</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>No uncertainty or inconsistency reported yet.</p>
<p>See table of empirical evidence.</p>
HighAll life stagesHighHigh<p>Support for the link between glutamate dyshomeostasis and cell injury /death can be found in rats, and mouse. However, as the neurotransmitter glutamate is already found in insects, it is plausible that this KER is valid throughout taxa (Harris et al., 2014).</p>
<p style="margin-left:36.0pt"><!--[if supportFields]><span
style='mso-bidi-font-family:Calibri;mso-bidi-theme-font:major-latin;color:windowtext;
mso-ansi-language:FR-CH'><span style='mso-element:field-begin'></span><span
style='mso-spacerun:yes'> </span>ADDIN EN.REFLIST <span style='mso-element:
field-separator'></span></span><![endif]-->Albrecht, J., Talbot, M., Kimelberg, H.K., Aschner, M. (1993) The role of sulfhydryl groups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures. <em>Brain Res</em> <strong>607</strong>, 249-254.</p>
<p style="margin-left:36.0pt">Anderson, C.M., Swanson, R.A. (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. <em>Glia</em> <strong>32</strong>, 1-14.</p>
<p style="margin-left:36.0pt">Aschner, M., Yao, C.P., Allen, J.W., Tan, K.H. (2000) Methylmercury alters glutamate transport in astrocytes. <em>Neurochem Int</em> <strong>37</strong>, 199-206.</p>
<p style="margin-left:36.0pt">Balazs, R. (2006) Trophic effect of glutamate. <em>Curr Top Med Chem</em> <strong>6</strong>, 961-968.</p>
<p style="margin-left:36.0pt">Brookes, N., Kristt, D.A. (1989) Inhibition of amino acid transport and protein synthesis by HgCl2 and methylmercury in astrocytes: selectivity and reversibility. <em>J Neurochem</em> <strong>53</strong>, 1228-1237.</p>
<p style="margin-left:36.0pt">Ceccatelli, S., Dare, E., Moors, M. (2010) Methylmercury-induced neurotoxicity and apoptosis. <em>Chem Biol Interact</em> <strong>188</strong>, 301-308.</p>
<p style="margin-left:36.0pt">Choi, D.W. (1992) Excitotoxic cell death. <em>J Neurobiol</em> <strong>23</strong>, 1261-1276.</p>
<p style="margin-left:36.0pt">Feng, S., Xu, Z., Liu, W., Li, Y., Deng, Y., Xu, B. (2014) Preventive effects of dextromethorphan on methylmercury-induced glutamate dyshomeostasis and oxidative damage in rat cerebral cortex. <em>Biol Trace Elem Res</em> <strong>159</strong>, 332-345.</p>
<p style="margin-left:36.0pt">Fonfria, E., Vilaro, M.T., Babot, Z., Rodriguez-Farre, E., Sunol, C. (2005) Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells. <em>J Neurosci Res</em> <strong>79</strong>, 545-553.</p>
<p style="margin-left:36.0pt">Harris, K.D., Weiss, M., Zahavi, A. (2014) Why are neurotransmitters neurotoxic? An evolutionary perspective. <em>F1000Res</em> <strong>3</strong>, 179.</p>
<p style="margin-left:36.0pt">Juarez, B.I., Martinez, M.L., Montante, M., Dufour, L., Garcia, E., Jimenez-Capdeville, M.E. (2002) Methylmercury increases glutamate extracellular levels in frontal cortex of awake rats. <em>Neurotoxicol Teratol</em> <strong>24</strong>, 767-771.</p>
<p style="margin-left:36.0pt">Lafon-Cazal, M., Pietri, S., Culcasi, M., Bockaert, J. (1993) NMDA-dependent superoxide production and neurotoxicity. <em>Nature</em> <strong>364</strong>, 535-537.</p>
<p style="margin-left:36.0pt">Liu, W., Xu, Z., Deng, Y., Xu, B., Wei, Y., Yang, T. (2013) Protective effects of memantine against methylmercury-induced glutamate dyshomeostasis and oxidative stress in rat cerebral cortex. <em>Neurotox Res</em> <strong>24</strong>, 320-337.</p>
<p style="margin-left:36.0pt">LoPachin, R.M., Schwarcz, A.I., Gaughan, C.L., Mansukhani, S., Das, S. (2004) In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release. <em>Neurotoxicology</em> <strong>25</strong>, 349-363.</p>
<p style="margin-left:36.0pt">Maragakis, N.J., Rothstein, J.D. (2001) Glutamate transporters in neurologic disease. <em>Arch Neurol</em> <strong>58</strong>, 365-370.</p>
<p style="margin-left:36.0pt">Meldrum, B.S. (2000) Glutamate as a neurotransmitter in the brain: review of physiology and pathology. <em>J Nutr</em> <strong>130</strong>, 1007S-1015S.</p>
<p style="margin-left:36.0pt">Moretto, M.B., Funchal, C., Santos, A.Q., Gottfried, C., Boff, B., Zeni, G., Pureur, R.P., Souza, D.O., Wofchuk, S., Rocha, J.B. (2005) Ebselen protects glutamate uptake inhibition caused by methyl mercury but does not by Hg2+. <em>Toxicology</em> <strong>214</strong>, 57-66.</p>
<p style="margin-left:36.0pt">Morken, T.S., Sonnewald, U., Aschner, M., Syversen, T. (2005) Effects of methylmercury on primary brain cells in mono- and co-culture. <em>Toxicol Sci</em> <strong>87</strong>, 169-175.</p>
<p style="margin-left:36.0pt">Ozawa, S., Kamiya, H., Tsuzuki, K. (1998) Glutamate receptors in the mammalian central nervous system. <em>Prog Neurobiol</em> <strong>54</strong>, 581-618.</p>
<p style="margin-left:36.0pt">Pivovarova, N.B., Andrews, S.B. (2010) Calcium-dependent mitochondrial function and dysfunction in neurons. <em>FEBS J</em> <strong>277</strong>, 3622-3636.</p>
<p style="margin-left:36.0pt">Porciuncula, L.O., Rocha, J.B., Tavares, R.G., Ghisleni, G., Reis, M., Souza, D.O. (2003) Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain. <em>Neuroreport</em> <strong>14</strong>, 577-580.</p>
<p style="margin-left:36.0pt">Reynolds, J.N., Racz, W.J. (1987) Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices. <em>Can J Physiol Pharmacol</em> <strong>65</strong>, 791-798.</p>
<p style="margin-left:36.0pt">Szydlowska, K., Tymianski, M. (2010) Calcium, ischemia and excitotoxicity. <em>Cell Calcium</em> <strong>47</strong>, 122-129.</p>
<p style="margin-left:36.0pt">Tao, X., West, A.E., Chen, W.G., Corfas, G., Greenberg, M.E. (2002) A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF. <em>Neuron</em> <strong>33</strong>, 383-395.</p>
<p style="margin-left:36.0pt">Tian, S.M., Ma, Y.X., Shi, J., Lou, T.Y., Liu, S.S., Li, G.Y. (2015) Acrylamide neurotoxicity on the cerebrum of weaning rats. <em>Neural Regen Res</em> <strong>10</strong>, 938-943.</p>
<p style="margin-left:36.0pt">Xu, B., Xu, Z.F., Deng, Y., Liu, W., Yang, H.B., Wei, Y.G. (2012) Protective effects of MK-801 on methylmercury-induced neuronal injury in rat cerebral cortex: involvement of oxidative stress and glutamate metabolism dysfunction. <em>Toxicology</em> <strong>300</strong>, 112-120.</p>
<p style="margin-left:36.0pt">Yin, Z., Milatovic, D., Aschner, J.L., Syversen, T., Rocha, J.B., Souza, D.O., Sidoryk, M., Albrecht, J., Aschner, M. (2007) Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes. <em>Brain Res</em> <strong>1131</strong>, 1-10.</p>
<p style="margin-left:36.0pt">Zhao, H., Alam, A., San, C.Y., Eguchi, S., Chen, Q., Lian, Q., Ma, D. (2017) Molecular mechanisms of brain-derived neurotrophic factor in neuro-protection: Recent developments. <em>Brain Res</em> <strong>1665</strong>, 1-21.</p>
<p><!--[if supportFields]><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;
mso-ascii-theme-font:major-latin;mso-fareast-font-family:"MS Mincho";
mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:major-latin;
mso-bidi-theme-font:major-latin;color:windowtext;mso-ansi-language:FR-CH;
mso-fareast-language:FR;mso-bidi-language:AR-SA'><span style='mso-element:field-end'></span></span><![endif]--></p>
2017-11-09T04:10:562020-02-06T11:20:03eb624ffb-7315-4087-82d2-94e1a4f9d480d61927c3-2b15-4805-bacf-589e50a64ba6<p>The pioneering work of Kreutzberg and coworkers (1995, 1996) has shown that neuronal injury leads to neuroinflammation, with microglia and astrocyte reactivities. Several chemokines and chemokines receptors (fraktalkine, CD200) control the neuron-microglia interactions, and a loss of this control can trigger microglial reactivity (Blank and Prinz, 2013; Chapman et al., 2000; Streit et al., 2001). Upon injury causing neuronal death (mainly necrotic), signals termed Damage-Associated Molecular Patterns (DAMPs) are released by damaged neurons and promote microglial reactivity (Marin-Teva et al., 2011; Katsumoto et al., 2014). Toll-like receptors (TLRs) are pattern-recognition receptors that recognize specific pathogen- and danger-associated molecular signatures (PAMPs and DAMPs) and subsequently initiate inflammatory and immune responses. Microglial cells express TLRs, mainly TLR-2, which can detect neuronal cell death (for review, see Hayward and Lee, 2014). TLR-2 functions as a master sentry receptor to detect neuronal death and tissue damage in many different neurological conditions including nerve trans-section injury, traumatic brain injury and hippocampal excitotoxicity (Hayward and Lee, 2014). Astrocytes, the other cellular mediator of neuroinflammation (Ranshoff and Brown, 2012) are also able to sense tissue injury via TLR-3 (Farina et al., 2007; Rossi, 2015).</p>
<p>It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain. In the developing brain, neuroinflammation was observed after neurodegeneration induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003) or after ethanol exposure (Tiwari et al., 2012; Ahmad et al., 2016). It is important to note that physiological activation of microglial cells is observed during normal brain development for removal of apoptotic debris (Ashwell 1990, 1991). But exposure to toxicant (ethanol), excitotoxic insults (kainic acid) or traumatic brain injury during development can also induce apoptosis in hippocampus and cerebral cortex, as measured either by TUNEL, BID or caspase 3 upregulation associated to an inflammatory response, as evidenced by increased level of pro- inflammatory cytokines IL-1b, TNF-a, of NO, of p65 NF-kB or of the marker of astrogliosis, glial fibrillary acidic protein (GFAP), suggesting that, during brain development, neuroinflammation can also be triggerred by apoptosis induced by several types of insult (Tiwari and Chopra, 2012; Baratz et al., 2015; Mesuret et al., 2014).</p>
<p><em>Include consideration of temporal concordance here </em></p>
<p>Pb</p>
<p>In 3D cultures prepared from fetal rat brain cells exposed to Pb (10<sup>-6</sup> - 10<sup>-4</sup> M for 10 days), Pb-induced neuronal death was evidenced by a decrease of cholinergic and GABAergic markers associated to a decrease in protein content, and was accompanied by microglial and astrocyte reactivities (Zurich et al., 2002). These effects were more pronounced in immature than in differentiated cultures (Zurich et al., 2002). In adult rats, exposure to 100 ppm of Pb for 8 weeks caused neuronal death, evidenced by an increase in apoptosis (TUNEL) that was associated with microglial reactivity and an increase in IL-1b, TNF-a and i-NOS expression (Liu et al., 2012). Acute exposure to Pb (25 mg/kg, ip, for 3 days) increased GFAP and glutamate synthetase expression with impaiment of glutamate uptake and probable neuronal injury (Struzynska, 2000; Struzynska et al., 2001).</p>
<p>It is interesting to note that glial cells and in particular astrocytes are able to accumulate lead, suggesting that thes cells may be also a primary target of lead neurotoxic effects (Zurich et al., 1998; Lindhal et al., 1999). </p>
<p>Domoic acid</p>
<ul>
<li>Astrogliosis is one of the histopathological findings revealed by the assessment of brains derived from patients diagnosed with Amnesic Shellfish Poisoning (ASP) (reviewed in Pulido, 2008). In a reference study, where the brain of a patient after acute <span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">domoic acid (kainic acid-type neurotoxin that causes amnesic shellfish poisoning) </span></span>intoxication has been examined in great detail gliosis has been detected in the overlying cortex, dorsal and ventral septal nuclei, the secondary olfactory areas and the nucleus accumbens (Cendes et al., 1995). Reactive astrogliosis has also been confirmed in the sixth cortical layer and subjacent white matter in the orbital and lateral basal areas, the first and second temporal gyri, the fusiform gyrus, the parietal parasagittal cortex, and the insula (Cendes et al., 1995).</li>
</ul>
<ul>
<li>Adult rats have been assessed seven days after the administration of DomA (2.25 mg/kg i.p.) and revealed astrocytosis identified by glial fibrillary acidic protein (GFAP)-immunostaining and activation of microglia by GSI-B4 histochemistry (Appel et al., 1997). More investigators have suggested that DomA can activate microglia (Ananth et al., 2001; Chandrasekaran et al., 2004).</li>
</ul>
<ul>
<li>DomA treatment (2 mg/kg once a day for 3 weeks) in mice significantly stimulates the expression of inflammatory mediators, including IL-1β (1.7 fold increase), TNF-α (2 fold increase), GFAP (1.4 fold increase), Cox-2 (3 fold increase), and iNOS (1.6 fold increase) compared to controls (Lu et al, 2013).</li>
</ul>
<ul>
<li>Adult female and male mice have been injected i.p. with 4mg/kg (LD50) of DomA and Real-time PCR has been performed in the brain derived at 30, 60 and 240 min post-injection. The inflammatory response element cyclooxygenase 2 (COX-2) has been found to be 8 fold increased at the 30 and 60 min time points and then showed a descent back toward basal expression levels by 240 min (Ryan et al., 2005).</li>
</ul>
<ul>
<li>Adult male rats treated with 2 mg/kg DomA i.p. have been sacrificed after 3 or 7 d and shown that GFAP and lectin staining could identify regions of reactive gliosis within areas of neurodegeneration however observed at higher magnifications compared to the ones used for neurodegeneration (Appel et al., 1997; Scallet et al., 2005).</li>
</ul>
<ul>
<li>At 5 days and 3 months following DomA administration of male Wistar rats, a large number of OX-42 positive microglial cells exhibiting intense immunoreactivity in CA1 and CA3 regions of the hippocampus have been detected. With an antibody against GFAP, immunoreactive astrocytes have been found to be sparsely distributed in the hippocampus derived from DomA treated rats after 3 months' time interval (Ananth et al., 2003). At 5 days after the administration of DomA, GFAP positive astrocytes have been found increased in the hippocampus (Ananth et al., 2003).</li>
</ul>
<p> </p>
<p><strong>Mercury</strong></p>
<p>Young mice receiving a fish diet (MeHgCl) for 3 months exhibited in cortex a decrease of the chemokine Ccl<sub>2</sub> and neuronal death, as measured by a decrease in cell density, as well as microglial reactivity (increase in Iba1-labelled cells) (<strong>Godefroy et al., 2012</strong>)</p>
<p>Perinatal exposure to MeHgCl (GD7-PD21, 0.5 mg/kg bw/day in drinking water) lead to a delayed decrease (PD 36) of cholinergic muscarinic receptors in cerebellum accompanied by astrogliosis (<strong>Roda et al., 2008</strong>).</p>
<p>Immature rat brain cell cultures maintained in 3D conditions were exposed to either MeHgCl or HgCl<sub>2</sub> (10<sup>-9</sup> – 10<sup>-6</sup> M, for 10 days). This treatment caused microglial and astrocyte activation without neuronal death, but a reversible decrease of the expression of the neuronal marker MAP2 (<strong>Monnet-Tschudi et al., 1996 ; Eskes et al., 2002</strong>).</p>
<p>Adult marmoset exposed acutely to 5 mg Hg/kg/day p.o. exhibited apoptosis in occipital cortex, as well as glial reactivity (GFAP and Iba1 increased). Mercury content in occipital cortex was 31 mg/g (<strong>Yamamoto et al., 2012</strong>).</p>
<p>Monkeys exposed to MeHgCl (50 mg/bw for 6,12,18 months) showed microglial and astrocyte activation without any change in neuronal number. Both astrocyte and microglia accumulated elevated levels of inorganic mercury, suggesting a direct effect of mercury on glial cells (<strong>Charleston et al., 1996</strong>).</p>
<p>Human LUHMES cells as model of dopaminergic neurons and the human astrocyte cell line CFF-STTG1 were exposed to MeHgCl (0.25 -5 mM), thiomersal (0.25 – 5 mM) or HgCl2 (5-35 mM), what affected their cell viability. Neurons were much more sensitive than astrocytes (<strong>Lohren et al., 2015</strong>).</p>
<p>A direct activation of rat primary microglial cells and astrocytes was observed after exposure to MeHgCl (10<sup>-10</sup>-10<sup>-6</sup> M, for 5 days). (<strong>Eskes et al., 2002</strong>).</p>
<p>Astrocyte + microglia in co-cultures exposed to mercury (1-5 mM for 30 min to 6 days) showed lower levels of GSH in microglia than in astrocytes (<strong>Ni et al., 2011 ; 2012</strong>).</p>
<p>Human primary astrocyte cell line exposed to MeHgCl (1.125 mM) for 24h and 72h did not exhibit an increase of GFAP, but of NfkB after the 72h (<strong>Malfa et al., 2014</strong>).</p>
<p> </p>
<p><em>Sex dependency</em></p>
<p>In prairie voles 10 weeks exposure to 600 ppm HgCl<sub>2</sub> in drinking water lead to an increase of TNF-a in hippocampus of male, but not in female (<strong>Curtis et al., 2011</strong>).</p>
<p> </p>
<p><strong>Acrylamide</strong> (acrylamide is a common food contaminant generated by heat processing)</p>
<p>Adult mice received 10, 20, 30 mg/kg bw for 4 weeks. The dose of 20 mg/kg bw caused neurological symptoms (ex. cognitive impairment) associated to an increased oxidative stress, a decrease of GSH and glial reactivity (GFAP and Iba1 increased) in cortex, hippocampus and striatum. An increase in TNF-a, IL-1b and i-NOS expression in all 3 brain regions was also observed. (<strong>Santhanasabepathy et al., 2015</strong>)</p>
<p>Isolated and/or co-cultures of microglial cells or astrocytes treated with acrylamide 0-1mM for 24-96h exhibited an increased release of TNF-a, IL-1b, IL-6 and G-CSF, suggesting a direct effect of acrylamide on glial cells (<strong>Zhao et al., 2017a,b,c</strong>).</p>
<p>Neonatal rat astrocytes treated with acrylamide (0.1-1mM) for 7, 11, 15, or 20 days increased their proliferation rate as measured by PCNA staining. Astrocyte proliferation is also a sign of reactivity. (<strong>Aschner et al., 2005)</strong>.</p>
<p> </p>
<p>Pb</p>
<p>It is interesting to note that glial cells and in particular astrocytes are able to accumulate lead, suggesting that thes cells may be also a primary target of lead neurotoxic effects (Zurich et al., 1998; Lindhal et al., 1999). </p>
<p>Sobin and coworkers (2013) described a Pb-induced decrease in dentate gyrus volume associated with microglial reactivity at low dose of Pb (30 ppm), but not at high doses (330 ppm), plausibly due to the death of microglial cells at the high dose of Pb.</p>
<p>Pb decreased IL-6 secretion by isolated astrocytes (Qian et al., 2007). Such a decrease was also observed in isolated astrocytes treated with methylmercury, and was reverted in microglia astrocyte co-cultures, suggesting that cell-cell interactions can modify the response to a toxicant and that cultures of a single cell type may not be representative of the organ toxicity (Eskes et al., 2002). </p>
<p><br />
Domoic acid</p>
<p>Adult male and female Sprague Dawley rats have received a single intraperitoneal (i.p.) injection of DomA (0, 1.0, 1.8 mg/kg) and have been sacrificed 3 h after the treatment. Histopathological analysis of these animals has shown no alterations for GFAP immunostaining in the dorsal hippocampus and olfactory bulb, indicating absence of reactive gliosis (Baron et al., 2013).</p>
<p>The exposed zebrafish from the 36-week treatment with DomA showed no neuroinflammation in brain (Hiolski et al., 2014). At the same time, microarray analysis revealed no significant changes in <em>gfap</em> gene expression, a marker of neuroinflammation and astrocyte activation (Hiolski et al., 2014).</p>
<p><strong>Mercury</strong></p>
<p>Mouse developmental exposure to 50 mM of HgCl<sub>2</sub> in maternal drinking water from GD8 to PD21 did not induce any change in GM-CSF, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70. IL-13, IL-17, MCP1, MIP2 and TNF-a measured by Luminex in brain slices of PD21 and PD70. No sex differences, but brain increase of IgG and increased sociability in females (Zhang et al., 2012).</p>
<p>3D rat brain cell cultures treated for 10 days with HgCl2 or MeHgCl (10-10 - 10-6 M) exhibited increased apotosis measured by TUNEL, but exclusively in immature cultures. The proportion of cells undergoing apoptotis was highest for astrocytes than for neurons. But the apoptotic nuclei were not associated with reactive microglial cells as evidenced by double staining (Monnet-Tschudi, 1998).</p>
<p><strong>Acrylamide</strong></p>
<p>A 2 weeks exposure to acrylamide in drinking water (44mg/kg/day) induced behavioral effects, such a decreased in locomotor activity, but with no effect at gene level on neuronal and inflammatory markers analyzed in somatosensory and motor cortex (Bowyer et al., 2009).</p>
<p><em>Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships? </em></p>
<p>Quantitative evalutation of this KER does not exist (gap of knowledge).</p>
Not SpecifiedMaleNot SpecifiedFemaleHighAll life stagesHighHighHighHigh<p>California sea lions that have been exposed to the marine biotoxin DomA developed an acute or chronic toxicosis marked by seizures, whereas histopathological analysis revealed neuroinflammation characterised by gliosis (Kirkley et al., 2014).</p>
<p>Neuroinflammation has been described in mammals (rat, mouse, monkey, human).</p>
<p>Acarin L, González B, Castellano B, Castro AJ. 1997. Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain. ExpNeurol 147: 410-417.</p>
<p>Ahmad A, Shah SA, Badshah H, Kim MJ, Ali T, Yoon GH, et al. 2016. Neuroprotection by Vitamin C Against Ethanol-Induced Neuroinflammation Associated Neurodegeneration in the Developing Rat Brain. CNS Neurol Disord Drug Targets 15(3): 360-370.</p>
<p>Ananth C, Thameem DS, Gopalakrishnakone P, Kaur C. Domoic acid-induced neuronal damage in the rat hippocampus: changes in apoptosis related genes (bcl-2, bax, caspase-3) and microglial response. J Neurosci Res., 2001, 66: 177-190.</p>
<p>Ananth C, Gopalakrishnakone P, Kaur C. Induction of inducible nitric oxide synthase expression in activated microglia following domoic acid (DA)-induced neurotoxicity in the rat hippocampus. Neurosci Lett., 2003, 338: 49-52.</p>
<p>Appel NM, Rapoport SI, O’Callaghan JP, Bell JM, Freed LM. Sequelae of parenteral domoic acid administration in rats: comparison of effects on different metabolic markers in brain. Brain Res., 1997, 754: 55-64.</p>
<p>Aschner, M., Wu, Q., Friedman, M.A., 2005. Effects of acrylamide on primary neonatal rat astrocyte functions. Ann N Y Acad Sci. 1053<strong>,</strong> 444-54.</p>
<p>Ashwell K. 1990. Microglia and cell death in the developing mouse cerebellum. DevBrain Res 55: 219-230.</p>
<p>Ashwell K. 1991. The distribution of microglia and cell death in the fetal rat forebrain. DevBrain Res 58: 1-12.</p>
<p>Baratz R, Tweedie D, Wang JY, Rubovitch V, Luo W, Hoffer BJ, et al. 2015. Transiently lowering tumor necrosis factor-alpha synthesis ameliorates neuronal cell loss and cognitive impairments induced by minimal traumatic brain injury in mice. J Neuroinflammation 12: 45.</p>
<p>Baron AW, Rushton SP, Rens N, Morris CM, Blain PG, Judge SJ. Sex differences in effects of low level domoic acid exposure. Neurotoxicology, 2013, 34: 1-8.</p>
<p>Blank T, Prinz M. Microglia as modulators of cognition and neuropsychiatric disorders. Glia, 2013, 61: 62-70.</p>
<p>Bowyer, J.F., et al., 2009. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water. Toxicol Appl Pharmacol. 240<strong>,</strong> 401-11.</p>
<p>Cendes F, Andermann F, Carpenter S, Zatorre RJ, Cashman NR. Temporal lobe epilepsy caused by domoic acid intoxication: evidence for glutamate receptor-mediated excitotoxicity in humans. Ann Neurol., 1995, 37: 123-126.</p>
<p>Chandrasekaran A, Ponnambalam G, Kaur C. Domoic acid-induced neurotoxicity in the hippocampus of adult rats. Neurotox Res., 2004, 6:1 05-117.</p>
<p>Chapman GA, Moores K, Harrison D, Campbell CA, Stewart BR, Strijbos PJLM. Fractalkine Cleavage from Neuronal Membrans Represents an Acute Event in Inflammatory Response to Excitotoxic Brain Damage. J Neurosc., 2000, 20 RC87: 1-5.</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM: Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. <em>NeuroToxicology </em>1996, <strong>17:</strong>127-138.</p>
<p>Curtis, TJ., et al., 2011. Chronic inorganic mercury exposure induces sex-specific changes in central TNFalpha expression: importance in autism? Neurosci Lett. 504<strong>,</strong> 40-4.</p>
<p>Dommergues MA, Plaisant F, Verney C, Gressens P. 2003. Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection. Neuroscience 121(3): 619-628.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity. Trends Immunol, 2007, 28(3): 138-145.</p>
<p>Godefroy, D., et al., 2012. The chemokine CCL2 protects against methylmercury neurotoxicity. Toxicol Sci. 125<strong>,</strong> 209-18.</p>
<p>Hayward JH, Lee SJ. A Decade of Research on TLR2 Discovering Its Pivotal Role in Glial Activation and Neuroinflammation in Neurodegenerative Diseases. Experimental Neurobiology, 2014, 23(2): 138-147.</p>
<p>Hiolski EM, Kendrick PS, Frame ER, Myers MS, Bammler TK, Beyer RP, Farin FM, Wilkerson HW, Smith DR, Marcinek DJ, Lefebvre KA., Chronic low-level domoic acid exposure alters gene transcription and impairs mitochondrial function in the CNS. Aquat Toxicol., 2014, 155: 151-159.</p>
<p>Katsumoto A, Lu H, Miranda AS, Ransohoff RM. Ontogeny and functions of central nervous system macrophages. J Immunol., 2014, 193(6): 2615-2621.</p>
<p>Kirkley KS, Madl JE, Duncan C, Gulland FM, Tjalkens RB. Domoic acid-induced seizures in California sea lions (Zalophus californianus) are associated with neuroinflammatory brain injury. Aquat Toxicol., 2014, 156C: 259-268.</p>
<p>Kreutzberg GW. Microglia, the first line of defence in brain pathologies. Arzneimttelforsch, 1995, 45: 357-360.</p>
<p>Kreutzberg GW. Microglia : a sensor for pathological events in the CNS. Trends Neurosci., 1996, 19: 312-318.</p>
<p>Lindhal LS, Bird L, Legare ME, Mikeska G, Bratton GR, Tiffany-Castiglioni E. 1999. Differential ability of astroglia and neuronal cells to accumulate lead: Dependence on cell type and on degree of differentiation. ToxSci 50: 236-243.</p>
<p>Liu MC, Liu XQ, Wang W, Shen XF, Che HL, Guo YY, et al., Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One, 2012, 7(8): e43924.</p>
<p>Lohren, H., et al., 2015. Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes. J Trace Elem Med Biol. 32<strong>,</strong> 200-8.</p>
<p>Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ. Troxerutin counteracts domoic acid-induced memory deficits in mice by inhibiting CCAAT/enhancer binding protein β-mediated inflammatory response and oxidative stress. J Immunol., 2013, 190: 3466-3479.</p>
<p>Malfa, G.A., et al., 2014. "Reactive" response evaluation of primary human astrocytes after methylmercury exposure. J Neurosci Res. 92<strong>,</strong> 95-103.</p>
<p>Marin-Teva JL, Cuadros MA, Martin-Oliva D, Navascues J., Microglia and neuronal cell death. Neuron glia biology, 2011, 7(1): 25-40.</p>
<p>Mesuret G, Engel T, Hessel EV, Sanz-Rodriguez A, Jimenez-Pacheco A, Miras-Portugal MT, et al. 2014. P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage. CNS neuroscience & therapeutics 20(6): 556-564.</p>
<p>Monnet-Tschudi F, Zurich MG, Honegger P (1996) Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res 741:52-59</p>
<p>Monnet-Tschudi F (1998) Induction of apoptosis by Mercury Compounds depends on maturation and is not associated with microglial activation. JNeurosciRes 53:361-367</p>
<p>Ni, M., et al., 2011. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity. Glia. 59<strong>,</strong> 810-20.</p>
<p>Ni, M., et al., 2012. Glia and methylmercury neurotoxicity. J Toxicol Environ Health A. 75<strong>,</strong> 1091-101.</p>
<p>Pulido OM. Domoic acid toxicologic pathology: a review. Mar Drugs, 2008, 6: 180-219.</p>
<p>Qian Y, Zheng Y, Weber D, Tiffany-Castiglioni E. 2007. A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes. American journal of physiology Cell physiology 293(3): C897-905.</p>
<p>Ransohoff RM, Brown MA. Innate immunity in the central nervous system. J Clin Invest., 2012, 122(4): 1164-1171.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Rossi D. Astrocyte physiopathology: At the crossroads of intercellular networking, inflammation and cell death. Prog Neurobiol., 2015, 130: 86-120.</p>
<p>Ryan JC, Morey JS, Ramsdell JS, Van Dolah FM. Acute phase gene expression in mice exposed to the marine neurotoxin domoic acid. Neuroscience, 2005, 136: 1121-1132.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Scallet AC, Schmued LC, Johannessen JN. Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid. Neurotoxicol Teratol., 2005, 27: 745-752.</p>
<p>Sobin C, Montoya MG, Parisi N, Schaub T, Cervantes M, Armijos RX. 2013. Microglial disruption in young mice with early chronic lead exposure. Toxicol Lett 220(1): 44-52.</p>
<p>Streit WJ, Conde J, Harrison JK. Chemokines and Alzheimer's disease. Neurobiol Aging., 2001, 22: 909-913.</p>
<p>Struzynska L. 2000. The protective role of astroglia in the early period of experimental lead toxicity in the rat. Acta Neurobiol Exp (Wars) 60(2): 167-173.</p>
<p>Struzynska L, Bubko I, Walski M, Rafalowska U. 2001. Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain. Toxicology 165: 121-131.</p>
<p>Tiwari V, Chopra K. 2012. Attenuation of oxidative stress, neuroinflammation, and apoptosis by curcumin prevents cognitive deficits in rats postnatally exposed to ethanol. Psychopharmacology (Berl) 224(4): 519-535.</p>
<p>Wang, Y.T., et al., 2017. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of alpha-synuclein aggregation and programmed cell death. Sci Rep. 7<strong>,</strong> 45741.</p>
<p>Yamamoto, M., et al., 2012. Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset. J Toxicol Sci. 37<strong>,</strong> 749-63.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2012. Developmental exposure to mercury chloride does not impair social behavior of C57BL/6 x BTBR F(1) mice. J Immunotoxicol. 9<strong>,</strong> 401-10.</p>
<p>Zhao, M., et al., 2017a. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model. Toxicol In Vitro. 39<strong>,</strong> 119-125.</p>
<p>Zhao, M., et al., 2017b. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-kappaB pathways. Food Chem Toxicol. 106<strong>,</strong> 25-35.</p>
<p>Zhao, W.Z., et al., 2017c. Neuroprotective Effects of Baicalein on Acrolein-induced Neurotoxicity in the Nigrostriatal Dopaminergic System of Rat Brain. Mol Neurobiol.</p>
<p>Zurich MG, Monnet-Tschudi F, Berode M, Honegger P. 1998. Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures. Toxicol In Vitro 12(2): 191-196.</p>
<p>Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.</p>
2016-11-29T18:41:332022-07-15T08:26:25eb624ffb-7315-4087-82d2-94e1a4f9d48073abd993-70e8-4491-8ac4-31bec7d54368<p>The pioneering work of Kreutzberg and coworkers (1995, 1996) has shown that neuronal injury leads to neuroinflammation, with microglia and astrocyte reactivity. Several chemokines and chemokines receptors (fraktalkine, CD200) control the neuron-microglia interactions, and a loss of this control can trigger microglial reactivity (Blank and Prinz, 2013; Chapman et al., 2000; Streit et al., 2001). Upon injury causing neuronal death (mainly necrotic), signals termed Damage-Associated Molecular Patterns (DAMPs) are released by damaged neurons and promote microglial reactivity (Marin-Teva et al., 2011; Katsumoto et al., 2014). Toll-like receptors (TLRs) are pattern-recognition receptors that recognize specific pathogen- and danger-associated molecular signatures (PAMPs and DAMPs) and subsequently initiate inflammatory and immune responses. Microglial cells express TLRs, mainly TLR-2, which can detect neuronal cell death (for review, see Hayward and Lee, 2014). TLR-2 functions as a master sentry receptor to detect neuronal death and tissue damage in many different neurological conditions including nerve trans-section injury, traumatic brain injury and hippocampal excitotoxicity (Hayward and Lee, 2014). Astrocytes, the other cellular mediator of neuroinflammation (Ranshoff and Brown, 2012) are also able to sense tissue injury via TLR-3 (Farina et al., 2007; Rossi, 2015).</p>
<p><strong><span style="font-size:14px">LIVER:</span></strong></p>
<p>Damaged hepatocytes release reactive oxygen species (ROS), cytokines such as TGF-β1 and TNF-α, and chemokines which lead to oxidative stress, inflammatory signalling and finally activation of the resident macrophages in the liver, Kupffer cells (KCs). ROS generation in hepatocytes results from oxidative metabolism by NADH oxidase (NOX) and cytochrome 2E1 activation as well as through lipid peroxidation. Damaged liver cells trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (DAMPs), which activate KCs through toll-like receptors and recruit activated neutrophils and monocytes into the liver. Central to this inflammatory response is the promotion of ROS formation by these phagocytes. Upon initiation of apoptosis hepatocytes undergo genomic DNA fragmentation and formation of apoptotic bodies; these apoptotic bodies are consecutively engulfed by KCs and cause their activation. This increased phagocytic activity strongly up-regulates NOX expression in KCs, a superoxide producing enzyme of phagocytes with profibrogenic activity, as well as nitric oxide synthase (iNOS) mRNA transcriptional levels with consequent harmful reaction between ROS and nitricoxide (NO), like the generation of cytotoxic peroxinitrite (N2O3). ROS and/or diffusible aldehydes also derive from liver sinusoidal endothelial cells (LSECs) which are additional initial triggers of KC activation. [Winwood and Arthur,1993; Luckey and Petersen, 2001; Roberts et al., 2007; Malhi, H. et al., 2010; Canbay et al., 2004; Orrenius et al., 2012; Kisseleva and Brenner, 2008; Jaeschke, 2011; Li et al., 2008; Poli, 2000]</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There is convincing theoretical evidence that hepatocyte injury and apoptosis causes KC activation, as well as inflammation and oxidative stress. But there are only limited experimental studies which could show that there is a direct relationship between these two events with temporal concordance. Specific markers for activated KCs have not been identified yet. KC activation cannot be detected morphologically by staining techniques since cell morphology does not change, but cytokines release can be measured (with the caveat that KCs activate spontaneously in vitro) and used as marker for KC activation. [Canbay et al., 2003; Soldatow et al., 2013] Tukov et al. examined the effects of KCs cultured in contact with rat hepatocytes. They found that by adding KCs to the cultures they could mimic <em>in vivo</em> drug-induced inflammatory responses. Experiments on cells of the macrophage lineage showed significant aldehyde-induced stimulation of the activity of protein kinase C, an enzyme involved in several signal transduction pathways. Further, 4-Hydroxynonenal (HNE) was demonstrated to up-regulate TGF-β1 expression and synthesis in isolated rat KCs. [Tukov et al., 2006] Canbay et al could prove that engulfment of hepatocyte apoptotic bodies stimulated KC generation of cytokines. [LeCluyse et al., 2012] </p>
<p>It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain. In the developing brain, neuroinflammation was observed after neurodegeneration induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003) or after ethanol exposure (Tiwari et al., 2012; Ahmad et al., 2016). It is important to note that physiological activation of microglial cells is observed during normal brain development for removal of apoptotic debris (Ashwell 1990, 1991). But exposure to toxicant (ethanol), excitotoxic insults (kainic acid) or traumatic brain injury during development can also induce apoptosis in hippocampus and cerebral cortex, as measured either by TUNEL, BID or caspase 3 upregulation associated to an inflammatory response, as evidenced by increased level of pro- inflammatory cytokines IL-1b, TNF-a, of NO, of p65 NF-kB or of the marker of astrogliosis, glial fibrillary acidic protein (GFAP), suggesting that, during brain development, neuroinflammation can also be triggerred by apoptosis induced by several types of insult (Tiwari and Chopra, 2012; Baratz et al., 2015; Mesuret et al., 2014).</p>
<p><strong>Mercury</strong></p>
<p>Young mice receiving a fish diet (MeHgCl) for 3 months exhibited in cortex a decrease of the chemokine Ccl<sub>2</sub> and neuronal death, as measured by a decrease in cell density, as well as microglial reactivity (increase in Iba1-labelled cells) (Godefroy et al., 2012)</p>
<p>Perinatal exposure to MeHgCl (GD7-PD21, 0.5 mg/kg bw/day in drinking water) lead to a delayed decrease (PD 36) of cholinergic muscarinic receptors in cerebellum accompanied by astrogliosis (Roda et al., 2008).</p>
<p>Immature rat brain cell cultures maintained in 3D conditions were exposed to either MeHgCl or HgCl<sub>2</sub> (10<sup>-9</sup> – 10<sup>-6</sup> M, for 10 days). This treatment caused microglial and astrocyte activation without neuronal death, but a reversible decrease of the expression of the neuronal marker MAP2 (Monnet-Tschudi et al., 1996 ; Eskes et al., 2002).</p>
<p>Adult marmoset exposed acutely to 5 mg Hg/kg/day p.o. exhibited apoptosis in occipital cortex, as well as glial reactivity (GFAP and Iba1 increased). Mercury content in occipital cortex was 31 mg/g (Yamamoto et al., 2012).</p>
<p>Monkeys exposed to MeHgCl (50 mg/bw for 6,12,18 months) showed microglial and astrocyte activation without any change in neuronal number. Both astrocyte and microglia accumulated elevated levels of inorganic mercury, suggesting a direct effect of mercury on glial cells (Charleston et al., 1996).</p>
<p>Human LUHMES cells as model of dopaminergic neurons and the human astrocyte cell line CFF-STTG1 were exposed to MeHgCl (0.25 -5 mM), thiomersal (0.25 – 5 mM) or HgCl2 (5-35 mM), what affected their cell viability. Neurons were much more sensitive than astrocytes (Lohner et al., 2015).</p>
<p>A direct activation of rat primary microglial cells and astrocytes was observed after exposure to MeHgCl (10<sup>-10</sup>-10<sup>-6</sup> M, for 5 days). (Eskes et aé., 2002).</p>
<p>Astrocyte + microglia in co-cultures exposed to mercury (1-5 mM for 30 min to 6 days) showed lower levels of GSH in microglia than in astrocytes (Ni et al., 2011 ; 2012).</p>
<p>Human primary astrocyte cell line exposed to MeHgCl (1.125 mM) for 24h and 72h did not exhibit an increase of GFAP, but of NfkB after the 72h (Malfa et al., 2014).</p>
<p>Human mast cells (leukemic LAD2, derived from umbilical cord blood) showed an increase of IL-6 release when exposed to HgCl<sub>2</sub> (0.1-10 mM, for 10 min to 24h). It is hypothesized that mast cell activation could lead to BBB disruption and to neuroinflammation. (Kempurai et al., 2010).</p>
<p> </p>
<p><em>Sex dependency</em></p>
<p>In prairie voles 10 weeks exposure to 600 ppm HgCl<sub>2</sub> in drinking water lead to an increase of TNF-a in hippocampus of male, but not in female (Curtis et al., 2011).</p>
<p> </p>
<p><strong>Acrylamide</strong> (acrylamide is a common food contaminant generated by heat processing)</p>
<p>Adult mice received 10, 20, 30 mg/kg bw for 4 weeks. The dose of 20 mg/kg bw caused neurological symptoms (ex. cognitive impairment) associated to an increased oxidative stress, a decrease of GSH and glial reactivity (GFAP and Iba1 increased) in cortex, hippocampus and striatum. An increase in TNF-a, IL-1b and i-NOS expression in all 3 brain regions was also observed. (Santhanasabepathy et al., 2015)</p>
<p>Isolated and/or co-cultures of microglial cells or astrocytes treated with acrylamide 0-1mM for 24-96h exhibited an increased release of TNF-a, IL-1b, IL-6 and G-CSF, suggesting a direct effect of acrylamide on glial cells (Zhao et al., 2017a,b).</p>
<p>Neonatal rat astrocytes treated with acrylamide (0.1-1mM) for 7, 11, 15, or 20 days increased their proliferation rate as measured by PCNA staining. Astrocyte proliferation is also a sign of reactivity. (Aschner et al., 2005).</p>
<p> </p>
<p><strong>Acrolein</strong></p>
<p>Adult rat received an infusion of acrolein (15, 50, 150 nmoles/0.5 ml) directly in substantia nigra which caused a decrease of Tyrosine hydroxylase immunostaining, an increase in caspase 1 and an activation of microglial cells and astrocytes (Wang et al., 2017).</p>
<p>Similar treatment as above induced an increase in lipid peroxidation, of hsp32 and of caspase 1 with an increase in GFAP and in ED1 (marker of macrophagic microglial cells) as well as of IL-1b (Zhao et al., 2017).</p>
<p> </p>
<p><strong>Mercury</strong></p>
<p>Mouse developmental exposure to 50 mM of HgCl<sub>2</sub> in maternal drinking water from GD8 to PD21 did not induce any change in GM-CSF, IFN-g, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70. IL-13, IL-17, MCP1, MIP2 and TNF-a measured by Luminex in brain slices of PD21 and PD70. No sex differences, but brain increase of IgG and increased sociability in females (Zhang et al., 2012).</p>
<p>3D rat brain cell cultures treated for 10 days with HgCl2 or MeHgCl (10-10 - 10-6 M) exhibited increased apotosis measured by TUNEL, but exclusively in immature cultures. The proportion of cells undergoing apoptotis was highest for astrocytes than for neurons. But the apoptotic nuclei were not associated with reactive microglial cells as evidenced by double staining (Monnet-Tschudi, 1998).</p>
<p><strong>Acrylamide</strong></p>
<p>A 2 weeks exposure to acrylamide in drinking water (44mg/kg/day) induced behavioral effects, such a decreased in locomotor activity, but with no effect at gene level on neuronal and inflammatory markers analyzed in somatosensory and motor cortex (Bowyer et al., 2009).</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>The detailed mechanisms of the KC - hepatocyte interaction and its consequences for both normal and toxicant-driven liver responses remain to be determined. KC activation followed by cytokine release is associated in some cases with evident liver damage, whereas in others this event is unrelated to liver damage or may be even protective; apparently this impact is dependent on the quantity of KC activation; excessive or prolonged release of KC mediators can switch an initially protective mechanism to a damaging inflammatory response. Evidence suggests that low levels of cytokine release from KCs constitute a survival signal that protects hepatocytes from cell death and in some cases, stimulates proliferation. [Roberts et al., 2007] </p>
HighUnspecificHighDuring brain development, adulthood and agingHighAll life stagesHighHighHighHighHigh<p><span style="font-size:14px"><strong>Liver:</strong></span></p>
<p>Human [Winwood and Arthur,1993; Roberts et al., 2007; Kolios et al., 2006] </p>
<p>Rat [Tukov et al., 2006; Roberts et al., 2007]</p>
<p>Acarin L, González B, Castellano B, Castro AJ. 1997. Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain. ExpNeurol 147: 410-417.</p>
<p>Ahmad A, Shah SA, Badshah H, Kim MJ, Ali T, Yoon GH, et al. 2016. Neuroprotection by Vitamin C Against Ethanol-Induced Neuroinflammation Associated Neurodegeneration in the Developing Rat Brain. CNS Neurol Disord Drug Targets 15(3): 360-370.</p>
<p>Aschner, M., Wu, Q., Friedman, M.A., 2005. Effects of acrylamide on primary neonatal rat astrocyte functions. Ann N Y Acad Sci. 1053<strong>,</strong> 444-54.</p>
<p>Ashwell K. 1990. Microglia and cell death in the developing mouse cerebellum. DevBrain Res 55: 219-230.</p>
<p>Ashwell K. 1991. The distribution of microglia and cell death in the fetal rat forebrain. DevBrain Res 58: 1-12.</p>
<p>Baratz R, Tweedie D, Wang JY, Rubovitch V, Luo W, Hoffer BJ, et al. 2015. Transiently lowering tumor necrosis factor-alpha synthesis ameliorates neuronal cell loss and cognitive impairments induced by minimal traumatic brain injury in mice. J Neuroinflammation 12: 45.</p>
<p>Blank T, Prinz M. Microglia as modulators of cognition and neuropsychiatric disorders. Glia, 2013, 61: 62-70.</p>
<p>Bowyer, J.F., et al., 2009. The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water. Toxicol Appl Pharmacol. 240<strong>,</strong> 401-11.</p>
<p>Chapman GA, Moores K, Harrison D, Campbell CA, Stewart BR, Strijbos PJLM. Fractalkine Cleavage from Neuronal Membrans Represents an Acute Event in Inflammatory Response to Excitotoxic Brain Damage. J Neurosc., 2000, 20 RC87: 1-5.</p>
<p>Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM: Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. <em>NeuroToxicology </em>1996, <strong>17:</strong>127-138.</p>
<p>Thomas Curtis, J., et al., 2011. Chronic inorganic mercury exposure induces sex-specific changes in central TNFalpha expression: importance in autism? Neurosci Lett. 504<strong>,</strong> 40-4.</p>
<p>Dommergues MA, Plaisant F, Verney C, Gressens P. 2003. Early microglial activation following neonatal excitotoxic brain damage in mice: a potential target for neuroprotection. Neuroscience 121(3): 619-628.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Farina C, Aloisi F, Meinl E. Astrocytes are active players in cerebral innate immunity. Trends Immunol, 2007, 28(3): 138-145.</p>
<p>Godefroy, D., et al., 2012. The chemokine CCL2 protects against methylmercury neurotoxicity. Toxicol Sci. 125<strong>,</strong> 209-18.</p>
<p>Hayward JH, Lee SJ. A Decade of Research on TLR2 Discovering Its Pivotal Role in Glial Activation and Neuroinflammation in Neurodegenerative Diseases. Experimental Neurobiology, 2014, 23(2): 138-147.</p>
<p>Katsumoto A, Lu H, Miranda AS, Ransohoff RM. Ontogeny and functions of central nervous system macrophages. J Immunol., 2014, 193(6): 2615-2621.</p>
<p>Kempuraj, D., et al., 2010. Mercury induces inflammatory mediator release from human mast cells. J Neuroinflammation. 7<strong>,</strong> 20.</p>
<p>Kreutzberg GW. Microglia, the first line of defence in brain pathologies. Arzneimttelforsch, 1995, 45: 357-360.</p>
<p>Kreutzberg GW. Microglia : a sensor for pathological events in the CNS. Trends Neurosci., 2006, 19: 312-318.</p>
<p>Lohren, H., et al., 2015. Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes. J Trace Elem Med Biol. 32<strong>,</strong> 200-8.</p>
<p>Malfa, G.A., et al., 2014. "Reactive" response evaluation of primary human astrocytes after methylmercury exposure. J Neurosci Res. 92<strong>,</strong> 95-103.</p>
<p>Marin-Teva JL, Cuadros MA, Martin-Oliva D, Navascues J., Microglia and neuronal cell death. Neuron glia biology, 2011, 7(1): 25-40.</p>
<p>Mesuret G, Engel T, Hessel EV, Sanz-Rodriguez A, Jimenez-Pacheco A, Miras-Portugal MT, et al. 2014. P2X7 receptor inhibition interrupts the progression of seizures in immature rats and reduces hippocampal damage. CNS neuroscience & therapeutics 20(6): 556-564.</p>
<p>Ni, M., et al., 2011. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity. Glia. 59<strong>,</strong> 810-20.</p>
<p>Ni, M., et al., 2012. Glia and methylmercury neurotoxicity. J Toxicol Environ Health A. 75<strong>,</strong> 1091-101.</p>
<p>Ransohoff RM, Brown MA. Innate immunity in the central nervous system. J Clin Invest., 2012, 122(4): 1164-1171.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Rossi D. Astrocyte physiopathology: At the crossroads of intercellular networking, inflammation and cell death. Prog Neurobiol., 2015, 130: 86-120.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Streit WJ, Conde J, Harrison JK. Chemokines and Alzheimer's disease. Neurobiol Aging., 2001, 22: 909-913.</p>
<p>Tiwari V, Chopra K. 2012. Attenuation of oxidative stress, neuroinflammation, and apoptosis by curcumin prevents cognitive deficits in rats postnatally exposed to ethanol. Psychopharmacology (Berl) 224(4): 519-535</p>
<p>Wang, Y.T., et al., 2017. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of alpha-synuclein aggregation and programmed cell death. Sci Rep. 7<strong>,</strong> 45741.</p>
<p>Yamamoto, M., et al., 2012. Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset. J Toxicol Sci. 37<strong>,</strong> 749-63.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2012. Developmental exposure to mercury chloride does not impair social behavior of C57BL/6 x BTBR F(1) mice. J Immunotoxicol. 9<strong>,</strong> 401-10.</p>
<p>Zhao, M., et al., 2017. Effect of acrylamide-induced neurotoxicity in a primary astrocytes/microglial co-culture model. Toxicol In Vitro. 39<strong>,</strong> 119-125.</p>
<p>Zhao, M., et al., 2017. Acrylamide-induced neurotoxicity in primary astrocytes and microglia: Roles of the Nrf2-ARE and NF-kappaB pathways. Food Chem Toxicol. 106<strong>,</strong> 25-35.</p>
<p>Zhao, W.Z., et al., 2017. Neuroprotective Effects of Baicalein on Acrolein-induced Neurotoxicity in the Nigrostriatal Dopaminergic System of Rat Brain. Mol Neurobiol.</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<ul style="list-style-type:circle">
<li>Canbay, A. et al. (2003), Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression, Hepatology, vol. 38, no. 5, pp. 1188-1198.</li>
<li>Canbay, A., S.L. Friedman and G.J. Gores (2004), Apoptosis: the nexus of liver injury and fibrosis, Hepatology, vol. 39, no. 2, pp. 273-278.</li>
<li>Jaeschke, H. (2011), Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts, J Gastroenterol Hepatol. vol. 26, suppl. 1, pp. 173-179.</li>
<li>Kisseleva T and Brenner DA, (2008), Mechanisms of Fibrogenesis, Exp Biol Med, vol. 233, no. 2, pp. 109-122.</li>
<li>Kolios, G., V. Valatas and E. Kouroumalis (2006), Role of Kupffer cells in the pathogenesis of liver disease, World J.Gastroenterol, vol. 12, no. 46, pp. 7413-7420.</li>
<li>LeCluyse, E.L. et al. (2012), Organotypic liver culture models: meeting current challenges in toxicity testing, Crit Rev Toxicol, vol. 42, no. 6, 501-548.</li>
<li>Li, Jing-Ting et al. (2008), Molecular mechanism of hepatic stellate cell activation and antifibrotic therapeutic strategies, J Gastroenterol, vol. 43, no. 6, pp. 419–428.</li>
<li>Luckey, S.W., and D.R. Petersen (2001), Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats, Exp Mol Pathol, vol. 71, no. 3, pp. 226-240.</li>
<li>Malhi, H. et al. (2010), Hepatocyte death: a clear and present danger, Physiol Rev, vol. 90, no. 3, pp. 1165-1194.</li>
<li>Orrenius, S., P. Nicotera and B. Zhivotovsky (2011), Cell death mechanisms and their implications in toxicology, Toxicol. Sci, vol. 119, no. 1, pp. 3-19.</li>
<li>Poli, G. (2000), Pathogenesis of liver fibrosis: role of oxidative stress, Mol Aspects Med, vol. 21, no. 3, pp. 49 – 98.</li>
<li>Roberts, R.A. et al. (2007), Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis, Toxicol Sci, vol. 96, no. 1, pp. 2-15.</li>
<li>Soldatow, V.Y. et al. (2013), In vitro models for liver toxicity testing, Toxicol Res, vol. 2, no.1, pp. 23–39.</li>
<li>Tukov, F.F. et al. (2006), Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte co-culture system, Toxicol In Vitro, vol. 20, no. 8, pp. 1488-1499.</li>
<li>Winwood, P.J., and M.J. Arthur (1993), Kupffer cells: their activation and role in animal models of liver injury and human liver disease, Semin Liver Dis, vol. 13, no. 1, pp. 50-59.</li>
</ul>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
2018-02-01T05:53:152018-08-02T03:02:05d61927c3-2b15-4805-bacf-589e50a64ba6eb624ffb-7315-4087-82d2-94e1a4f9d480<p>Cells of the innate (microglia and astrocytes) and of the adaptive (infiltrating monocytes and lymphocytes) immune system of the brain have various ways to kill neighboring cells. This is in part due to evolutionary-conserved mechanisms evolved to kill virus-infected cells or tumor cells; in part it is a bystander phenomenon due to the release of mediators that should activate other cells and contribute to the killing of invading micro-organisms. An exaggerated or unbalanced activation of immune cells can thus lead to parenchymal (neuronal) cell death (Gehrmann et al., 1995). Mediators known to have such effects comprise components of the complement system and cytokines/death receptor ligands triggering programmed cell death (Dong and Benveniste, 2001). Various secreted proteases (e.g. matrix metalloproteases), lipid mediators (e.g. ceramide or gangliosides) or reactive oxygen species can contribute to bystander death of neurons (Chao et al., 1995; Nakajima et al., 2002; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetzsch and Block, 2013). The equimolar production of superoxide and NO from glial cells can lead to high steady levels of peroxynitrite, which is a very potent cytotoxicant (Yuste et al., 2015). Already stressed neurons, with an impaired anti-oxidant defence system, are more sensitive to such mediators (Xu et al., 2015). Healthy cells continuously display anti "eat-me" signals, while damaged and stressed neurons/neurites display "eat-me" signals that may be recognized by microglia as signals to start phagocytosis (Neher et al., 2012) or by astrocytes (Wakida et al., 2018; Byun and Chung, 2018; Gomez-Arboledas et al., 2018; Morizawa et al., 2017). Reactive astrocytes are also able to release neurotoxic molecules (Mena and Garcia de Ybenes, 2008; Niranjan, 2014). However, astrocytes may also be protective due to their capacity to quench free radicals and secrete neurotrophic factors. The activation of astrocytes may reduce neurotrophic support to neurons (for review, Mena and Garcia de Ybenes, 2008).</p>
<p>In vitro co-culture experiments have demonstrated that reactive glial cells (microglia and astrocytes) can kill neurons (Chao et al., 1995; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetzsch and Block, 2013) and that interventions with e.g. i-NOS inhibition can rescue the neurons (Yadav et al., 2012; Brzozowski et al., 2015). Drugs that block Toll like receptor pathways, which are expressed by glial cells have been proven to be protective by decreasing ROS and RNS production (Lucas et al., 2013).</p>
<p>Reactive microglia can remove synapses, a process known as synapse stripping (Banati et al., 1993; Kettenmann et al., 2013). Reactive astrocytes were also associated with neurite and synapse reduction (Calvo-Ochoa et al., 2014). Microglia can modulate synapse plasticity, an effect mediated by cytokines. During development, microglia can promote synaptogenesis or engulf synapses, a process known as synaptic pruning (for review, Jebelli et al., 2015). It is hypothesized that alterations in microglia functioning during synapse formation and maturation of the brain can have significant long-term effects on the final established neural circuits (for review, Harry and Kraft, 2012). The fact that astrocytes can receive and respond to the synaptic information produced by neuronal activity, owing to their expression of a wide range of neurotransmitter receptors, has given rise to the concept of tripartite synapse (for review, Perez-Alvarez and Araque, 2013; Bezzi and Volterra, 2001). Pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6, which are produced by reactive astrocytes, are on one side implicated in synapse formation and scaling, long-term potentiation and neurogenesis (for review, Bilbo and Schwartz, 2009) and on the other side can kill neurons (Chao et al., 1995; Kraft and Harry, 2011). Taken together, this suggests that neuron-glia interactions are tightly regulated and that an imbalance, such as increased or long-term release of these inflammatory mediators may lead to deleterious effects on neurons.</p>
<p><strong>Mercury</strong></p>
<p>Mercury accumulates in the brain particularly in astrocytes and induce astrocyte swelling, excitatory amino acid release and decreased anti-oxidant protections (Shanker et al., 2003; Allen et al., 2001), features that are also observed in reactive astrocytes. Due to the central role of astrocytes for neuronal function (control of water transport, production of trophic factors, of anti-oxidants, tri-partite synapse,… (Ximeres da Silva, 2016; Bezzi and Volterra, 2001; Hertz and Zielke, 2004; Sidoryk-Wegrzynowicz et al., 2011), it is thought that neuronal dysfunction may be secondary to disturbance in astrocytes (Aschner et al., 2007).</p>
<p>Perinatal exposure (GD7-PD21) of rat to MeHgCl (0.5 mg/kg bw/day) in drinking water lead to gliosis in cerebellum of immature rats (PD21) without affecting the cholinergic system. In contrast, at PD36, astrogliosis was accompanied by an increase of muscarinic M2-immunopositive Bergman cells and a lack of M3 muscarinic receptors in the molecular layer. These results suggest that astrogliosis which is observed first at PD21 may be responsible of the delayed effects of mercury on neurons (<strong>Roda et al., 2008</strong>).</p>
<p>Developmental exposure of mice from GD8 to PD21 to 50 mM HgCl<sub>2</sub> in maternal drinking water: Female offsprings exhibited higher neuroinflammation which is associated with altered social behavior (<strong>Zhang et al., 2013</strong>).</p>
<p>MG17, a novel triazole derivative, was able to reduce mercury-induced upregulation of IL-1b, IL-6 and TNF-a (measured by RT-PCR) and proved to be protective against mercury-induced neurodegeneration (<strong>Matharasala et al., 2017</strong>).</p>
<p>Adult rats exposed to MeHg (5mg/kg bw) for 12 consecutive days exhibited piknotic nuclei in cerebellar granule cells, what was reverted by a co-administration of CA074 an inhibitor of cathepsin released by activated microglia. These observations strongly suggest that the mercury–induced neuronal pathological changes are secondary to microglial activation (<strong>Sakamoto et al., 2008</strong>).</p>
<p> </p>
<p><strong>Acrylamide</strong></p>
<p>Rats exposed to acrylamide (20 mg/kg bw for 4 weeks) together with farmesol (sequiterpene) showed a downregulation of astrogliosis (i.e. decreased GFAP) and of microgliosis (i.e. decreased Iba1) and of TNF-a, Il-1b and i-NOS in cortex, hippocampus and striatum. This was associated with a marked improvement in motor coordination and a decrease in markers of oxidative stress, as compared to rats exposed to acrylamide alone (<strong>Santhanasabapathy et al., 2015</strong>).</p>
<p> </p>
<p>In 3D rat brain cell-cultures, co-administration of the pro-inflammatory cytokine IL-6 (10 ng/ml) together with non-cytotoxic concentrations of MeHgCl (3 x 10<sup>-7</sup> M) for 10 days protected from the mercury-induced decreased in MAP2 immunostaining, suggesting a positive effect of IL-6, in accord with its descibed trophic activity (<strong>Eskes et al., 2002</strong>).</p>
<p>The consequences of neuroinflammation depends rather on the balance between the pro-inflammatory/neurodegenerative and anti-inflammatory/alternative/neuro-reparative side, of the duration and probably of the cellular context. There is not enough literature describing an inhibition of mercury-induced neuroinflammation and the potential protection on neurons.</p>
Not SpecifiedMaleNot SpecifiedFemaleNot SpecifiedAll life stagesHighHigh<p>Most experimental evidences derived from mouse and rat studies.</p>
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<p>Aschner, M., et al., 2007. Involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity. Braz J Med Biol Res. 40<strong>,</strong> 285-91.</p>
<p>Banati, R.B., et al., 1993. Cytotoxicity of microglia. Glia. 7<strong>,</strong> 111-8.</p>
<p>Bezzi, P., Volterra, A., 2001. A neuron-glia signalling network in the active brain. Curr Opin Neurobiol. 11<strong>,</strong> 387-94.</p>
<p>Bilbo, S.D., Schwarz, J.M., 2009. Early-life programming of later-life brain and behavior: a critical role for the immune system. Front Behav Neurosci. 3<strong>,</strong> 14.</p>
<p>Brown GC, Bal-Price A. 2003. Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27(3): 325-355.</p>
<p>Brzozowski MJ, Jenner P, Rose S. 2015. Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity. J Neural Transm 122(6): 779-788.</p>
<p>Byun YG, Chung WS., A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes., J Vis Exp. 2018 Feb 5;(132). doi: 10.3791/56647.</p>
<p>Calvo-Ochoa, E., et al., 2014. Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus. J Cereb Blood Flow Metab. 34<strong>,</strong> 1001-8.</p>
<p>Chao CC, Hu S, Peterson PK. 1995. Glia, cytokines, and neurotoxicity. CritRevNeurobiol 9: 189-205.</p>
<p>Dong Y, Benveniste EN. 2001. Immune Function of Astrocytes. Glia 36: 180-190.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Gehrmann J, Banati RB, Wiessnert C, Hossmann KA, Kreutzberg GW. 1995. Reactive microglia in cerebral ischaemia: An early mediator of tissue damage? NeuropatholApplNeurobiol 21: 277-289.</p>
<p>Gomez-Arboledas A, Davila JC, Sanchez-Mejias E, Navarro V, Nuñez-Diaz C, Sanchez-Varo R, Sanchez-Mico MV, Trujillo-Estrada L, Fernandez-Valenzuela JJ, Vizuete M, Comella JX, Galea E, Vitorica J, Gutierrez A., Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease., Glia. 2018 Mar;66(3):637-653. doi: 10.1002/glia.23270. Epub 2017 Nov 27.</p>
<p>Harry, G.J., Kraft, A.D., 2012. Microglia in the developing brain: a potential target with lifetime effects. Neurotoxicology. 33<strong>,</strong> 191-206.</p>
<p>Hertz, L., Zielke, H.R., 2004. Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci. 27<strong>,</strong> 735-43.</p>
<p>Jebelli, J., et al., 2015. Glia: guardians, gluttons, or guides for the maintenance of neuronal connectivity? Ann N Y Acad Sci. 1351<strong>,</strong> 1-10.</p>
<p>Kettenmann, H., Kirchhoff, F., Verkhratsky, A., 2013. Microglia: new roles for the synaptic stripper. Neuron. 77<strong>,</strong> 10-8.</p>
<p>Kraft AD, Harry GJ. 2011. Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International journal of environmental research and public health 8(7): 2980-3018.</p>
<p>Lucas, K., Maes, M., 2013. Role of the Toll Like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway. Mol Neurobiol. 48<strong>,</strong> 190-204.</p>
<p>Matharasala, G., Samala, G., Perumal, Y., 2017. MG17, a novel triazole derivative abrogated neuroinflammation and related neurodegenerative symptoms in rodents. Curr Mol Pharmacol.</p>
<p>Mena MA, Garcia de Yebenes J. 2008. Glial cells as players in parkinsonism: the "good," the "bad," and the "mysterious" glia. Neuroscientist 14(6): 544-560.</p>
<p>Morizawa YM, Hirayama Y, Ohno N, Shibata S, Shigetomi E, Sui Y, Nabekura J, Sato K, Okajima F, Takebayashi H, Okano H, Koizumi S., Reactive astrocytes function as phagocytes after brain ischemia via ABCA1-mediated pathway., Nat Commun. 2017 Jun 22;8(1):28. doi: 10.1038/s41467-017-00037-1. Erratum in: Nat Commun. 2017 Nov 14;8(1):1598.</p>
<p>Nakajima K, Tohyama Y, Kohsaka S, Kurihara T. 2002. Ceramide activates microglia to enhance the production/secretion of brain-derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro. J Neurochem 80: 697-705.</p>
<p>Niranjan R. 2014. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease: focus on astrocytes. Mol Neurobiol 49(1): 28-38.</p>
<p>Neher JJ, Neniskyte U, Brown GC. 2012. Primary phagocytosis of neurons by inflamed microglia: potential roles in neurodegeneration. Frontiers in pharmacology 3: 27.</p>
<p>Perez-Alvarez, A., Araque, A., 2013. Astrocyte-neuron interaction at tripartite synapses. Curr Drug Targets. 14<strong>,</strong> 1220-4.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Sakamoto, M., et al., 2008. Possible involvement of cathepsin B released by microglia in methylmercury-induced cerebellar pathological changes in the adult rat. Neurosci Lett. 442<strong>,</strong> 292-6.</p>
<p>Shanker, G., Syversen, T., Aschner, M., 2003. Astrocyte-mediated methylmercury neurotoxicity. Biol Trace Elem Res. 95<strong>,</strong> 1-10.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Sidoryk-Wegrzynowicz, M., et al., 2011. Role of astrocytes in brain function and disease. Toxicol Pathol. 39<strong>,</strong> 115-23.</p>
<p>Taetzsch T, Block ML. 2013. Pesticides, microglial NOX2, and Parkinson's disease. J Biochem Mol Toxicol 27(2): 137-149.</p>
<p>Wakida NM, Cruz GMS, Ro CC, Moncada EG, Khatibzadeh N, Flanagan LA, Berns MW., Phagocytic response of astrocytes to damaged neighboring cells., PLoS One. 2018 Apr 30;13(4):e0196153. doi: 10.1371/journal.pone.0196153. eCollection 2018.</p>
<p>Ximenes-da-Silva, A., 2016. Metal Ion Toxins and Brain Aquaporin-4 Expression: An Overview. Front Neurosci. 10<strong>,</strong> 233.</p>
<p>Xu, S., et al., 2015. Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro. Cell Mol Neurobiol. 35<strong>,</strong> 389-398.</p>
<p>Yadav, S., et al., 2012. Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson's disease phenotype in the mouse. Neurochem Res. 37<strong>,</strong> 875-84.</p>
<p>Yuste, J.E., et al., 2015. Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci. 9<strong>,</strong> 322.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2013. Maternal exposure to mercury chloride during pregnancy and lactation affects the immunity and social behavior of offspring. Toxicol Sci. 133<strong>,</strong> 101-11.</p>
2017-11-09T04:11:552019-11-07T10:27:595c1d31fd-b491-4910-9692-349d787fe960eb624ffb-7315-4087-82d2-94e1a4f9d480<p>Cells of the innate (microglia and astrocytes) and of the adaptive (infiltrating monocytes and lymphocytes) immune system of the brain have various ways to kill neighboring cells. This is in part due to evolutionary-conserved mechanisms evolved to kill virus-infected cells or tumor cells; in part it is a bystander phenomenon due to the release of mediators that should activate other cells and contribute to the killing of invading micro-organisms. An exaggerated or unbalanced activation of immune cells can thus lead to parenchymal (neuronal) cell death (Gehrmann et al., 1995). Mediators known to have such effects comprise components of the complement system and cytolkines/death receptor ligands triggering programmed cell death (Dong and Benveniste, 2001). Various secreted proteases (e.g. matrix metalloproteases), lipid mediators (e.g. ceramide or gangliosides) or reactive oxygen species can contribute to bystander death of neurons (Chao et al., 1995; Nakajima et al., 2002; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetsch and Block, 2013). The equimolar production of superoxide and NO from glial cells can lead to high steady levels of peoxynitrite, which is a very potent cytotoxicant (Yuste et al., 2015). Already stressed neurons, with an impaired anti-oxidant defence system, are more sensitive to such mediators (Xu et al., 2015). Healthy cells continuously display anti "eat-me" signals, while damaged and stressed neurons/neurites display "eat-me" signals that may be recognized by microglia as signals to start phagocytosis (Neher et al., 2012). Reactive astrocytes are also able to release neurotoxic molecules (Mena and Garcia de Ybenes, 2008; Niranjan, 2014). However, astrocytes may also be protective due to their capacity to quench free radicals and secrete neurotrophic factors. The activation of astrocytes may reduce neurotrophic support to neurons (for review, Mena and Garcia de Ybenes, 2008).</p>
<p>In vitro co-culture experiments have demonstrated that reactive glial cells (microglia and astrocytes) can kill neurons (Chao et al., 1995; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetzsch and Block, 2013) and that interventions with e.g. i-NOS inhibition can rescue the neurons (Yadav et al., 2012; Brzozowski et al., 2015). Drugs that block Toll like receptor pathways, which are expressed by glial cells have been proven to be protective by decreasing ROS and RNS production (Lucas et al., 2013).</p>
<p>Reactive microglia can remove synapses, a process known as synapse stripping (Banati et al., 1993; Kettenmann et al., 2013). Reactive astrocytes were also associated with neurite and synapse reduction (Calvo-Ochoa et al., 2014). Microglia can modulate synapse plasticity, an effect mediated by cytokines. During development, microglia can promote synaptogenesis or engulf synapses, a process known as synaptic pruning (for review, Jebelli et al., 2015). It is hypothesized that alterations in microglia functioning during synapse formation and maturation of the brain can have significant long-term effects on the final established neural circuits (for review, Harry and Kraft, 2012). The fact that astrocytes can receive and respond to the synaptic information produced by neuronal activity, owing to their expression of a wide range of neurotransmitter receptors, has given rise to the concept of tripartite synapse (for review, Perez-Alvarez and Araque, 2013; Bezzi and Volterra, 2001). Pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6, which are produced by reactive astrocytes, are on one side implicated in synapse formation and scaling, long-term potentiation and neurogenesis (for review, Bilbo and Schwartz, 2009) and on the other side can kill neurons (Chao et al., 1995; Kraft and Harry, 2011). Taken together, this suggests that neuron-glia interactions are tightly regulated and that an imbalance, such as increased or long-term release of these inflammatory mediators may lead to deleterious effects on neurons.</p>
<p><strong>Mercury</strong></p>
<p>Mercury accumulates in the brain particularly in astrocytes and induce astrocyte swelling, excitatory amino acid release and decreased anti-oxidant protections (Shanker et al., 2003; Allen et al., 2001), features that are also observed in reactive astrocytes. Due to the central role of astrocytes for neuronal function (control of water transport, production of trophic factors, of anti-oxidants, tri-partite synapse,… (Ximeres da Silva, 2016; Bezzi and Volterra, 2001; Hertz and Zielke, 2004; Sidoryk-Wegrzynowicz et al., 2011), it is thought that neuronal dysfunction may be secondary to disturbance in astrocytes (Aschner et al., 2007).</p>
<p>Perinatal exposure (GD7-PD21) of rat to MeHgCl (0.5 mg/kg bw/day) in drinking water lead to gliosis in cerebellum of immature rats (PD21) without affecting the cholinergic system. In contrast, at PD36, astrogliosis was accompanied by an increase of muscarinic M2-immunopositive Bergman cells and a lack of M3 muscarinic receptors in the molecular layer. These results suggest that astrogliosis which is observed first at PD21 may be responsible of the delayed effects of mercury on neurons (Roda et al., 2008).</p>
<p>Developmental exposure of mice from GD8 to PD21 to 50 mM HgCl<sub>2</sub> in maternal drinking water: Female offsprings exhibited higher neuroinflammation which is associated with altered social behavior (Zhang et al., 2013).</p>
<p>MG17, a novel triazole derivative, was able to reduce mercury-induced upregulation of IL-1b, IL-6 and TNF-a (measured by RT-PCR) and proved to be protective against mercury-induced neurodegeneration (Matharasala et al., 2017).</p>
<p>Adult rats exposed to MeHg (5mg/kg bw) for 12 consecutive days exhibited piknotic nuclei in cerebellar granule cells, what was reverted by a co-administration of CA074 an inhibitor of cathepsin released by activated microglia. These observations strongly suggest that the mercury–induced neuronal pathological changes are secondary to microglial activation (Sakamoto et al., 2008).</p>
<p> </p>
<p><strong>Acrylamide</strong></p>
<p>Rats exposed to acrylamide (20 mg/kg bw for 4 weeks) together with farmesol (sequiterpene) showed a downregulation of astrogliosis (i.e. decreased GFAP) and of microgliosis (i.e. decreased Iba1) and of TNF-a, Il-1b and i-NOS in cortex, hippocampus and striatum. This was associated with a marked improvement in motor coordination and a decrease in markers of oxidative stress, as compared to rats exposed to acrylamide alone (Santhanasabapathy et al., 2015).</p>
<p> </p>
<p>In 3D rat brain cell-cultures, co-administration of the pro-inflammatory cytokine IL-6 (10 ng/ml) together with non-cytotoxic concentrations of MeHgCl (3 x 10<sup>-7</sup> M) for 10 days protected from the mercury-induced decreased in MAP2 immunostaining, suggesting a positive effect of IL-6, in accord with its descibed trophic activity (Eskes et al., 2002).</p>
HighUnspecificHighDuring brain development, adulthood and agingHighHigh<p>Allen, J.W., Shanker, G., Aschner, M., 2001. Methylmercury inhibits the in vitro uptake of the glutathione precursor, cystine, in astrocytes, but not in neurons. Brain Res. 894<strong>,</strong> 131-40.</p>
<p>Aschner, M., et al., 2007. Involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity. Braz J Med Biol Res. 40<strong>,</strong> 285-91.</p>
<p>Banati, R.B., et al., 1993. Cytotoxicity of microglia. Glia. 7<strong>,</strong> 111-8.</p>
<p>Bezzi, P., Volterra, A., 2001. A neuron-glia signalling network in the active brain. Curr Opin Neurobiol. 11<strong>,</strong> 387-94.</p>
<p>Bilbo, S.D., Schwarz, J.M., 2009. Early-life programming of later-life brain and behavior: a critical role for the immune system. Front Behav Neurosci. 3<strong>,</strong> 14.</p>
<p>Brown GC, Bal-Price A. 2003. Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27(3): 325-355.</p>
<p>Brzozowski MJ, Jenner P, Rose S. 2015. Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity. J Neural Transm 122(6): 779-788.</p>
<p>Calvo-Ochoa, E., et al., 2014. Short-term high-fat-and-fructose feeding produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus. J Cereb Blood Flow Metab. 34<strong>,</strong> 1001-8.</p>
<p>Chao CC, Hu S, Peterson PK. 1995. Glia, cytokines, and neurotoxicity. CritRevNeurobiol 9: 189-205.</p>
<p>Dong Y, Benveniste EN. 2001. Immune Function of Astrocytes. Glia 36: 180-190.</p>
<p>Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.</p>
<p>Gehrmann J, Banati RB, Wiessnert C, Hossmann KA, Kreutzberg GW. 1995. Reactive microglia in cerebral ischaemia: An early mediator of tissue damage? NeuropatholApplNeurobiol 21: 277-289.</p>
<p>Harry, G.J., Kraft, A.D., 2012. Microglia in the developing brain: a potential target with lifetime effects. Neurotoxicology. 33<strong>,</strong> 191-206.</p>
<p>Hertz, L., Zielke, H.R., 2004. Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci. 27<strong>,</strong> 735-43.</p>
<p>Jebelli, J., et al., 2015. Glia: guardians, gluttons, or guides for the maintenance of neuronal connectivity? Ann N Y Acad Sci. 1351<strong>,</strong> 1-10.</p>
<p>Kettenmann, H., Kirchhoff, F., Verkhratsky, A., 2013. Microglia: new roles for the synaptic stripper. Neuron. 77<strong>,</strong> 10-8.</p>
<p>Kraft AD, Harry GJ. 2011. Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International journal of environmental research and public health 8(7): 2980-3018.</p>
<p>Lucas, K., Maes, M., 2013. Role of the Toll Like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway. Mol Neurobiol. 48<strong>,</strong> 190-204.</p>
<p>Matharasala, G., Samala, G., Perumal, Y., 2017. MG17, a novel triazole derivative abrogated neuroinflammation and related neurodegenerative symptoms in rodents. Curr Mol Pharmacol.</p>
<p>Mena MA, Garcia de Yebenes J. 2008. Glial cells as players in parkinsonism: the "good," the "bad," and the "mysterious" glia. Neuroscientist 14(6): 544-560.</p>
<p>Nakajima K, Tohyama Y, Kohsaka S, Kurihara T. 2002. Ceramide activates microglia to enhance the production/secretion of brain-derived neurotrophic factor (BDNF) without induction of deleterious factors in vitro. J Neurochem 80: 697-705.</p>
<p>Niranjan R. 2014. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease: focus on astrocytes. Mol Neurobiol 49(1): 28-38.</p>
<p>Neher JJ, Neniskyte U, Brown GC. 2012. Primary phagocytosis of neurons by inflamed microglia: potential roles in neurodegeneration. Frontiers in pharmacology 3: 27.</p>
<p>Perez-Alvarez, A., Araque, A., 2013. Astrocyte-neuron interaction at tripartite synapses. Curr Drug Targets. 14<strong>,</strong> 1220-4.</p>
<p>Roda, E., et al., 2008. Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: an immunohistochemical study in rat. J Chem Neuroanat. 35<strong>,</strong> 285-94.</p>
<p>Sakamoto, M., et al., 2008. Possible involvement of cathepsin B released by microglia in methylmercury-induced cerebellar pathological changes in the adult rat. Neurosci Lett. 442<strong>,</strong> 292-6.</p>
<p>Shanker, G., Syversen, T., Aschner, M., 2003. Astrocyte-mediated methylmercury neurotoxicity. Biol Trace Elem Res. 95<strong>,</strong> 1-10.</p>
<p>Santhanasabapathy, R., et al., 2015. Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence. Neuroscience. 308<strong>,</strong> 212-27.</p>
<p>Sidoryk-Wegrzynowicz, M., et al., 2011. Role of astrocytes in brain function and disease. Toxicol Pathol. 39<strong>,</strong> 115-23.</p>
<p>Taetzsch T, Block ML. 2013. Pesticides, microglial NOX2, and Parkinson's disease. J Biochem Mol Toxicol 27(2): 137-149.</p>
<p>Ximenes-da-Silva, A., 2016. Metal Ion Toxins and Brain Aquaporin-4 Expression: An Overview. Front Neurosci. 10<strong>,</strong> 233.</p>
<p>Xu, S., et al., 2015. Wogonin prevents rat dorsal root ganglion neurons death via inhibiting tunicamycin-induced ER stress in vitro. Cell Mol Neurobiol. 35<strong>,</strong> 389-398.</p>
<p>Yadav, S., et al., 2012. Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson's disease phenotype in the mouse. Neurochem Res. 37<strong>,</strong> 875-84.</p>
<p>Yuste, J.E., et al., 2015. Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci. 9<strong>,</strong> 322.</p>
<p>Zhang, Y., Bolivar, V.J., Lawrence, D.A., 2013. Maternal exposure to mercury chloride during pregnancy and lactation affects the immunity and social behavior of offspring. Toxicol Sci. 133<strong>,</strong> 101-11.</p>
2018-02-01T05:55:072018-02-12T04:58:24eb624ffb-7315-4087-82d2-94e1a4f9d48001b5a972-b7d4-4745-8a96-d3ad5fe5dd1b<p>Under physiological conditions, in the developing nervous system, apoptosis occurs during the process of synaptogenesis, where competition leads to the loss of excess neurons and to the connection of the appropriate neurons (Buss et al., 2006; Mennerick and Zorumski, 2000; Oppenheim, 1991). When a stressor increases the number of apoptotic cells this KE has a negative effect on synaptogenesis as the reduced number of neurons (besides the ones that have been already eliminated through the physiological process of apoptosis) provides limited dendritic fields for receiving synaptic inputs from incoming axons. At the same time the loss of neurons also means that there are less axons to establish synaptic contacts (Olney, 2014), leading to reduced synaptogenesis. The ability of a neuron to communicate is based on neural network formation that relies on functional synapse establishment (Colón-Ramos, 2009). The main roles of synapses are the regulation of intercellular communication in the nervous system, and the information flow within neural networks. The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation due to cell death during the process of synaptogenesis is critical and leads to decrease of neural network formation and function in the adult brain.</p>
<p>Synaptic transmission and plasticity require the integrity of the anatomical substrate. The connectivity of axons emanating from one set of cells to post-synaptic side of synapse on the dendrites of the receiving cells must be intact for effective communication between neurons. Changes in the placement of cells within the network due to delays in neuronal migration, the absence of a full formation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will individually and cumulatively impair synaptic function.</p>
<p>Therefore, chemicals inducing neuronal cell death by apoptosis or necrosis, or interfering with a particular system of neurotransmitters, will alter network structure and function.</p>
<p>Recently, Dekkers et al. 2013 have reviewed how under physiological conditions components of the apoptotic machinery in developing brain regulate synapse formation and neuronal connectivity. For example, caspase activation is known to be required for axon pruning during development to generate neuronal network (reviewed in Dekkers et al., 2013). Experimental work carried out in Drosophila melanogaster and in mammalian neurons shows that components of apoptotic machinery are involved in axonal degeneration that can consequently interfere with synapse formation (reviewed in Dekkers et al., 2013). Furthermore, Bax mutant mice studies indicate that the lack of this pro-apoptotic protein BAX leads to disruption of intrinsically photosensitive retinal ganglion cells spacing and dendritic stratification that affects synapse localization and function (Chen et al., 2013).</p>
<p>Neuronal network formation and function are established via the process of synaptogenesis. The developmental period of synaptogenesis is critical for the formation of the basic circuitry of the nervous system, although neurons are able to form new synapses throughout life (Rodier, 1995). The brain electrical activity dependence on synapse formation is critical for proper neuronal communication.</p>
<p>Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD-95 arrests the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).</p>
<p>Studies of the last 30 years demonstrated that astrocytes possess functional receptors for neurotransmitters and respond to their stimulation via release of gliotransmitters, including glutamate. These findings have led to a new concept of neuron–glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity (Rossi and Volterra, 2009). According to the concept termed "tripartite synapse", the emerging view is that brain function actually arises from the coordinated activity of a network comprising both neurons and astrocytes. Furthermore, myelinating glial cells are well-known to insulate axons and to speed up action potential propagation. Be it motor skill learning or social behaviors in higher vertebrates, proper myelination is critical in shaping brain functions. Neurons rely on their myelinating partners not only for setting conduction speed, but also for regulating the ionic environment and fueling their energy demands with metabolites. Also, long-term axonal integrity and neuronal survival are maintained by oligodendrocytes and loss of this well-coordinated axon-glial interplay contributes to brain diseases (Saab and Nave, 2017). Therefore, reduction in glial cell number and/or reduction in myelination of axons, will very much impact the neural network function.</p>
<p><strong>Mercury</strong></p>
<table border="1" cellpadding="0" cellspacing="0" class="Tabellenraster1" style="border-collapse:collapse; border:none; mso-border-alt:solid windowtext .5pt; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-table-layout-alt:fixed; mso-yfti-tbllook:1184; width:683px">
<tbody>
<tr>
<td style="width:81.5pt">
<p style="text-align:left"><strong><span style="font-size:9.0pt">KE<sub>up</sub></span></strong></p>
<p style="text-align:left"><strong><span style="font-size:8.0pt">Cell injury/death</span></strong></p>
</td>
<td style="width:88.55pt">
<p style="text-align:left"><strong><span style="font-size:9.0pt">KE<sub>down</sub></span></strong></p>
<p style="text-align:left"><strong><span style="font-size:8.0pt">Decreased network formation and function</span></strong></p>
</td>
<td style="width:91.95pt">
<p><strong><span style="font-size:9.0pt">species; developmental stage of exposure to stressor</span></strong></p>
</td>
<td style="width:42.55pt">
<p><strong><span style="font-size:9.0pt">Stressor</span></strong></p>
</td>
<td style="width:70.8pt">
<p style="text-align:left"><strong><span style="font-size:9.0pt">Dose or conc.</span></strong></p>
<p style="text-align:left"><strong><span style="font-size:9.0pt">Duration</span></strong></p>
</td>
<td style="width:70.85pt">
<p style="text-align:left"><strong><span style="font-size:9.0pt">Protective/ aggravating evidence</span></strong></p>
</td>
<td style="width:66.3pt">
<p><strong><span style="font-size:9.0pt">Reference</span></strong></p>
</td>
</tr>
<tr>
<td style="width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Apoptosis measured by levels of Cleaved caspase3 (2x CTR values)</span></p>
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Inhibition of hippocampal-dependent memory processes at P35</span></p>
<p style="text-align:left"><span style="font-size:8.0pt">(water maze)</span></p>
</td>
<td style="width:91.95pt">
<p><span style="font-size:8.0pt">Rat</span></p>
<p><span style="font-size:8.0pt">exposed at P7</span></p>
</td>
<td style="width:42.55pt">
<p><span style="font-size:8.0pt">MeHgCl</span></p>
</td>
<td style="width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">5 µg g<sup>-1</sup></span></p>
<p style="text-align:left"><span style="font-size:8.0pt">single injection</span></p>
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:66.3pt">
<p><span style="font-size:8.0pt">Falluel-Morel, 2007</span></p>
</td>
</tr>
<tr>
<td style="height:68.4pt; width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Apoptosis measured by DNA laddering and electron microscopy</span></p>
</td>
<td style="height:68.4pt; width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Nerve fibers degeneration in peripheral nerves, sensory ganglia, root nerve, spinal cord and cerebellum</span></p>
</td>
<td style="height:68.4pt; width:91.95pt">
<p><span style="font-size:8.0pt">Rat</span></p>
<p><span style="font-size:8.0pt">adult exposure</span></p>
</td>
<td style="height:68.4pt; width:42.55pt">
<p><span style="font-size:8.0pt">MeHgCl</span></p>
</td>
<td style="height:68.4pt; width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">4-10 mg kg<sup>-1</sup> day<sup>-1</sup></span></p>
<p style="text-align:left"><span style="font-size:8.0pt">7-20 days</span></p>
<p style="text-align:left"><span style="font-size:8.0pt">subcutaneous or oral</span></p>
</td>
<td style="height:68.4pt; width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="height:68.4pt; width:66.3pt">
<p><span style="font-size:8.0pt">Nagashima, 1997 (review)</span></p>
</td>
</tr>
<tr>
<td style="width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Apoptosis measured by in situ DNA strand breaks, DNA laddering and electron microscopy</span></p>
</td>
<td style="width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Nerve fibers degeneration in cerebellum</span></p>
</td>
<td style="width:91.95pt">
<p><span style="font-size:8.0pt">Rat</span></p>
<p><span style="font-size:8.0pt">adult exposure</span></p>
</td>
<td style="width:42.55pt">
<p><span style="font-size:8.0pt">MeHgCl</span></p>
</td>
<td style="width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">4 mg kg<sup>-1</sup> day<sup>-1</sup></span></p>
<p style="text-align:left"><span style="font-size:8.0pt">20 days</span></p>
<p style="text-align:left"><span style="font-size:8.0pt">oral</span></p>
</td>
<td style="width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:66.3pt">
<p><span style="font-size:8.0pt">Nagashima, 1996</span></p>
</td>
</tr>
<tr>
<td style="width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Necrosis and apoptosis measured by chromatin condensation on primary cultures of cortical neurons prepared from the F1 generation pups</span></p>
</td>
<td style="width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Fragmentation of the neuronal network (microtubule disruption) in vitro and long-term memory impairment in vivo (at P90)</span></p>
</td>
<td style="width:91.95pt">
<p><span style="font-size:8.0pt">Rat pregnant exposed to mercury at GD15</span></p>
<p><span style="font-size:8.0pt"> </span></p>
<p><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:42.55pt">
<p><span style="font-size:8.0pt">MeHgCl</span></p>
</td>
<td style="width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">4 and 8 mg kg<sup>-1</sup> single gavage</span></p>
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
</td>
<td style="width:66.3pt">
<p><span style="font-size:8.0pt">Ferraro, 2009</span></p>
</td>
</tr>
<tr>
<td style="height:30.25pt; width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Extensive neuronal cell loss (histopathology) in F1 generation pups (PND25)</span></p>
</td>
<td style="height:30.25pt; width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Decreased activity of acetylcholinesterase in F1 generation pups (PND24) and less time latency to fall in rotarod test, increased escape time latency in Morris water maze test, increased immobility time in forced-swim test</span></p>
</td>
<td style="height:30.25pt; width:91.95pt">
<p><span style="font-size:8.0pt">Rat pregnant</span></p>
<p><span style="font-size:8.0pt">exposed to mercury from GD5 till</span></p>
<p><span style="font-size:8.0pt">parturition</span></p>
</td>
<td style="height:30.25pt; width:42.55pt">
<p><span style="font-size:8.0pt">MeHgCl</span></p>
</td>
<td style="height:30.25pt; width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">1.5 mg kg<sup>-1</sup></span></p>
<p style="text-align:left"><span style="font-size:8.0pt">orally</span></p>
</td>
<td style="height:30.25pt; width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt">Co-administration of fisetin (plant flavonoid) alleviated all MeHgCl effects</span></p>
</td>
<td style="height:30.25pt; width:66.3pt">
<p><span style="font-size:8.0pt">Jacob, 2017</span></p>
</td>
</tr>
<tr>
<td style="height:30.25pt; width:81.5pt">
<p style="text-align:left"><span style="font-size:8.0pt">Apoptosis observed 7 days after exposure</span></p>
</td>
<td style="height:30.25pt; width:88.55pt">
<p style="text-align:left"><span style="font-size:8.0pt">Degeneration of the dopaminergic system observed 7 days after exposure</span></p>
</td>
<td style="height:30.25pt; width:91.95pt">
<p><span style="font-size:8.0pt">Rat</span></p>
<p><span style="font-size:8.0pt">adult exposure</span></p>
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<td style="height:30.25pt; width:42.55pt">
<p><span style="font-size:8.0pt">Acrolein</span></p>
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<td style="height:30.25pt; width:70.8pt">
<p style="text-align:left"><span style="font-size:8.0pt">Single intranigral infusion of 15, 50, 150 nmoles </span></p>
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<td style="height:30.25pt; width:70.85pt">
<p style="text-align:left"><span style="font-size:8.0pt"> </span></p>
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<td style="height:30.25pt; width:66.3pt">
<p><span style="font-size:8.0pt">Wang, 2017</span></p>
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</table>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p><strong>Acrylamide</strong></p>
<p>No publications found to support this KE</p>
<p> </p>
<p> </p>
<p>Ogawa et al. (2011) reported decreased apoptosis and an increase in the number of Gabaergic interneurons in the dentate gyrus of Sprague-Dawley pups either maternally exposed to acrylamide or directly injected with acrylamide.</p>
<p>Although it appears evident that a decrease in cell number, in dendritic arborization or in axonal growth, as well as synapse alterations may lead to decreased neuronal network formation and function, the exact mechanism remain to be elucidated.</p>
<p>Whereas the quantification of cell injury and death is straightforward, the quantification of the decreased network function is much more qualitative than quantitative, precluding a quantitative understanding of the linkage for this KER.</p>
Not SpecifiedMaleNot SpecifiedFemaleNot SpecifiedAll life stagesHighHighModerate<p>Support for the link between cell injury/death and decreased neuronal network formation and function can be found in rat, mouse and minnow. (for references, see empirical evidences)</p>
<p>Buss, R.R., Sun, W., Oppenheim, R.W., 2006. Adaptive roles of programmed cell death during nervous system development. Annu Rev Neurosci 29, 1-35.</p>
<p>Chen, S.K., Chew, K.S., McNeill, D.S., Keeley, P.W., Ecker, J.L., Mao, B.Q., Pahlberg, J., Kim, B., Lee, S.C., Fox, M.A., Guido, W., Wong, K.Y., Sampath, A.P., Reese, B.E., Kuruvilla, R., Hattar, S., 2013. Apoptosis regulates ipRGC spacing necessary for rods and cones to drive circadian photoentrainment. Neuron 77, 503-515.</p>
<p>Cline, H., Haas, K., 2008. The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis. J Physiol 586, 1509-1517.</p>
<p>Colon-Ramos, D.A., 2009. Synapse formation in developing neural circuits. Curr Top Dev Biol 87, 53-79.</p>
<p>Dekkers, M.P., Nikoletopoulou, V., Barde, Y.A., 2013. Cell biology in neuroscience: Death of developing neurons: new insights and implications for connectivity. J Cell Biol 203, 385-393.</p>
<p>Ehrlich, I., Klein, M., Rumpel, S., Malinow, R., 2007. PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A 104, 4176-4181.</p>
<p>Falluel-Morel, A., Sokolowski, K., Sisti, H.M., Zhou, X., Shors, T.J., Dicicco-Bloom, E., 2007. Developmental mercury exposure elicits acute hippocampal cell death, reductions in neurogenesis, and severe learning deficits during puberty. J Neurochem 103, 1968-1981.</p>
<p>Ferraro, L., Tomasini, M.C., Tanganelli, S., Mazza, R., Coluccia, A., Carratu, M.R., Gaetani, S., Cuomo, V., Antonelli, T., 2009. Developmental exposure to methylmercury elicits early cell death in the cerebral cortex and long-term memory deficits in the rat. Int J Dev Neurosci 27, 165-174.</p>
<p>Jacob, S., Thangarajan, S., 2017. Effect of Gestational Intake of Fisetin (3,3',4',7-Tetrahydroxyflavone) on Developmental Methyl Mercury Neurotoxicity in F1 Generation Rats. Biol Trace Elem Res 177, 297-315.</p>
<p>Mennerick, S., Zorumski, C.F., 2000. Neural activity and survival in the developing nervous system. Mol Neurobiol 22, 41-54.</p>
<p>Nagashima, K., 1997. A review of experimental methylmercury toxicity in rats: neuropathology and evidence for apoptosis. Toxicol Pathol 25, 624-631.</p>
<p>Nagashima, K., Fujii, Y., Tsukamoto, T., Nukuzuma, S., Satoh, M., Fujita, M., Fujioka, Y., Akagi, H., 1996. Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats. Acta Neuropathol 91, 72-77.</p>
<p>Ogawa, B., Ohishi, T., Wang, L., Takahashi, M., Taniai, E., Hayashi, H., Mitsumori, K., Shibutani, M., 2011. Disruptive neuronal development by acrylamide in the hippocampal dentate hilus after developmental exposure in rats. Arch Toxicol 85, 987-994.</p>
<p>Olney, J.W., 2014. Focus on apoptosis to decipher how alcohol and many other drugs disrupt brain development. Front Pediatr 2, 81.</p>
<p>Oppenheim, R.W., 1991. Cell death during development of the nervous system. Annu Rev Neurosci 14, 453-501.</p>
<p>Perea, G., Navarrete, M., Araque, A., 2009. Tripartite synapses: astrocytes process and control synaptic information. Trends Neurosci 32, 421-431.</p>
<p>Rodier, P.M., 1995. Developing brain as a target of toxicity. Environ Health Perspect 103 Suppl 6, 73-76.</p>
<p>Rossi, D., Volterra, A., 2009. Astrocytic dysfunction: insights on the role in neurodegeneration. Brain Res Bull 80, 224-232.</p>
<p>Saab, A.S., Nave, K.A., 2017. Myelin dynamics: protecting and shaping neuronal functions. Curr Opin Neurobiol 47, 104-112.</p>
<p>Wang, Y.T., Lin, H.C., Zhao, W.Z., Huang, H.J., Lo, Y.L., Wang, H.T., Lin, A.M., 2017. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of alpha-synuclein aggregation and programmed cell death. Sci Rep 7, 45741.</p>
2017-11-09T04:12:252020-02-05T12:26:4201b5a972-b7d4-4745-8a96-d3ad5fe5dd1b8a7262ff-2e32-43a4-a7e2-e3bde5e0b42f<p>Learning and memory is one of the outcomes of the functional expression of neurons and neural networks from mammalian to invertebrates. Damage or destruction of neurons by chemical compounds during development when they are in the process of synapses formation, integration and formation of neural networks, will derange the organization and function of these networks, thereby setting the stage for subsequent impairment of learning and memory. Exposure to the potential developmental toxicants during neuronal differentiation and synaptogenesis will increase risk of functional neuronal network damage leading to learning and memory impairment.</p>
<p>Impairments in learning and memory are measured using behavioral techniques. It is well accepted that these alterations in behavior are the result of structural or functional changes in neurocircuitry. Functional impairments are often measured using field potentials of critical synaptic circuits in hippocampus and cortex. A number of studies have been performed in rodent models that reveal deficits in both excitatory and inhibitory synaptic transmission in the hippocampus as a result of developmental thyroid insufficiency (Wang et al., 2012; Oerbeck et al., 2003; Wheeler et al., 2011; Wheeler et al., 2015; Willoughby et al., 2014; Davenport and Dorcey, 1972; Tamasy et al., 1986; Akaike, 1991; Axelstad et al., 2008; Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011; Gilbert et al., 2016). A well-established functional readout of memory at the synaptic level is known as long-term potentiation (LTP) (i.e., a persistent strengthening of synapses based on recent patterns of activity). Deficiencies in LTP are generally regarded as potential substrates of learning and memory impairments. In rodent models where synaptic function is impaired by TH deficiencies, deficits in hippocampus-mediated memory are also prevalent (Gilbert and Sui, 2006; Gilbert et al., 2016; Gilbert, 2011; Gilbert et al., 2016).</p>
<p>A number of studies have consistently reported alterations in synaptic transmission resulting from developmental TH disruption, and leading to decreased cognition.</p>
<p>Long-term potentiation (LTP) is a long-lasting increase in synaptic efficacy and its discovery suggested that changes in synaptic strength could provide the substrate for learning and memory (reviewed in Lynch, 2004). Moreover, LTP is intimately related to the theta rhythm, an oscillation long associated with learning. Learning-induced enhancement in neuronal excitability, a measurement of neural network function, has also been shown in hippocampal neurons following classical conditioning in several experimental approaches (reviewed in Saar and Barkai, 2003).</p>
<p>On the other hand, memory requires the increase in magnitude o<span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">f excitatory postsynaptic currents (</span></span>EPSCs) to be developed quickly and to be persistent for few weeks at least without disturbing already potentiated contacts. Once again, a substantial body of evidence has demonstrated that tight connection between LTP and diverse instances of memory exist (reviewed in Lynch, 2004).</p>
<p>A review on Morris water maze (MWM) as a tool to investigate spatial learning and memory in laboratory rats also pointed out that the disconnection between neuronal networks rather than the brain damage of certain regions is responsible for the impairment of MWM performance. Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and MWM performance<strong> </strong><span style="font-size:12px">(D'Hooge and De Deyn, 2001).</span></p>
<p>Moreover, it is well accepted that alterations in synaptic transmission and plasticity contribute to deficits in cognitive function. There are a number of studies that have linked exposure to TPO inhibitors (e.g., PTU, MMI), as well as iodine deficient diets, to changes in serum TH levels, which result in alterations in both synaptic function and cognitive behaviors (Akaike et al., 1991; Vara et al., 2002; Gilbert and Sui, 2006; Axelstad et al., 2008; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016), described in the indirect KER "Decrease of TH synthesis leads to learning and memory deficits".</p>
<p style="text-align:justify">Developmental hypothyroidism reduces the functional integrity in brain regions critical for learning and memory. Neurophysiological indices of synaptic transmission of excitatory and inhibitory circuitry are impaired in the hippocampus of hypothyroid animals. Both hippocampal regions (area CA1 and dentate gyrus) exhibit alterations in excitatory and inhibitory synaptic transmission following reductions in serum TH in the pre and early postnatal period (Vara et al., 2002; Sui and Gilbert, 2003; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). These alterations persist into adulthood despite a recovery to euthyroid conditions in blood. The latter observation indicates that these alterations represent permanent changes in brain function caused by transient hormones insufficiencies induced during critical window of development. </p>
<p style="text-align:justify">Because the adult hippocampus is involved in learning and memory, it is a brain region of remarkable plasticity. Use-dependent synaptic plasticity is critical during brain development for synaptogenesis and fine tuning of synaptic connectivity. In the adult brain, similar plasticity mechanisms underlie use-dependency that underlies learning and memory, as exhibited in LTP model of synaptic memory. Hypothyroidism during development reduces the capacity for synaptic plasticity in juvenile and adult offspring (Vara et al., 2002; Sui and Gilbert, 2003; Dong et al., 2005; Sui et al., 2005; Gilbert and Sui, 2006; Taylor et al., 2008; Gilbert, 2011; Gilbert et al., 2016). Decrease of neuronal network function and plasticity are observed coincident with deficits in learning tasks that require the hippocampus.</p>
<p style="text-align:justify"><strong>- Wang et al., 2012: </strong>This study showed that maternal subclinical hypothyroidism impairs spatial learning in the offspring, as well as the efficacy and optimal time of T4 treatment in pregnancy. Female adult Wistar rats were randomly divided into six groups: control, hypothyroid (H), subclinical hypothyroid (SCH) and SCH treated with T4, starting from GD10, GD13 and GD17, respectively, to restore normal TH levels. Results indicate that progenies of SCH and H groups demonstrated significantly longer mean latency in the water maze test (on the 2<sup>nd</sup> training day, latency was ~83% higher in H group, and ~50% higher in SCH), and a lower amplification percentage of the amplitude (~15% lower in H group, and 12% lower in SCH), and slope of the field excitatory postsynaptic potential (fEPSP) recording (~20% lower in H group, and 17% lower in SCH), compared to control group. T4 treatment at GD10 and GD13 significantly shortened mean latency and increased the amplification percentage of the amplitude and slope of the fEPSPs of the progeny of rats with subclinical hypothyroidism. However, T4 treatment at GD17 showed only minimal effects on spatial learning in the offspring. Altogether these data indicate direct correlation between decrease of neural network function and learning and memory deficits.</p>
<p style="text-align:justify"><strong>- Liu et al., 2010 </strong>This study assessed the effects of hypothyroidism in 60 female rats who were divided into three groups: (i) maternal subclinical hypothyroidism (total thyroidectomy with T4 infusion), (ii) maternal hypothyroidism (total thyroidectomy without T4 infusion), and (iii) control (sham operated). The Morris water maze tests revealed that pups from the subclinical hypothyroidism group showed long-term memory deficits, and a trend toward short-term memory deficits.</p>
<p style="text-align:justify"><strong>- Gilbert and Sui, 2006 </strong>Administration of 3 or 10 ppm PTU to pregnant and lactating dams via the drinking water from GD6 until PND30 caused a 47% and 65% reduction in serum T4, in the dams of the low and high-dose groups, respectively. Baseline synaptic transmission was impaired in PTU-exposed animals: mean EPSP slope (by ~60% with 10 ppm PTU) and population spike amplitudes (by ~70% with 10 ppm PTU) in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams. High-dose animals (10 ppm) demonstrated very little evidence of learning despite 16 consecutive days of training (~5-fold higher mean latency to find the hidden platform, used as an index of learning).</p>
<p style="text-align:justify"><strong>- Gilbert et al., 2016</strong> Exposure to PTU during development produced dose-dependent reductions in mRNA expression of nerve growth factor (Ngf) in whole hippocampus of neonates. These changes in basal expression persisted to adulthood despite the return to euthyroid conditions in blood. Developmental PTU treatment dramatically reduced the activity-dependent expression of neurotrophins and related genes in neonate hippocampus and was accompanied by deficits in hippocampal-based learning (e.g., mean latency to find a hidden platform, at 2<sup>nd</sup> trial resulted ~60% higher in rats treated with 10 ppm PTU).</p>
<p style="text-align:justify"><strong>- Gilbert, 2011</strong> Trace fear conditioning deficits to context and to cue reported in animals treated with PTU and who also displayed synaptic transmission and LTP deficits in hippocampus. Baseline synaptic transmission was impaired in PTU-exposed animals (by ~50% in animal treated with 3 ppm PTU). EPSP slope amplitudes in the dentate gyrus were reduced in a dose-dependent manner in adult offspring of PTU-treated dams.</p>
<p style="text-align:justify"> </p>
<p style="text-align:justify">BPA, an environmental toxicant known to inhibit NIS-mediated iodide uptake (Wu Y et al., 2016) has been found to cause learning and memory deficits in rodents as described below:</p>
<p style="text-align:justify">- <strong>Jang et al., 2012</strong> In this study, pregnant female C57BL/6 mice (F0) were exposed to BPA (0.1-10 mg/kg) from gestation day 6 to 17, and female offspring (F2) from F1 generation mice were analysed. Exposure of F0 mice to BPA (10 mg/kg) decreased hippocampal neurogenesis (~ 30% decrease of hippocampal BrdU<sup>+</sup> cells vs control) in F2 female mice. High-dose BPA (10 mg/kg) caused neurocognitive deficit (i.e., reduced memory retention) as shown by passive avoidance testing (~ 33% decrease vs control) in F2 mice. Furthermore, 10 mg/kg BPA decreased the hippocampal levels of BDNF (~ 35% lower vs control) in F2 mice. These results suggest that BPA exposure (NIS inhibitor) in pregnant mothers could decrease hippocampal neurogenesis (decreased number of neurons) and cognitive function in future generations.</p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><u>In humans</u>, the data linking these two specific KE are much more limited, but certainly clear reductions in IQ, with specific impairments in hippocampus-mediated functions have been observed.</p>
<p style="text-align:justify"><strong>- Wheeler et al., 2015</strong> This study assessed hippocampal functioning in adolescents with congenital hypothyroidism (CH), using functional magnetic resonance imaging (fMRI). 14 adolescents with CH and 14 typically developing controls (TDC) were studied. Hippocampal activation was greater for pairs than items in both groups, but this difference was only significant in TDC. When the groups were directly compared, the right anterior hippocampus was the primary region in which the TDC and CH groups differed for this pair memory effect. Results signify that adolescents with CH show abnormal hippocampal functioning during verbal memory processing, in order to compensate for the effects induced by TH deficit in the brain.</p>
<p style="text-align:justify"><strong>- Wheeler et al., 2012</strong> In this study hippocampal neuronal network function was measured based on synaptic performance using fMRI and was altered while subjects engaged in a memory task. Data showed paired word recognition deficits in adolescents with congenital hypothyroidism (N = 14; age range, 11.5-14.7 years) compared with controls (N = 15; age range, 11.2-15.5 years), with no impairment on simple word lists. Analysis of functional magnetic resonance imaging showed that adolescents with congenital hypothyroidism had both increased magnitude of hippocampal activation relative to controls and bilateral hippocampal activation when only the left was observed in controls. Furthermore, the increased activation in the congenital hypothyroidism group was correlated with the severity of the hypothyroidism experienced early in life.</p>
<p style="text-align:justify"><strong>- Willoughby et al., 2013</strong> Analogously, in this study, fMRI revealed increased hippocampus activation with word pair recognition task in CH and children born to women with hypothyroxinemia during midgestation. These differences in functional activation were not seen with single word recognition, but were revealed when retention of word pair associations was probed. The latter is a task requiring engagement of the hippocampus.</p>
<p style="text-align:justify">A series of important findings suggest that the biochemical changes that happen after induction of LTP also occur during memory acquisition, showing temporality between the two KEs (reviewed in Lynch, 2004).</p>
<p style="text-align:justify"><strong>- Morris et al., 1986</strong> This study found that blocking the NMDA receptor of the neuronal network with AP5 inhibits spatial learning in rats. Most importantly, in the same study they measured brain electrical activity and recorded that this agent also inhibits LTP, however, they have not proven that spatial learning and LTP inhibition are causally related.</p>
<p style="text-align:justify">Since then a number of NMDA receptor antagonists have been studied towards their ability to induce impairment of learning and memory. It is worth mentioning that similar findings have been found in human subjects:</p>
<p style="text-align:justify"><strong>- Grunwald et al., 1999</strong> By combining behavioural and electrophysiological data from patients with temporal lobe epilepsy exposed to ketamine, involvement of NMDA receptors in human memory processes was demonstrated.</p>
<p style="text-align:justify"> </p>
<p style="text-align:justify">The last KE preceding the AO (learning and memory deficits), i.e. "Decreased Neural Network Function", is also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (<a href="https://aopwiki.org/aops/13">https://aopwiki.org/aops/13</a>). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER described in the present AOP.</p>
<p style="text-align:justify"><strong>Pb2+:</strong> Exposure to low levels of Pb2+, during early development, has been implicated in long-lasting behavioural abnormalities and cognitive deficits in children (Needleman et al., 1975; Needleman and Gatsonis, 1990; Bellinger et al., 1991; 1992; Baghurst et al., 1992; Leviton et al., 1993; Needleman et al., 1996; Finkelstein et al., 1998; Lanphear et al., 2000; 2005; Canfield et al., 2003; Bellinger 2004; Lanphear et al., 2005; Surkan et al., 2007; Jusko et al., 2008; Neal and Guilarte, 2010) and experimental animals (Brockel and Cory-Slechta, 1998; Murphy and Regan, 1999; Moreira et al., 2001). Multiple lines of evidence suggest that Pb2+ can impair hippocampus-mediated learning in animal models (reviewed in Toscano and Guilarte, 2005).</p>
<p style="text-align:justify"><strong>- Jett et al., 1997</strong> Female rats exposed to Pb<sup>2+</sup> through gestation and lactation have shown more severe impairment of memory than male rats with similar Pb<sup>2+</sup> exposures.</p>
<p style="text-align:justify"><strong>- De Souza Lisboa et al., 2005</strong> This study reported that exposure to Pb<sup>2+ </sup>during both pregnancy and lactation caused depressive-like behaviour (detected in the forced swimming test) in female but not male rats.</p>
<p style="text-align:justify"><strong>- Anderson et al., 2012</strong> This study investigated the neurobehavioral outcomes in Pb<sup>2+</sup>-exposed rats (250, 750 and 1500 ppm Pb<sup>2+</sup> acetate in food) during gestation and through weaning and demonstrated that these outcomes are very much influenced by sex and rearing environment. In females, Pb<sup>2+</sup> exposure lessened some of the benefits of enriched environment on learning, whereas, in males, enrichment does help to overcome detrimental effects of Pb<sup>2+</sup> on learning. Regarding reference memory, environmental enrichment has not been beneficial in females when exposure to Pb<sup>2+</sup> occurs, in contrast to males.</p>
<p style="text-align:justify"><strong>- Jaako-Movits et al., 2005</strong> Wistar rat pups were exposed to 0.2% Pb<sup>2+</sup> via their dams' drinking water from PND 1 to PND 21 and directly via drinking water from weaning until PND 30. At PND 60 and 80, the neurobehavioural assessment has revealed that developmental Pb<sup>2+</sup> exposure induces persistent increase in the level of anxiety and inhibition of contextual fear conditioning. The same behavioural syndrome in rats has been described in Salinas and Huff, 2002.</p>
<p style="text-align:justify"><strong>- Finkelstein et al., 1998</strong> These observations are in agreement with observations on humans, as children exposed to low levels of Pb<sup>2+</sup> displayed attention deficit, increased emotional reactivity and impaired memory and learning.</p>
<p style="text-align:justify"><strong>- Kumar and Desiraju, 1992</strong> In Wistar rats fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings showed statistically significant reduction in the delta, theta, alpha and beta band EEG spectral power in motor cortex and hippocampus, but not in delta and beta bands power of motor cortex in wakeful state. After 40 days of recovery, animals were assessed for their neurobehaviour, and revealed that Pb<sup>2+</sup> treated animals showed more time and sessions in attaining criterion of learning than controls.</p>
<p style="text-align:justify">Further data obtained using animal behavioral techniques demonstrate that NMDA mediated synaptic transmission is decreased by Pb<sup>2+</sup> exposure (Cory-Slechta, 1995; Cohn and Cory-Slechta, 1993 and 1994).</p>
<p style="text-align:justify"><strong>- Xiao et al., 2014</strong> Rat pups from parents exposed to 2 mM PbCl<sub>2</sub> three weeks before mating until their weaning (pre-weaning Pb<sup>2+</sup>) and weaned pups exposed to 2 mM PbCl<sub>2 </sub>for nine weeks (post-weaning Pb<sup>2+</sup>) were assessed for their spatial learning and memory by MWM on PND 85-90. The study revealed that both rat pups in pre-weaning Pb<sup>2+</sup> and post-weaning Pb<sup>2+</sup> groups performed significantly worse than those in the control group. The number of synapses in pre-weaning Pb<sup>2+</sup> group increased significantly, but it was still less than that of control group. The number of synapses in post-weaning Pb<sup>2+</sup> group was also less than that of control group, although the number of synapses had no differences between post-weaning Pb<sup>2+</sup> and control groups before MWM. In both pre-weaning Pb<sup>2+</sup> and post-weaning Pb<sup>2+</sup> groups, synaptic structural parameters such as thickness of postsynaptic density (PSD), length of synaptic active zone and synaptic curvature increased, whereas width of synaptic cleft decreased compared to controls.</p>
<p style="text-align:justify"><strong>The last KE preceding the AO (learning and memory deficits), i.e. "Decreased Neural Network Function", is also common to the AOP 17, entitled "</strong><strong> Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins during brain development leads to impairment of learning and memory" (</strong><a href="https://aopwiki.org/aops/13"><strong>https://aopwiki.org/aops/17</strong></a><strong>). In this AOP 17, data on mercury exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER described in the present AOP.</strong></p>
<p style="text-align:justify"><strong>Sokolowski et al. 2013</strong>. Rats at postnatal day 7 received a single injection of methylmercury (0.6 microgr/g, that caused caspase activation in the hilus of granule cell layer in hippocampus. At PD 21, a decrease in cell number or 22% in hilus and of 27% in granule cell layer, as well as a decreased proliferation of neural precursor cells of 25% were observed. This was associated with a decrease of spatial memory as assessed by Morris water maze.</p>
<p style="text-align:justify"><strong>Eddins et al., 2008</strong>. Mice exposed during postnatal week 1-3 to 2-5 mg/kg mercury chloride in 0.01 ml/g of NaCl injectd s.c. The behavioral tests at 3 months of age revealed learning deficits (radial maze), which was associated with increased levels of monoamines in frontal cortex.</p>
<p style="text-align:justify"><strong>Zanoli et al., 1994.</strong> Single injection of methylmercury (8 mg/kg by gavage) at gestational day 15. Offsprings analyzed at 14, 21, and 60 days of age exhibited a decrease in the number of muscarinic receptors at 14 and 21 days and a decrease in avoidance latency at 60 days, indicating learning and memory deficits.</p>
<p style="text-align:justify"><strong>Zanoli et al., 2001.</strong> Single injection of methylmercury (8 mg/kg) at gestational day 8. Brain was removed at PD 21 and 60. An increase in tryptophan level in hippocampus was detected at both days. At PD 21, a decrease in anthranilic acid and an increase in quinolinic acid was found. No change in glutamic acid nor in aspartic acid were detected.</p>
<p style="text-align:justify"><strong>Montgomery et al., 2008.</strong> C57/B6 mice exposed during pregnancy (GD 8-18) with food containing methylmercury (0.01 mg/kg body wheight). Tested when adult, they showed deficits in motor function, coordination, overall activity and impairment in reference memory.</p>
<p style="text-align:justify"><strong>Glover et al., 2009.</strong> Balb mice exposed to methylmercury in diet (low dose: 1.5 mg/kg; high dose: 4.5 mg/kg) during 11 weeks (6 weeks prior mating, 3 weeks during gestation and 2 weeks post-partum). Offsprings tested at PD 15 showed an accumulation of Hg in brain (0.08 mg/kg for low dose and 0.25 mg/kg for the high dose). At hte cellular level, there was alterations in gene expression for cytoskeleton, cell processes, cell adhesion, cell differentiation, development), which could be all involved in cellular network formation. This was associated with behavioral impairment, i.e. a decrease in exploratory activity measured in open field.</p>
<p style="text-align:justify"><strong>Onishchenko et al., 2007</strong>. Pregnant mice received 0.5 mg methylmercury/kg/day in drinking water from gestational dy 7 until day 7 after delivery. Offspring behavior was monitored at 5-15 and 26-36 weeks of age. Mercury-induced alterations in reference memory were detected.</p>
<p style="text-align:justify"><strong>Cagiano et al., 1990.</strong> Pregnant rat received at GD 15 8mg/kg of methylmercury by gavage. Offsprings were tested at day 16, 21 and 60. A reduced functional activity of glutamatergic system associated with disturbances in learning and memory were observed.</p>
<p style="text-align:justify"><strong>Rice, 1992.</strong> Female monkeys exposed to 10, 25 and 50 microg/kg/day to methylmercury. Male unexposed. Infants separated from mother at birth and exposed to similar doses did not show gross intellectual impairment, but interferences with temporal discrimination.</p>
<p style="text-align:justify"><strong>Sahin et al., 2016.</strong> Exposure of rat pups for 5 weeks or 5 months with mercury chloride (4.6 microg/kg as first injection, followed each day by 0.07 microg/kg/day). Learning and memory impairment measured by passive avoidance and Morris-water-maze was found in 5-weeks group, but not in the 5-month group. This was accompanied by hearing loss.</p>
<p style="text-align:justify"><strong>In humans:</strong></p>
<p style="text-align:justify"><strong>Orenstein et al., 2014. </strong>Maternal peripartum hair mercury level was measured to assess prenatal mercury exposure. The concentrations of mercury was found in the range of 0.3-5.1 microg/g, similar to fish eating population in US. However, statistical analyses revealed that each microg/g increase in hair Hg was associated with a decrement in visula memory, learning and verbal memory.</p>
<p style="text-align:justify"><strong>Yorifuji et al., 2011</strong>. A survey of the Minamata exposed population made in 1971 to assess pre- and post-natal exposure revealed a methylmercury-induced impairment of intelligence as well as behavioral dysfunction.</p>
<p>One of the most difficult issues for neuroscientists is to link neuronal network function to cognition, including learning and memory. It is still unclear what modifications of neuronal circuits need to happen in order to alter motor behaviour as it is recorded in a learning and memory test (Mayford et al., 2012), meaning that there is no clear understanding about how these two KEs are connected.</p>
<p style="text-align:justify">The direct relationship of alterations in neural network function and specific cognitive deficits is difficult to ascertain given the many forms that learning and memory can take and the complexity of synaptic interactions in even the simplest brain circuit. Linking of neurophysiological assessments to learning and memory processes have, by necessity, been made across simple monosynaptic connections and largely focused on the hippocampus. Alterations in synaptic function have been found in the absence of behavioral impairments. This may result from measuring only one component in the complex brain circuitry that underlies 'cognition', behavioral tests that are not sufficiently sensitive for the detection of subtle cognitive impairments, and behavioral plasticity whereby tasks are solved by the animal via different strategies developed as a consequence of developmental insult.</p>
<p style="text-align:justify">Finally, in order to provide empirical support for this KER, data on the effects of lead (Pb) exposure are reported. Several epidemiological studies where Pb2+ exposure levels have been studied in relation to neurobehavioural alterations in children have been reviewed in Koller et al. 2004. This review has concluded that in some occasions there is negative correlation between Pb2+ dose and cognitive deficits of the subjects due to high influence of social and parenting factors in cognitive ability like learning and memory (Koller et al. 2004), meaning that not always Pb2+ exposure is positively associated with learning and memory impairment in children.</p>
<p style="text-align:justify"><strong>Mercury</strong></p>
<p style="text-align:justify"><strong>Olczak et al., 2001. </strong>Postnatal exposure of rats to Thimerosal (4 injections with 12, 240, 1440 and 3000 microgHg/kg per injection). Effects were measured in adult, which exhibited alterations in dopaminergic system with decline in the density of striatal D2 receptors, with a higher sensitivity for males. No alterations in spatial learning and memory was observed, but impairments of motor activity, increased anxiety (open fiel measurment), which are other symptoms of autism spectrum disorder.</p>
<p style="text-align:justify"><strong>Franco et al., 2006.</strong> Lactational exposure of mice to methylmercury in drinking water (10 mg/L). Analysis at weaning revealed only impairment in motor performances.</p>
<p><strong>Franco et al., 2007.</strong> Lactational exposure of mice with mercury chloride (0.5 and 1.5 mg/kg, i.p. injection once a day).. At weaning , animals exhibited an increased level of mercury in cerebellum associated with motor deficit.</p>
<p style="text-align:justify"><strong>Cardenas et al., 2017</strong> showed that maternal red blood cell mercury of 3.8 ng/g was associated to increased DNA methylation of PON1 in umbilical cord blood only in male and observed deficit in cognitive performances, such as visual motor ability, vocabiary and verbal intellgence.</p>
<p>There is not enough quantitative information how much change decrease of neuronal network functions leads to learning and memory deficits. However, qualitatively is well documented that decrease of LTP is directly linked to learning and memory deficits.</p>
<p>There is very limited information on the degree of quantitative change in neural network function required to alter cognitive behaviors. This is a result of the diversity of methods for measuring both neuronal network function and learning and memory deficits, which hamper cross-study analyses. This highlights the need to develop empirical data based models of this KER. It is well known that the altered balance between excitatory and inhibitory synapses affects learning and memory, although no quantitative data are available.</p>
HighMixedHighDuring brain developmentHighHighHigh<p style="text-align:justify">Synaptic transmission and plasticity are achieved via mechanisms common across taxonomies. LTP has been recorded in aplysia, lizards, turtles, birds, mice, guinea pigs, rabbits and rats. Deficiencies in hippocampally based learning and memory following developmental hypothyroidism have been documented mainly in rodents and humans.</p>
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<p style="text-align:justify">Zanoli, P., et al. (1994). "Methyl mercury during late gestation affects temporarily the development of cortical muscarinic receptors in rat offspring." Pharmacol Toxicol <strong>75</strong>(5): 261-264.</p>
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2016-11-29T18:41:332022-07-15T08:41:52027ac0d2-2c92-4699-8e71-c493895739f9eb624ffb-7315-4087-82d2-94e1a4f9d480<p>Oxidative stress (OS) as a concept in redox biology and medicine has been formulated in 1985 (Sies, 2015). OS is intimately linked to cellular energy balance and comes from the imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS) or from a decay of the antioxidant protective ability. OS is characterized by the reduced capacity of endogenous systems to fight against the oxidative attack directed towards target biomolecules (Wang and Michaelis, 2010; Pisoschi and Pop, 2015). Glutathione, the most important redox buffer in cells (antioxidant), cycles between reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), and serves as a vital sink for control of ROS levels in cells (Reynolds <em>et al.</em>, 2007). Several case-control studies have reported the link between lower concentrations of GSH, higher levels of GSSG and the development of diseases (Rossignol and Frye, 2014). OS can cause cellular damage and subsequent cell death because the ROS oxidize vital cellular components such as lipids, proteins, and nucleic acids (Gilgun-Sherki, Melamed and Offen, 2001; Wang and Michaelis, 2010).</p>
<p>The central nervous system is especially vulnerable to free radical damage since it has a high oxygen consumption rate, an abundant lipid content and reduced levels of antioxidant enzymes (Coyle and Puttfarcken, 1993; Markesbery, 1997). It has been show that the developing brain is particularly vulnerable to neurotoxicants and OS due to differentiation processes, changes in morphology, lack of physiological barriers and less intrinsic capacity to cope with cellular stress (Grandjean and Landrigan, 2014; Sandström <em>et al.</em>, 2017). However, it has to be noted that neural stem cells distinguish themeselves from post-mitotic neural cells by their lower ROS levels and higher expression of the key antioxidant enzymes glutathione peroxidase. This increased "vigilance" of antioxidant mechanisms might represent an innate characteristic of NSCs, which not only defines their cell fate, but also helps them to encounter oxidative stress (Madhavan et al., 2006).</p>
<p>OS has been linked to brain aging, neurodegenerative diseases, and other related adverse conditions. There is evidence that free radicals play a role in cerebral ischemia-reperfusion, head injury, Parkinson’s disease, amyotrophic lateral sclerosis, Down’s syndrome, and Alzheimer’s disease due to cellular damage (Markesbery, 1997; Gilgun-Sherki, Melamed and Offen, 2001; Wang and Michaelis, 2010). OS has also been linked to neurodevelopmental diseases and deficits like autism spectrum disorder and postnatal motor coordination deficits (Wells <em>et al.</em>, 2009; Rossignol and Frye, 2014; Bhandari and Kuhad, 2015).</p>
<p> </p>
<p>A noteworthy insight, early on, was the perception that oxidation-reduction (redox) reactions in living cells are utilized in fundamental processes of redox regulation, collectively termed ‘redox signaling’ and ‘redox control’ (Sies, 2015).</p>
<p>Free radical-induced damage in OS has been confirmed as a contributor to the pathogenesis and patho-physiology of many chronic diseases, such as Alzheimer, atherosclerosis, Parkinson, but also in traumatic brain injury, sepsis, stroke, myocardial infraction, inflammatory diseases, cataracts and cancer (Bar-Or <em>et al.</em>, 2015; Pisoschi and Pop, 2015). It has been assessed that oxidative stress is correlated with over 100 diseases, either as source or outcome (Pisoschi and Pop, 2015).</p>
<p>Therefore, the fact that ROS over-production can kill neurons is well accepted (Brown and Bal-Price, 2003; Taetzsch and Block, 2013). This ROS over-production can occur in the neurons themselves or can also have a glial origin (Yuste et al., 2015).</p>
<p><strong>Mercury</strong></p>
<p>Oxidative stress has been implicated in the pathogenesis of methylmercury (MeHg) neurotoxicity. Studies of mature neurons suggest that the mitochondrion may be a major source of MeHg-induced reactive oxygen species and a critical mediator of MeHg-induced neuronal death, likely by activation of apoptotic pathways. (Polunas <em>et al.</em>, 2011)</p>
<p><strong>(Lu <em>et al.</em>, 2011)</strong> - MeHg in the mouse cerebrum (in vivo) and in cultured Neuro-2a cells (in vitro).</p>
<ul>
<li><em>In vivo</em> - <u>50µg/kg/day MeHg for 7 consecutive weeks</u> - increased levels of lipid peroxidation in the plasma and cerebral cortex. Decreased GSH level and increase the expressions of caspase-3, -7, and -9, accompanied by Bcl-2 down-regulation and up-regulation of Bax, Bak, and p53.</li>
<li><em>In vitro</em> – <u>3 and 5 µM MeHg</u> - reduced cell viability, increased oxidative stress damage, and induced several features of mitochondria-dependent apoptotic signals, including increased sub-G1 hypodiploids, mitochondrial dysfunctions, and the activation of PARP, and caspase cascades. </li>
<li>These MeHg-induced apoptotic-related signals could be remarkably reversed by <u>antioxidant NAC</u>.</li>
</ul>
<p><strong>(Sarafian <em>et al.</em>, 1994)</strong> - Hypothalamic mouse neural cell line GT1-7 without and with expression construct for the anti-apoptotic proto-oncogene, bcl-2.</p>
<ul>
<li><u>3h exposure, 10 µM MeHg </u>- increased formation of reactive ROS, and decreased levels of GSH, associated with 20% cell death. Cells transfected with an expression construct bcl-2, displayed attenuated ROS induction and negligible cell death.</li>
<li><u>24h exposure, 5 µM MeHg</u> - killed 56% of control cells, but only 19% of bcl-2-transfected cells.</li>
<li>By using diethyl maleate to deplete cells of GSH, we demonstrate that the differential sensitivity to MeHg was not due solely to intrinsically different GSH levels. The data suggest that MeHg-mediated cell killing correlates more closely with ROS generation than with GSH levels and that bcl-2 protects MeHg-treated cells by suppressing ROS generation.</li>
</ul>
<p><strong>(Castoldi <em>et al.</em>, 2000)</strong> - In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death.</p>
<ul>
<li><u>1 hr exposure, 5–10 µM MeHg</u> - impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death.</li>
<li><u>1hr exposure, 0.5–1 µM MeHg</u> - did not compromise cell viability, mitochondrial membrane potential and function at early time points.</li>
<li><u>1hr exposure, 1 µM MeHg</u> - only a small population of neurons (+-20%) dies by necrosis. The surviving neurons show network damage, but maintain membrane integrity, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively display the morphological signs of apoptosis.</li>
<li><u>18hr exposure, 0.5–1 µM MeHg</u> – cells progressively underwent apoptosis reaching the 100% cell death</li>
<li>insulin-like growth factor-I partially <em>rescued </em>CGCs from MeHg-triggered apoptosis.</li>
</ul>
<p>(<strong>Kaur,et al., 2006</strong>) - primary cell cultures of cerebellar neurons and astrocytes from 7-day-old NMRI mice. 5 mM MeHg for 30 min.</p>
<ul>
<li>Twenty-one days post-astrocyte isolation - 250mM N-acetyl cysteine (NAC) or 3mM di-ethyl maleate (DEM) added to the wells 12 h prior to MeHg exposure</li>
<li>7 days post-neurons isolation - 200mM of NAC or 1.8mM of DEM added to the wells 12 h prior to MeHg exposure</li>
<li>The intracellular GSH content was modified by pretreatment with NAC or DEM for 12 h.</li>
<li>Treatment with 5 mM Me Hg for 30 min led to significant (p < 0.05) increase in ROS and reduction (p < 0.001) in GSH content.</li>
<li>Depletion of intracellular GSH by DEM further increased the generation of MeHg-induced ROS in both cell cultures.</li>
<li>NAC supplementation increased intracellular GSH and provided protection against MeHg-induced oxidative stress in both cell cultures.</li>
</ul>
<p><strong>(Franco <em>et al.</em>, 2007)</strong> – Mitochondrial enriched fractions from adult (2 months old) Swiss Albino male mice.</p>
<ul>
<li>MeHg and HgCl2 (10–100 µM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation.</li>
<li>Both mercurials induced a significant reduction of GSH in a dose-dependent manner.</li>
<li>Correlation analyses showed significant positive correlations between mitochondrial viability and glutathione content for MeHg (Pearson coefficient) 0.933; P < 0.01) and or HgCl2 (Pearson coefficient ) 0.854; P < 0.01).</li>
<li>Quercetin (100–300 µM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl2, prevented mercurial-induced glutathione oxidation.</li>
</ul>
<p><strong>(Polunas <em>et al.</em>, 2011)</strong> - Murine embryonal carcinoma (EC) cells, which differentiate into neurons following exposure to retinoic acid.</p>
<ul>
<li><u>4h exposure, 1.5 mM MeHg </u>- earlier and significantly higher levels of ROS production and more extensive mitochondrial depolarization in neurons than in undifferentiated EC cells. cyclosporin A (CsA) completely inhibited mitochondrial depolarization by MeHg in EC cells but only delayed this response in the neurons. In contrast, CsA significantly inhibited MeHg-induced neuronal ROS production. Cyt c release was also more extensive in neurons, with less protection afforded by CsA.</li>
</ul>
<p>(<strong>Sandström <em>et al.</em>, 2016</strong>) - in vitro 3D human neural tissues from neural progenitor cells derived from human embryonic stem cells. Single MeHg exposure at day 42 of 3D culturing (week 6) and material was collected 72 h after.</p>
<ul>
<li>1-10 μM - LDH activity increased, confirming induced cell death.</li>
<li>5 and 10 μM - increased HMOX1 gene expression as indirect marker of oxidative stress.</li>
</ul>
<p> </p>
<p><strong>Acrylamide</strong></p>
<p><strong>(Allam <em>et al.</em>, 2011)</strong> - sixty albino <em>Rattus norvegicus</em>, 45 virgin females and 15 mature males. This study examined its effects on the development of external features in cubs.</p>
<ul>
<li><u>prenatal intoxicated group</u> - newborns from mothers treated with ACR (10 mg/kg/day by gastric intubation) from day 7 (GD 7) of gestation till birth</li>
<li><u>perinatal intoxicated group</u> - newborns from mothers treated with ACR (10 mg/kg/day by gastric intubation) from GD7 of gestation till D28 after birth</li>
</ul>
<p style="text-align:justify">ACR administered either prenatally or perinatally has been shown to induce significant retardation in the new- borns’ body weights development, increase of thiobarbituric acid- reactive substances (TBARS) and oxidative stress (significant reductions in reduced glutathione, total thiols, superoxide dismutase and peroxidase activities) in the developing cerebellum. ACR treatment delayed the proliferation in the granular layer and delayed both cell migration and differentiation. Purkinje cell loss was also seen in acrylamide-treated animals. Ultrastructural studies of Purkinje cells in the perinatal group showed microvacuolations and cell loss.</p>
<p>(<strong>Lakshmi <em>et al.</em>, 2012</strong>) - Wistar male albino rats, four groups (n = 6 per group)</p>
<ul>
<li><u>II – (Acrylamide) ACR - 30 mg/kg ACR for 30 days</u>: increase in the lipid peroxidative (LPO), protein carbonyl, hydroxyl radical and hydroperoxide levels with subsequent decrease in the activities of enzymic antioxidants and level of GSH. Cortex showed condensed nuclei along with damaged cells. Decrease in the expression of Bcl2 along with simultaneous increase in the expressions of Bax and Bad as compared to control.</li>
<li><u>II rats – ACR + Fish oil -0.5 ml/kg b.w.fish oil orally 10 min before ACR induction with 30 mg/kg for 30 days </u>– reversed significantly all the OS markers.</li>
</ul>
<p>Mercury-induced upregulation of GSH level and GR activity as an adaptive mechanism following lactational exposure to methylmercury (10 mg/L in drinking water) associated with motor deficit, suggesting neuronal impairment (Franco <em>et al.</em>, 2006).</p>
<table align="center" border="1" cellpadding="0" cellspacing="0" style="width:657px">
<tbody>
<tr>
<td style="width:103px">
<p><strong>Reference</strong></p>
</td>
<td style="width:109px">
<p><strong>Chemical Concentration</strong></p>
</td>
<td style="width:291px">
<p><strong>OS</strong></p>
</td>
<td style="width:153px">
<p><strong>Cell injury/death</strong></p>
</td>
</tr>
<tr>
<td rowspan="3" style="width:103px">
<p>(Sarafian <em>et al.</em>, 1994)</p>
</td>
<td style="width:109px">
<p>MeHg 0 µM</p>
</td>
<td style="width:291px">
<p><strong>ROS</strong> – ±<strong>100%</strong> DCF Fluorescence</p>
<p><strong>GSH</strong> – ±<strong>150%</strong> MCB Fluorescence</p>
</td>
<td style="width:153px">
<p><strong>±90%</strong> Viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 5 µM</p>
</td>
<td style="width:291px">
<p><strong>ROS</strong> – ±<strong>150%</strong> DCF Fluorescence</p>
<p><strong>GSH</strong> – ±<strong>100%</strong> MCB Fluorescence</p>
</td>
<td style="width:153px">
<p><strong>±80%</strong> Viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 10 µM</p>
</td>
<td style="width:291px">
<p><strong>ROS</strong> – ±<strong>200%</strong> DCF Fluorescence</p>
<p><strong>GSH</strong> – ±<strong>70%</strong> MCB Fluorescence</p>
</td>
<td style="width:153px">
<p><strong>±70%</strong> Viability</p>
</td>
</tr>
<tr>
<td rowspan="5" style="width:103px">
<p>(Lu <em>et al.</em>, 2011) <em>in vitro</em></p>
</td>
<td style="width:109px">
<p>MeHg 0µM</p>
</td>
<td style="width:291px">
<p>(2h) <strong>ROS</strong> – ±<strong>100%</strong> DCF Fluorescence</p>
<p>(24h) <strong>100%</strong> intracellular <strong>GSH</strong> levels</p>
</td>
<td style="width:153px">
<p><strong>100%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 3µM</p>
</td>
<td style="width:291px">
<p>(2h)<strong> ROS </strong>– ±<strong>160 </strong>DCF Fluorescence</p>
<p>(24h) ±<strong>60%</strong> intracellular <strong>GSH</strong> levels</p>
</td>
<td style="width:153px">
<p><strong>±50%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 5µM</p>
</td>
<td style="width:291px">
<p>(2h) <strong>ROS</strong> – ±<strong>230</strong> DCF Fluorescence</p>
<p> (24h) ±<strong>30%</strong> intracellular <strong>GSH</strong> levels</p>
</td>
<td style="width:153px">
<p><strong>±10%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 3µM + NAC 1mM</p>
</td>
<td style="width:291px">
<p>(2h) <strong>ROS</strong> – ±<strong>70</strong> DCF Fluorescence</p>
<p>(24h) ±<strong>90%</strong> intracellular <strong>GSH</strong> levels</p>
</td>
<td style="width:153px">
<p><strong>±90%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>MeHg 5µM + NAC 1mM</p>
</td>
<td style="width:291px">
<p>(2h) <strong>ROS% </strong>– ±<strong>70</strong> DCF Fluorescence</p>
<p>(24h) ±<strong>90%</strong> intracellular <strong>GSH</strong> levels</p>
</td>
<td style="width:153px">
<p><strong>±90%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td rowspan="6" style="width:103px">
<p>(Kaur <em>et al.</em>, 2006)</p>
</td>
<td style="width:109px">
<p>0 mM MeHg</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – <strong>100v</strong> MCB Fluorescence</p>
<p><strong>ROS</strong> – <strong>100%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH </strong>– <strong>100v</strong> MCB Fluorescence</p>
<p><strong>ROS</strong> – <strong>100%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>100%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>100%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>5 mM MeHg</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – ± <strong>50v</strong> MCB Fluorescence</p>
<p><strong>ROS </strong>– ± <strong>400%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH </strong>– ± <strong>70%</strong> MCB Fluorescence</p>
<p><strong>ROS</strong> – ± <strong>120%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>±60%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>±75%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>5 mM MeHg + NAC</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – ± <strong>80%</strong> MCB Fluorescence</p>
<p><strong>ROS </strong>– ± <strong>200%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH</strong> – ± <strong>80%</strong> MCB Fluorescenc e</p>
<p><strong>ROS</strong> – ± <strong>90%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>±90%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>±90%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>5 mM MeHg + DEM</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – ± <strong>50</strong><strong>%</strong> MCB Fluorescenc e</p>
<p><strong>ROS</strong> – ± <strong>470</strong><strong>%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH </strong>– ± <strong>70% </strong>MCB Fluorescence</p>
<p><strong>ROS</strong> – ± <strong>120% </strong>CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>±55%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>±65%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>NAC</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – ± <strong>110v</strong> MCB Fluorescence</p>
<p><strong>ROS </strong>– ± <strong>100%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH</strong> – ±<strong>100% </strong>MCB Fluorescence</p>
<p><strong>ROS </strong>– ± <strong>60%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>±110%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>±110%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>DEM</p>
</td>
<td style="width:291px">
<p><u>(Neurons) </u></p>
<p><strong>GSH</strong> – ±<strong> 60% </strong>MCB Fluorescence</p>
<p><strong>ROS</strong> – ± <strong>250%</strong> CMH<sub>2</sub>DCFDA Fluorescence</p>
<p><u>(Astrocytes) </u></p>
<p><strong>GSH </strong>– ± <strong>80</strong> MCB Fluorescence</p>
<p><strong>ROS </strong>– ± <strong>110 </strong>CMH<sub>2</sub>DCFDA Fluorescence</p>
</td>
<td style="width:153px">
<p>(Neurons)</p>
<p><strong>±80%</strong> Cell viability</p>
<p>(Astrocytes)</p>
<p><strong>±85%</strong> Cell viability</p>
</td>
</tr>
<tr>
<td rowspan="4" style="width:103px">
<p>(Franco <em>et al.</em>, 2007)</p>
</td>
<td style="width:109px">
<p>0 µM MeHg</p>
</td>
<td style="width:291px">
<p><strong>100% GSH</strong></p>
</td>
<td style="width:153px">
<p><strong>100%</strong> mitochondrial viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>30 µM MeHg</p>
</td>
<td style="width:291px">
<p><strong>± 70% GSH</strong></p>
</td>
<td style="width:153px">
<p><strong>± 70%</strong> mitochondrial viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>0 µM HgCl<sub>2</sub></p>
</td>
<td style="width:291px">
<p><strong>100% GSH</strong></p>
</td>
<td style="width:153px">
<p><strong>100%</strong> mitochondrial viability</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>30 µM HgCl<sub>2</sub></p>
</td>
<td style="width:291px">
<p><strong>± 65% GSH</strong></p>
</td>
<td style="width:153px">
<p><strong>± 65%</strong> mitochondrial viability</p>
</td>
</tr>
<tr>
<td rowspan="4" style="width:103px">
<p>(Lakshmi <em>et al.</em>, 2012)</p>
</td>
<td style="width:109px">
<p>Control</p>
</td>
<td style="width:291px">
<p><strong>GSH – 0.5 µmoles</strong>/mg of protein</p>
</td>
<td style="width:153px">
<p><strong>± 6</strong> Damaged cells/Field</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>Acrylamid</p>
</td>
<td style="width:291px">
<p><strong>GSH – 0.2 µmoles</strong>/mg of protein</p>
</td>
<td style="width:153px">
<p><strong>± 20</strong> Damaged cells/Field</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>Acrylamid + Fish Oil</p>
</td>
<td style="width:291px">
<p><strong>GSH – 0.4 µmoles</strong>/mg of protein</p>
</td>
<td style="width:153px">
<p><strong>± 11</strong> Damaged cells/Field</p>
</td>
</tr>
<tr>
<td style="width:109px">
<p>Fish Oil</p>
</td>
<td style="width:291px">
<p><strong>GSH – 0.5 µmoles</strong>/mg of protein</p>
</td>
<td style="width:153px">
<p><strong>± 5</strong> Damaged cells/Field</p>
</td>
</tr>
</tbody>
</table>
<div> </div>
Not SpecifiedMaleNot SpecifiedFemaleHighAll life stagesHighHighHighHigh<p><strong>Rat, Mouse: </strong>(Sarafian <em>et al.</em>, 1994; Castoldi <em>et al.</em>, 2000; Kaur <em>et al</em>., 2006; Franco <em>et al.</em>, 2007; Lu <em>et al.</em>, 2011; Polunas <em>et al.</em>, 2011)</p>
<p><strong>(Richetti <em>et al.</em>, 2011)</strong> - Adult and healthy zebrafish of both sexes (12 animals and housed in 3 L) mercury chloride final concentration of 20 mg/L. Mercury chloride promoted a significant decrease in acetylcholinesterase activity and the antioxidant competence was also decreased.</p>
<p><strong>(Berntssen, Aatland and Handy, 2003)</strong> - Atlantic salmon (<em>Salmo salar L.</em>) were supplemented with mercuric chloride (0, 10, or 100 mg Hg per kg) or methylmercury chloride (0, 5, or 10 mg Hg per kg) for 4 months.</p>
<p><em><u>Methylmercury chloride </u></em></p>
<ul>
<li>accumulated significantly in the brain of fish fed 5 or 10 mg/kg</li>
<li>No mortality or growth reduction</li>
<li>- 2-fold increase in the antioxidant enzyme super oxide dismutase (SOD) in the brain</li>
<li><u>10 mg/kg</u> - 7-fold increase of lipid peroxidative products (thiobarbituric acid reactive substances, TBARS) and a subsequently 1.5-fold decrease in anti oxidant enzyme activity (SOD and glutathione peroxidase, GSH-Px). Fish also had pathological damage (vacoulation and necrosis), significantly reduced neural enzyme activity (5-fold reduced monoamine oxidase, MAO, activity), and reduced overall post-feeding activity behaviour.</li>
</ul>
<p><em><u>Mercuric chloride</u></em></p>
<ul>
<li>accumulated significantly in the brain only at 100 mg/kg</li>
<li>No mortality or growth reduction</li>
<li><u>100 mg/kg</u> - significant reduced neural MAO activity and pathological changes (astrocyte proliferation) in the brain, however, neural SOD and GSH-Px enzyme activity, lipid peroxidative products (TBARS), and post feeding behaviour did not differ from controls.</li>
</ul>
<p> </p>
<p> </p>
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<p>Wang, X. and Michaelis, E. K. (2010) ‘Selective neuronal vulnerability to oxidative stress in the brain’, <em>Frontiers in Aging Neuroscience</em>, 2(MAR), pp. 1–13. doi: 10.3389/fnagi.2010.00012.</p>
<p>Wells, P. G. <em>et al.</em> (2009) ‘Oxidative stress in developmental origins of disease: Teratogenesis, neurodevelopmental deficits, and cancer’, <em>Toxicological Sciences</em>, 108(1), pp. 4–18. doi: 10.1093/toxsci/kfn263.</p>
<p>Yuste, J.E., et al., 2015. Implications of glial nitric oxide in neurodegenerative diseases. Front Cell Neurosci. 9<strong>,</strong> 322.</p>
2017-11-09T04:13:462020-02-07T09:32:24Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memoryOxidative stress and Developmental impairment in learning and memory<p>Florianne Tschudi-Monnet, Department of Biological Sciences, University of Lausanne, Switzerland, and Swiss Centre for Applied Human Toxicology (SCAHT), Florianne.Tschudi-Monnet@unil.ch</p>
<p>Marie-Gabrielle Zurich, Department of Biological Sciences, University of Lausanne and SCAHT, Switzerland, mzurich@unil.ch</p>
<p>Carolina Nunes, Department of Biological Sciences, University of Lausanne, Switzerland, carolina.nunes@unil.ch</p>
<p>Jenny Sandström, SCAHT, Switzerland, jsm.sandstrom@gmail.com</p>
<p>Rex FitzGerald, SCAHT, Switzerland, rex.fitzgerald@unibas.ch</p>
<p>Michael Aschner, Albert Einstein College of Medecine, New York, USA, michael.aschner@einstein.yu.edu</p>
<p>Joao Rocha, Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, Brazil, jbtrocha@gmail.com</p>
<p> </p>
<p>The authors of KEs AOPwiki ID 1392 (oxidative stress), 55 (Cell injury/death), 386 (Decrease network function), of the AO (Learning and memory, impairment), and of KER 359 (decrease network function leads to impairment in learning and memory) are greatly acknowledged.</p>
<p style="margin-left:36.0pt"> </p>
Open for citation & commentWPHA/WNT EndorsedIncluded in OECD Work Plan1.13<p style="text-align:justify">This Adverse Outcome Pathway (AOP) describes the linkage between binding to sulfhydryl(SH)-/seleno-proteins involved in protection against oxidative stress and impairment in learning and memory, the Adverse Outcome (AO). Binding to SH-/ seleno-proteins involved in protection against oxidative stress has been defined as the Molecular Initiating Event (MIE). Production, binding and degradation of Reactive Oxygen Radicals (ROS) are tightly regulated, and an imbalance between production and protection may cause oxidative stress, which is common to many toxicity pathways. Oxidative stress may lead to an imbalance in glutamate neurotransmission, which is involved in learning and memory. Oxidative stress may also cause cellular injury and death. During brain development and in particular during the establishment of neuronal connections and networks, such perturbations may lead to functional impairment in learning and memory. Neuroinflammation (Resident cell activation; Increased pro-inflammatory mediators) is triggered early in cell injury cascades and is considered as an exacerbating factor. The weight-of-evidence supporting the relationship between the described key events is based mainly on developmental effects observed after an exposure to the heavy metal, mercury, known for its strong affinity to many SH-/seleno-containing proteins, but in particular to those having anti-oxidant properties, such as glutathione (GSH). The overall assessment of this AOP is considered as strong, based on the biological plausibility, the empirical support and on the essentiality of the Key Events (KEs), which are moderate to strong, since blocking, preventing or attenuating an upstream KE is mitigating the downstream KE. The gap of knowledge is mainly due to limited quantitative evaluations, impeding thus the development of predictive models.</p>
<p style="text-align:justify">This AOP was originally started in a workshop report entitled: Adverse Outcome Pathways (AOP) relevant to Neurotoxicity and published in Critical Review in Toxicol: Bal-Price, A., Crofton, K.M., Sachana, M., Shafer, T.J., Behl, M., Forsby, A., Hargreaves, A., Landesmann, B., Lein, P.J., Louisse, J., Monnet-Tschudi, F., Paini, A., Rolaki, A., Schrattenholz, A., Sunol, C., van Thriel, C., Whelan, M., Fritsche, E., 2015. Putative adverse outcome pathways relevant to neurotoxicity. Crit Rev Toxicol 45(1), 83-91.</p>
<p style="text-align:justify">The process of inflammation is common to many tissues and can be described by several KEs, as proposed in a dedicated workshop (Villeneuve et al., 2018). Brain inflammation called Neuroinflammation can be described by the two common KEs: Tissue resident cell, activation and pro-inflammatory mediators, increased. However, Neuroinflammation is a concept accepted by the regulators and is found in the whole literature describing brain inflammation. Therefore, in accord with the external reviewers, we decided to use the KE Neuroinflammation for building the KERs of this AOP, but we introduced in the list of the KEs the two KEs common to the inflammatory process, as proposed in Villeneuve et al., 2018.</p>
<p style="margin-right:28.05pt; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:12pt">Mercury (Methylmercury, mercury chloride)</span></strong></span></p>
<p style="margin-right:28.05pt; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">The binding of Methylmercury (MeHg) to redox sensitive thiol- or selenol-groups can disrupt the activity of enzymes or the biochemical role of non-enzymatic proteins. The stable or transitory interaction (binding) of MeHg with critical thiol and selenol groups in target enzymes can disrupt the biological function of different types of enzymes, particularly of the antioxidant selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase isoforms. The dysregulation of cerebral glutathione (GSH and GSSG) and thioredoxin [Trx or Trx(SH)<sub>2</sub>] systems by MeHg (Farina et al. 2011; Branco et al. 2017) can impair the fine cellular redox balance via disruption of sensitive cysteinyl- or thiol-containing proteins (Go etal., 2013; Go et al. 2014; Jones 2015). </span></span></p>
<p> </p>
<p><a href="https://aopwiki.org/system/dragonfly/production/2018/02/07/3zeb7785qn_Diapositive1.jpg"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2018/02/07/3zeb7785qn_Diapositive1.jpg" style="height:540px; width:720px" /></a></p>
<p><strong><u>Figure 1</u></strong> – Hypothetical Binding of MeHg to different types of target proteins. The binding of MeHg to proteins can cause either a transitory inhibition of the protein fucntion (first line, the yellow protein was reactivated by interacting with LMM-SH or R-SH). The pink protein is an example of protein that after the binding of MeHg suffered a change in the structure in such a way that it cannot be reactivated by LMM-SH or R-SH. The third protein (blue) is an example of protein that was permanently denaturated after MeHg binding and even after the removal of MeHg the activity was not recovered. The same type of interactions can be applied to the selenol-containing proteins (i.e., the selenoproteins).</p>
<p style="text-align:justify">The affinity of Mercury chloride (Hg<sup>2+</sup>) for thiol and selenol groups is higher than that of MeHg (compare Table 2 with Table 1). The constants described in Table 1 and 2 indicate that MeHg and Hg<sup>2+</sup> behave as strong soft electrophiles, i.e., theyhave much higher affinity for the soft nucleophiles centers of thiol- and selenol-containing molecules (Rabenstein 1978a; Arnold et al. 1986; Sugiura et al., 1976).Furthermore, the rate constant for the reaction of MeHg with thiol/thiolate (R-SH/R-S<sup>-</sup>) has been estimated to be about 6 x 10<sup>8 </sup>M<sup>-</sup>1.sec<sup>-1</sup>, indicating that the reactions of electrophilic forms of Hg (EpHg<sup>+ </sup>; here MeHg and Hg<sup>2+</sup>) with thiolate and selenolate groups are diffusion controlled reactions (Rabenstein and Fairhurst, 1975). The constant indicates that the binding of EpHg<sup>+</sup> to thiolate (-S<sup>-</sup>) or selenolate (-Se<sup>-</sup>) groups will occurr almost instaneously, when an EpHg<sup>+</sup> collides with –S<sup>-</sup> or -Se<sup>-</sup> groups.</p>
<p style="text-align:justify">The studies of Rabenstein and others have also pointed out that the affinity of MeHg for –SeH groups is higher than for –SH groups (Sugira et al. 1976; Arnold et al. 1986). Consequently, –SeH-containing molecules (i.e., selenoproteins) should be the preferential targets for MeHg (Farina et al. 2011). Accordingly, several studies have demonstrate that the selenoenzymes glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) were inhibited after <em>in vitro</em> and <em>in vivo</em> exposure to MeHg or Hg<sup>2+</sup> (Carvalho et al., 2008a; 2011, Farina et al., 2009; Franco et al., 2009; Wagner et al., 2010; Branco et al., 2011; 2012; 2014, 2017; Dalla Corte et al., 2013; Meinerz et al., 2017).</p>
<p style="text-align:justify">As corollary, the occurrence of free MeHg and Hg<sup>2+</sup> or bound to other ligands such as carboxylates, amines, chloride or hydroxyl anions in the physiological media of living cells is insignificant or nonexistent (George et al. 2008). The binding of MeHg to abundant low molecular mass thiols or LMM-SH (e.g., cysteine and reduced glutathione-GSH) and high molecular mass thiol-containing proteins or HMM-SH (e.g., albumin, hemoglobin, etc) is critical for the MeHg distribution from non-target to target organs and cells (Farina et al. 2017). The coordination of MeHg with one –S<sup>-</sup> group of a LMM-SH will determine MeHg distribution to its targets organs, including the brain. The coordination of Hg<sup>2+</sup> with two –S<sup>-</sup> of LMM-SH molecules (particularly, cysteine or Cys) will determine the distribution of Hg<sup>2+</sup> to kidney (which is its main target) and to non-classical targets organs, such as the brain (Oliveira et al. 2017). The entrance of Hg<sup>2+</sup> into the brain is proportionally small, but recent literature data have indicated the neurotoxicity of very low and environmentally relevant doses of Hg<sup>2+</sup> in rodents (Mello-Carpes et al. 2013 ), which confirms data obtained with toxic doses in rodents (Peixoto et al. 2007 ; Franciscato et al. 2009 ; Chehimi et al. 2012).</p>
<p style="text-align:justify"><strong>Table 1</strong> - Affinity constants of methylmercury for important chemical groups found in biomolecules (adapted from <sup>a</sup>Rabestein, 1978a, <sup>b</sup>Rabestein and Bravo, 1987, using different thiol-containing molecules with the arylmercurialpara-mercurybenzenosulfonate, and from <sup>c</sup>Arnold et al. 1986 taking into consideration that the calculated formation constant of –Se-MeHg conjugates was 0.1 to 1.2 order greater than that of –S-MeHg). The values represent the Log of the constants.</p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:554px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:23px; width:188px">
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Functional Group</span></span></strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:23px; width:246px">
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Occurrence</span></span></strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:23px; width:120px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Formation constant</span></span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; width:188px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Thiol/thiolate (-SH/-S<sup>-</sup>)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; width:246px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Cysteine, glutathione, proteins</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; width:120px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">≈14-18<sup> a,b</sup></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; width:188px">
<p style="margin-right:-6px; text-align:justify"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Selenol/selenolate (-SeH/Se<sup>-</sup>)</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; width:246px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">Selenocysteinyl residues in selenoproteins</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; width:120px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif">≈ 16-18<sup>c</sup></span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><strong>Table 2. </strong>Formation constants of Hg<sup>2+</sup> with some representative nucleophilic centers from biomolecules.</p>
<table border="1" cellpadding="1" cellspacing="1" style="width:500px">
<tbody>
<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Functional group</strong></span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Hg<sup>2+</sup></strong></span></span></td>
</tr>
<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">R-S-R</span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">≈ 6-12</span></span></td>
</tr>
<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">R-SH</span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">≈ 40-50</span></span></td>
</tr>
<tr>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">R-SeH</span></span></td>
<td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">≈ 50-60</span></span></td>
</tr>
</tbody>
</table>
<p>The approximate (≈) Log of the constants. The values were adapted from Stricks and Kolthoff 1953; Mousavi 2011 and Liem-Nguyem et al. 2017.</p>
<p style="margin-right:28.05pt; text-align:justify"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">We have to emphasize that what we call of binding to –SH or –SeH groups is, in fact, an exchange reaction of MeHg from MeHg-S conjugates (e.g., MeHg-cysteine or MeHg-Cys and MeHg-glutathione or MeHg-SG. conjugates) to a free thiol/thiolate- or selenol/selenolate-group from non-target or target proteins. Thus, the interaction of MeHg with its target proteins in the brain usually involves the exchange of MeHg from low-molecular mass conjugates (LMM-S-conjugates) to a thiol or selenol group in different types of proteins (Rabenstein 1978b; Rabenstein and Fairhurst, 1975; Reid and Rabenstein et al.; 1982; Rabenstein and Reid, 1984; Arnold et al. 1986; Farina et al. 2011, 2017; Dórea et al. 2013). </span></span></p>
<p><a href="https://aopwiki.org/system/dragonfly/production/2018/02/07/44zji8zmr8_Diapositive1.jpg"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2018/02/07/44zji8zmr8_Diapositive1.jpg" style="height:540px; width:720px" /></a></p>
<p><strong>Figure 2</strong> – Binding of MeHg (CH3Hg+) to target thiol- (HMM-SH) or selenol-containing proteins (HMM-SeH). Note that, in fact, the binding of MeHg to their high molecular mass target proteins is mediated by exchange reactions of MeHg from low molecular mass thiol (LMM-SH) molecules to HMM-SH (represented by Prot-SH) or HMM-SeH (represented by Prot-SeH). The scheme also demonstrated that MeHg conjugated with one LMM-SH (here represented by either Cys<sub>1</sub>-SHgCH<sub>3</sub> or G<sub>1</sub>SHgCH<sub>3</sub>) can exchange with others LMM-SH (here represented by Cys<sub>2</sub>-SH or G<sub>2</sub>SH). After one exchange reaction, the conjugated Cys<sub>1</sub>-SHgCH<sub>3</sub> and G<sub>1</sub>SHgCH<sub>3</sub> release the free LMM-SH molecules Cys<sub>1</sub>-SH or G<sub>1</sub>SH.</p>
<p> </p>
<p><strong>Table 3:</strong> References for the inhibition by MeHg and Hg<sup>2+</sup> of SH-/seleno-proteins involved in protection against oxidative stress</p>
<table border="1" cellpadding="0" cellspacing="0" style="height:1399px; width:1217px">
<tbody>
<tr>
<td style="width:123px">
<p><strong>Protein activity inhibited by MeHg</strong></p>
</td>
<td style="width:94px">
<p><strong>Exposure</strong></p>
</td>
<td style="width:95px">
<p><strong>Functional group likely involved in the inhibition</strong></p>
</td>
<td style="width:142px">
<p><strong>Organism-preparation</strong></p>
</td>
<td style="width:142px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Glutathione peroxidase (total GPx)</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Glasser et al. 2013</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Glasser et al. 2010a</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Mitochondrial total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Franco et al. 2009</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vitro</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>SH-SY5Y cells</p>
</td>
<td style="width:142px">
<p>Franco et al. 2009</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>GPx1 and GPx4</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Zemolin et al. 2012</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult male mice</p>
</td>
<td style="width:142px">
<p>Malagutti et al. 2009</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vitro</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>PC12 cells</p>
</td>
<td style="width:142px">
<p>Li et al. 2008</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Mice gestational exposure</p>
</td>
<td style="width:142px">
<p>Stringari et al. 2008</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult rats</p>
</td>
<td style="width:142px">
<p>Cheng et al. 2005</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vitro</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Fetal Telencepalic cells from rats</p>
</td>
<td style="width:142px">
<p>Sorg et al. 1998</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total GPx</strong></p>
</td>
<td style="width:94px">
<p><em>in vitro</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Mice neuroblastoma cells</p>
</td>
<td style="width:142px">
<p>Kromidas et al. 1990</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Thioredoxin Reductase (TrxR)</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH and –SH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Zemolin et al. 2012</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>TrxR</strong></p>
</td>
<td style="width:94px">
<p><em>in vitro</em></p>
</td>
<td style="width:95px">
<p>-SeH and –SH</p>
</td>
<td style="width:142px">
<p>Adult mice</p>
</td>
<td style="width:142px">
<p>Wagner et al. 2010</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>TrxR</strong></p>
</td>
<td style="width:94px">
<p><em>in vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH- and –SH</p>
</td>
<td style="width:142px">
<p>Adult rats</p>
</td>
<td style="width:142px">
<p>Dalla Corte et al. 2013</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Mitochondrial total Gpx</strong></p>
</td>
<td style="width:94px">
<p><em>In vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult rat</p>
</td>
<td style="width:142px">
<p>Mori et al., 2007</p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Mitochondrial total Gpx</strong></p>
</td>
<td style="width:94px">
<p><em>In vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH</p>
</td>
<td style="width:142px">
<p>Adult Swiss male mice brain</p>
</td>
<td style="width:142px">
<p>Franco et al., 2009</p>
</td>
</tr>
<tr>
<td style="width:123px"> </td>
<td style="width:94px"> </td>
<td style="width:95px"> </td>
<td style="width:142px"> </td>
<td style="width:142px"> </td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total brain TrxR</strong></p>
</td>
<td style="width:94px">
<p><em>In vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH and -SH</p>
</td>
<td style="width:142px">
<p>Juvenile fish (zebra-seabreams)</p>
</td>
<td style="width:142px">
<p>Branco et al. 2011</p>
<p>Branco et al. 2012a,b</p>
</td>
</tr>
<tr>
<td style="width:123px"> </td>
<td style="width:94px"> </td>
<td style="width:95px"> </td>
<td style="width:142px"> </td>
<td style="width:142px"> </td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Protein activity inhibited by Hg2+</strong></p>
</td>
<td style="width:94px">
<p> </p>
<p><strong>exposure</strong></p>
</td>
<td style="width:95px">
<p><strong>Functional group likely involved in the inhibition</strong></p>
</td>
<td style="width:142px">
<p> </p>
<p><strong>organism-preparation</strong></p>
</td>
<td style="width:142px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:123px">
<p><strong>Total brain TrxR</strong></p>
</td>
<td style="width:94px">
<p><em>In vivo</em></p>
</td>
<td style="width:95px">
<p>-SeH and -SH</p>
</td>
<td style="width:142px">
<p>Juvenile fish (zebra-seabreams)</p>
</td>
<td style="width:142px">
<p>Branco et al. 2012a,b</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Acrylamide </strong></p>
<p style="text-align:justify">Acrylamide is an a,β-unsaturated (conjugated) reactive molecule, which can react with thiol (-SH) and amino (-NH2) groups in proteins (LoPachin, 2004; LoPachin et al. 2007; 2009; 2011; Friedman, 2003; Bent et al. 2016; Martyniuk et al.2011; LoPachin and Gavin, 2014 ). However, the rate constant for the reaction between acrylamide with thiol/thiolate groups is much lower than that for MeHg. The rate of reaction of this compound with HMM-SH and LMM-SH is slow but can occur under physiological conditions (Tong et al. 2004; LoPachin, 2004). The inhibition of brain enzymes by acrylamide have been studied and the inhibition caused by acrylamide in some HMM-SH can be reversible (Howland et al. 1980). Despite of this, we can infer that some targets of MeHg and acrylamide can overlap, in particular GSH,where the rate constant for MeHg and acrylamide are ≈6.0 x 10<sup>8 </sup>M<sup>-1</sup>.sec<sup>-1</sup> and ≈0.15-2.1 x 10<sup>-2 </sup>M<sup>-1</sup>.sec<sup>-1</sup>, respectively (Yousef and Demerdash, 2006; Lapadula et al. 1989; Kopańska et al. 2015). Acrylamide can also be metabolized to an epoxide intermediate (glycidamide), which can also form adducts with cysteinyl residues in HMM-SH target proteins (Bergmark et al. 1991).</p>
<p> </p>
<p>A prime example of impairments in learning and memory as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD TG 426) <span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">as well as OECD TG 443 (OECD, 2018)</span></span> both require testing of learning and memory (USEPA, 1998; OECD, 2007) advising to use the following tests passive avoidance, delayed-matching-to-position for the adult rat and for the infant rat, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and acquisition and retention of schedule-controlled behaviour. These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009).</p>
<p>Also, in the frame of the OECD GD 43 (2008) on reproductive toxicity, learning and memory testing may have potential to be applied in the context of developmental neurotoxicity studies. However, many of the learning and memory tasks used in guideline studies may not readily detect subtle impairments in cognitive function associated with modest degrees of developmental thyroid disruption (Gilbert et al., 2012).</p>
adjacentModerateModerateadjacentHighHighadjacentLowLowadjacentModerateHighadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedHighnon-adjacentHighHigh<table align="left" cellspacing="0" class="MsoTable15Grid5DarkAccent1" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td rowspan="2" style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Defining Question</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:125px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">High (Strong)</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:none; border-top:1px solid white; height:30px; vertical-align:top; width:111px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Moderate</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:1px solid white; height:30px; vertical-align:top; width:114px">
<p style="margin-left:8px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Low (Weak)</span></span></span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:125px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, KO models, etc.)</span></span></span></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:111px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO</span></span></span></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:114px">
<p style="margin-left:8px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">No or contradictory experimental evidence on the essentiality of any of the KEs</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE1</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased protection against oxidative stress</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: The fact that a decrease in anti-oxidant properties causes oxidative stress is well accepted. In addition, experimental evidences of knocking out proteins involved in protection against oxidative stress incresed the susceptibilty to oxidative stress.</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE2</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Oxidative stress</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: The deleterious consequences of oxidative stress are well accepted in various animal models. Oxygen radical scavengers, such as glutathione, catalase, selenium and cysteine can block the deleterious effects of oxidative stress.</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE3 </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Glutamate dyshomeostasis</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate is the main excitatory transmitter, and is involved in memory processes, it is well accepted that perturbation of glutamate homeostasis has deleterious functional consequences. Disruption of glutamate signaling is thought to play a role, at least in part, in the etiology underlying several neurodevelopmental disorders, including memory dysfunction.</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE4</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Cell Injury/death, increased</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Cell injury/death is a highly converging node in AOPs. Decrease in synaptic connectivity or cell loss will in turn induce perturbations in the establishment of neuronal connections and trigger inflammatory responses, which through a feedback loop can exacerbate this KE. Therefore, prevention of cell injury/death by anti-oxidant or by inhibitors of NMDA receptors prevents the downstream KEs.</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Neuroinflammation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5' Tissue resident cell activation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5'' Pro-inflammatory mediators, increased</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MODERATE</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: It is widely accepted in different experimental animal models that the use of minocycline, an antibiotic, which blocks microglial reactivity has protective effects, as have other interferences with any inflammatory mediators. However, we rate the essentiality of this KE as moderate given the complexity of the neuroinflammatory response, having either protective/reparative or aggravating consequences,</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE6</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased network formation and function</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate neurotransmission is an important mechanism underlying memory function (for review: Featherstone, 2010). During brain development, glutamate has also trophic effects, by stimulating BDNF production or through the activation of the different glutamate receptors. The trophic effect of glutamate receptor activation is developmental stage-dependent and may play an important role in determining the selective survival of neurons that made proper connections (Balazs, 2006).</span></span></span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:145px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">AO </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Impairment of learning and memory</span></span></span></strong></span></span></p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:138px">
<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Impairment in learning and memory is a converging KE in several AOPs related to brain development. Regarding this AOP and its chemical initiators, it was shown that the neurocognitive domain, in particular dentate gyrus, hippocampus and cortex are susceptible to the neurotoxicity of mercury in the developing brain (Sokolowski et al., 2011, 2013; Ceccatelli et al., 2013). Chronic, low-dose prenatal MeHg exposure from maternal consumption of fish has been associated with endpoints of neurotoxicity in children, including poor performance on neurobehavioral tests, particularly on tests of attention, fine-motor function, language, visual-spatial abilities (e.g., drawing), and verbal memory (NRC, 2000). Prenatal MeHg exposure is associated with childhood memory and learning deficits, particularly visual memory performance in school-aged children (Orenstein, 2014).</span></span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p> </p>
Not SpecifiedMaleNot SpecifiedFemaleNot SpecifiedDuring brain developmentHighHighModerate<p style="text-align:justify">Experimental and epidemiological evidences indicate that compared to the adult central nervous system (CNS), the developing CNS is generally more susceptible to toxicant exposure (Costa et al., 2004; Grandjean and Landrigan, 2006). Pre-natal and post-natal exposure may have long-term consequences, i.e. not detected immediately at the end of the exposure period. Such effects on visuospatial memory for example have been described on child development in communities with chronic low level mercury exposure (Castoldi et al., 2008a; Debes et al., 2006; Grandjean et al., 2014; Lam et al., 2013).</p>
<p style="text-align:justify">The aim of this AOP is to capture the KEs and the KERs that occur after binding to thiol- and selenol groups of proteins involved in protection against oxidative stress, the MIE, and impairment in learning and memory, the AO, which is a neurotoxicity marker belonging to the OECD regulatory tool box. The chemical initiators used for the empirical support are methylmercury and mercury chloride, and acrylamide. Data are most extensive for mercury as stressor during development; data for acrylamide are much more limited and restricted to some KEs. Chronic, low-dose prenatal MeHg exposure from maternal consumption of fish has been associated with endpoints of neurotoxicity in children, including poor performance on neurobehavioral tests, particularly on tests of attention, fine-motor function, language, visual-spatial abilities (e.g., drawing), and verbal memory (NRC, 2000). However, it is important to note that some uncertainties remain about the effects of low dose of mercury during brain development (Grandjean et al., 1999). Epidemiological studies in Seychelles on prenatal exposure through fish consumption did not evidenced adverse effects on memory when analyses were performed at 22 and 24 years (Van Wyngaarden et al., 2017), whereas similar experiments made in the Faroe Islands revealed dysfunctions in language, attention and memory at 7 years (Grandjean et al., 1997). And a clear association was observed between mercury cord blood level and memory deficit (Grandjean et al., 1997; Debes et al., 2006). Castoldi and coworkers (2008) proposed that modulating factors, such as diet, nutrition, gender, pattern of exposure and co-exposure could explain the discrepancies of these epidemiological studies. Nevertheless, there are experimental evidences showing that the neurocognitive domain, in particular dentate gyrus, hippocampus and cortex are susceptible to the neurotoxicity of mercury in the developing brain (Sokolowski et al., 2011, 2013; Ceccatelli et al., 2013); therefore, we focus on impairment in learning and memory as the AO. Some –SH- or –SeH-containing proteins involved in protection against oxidative stress have been demonstrated to be inhibited by MeHg either <em>in vitro</em> or <em>in vivo</em>, but a causal relationship has not been established between these inhibitory effects and the final pathological events (Oliveira, 2017). However, the analysis of the essentiality of the KEs and of the weight of evidence for the KERs supports a plausible mechanistic link between the MIE and the AO.</p>
<p style="text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12pt">This AOP is mainly focused on the developmental period, although it cannot be excluded that long-term exposure in adult may trigger a similar cascade of KEs leading also to impairment in learning and memory, as observed in neurodegenerative diseases such as Alzheimer's disease (Mutter et al., 2004). While no specific sex differences have been analyzed/described for most KEs, Curtis and coworkers (2010) observed a higher level of TNF-</span><span style="font-size:12pt">a</span><span style="font-size:12pt"> in hippocampus of male prairie wolf than in female, both treated for 10 weeks with inorganic mercury, in the form of HgCl<sub>2</sub>; whereas Zhang and coworkers (2013) found a higher neuroinflammatory response associated with altered social behavior in female mice offspring than in male, following gestational exposure to HgCl<sub>2</sub>. However, after developmental methylmercury exposure, long-lasting behavioral alterations were more prominent in males (Ceccatelli et al., 2013; Castoldi et al., 2008b). These discrepancies may be due to sex differences in kinetics or susceptibility (Vahter et al., 2006).</span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Defining Question</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">High (Strong)</span></span></span></strong></span></span></p>
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<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Moderate</span></span></span></strong></span></span></p>
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<p style="margin-left:8px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Low (Weak)</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></span></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, KO models, etc.)</span></span></span></span></span></p>
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<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO</span></span></span></span></span></p>
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<p style="margin-left:8px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">No or contradictory experimental evidence on the essentiality of any of the KEs</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE1</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased protection against oxidative stress</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: The fact that a decrease in anti-oxidant properties causes oxidative stress is well accepted. In addition, experimental evidences of knocking out proteins involved in protection against oxidative stress incresed the susceptibilty to oxidative stress.</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE2</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Oxidative stress</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: The deleterious consequences of oxidative stress are well accepted in various animal models. Oxygen radical scavengers, such as glutathione, catalase, selenium and cysteine can block the deleterious effects of oxidative stress.</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE3 </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Glutamate dyshomeostasis</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate is the main excitatory transmitter, and is involved in memory processes, it is well accepted that perturbation of glutamate homeostasis has deleterious functional consequences. Disruption of glutamate signaling is thought to play a role, at least in part, in the etiology underlying several neurodevelopmental disorders, including memory dysfunction.</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE4</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Cell Injury/death, increased</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Cell injury/death is a highly converging node in AOPs. Decrease in synaptic connectivity or cell loss will in turn induce perturbations in the establishment of neuronal connections and trigger inflammatory responses, which through a feedback loop can exacerbate this KE. Therefore, prevention of cell injury/death by anti-oxidant or by inhibitors of NMDA receptors prevents the downstream KEs.</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Neuroinflammation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5' Tissue resident cell activation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5'' Pro-inflammatory mediators, increased</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MODERATE</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: It is widely accepted in different experimental animal models that the use of minocycline, an antibiotic, which blocks microglial reactivity has protective effects, as have other interferences with any inflammatory mediators. However, we rate the essentiality of this KE as moderate given the complexity of the neuroinflammatory response, having either protective/reparative or aggravating consequences,</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE6</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased network formation and function</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate neurotransmission is an important mechanism underlying memory function (for review: Featherstone, 2010). During brain development, glutamate has also trophic effects, by stimulating BDNF production or through the activation of the different glutamate receptors. The trophic effect of glutamate receptor activation is developmental stage-dependent and may play an important role in determining the selective survival of neurons that made proper connections (Balazs, 2006).</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">AO </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Impairment of learning and memory</span></span></span></strong></span></span></p>
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<p style="margin-left:4px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:351px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Impairment in learning and memory is a converging KE in several AOPs related to brain development. Regarding this AOP and its chemical initiators, it was shown that the neurocognitive domain, in particular dentate gyrus, hippocampus and cortex are susceptible to the neurotoxicity of mercury in the developing brain (Sokolowski et al., 2011, 2013; Ceccatelli et al., 2013). Chronic, low-dose prenatal MeHg exposure from maternal consumption of fish has been associated with endpoints of neurotoxicity in children, including poor performance on neurobehavioral tests, particularly on tests of attention, fine-motor function, language, visual-spatial abilities (e.g., drawing), and verbal memory (NRC, 2000). Prenatal MeHg exposure is associated with childhood memory and learning deficits, particularly visual memory performance in school-aged children (Orenstein, 2014).</span></span></span></span></span></p>
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<p><strong>Dose-response and temporal concordance of KEs</strong></p>
<p>There is no study where all KEs are measured simultaneously after exposure to several doses, impeding a dose-response and concordance analysis. In one single study (in blue in the table), three downstream KEs were measured following pre-natal exposure to methylmercury. Comparisons of all animal studies show that doses used are ranging from 0.5 - 5 mg/kg; but dose-response was seldom performed. In these studies, the time (pre-natal, post-natal, lactation,...) and duration of exposure are quite diverse and no analysis of brain mercury content was made, so it is not possible to compare doses between studies. Therefore, based on the present data, it is impossible to define whether KEs up occur at lower doses and earlier time points than KEs down.</p>
<p>For <em>in vitro</em> studies, KEs up are often measured after acute exposure to high concentrations.</p>
<p>The following table summarizes concentrations/doses, time, and duration of exposure for the various test systems and KEs.</p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KEs</span></span></span></strong></span></span></p>
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<p style="margin-right:-203px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">In vivo</span></span></span></strong></span></span></p>
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<p style="margin-right:-6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">In vitro</span></span></span></strong></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">MIE</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Binding to SH-/seleno-proteins</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Binding of Hg to thiol groups and to various selenium-containing proteins:</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutathione, thioredoxin reductase, thioredoxin, glutaredoxin, glutathione reductase was measured using purified proteins</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Carvahlo et al., 2008, 2011; Wiederhold et al., 2010; Sugiura et al., 1978; Arnold et al., 1986; Han et al., 2001; Qiao et al., 2017)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE1</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased protection against oxidative stress</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Cytoplasmic and nuclear TrxR and Cytoplasmic Gpx were reduced in cerebral and cerebellar cortex of 22 days-old offspring (Ruszkiewicz, 2016)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Male C57BL/6NJcl mice exposed to methylmercury (1.5 mg/kg/day for 6-weeks) (Fujimura, 2017)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Adult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Zebra fish brain exposed to Hg2+, MeHg 1.8 molar (measured in brain tissue), for 28 days (Branco, 2012)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Prenatal and postnatal exposure of mice to 40 ppm of HgCl<sub>2</sub> decreased the activity of catalase, thioredoxin reductase, Gpx, superoxide dismutase (Malqui et al., 2017)</span></span></span></span></span></p>
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<p style="margin-right:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mouse primary cortical cultures exposed to 5 mM of methylmercury for 24h (Rush, 2012)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MeHg inhibits ex vivo rat thioredoxin reductase; IC50 0.158 </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">μ</span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">M (cerebral) (Wagner et al., 2010)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Human neuroblastoma cells (SH-SY5Y)exposed to 1 µM of methylmercury for 6 or 24 h (Branco, 2017; Franco, 2009)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE2</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Oxidative stress</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Male C57BL/6NJcl mice exposed to methylmercury (1.5 mg/kg/day for 6-weeks) (Fujimura, 2017)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Adult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Adult male Sprague-Dawley rats exposed to methylmercury (1 mg/kg orally for 6 months) (Joshi, 2014)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Zebra fish brain exposed to Hg2+, MeHg 1.8 molar (measured in brain tissue), for 28 days (Branco, 2012)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Prenatal and postnatal exposure of mice to 40 ppm of HgCl<sub>2</sub> caused oxidative stress evaluated by increased lipid peroxidation (Malqui et al., 2017)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mouse primary cortical cultures exposed to 5 mM of methylmercury for 24h (Rush, 2012)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Methylmercury (2-10 µM) in synaptic vesicles isolated from rat brain (with LD<sub>50</sub> at 50 µM) (Porciuncula et al., 2003)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Human neuroblastoma cells (SH-SY5Y)exposed to 1 µM of methylmercury for 6-24 h (Franco, 2009)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE3 </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Glutamate dyshomeostasis</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Rat Young (3-4 weeks) dosed with acrylamide by gavage (5, 15, 30 mg/kg, 5 applications per week during 4 weeks) (Tian, 2018)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Microdialysis probe in adult Wistar rats showed that acute exposure to methylmercury (10, 100 mM) induced an increase release of extracellular glutamate (9.8 fold at 10 mM and 2.4 fold at 100 mM). This extracellular glutamate level remained elevated at least 90 min (Juarez et al., 2002)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mouse astrocytes, neurons in mono- or co-cultures exposed to methylmercury 1-50 µM for 24h (Morken, 2005)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Methylmercury (2-10 µM) in synaptic vesicles isolated from rat brain (with LD<sub>50</sub> at 50 µM) (Porciuncula et al., 2003</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE4</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Cell Injury/death, increased</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Rat, perinatal exposure to methylmercury (GD7-PD21, i.e. 35 days) 0.5 mg/kg bw/day in drinking water (Roda et al., 2008)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Rat Young (3-4 weeks) exposed to acrylamide by gavage (5, 15, 30 mg/kg, 5 applications per week during 4 weeks) (Tian, 2018)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mouse astrocytes, neurons in mono- or co-cultures exposed to methylmercury 1-50 µM for 24h (Morken, 2005)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Neuroinflammation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5' Tissue resident cell activation</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5'' Pro-inflammatory mediators, increased</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Rat, perinatal exposure to methylmercury (GD7-PD21, i.e. 35 days) 0.5 mg/kg bw/day in drinking water (Roda et al., 2008)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Monkeys, 6,12,18 months oral exposure 50 mg/kg bw (Charleston et al., 1996)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">3D rat brain cell cultures 10 day treatmentHgCl<sub>2</sub> 10<sup>-9</sup>-10<sup>-6</sup>M</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MeHgCl 10<sup>-9</sup>-3x10<sup>-7</sup>M (Monnet-Tschudi et al., 1996; Eskes et al., 2002)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE6</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased network formation and function</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mice dosed during postnatal week 1-3 with subcutaneous 2-5 mg mercury chloride/kg/once per week (Eddins et al., 2008)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant rat dosed on GD 15 with 8 mg/kg of methylmercury by gavage. Offsprings were tested at day 16, 21 and 60. (Cagiano et al., 1990)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">AO </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Impairment of learning and memory</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Mice dosed during postnatal week 1-3 with subcutaneous 2-5 mg mercury chloride/kg/once per week (Eddins et al., 2008)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant rat dosed on GD 15 with 8 mg/kg of methylmercury by gavage. Offsprings were tested at day 16, 21 and 60 (Cagiano et al., 1990)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant rat exposed to methylmercury (1.5 mg/kg orally) from GD5 till parturition (Jacob, 2017)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Pregnant mice received 0.5 mg methylmercury/kg/day in drinking water from gestational day 7 until day 7 after delivery. Offspring behavior was monitored at 5-15 and 26-36 weeks of age (Onishchenko et al., 2007)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Balb mice exposed to methylmercury in diet (low dose: 1.5 mg/kg; high dose: 4.5 mg/kg) during 11 weeks (6 weeks prior mating, 3 weeks during gestation and 2 weeks post-partum). Offsprings tested at PD 15 showed an accumulation of Hg in brain (0.08 mg/kg for low dose and 0.25 mg/kg for the high dose) (Glover et al., 2009)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Prenatal and postnatal exposure of mice to 40 ppm of HgCl<sub>2</sub> caused impairment of memory (object recognition, Y maze) Malqui et al., 2017)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Maternal peripartum hair mercury level was measured to assess prenatal mercury exposure. The concentrations of mercury was found in the range of 0.3-5.1 µg/g, similar to fish-eating population in US. Statistical analyses revealed that each ug/g increase in hair Hg was associated with a decrement in visual memory, learning and verbal memory <strong>(</strong>Orenstein et al., 2014)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Epidemiological studies in the Faroe Islands revealed that mercury exposure through fish consumption (maternal hair conc. 10 ug/g) dysfunctions in memory, language and attention at age 7 (Grandjean et al., 1997; Debes et al., 2006)</span></span></span></span></span></p>
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<p> </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:12pt">Biological Plausibility and Empirical Support of the KERs</span></strong> </span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KERs</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Defining Question</span></span></span></strong></span></span></p>
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Is there a mechanistic (i.e. structural or functional) relationship between KEup and KEdown consistent with established biological knowledge?</span></span></span></strong></span></span></p>
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<p style="margin-left:13px; margin-right:-47px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">High (Strong)</span></span></span></strong></span></span></p>
<p style="margin-left:4px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Extensive understanding of the KER based on extensive previous documentation and broad acceptance</span></span></span></strong></span></span></p>
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<p style="margin-left:7px; margin-right:-47px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Moderate</span></span></span></strong></span></span></p>
<p style="margin-left:7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">The KER is plausible based on analogy to accept biological relationship but scientific understanding is not completely established</span></span></span></strong></span></span></p>
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<p style="margin-left:6px; margin-right:-47px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Low (Weak)</span></span></span></strong></span></span></p>
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">There is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood</span></span></span></strong></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">MIE to KE Decrease protection against oxidative stress</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MODERATE</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Thiol- and selenol containing proteins, which mainly belong to the anti-oxidant protections, have a high affinity for binding soft metals such as mercury (Farina, 2011). Binding to these thiol/sulfhydryl/SH/SeH groups results in structural modifications affecting the catalytic capacity, and thereby reducing the capacity to neutralize ROS. However, binding to other SH/SeH groups of other proteins not involved in protection against oxidative stress can occur and trigger other neurotoxicity pathways. Alternatively, binding to SH groups of electrophilic compounds may also induce cyto-protective reactions (e.g. via Nrf2).</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Decrease protection against oxidative stress to KE Oxidative stress</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. Several studies have shown depletion of GSH, the main anti-oxidant, and an increase in oxidative stress following methylmercury or mercury chloride exposures (Meinerz, 2011; Rush, 2012; Agrawal, 2015). Protection against oxidative stress was observed by supplementation with diphenyl selenide (Meinerz, 2011) or by glutathione ester (Rush, 2012). </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Limited conflicting data.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Oxidative stress to KE Glutamate (Glu) dyshomeostasis</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">LOW</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate transport is driven by the Na<sup>+</sup> ion gradient, which is dependent on the Na/K ATPase, which, in turn, requires energy. Glutamate enters the cells accompanied by 2 Na<sup>+ </sup>and one H<sup>+</sup>. Perturbations of energy metabolism such as mitochondrial dysfunction and increased production of ROS will lead to glutamate dyshomeostasis, due to the indirect coupling of glutamate transporters with ATP level, and to the important role of glutamate transporters in glutamate homeostasis. (Boron and Boulpaep, 2003). Methylmercury was shown to inhibit both the H<sup>+</sup>-ATPase activity and vesicular glutamate uptake (Porciuncula et al., 2003). As, on one hand, ROS production can interfere with glutamate uptake, and on the other hand, glutamate accumulation leads to excitotoxicity and ROS production, the exact sequence of the KER is difficult to assess. But the fact that both KEs are involved in mercury-induced neurotoxicity is broadly accepted (Farina et al., 2011; Antunes dos Santos et al., 2016; Morris et al., 2017; Kern et al., 2016).</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Glutamate dyshomeostasis to KE Cell injury/death</span></span></span></strong></span></span></p>
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<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Glutamate dyshomeostasis, in particular excess of glutamate in the synaptic cleft, leads to overactivation of ionotropic glutamate receptors, referred to as excitotoxicity. This, in turn, will cause cell injury/death via ROS production. This KER is also inherent to the developing brain, where glutamate ionotropic receptors are expressed early in various neural cells and when NMDA receptors are expressed in neurons. There is empirical support for all three chemical initiators (mercury, acrylamide, acrolein). In addition, several experiments aiming at blocking glutamate excitotoxicity and the resulting ROS production are protective for cell injury/death. </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Limited conflicting data.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Cell injury/death to KE Neuroinflammation</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MODERATE</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: It is widely accepted that cell/neuronal injury and death lead to neuroinflammation (microglial and astrocyte reactivities) in adult brain, and in the developing brain, where neuroinflammation was observed after cell injury/death induced by excitotoxic lesions (Acarin et al., 1997; Dommergues et al., 2003). Empirical support is available for all three chemical initiators (mercury, acrylamide, acrolein). Few experiments, showing a protection when blocking any feature of neuroinflammation have been described. There are some contradicting data showing an absence of neuroinflammatory response despite the occurrence of mercury-induced apotosis and slight behavioral alterations.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Neuroinflammation to KE Cell injury/death</span></span></span></strong></span></span></p>
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<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">MODERATE</span></span></span></span></span></p>
</td>
<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: In vitro co-culture experiments have demonstrated that reactive glial cells (microglia and astrocytes) can kill neurons via the release of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6 and/or ROS/RNS (Chao et al., 1995; Brown and Bal-Price, 2003; Kraft and Harry, 2011; Taetzsch and Block, 2013) and that interventions aiming at blocking these inflammatory biomolecules can rescue the neurons (Yadav et al., 2012; Brzozowski et al., 2015). Several reports showed that modulating mercury or acrylamide-induced neuroinflammation was protective for neurons. Because of the complexity of the neuroinflammatory response, that can have neuroprotective or neurodegenerative consequences depending on the duration, local environment or still unknown factors, the rating of this KER was kept as moderate. The vicious cycle between cell injury/death and neuroinflammation is well known and was described in other AOPs. Neuroinflammation could be considered as a modulating factor, but because of the numerous inhibiting experiments, it is considered as an essential KE. Some conflicting data due to the dual role of some inflammatory mediators have been reported.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Cell injury/death to KE Decreased network formation and function</span></span></span></strong></span></span></p>
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<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:121px; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: Neuronal network formation and functional crosstalk are established via synaptogenesis. It was shown that under physiological conditions components of the apoptotic machinery in the developing brain regulate synapse formation and neuronal connectivity (Dekkers et al., 2013). The brain’s electrical activity dependence on synapse formation is critical for proper neuronal communication. Glial cells are also involved in the establishment and stabilization of the neuronal network. Extensive experimental support for the adverse effects of mercury on synaptogenesis exist, establishing a strong link between mercury-induced apoptosis and/or neuronal loss and perturbations in a number of neurotransmitter systems (Jacob, 2017; Bridges, 2017) and perturbations of functionality (Falluel-Morel, 2007; Ferraro, 2009; Teixera, 2014; Onishchenko, 2007). </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Limited protective experiments and conflicting data reported.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE Decreased network formation and function to AO Impairment in learning and memory</span></span></span></strong></span></span></p>
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<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: A review on the Morris water maze (MWM) (Morris, 1981), as an investigative tool of spatial learning and memory in laboratory rats (Vorhees and Williams, 2006) pointed out that perturbed neuronal networks rather than neuronal death per se in certain regions is responsible for the impairment in MWM performance. Functional integrated neural networks that involve the coordination action of different brain regions are consequently important for spatial learning and memory performance (D'Hooge and De Deyn, 2001). Broad empirical support showing mercury-induced effects on learning and memory as consequence of network disruption (Sokolowski et al. 2013; Eddins et al., 2008; Glover et al., 2009). Similar observations were made in humans (Orenstein et al., 2014; Yorifuji et al., 2011). Interestingly, behavioral alterations were detected long time after exposure (delayed effects). Few conflicting data have been reported, but other behavioral deficits, such as alterations in motor activity and increased anxiety suggest that systems other than hippocampus-related learning and memory are also affected.</span></span></span></span></span></p>
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<p style="margin-left:8px; margin-right:-1px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE oxidative stress to KE Cell injury/death</span></span></span></strong></span></span></p>
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<td style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:142px">
<p style="margin-left:9px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">HIGH</span></span></span></span></span></p>
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<td colspan="3" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:548px">
<p style="margin-left:6px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">RATIONALE: The central nervous system is especially vulnerable to free radical damage since it has a high oxygen consumption rate, an abundant lipid content and reduced levels of antioxidant enzymes (Coyle and Puttfarcken, 1993; Markesbery, 1997). The developing nervous system is particularly vulnerable to chemical insults (Grandjean and Landrigan, 2014). One reason for this higher vulnerability is the incapacity of immature neural cells to cope with oxidative stress by increasing glutathione (GSH) production (Sandström et al., 2017a). Broad empirical support for mercury and acrylamide showing an association between increased ROS production and/or decreased protection against oxidative stress and apoptosis and/or necrosis (Lu <em>et al.</em>, 2011; Sarafian <em>et al.</em>, 1994; Allam <em>et al.</em>, 2011; Lakshmi <em>et al.</em>, 2012). Anti-oxidant treatments proved to be protective. Few conflicting data, except a mercury-induced upregulation of GSH level and GR activity as an adaptive mechanism following lactational exposure to methylmercury (10 mg/L in drinking water) associated with motor deficit, suggesting neuronal impairment (Franco et al., 2006).</span></span></span></span></span></p>
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<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Some quantitative relationships have been described between the upstream early KEs (MIE, oxidative stress, Cell injury/death), although the diversity of test systems and posology (dosing/exposure amount and duration) hampers comparison between studies. It is more difficult to evaluate quantitative relationships between later downstream KEs, such as Neuroinflammation and Decreased Network Function. Neuroinflammation is a complex adaptive mechanism which is not yet completely understood; it can have neuroprotective or neurodegenerative consequences, depending on triggering signals, duration, microenvironment or other unknown influences, which may determine the outcome of the neuroinflammatory process. Decreased network function is currently difficult to quantify because quantitative technologies for mapping and understanding of brain networks (and their plasticity) are still under development.</p>
<p>Optimally, we would like data from a single type of test system showing that exposure to stressor, e.g. mercury, is correlated with changes in all KEs. Such models are emerging, using cells of human origin (Pamies et al., 2016; Sandström et al., 2017b; Fritsche et al., 2017) and/or non-mammalian models, such as zebrafish (Geier et al., 2018; Padilla et al., 2018) and will allow in the future generation of quantitative data which may be used for <em>in silico</em> hazard prediction.</p>
<p>Summary table of Quantitative Evaluations</p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KEs</span></span></span></strong></span></span></p>
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<p style="margin-right:-203px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Methylmercury<strong> (</strong>MeHg,<strong> </strong>CH<sub>3</sub>Hg)</span></span></span></span></span></p>
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<p style="margin-right:-7px"> </p>
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<p style="margin-right:-6px"> </p>
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<p style="margin-right:-6px"> </p>
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<p style="margin-left:2px"> </p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">5 µM</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">mouse brain <em>in vitro</em></span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Rush, 2012)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">15–30 µM</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">mouse brain, after 40 mg/L in drinking water for 21 days</span></span></span></span></span></p>
<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Glaser, 2013)</span></span></span></span></span></p>
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<p style="margin-right:-2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">1 µM</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">mouse cerebral cortex ex vivo after oral dosing</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Lu et al 2011; conc. from Huang et al 2008)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">17-24, 75-µM (rat cerebral cortex <em>ex vivo</em> after 4w ip dosing)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">4w</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Xu, 2012; Liu 2013; Feng, 2014)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE1</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased protection against oxidative stress</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">GSH reduced 80% of control</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">24h</span></span></span></span></span></p>
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<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Cortical mitochondrial GPx activity decreased (70% of control), GR increased (170% of control)</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">GSH decreased (ca 50% of control)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">7 weeks</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Antioxidants NPSH, SOD, GSH-Px decreased (ca 80% and 50% of control)</span></span></span></span></span></p>
<p> </p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE2</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Oxidative stress</span></span></span></strong></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">ROS increased 120-150% of control</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">24h</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Cortical mitochondrial TBA-RS increased (ca 140% of control) and complex I, II-III, and IV activity decreased (ca 50% of control).</span></span></span></span></span></p>
<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Brain 8-OHdG content increased (ca 400% of control).</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">LPO increased (ca 200% of control)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">7 weeks</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">ROS (DCF) increased (190 and 400% of control at 22,87 </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">μ</span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">M)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE3 </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Glutamate dyshomeostasis</span></span></span></strong></span></span></p>
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<p> </p>
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<p style="margin-right:-7px"> </p>
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<p> </p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutamine synthetase decreased (80 and 50% of control at 24,89 </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">μ</span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">M)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutamate content increased (100 and 120% of control at 24,89 µM)</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutamine content decreased (80 and 50% of control at 24,89 </span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">μ</span></span></span><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">M)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE4</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Cell Injury/death, increased</span></span></span></strong></span></span></p>
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<p> </p>
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<p style="margin-right:-7px"> </p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Apoptosis-related gene expression: Bcl-2 decreased, ca 50% of control; Bax, Bak, p53, caspase-3,-5,-7 increased, ca 200-350% of control</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">7 weeks</span></span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Apoptosis increased dose-dependently (300 and 853% of control at 24,89 µM).</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">8-OHdG expression increased (200 and 450% of control at 24,89 µM)</span></span></span></span></span></p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Neuroinflammation</span></span></span></strong></span></span></p>
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<p> </p>
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<p style="margin-right:-7px"> </p>
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<p> </p>
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<p> </p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE6</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased network formation and function</span></span></span></strong></span></span></p>
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<p> </p>
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<p style="margin-right:-7px"> </p>
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<p> </p>
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<p> </p>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">AO </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Impairment of learning and memory</span></span></span></strong></span></span></p>
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<p> </p>
</td>
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<p style="margin-right:-7px"> </p>
</td>
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<p> </p>
</td>
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<p> </p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<table align="left" cellspacing="0" class="MsoTable15Grid5DarkAccent1" style="border-collapse:collapse; border:none">
<tbody>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KEs</span></span></span></strong></span></span></p>
</td>
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<p style="margin-right:-203px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Mercuric chloride<strong> (</strong>HgCl<sub>2</sub>)</span></span></span></span></span></p>
</td>
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<p style="margin-right:-7px"> </p>
</td>
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<p style="margin-right:-6px"> </p>
</td>
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<p style="margin-right:-6px"> </p>
</td>
</tr>
<tr>
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<p style="margin-left:2px"> </p>
</td>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">6 µM</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">rat brain, 1.13 µg Hg/g</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">6 mo</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Agrawal, 2015)</span></span></span></span></span></p>
</td>
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<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">0.1-100 µM cultured mouse cerebellar granule cells</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">10 min</span></span></span></span></span></p>
<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">(Fonfria, 2005)</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:84px; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE1</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased protection against oxidative stress</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Blood GSH decreased (ca 90% of control)</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p style="margin-right:-7px"> </p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE2</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Oxidative stress</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p style="margin-right:-7px"> </p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE3 </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Glutamate dyshomeostasis</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutamate (3H-aspartate) uptake inhibited (IC50 3.5 uM).</span></span></span></span></span></p>
<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Glutamate release stimulated (47% of total endogenous glutamate at 10 µM)</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE4</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Cell Injury/death, increased</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Serum AST increased (ca 140% of control).</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p style="margin-right:-7px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Cell viability (MTT) decreased (ca 10% of control at 10 µM)</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE5</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Neuroinflammation</span></span></span></strong></span></span></p>
</td>
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<p> </p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px">
<p style="margin-right:-7px"> </p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:74px; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
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<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">KE6</span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Decreased network formation and function</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px">
<p><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Brain noradrenaline and dopamine content decreased (ca 30% of control).</span></span></span></span></span></p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px">
<p style="margin-right:-7px"> </p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; height:95px; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
<tr>
<td style="background-color:#4472c4; border-bottom:1px solid white; border-left:1px solid white; border-right:1px solid white; border-top:none; vertical-align:top; width:136px">
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">AO </span></span></span></strong></span></span></p>
<p style="margin-left:2px"><span style="font-size:12pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:9.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:white">Impairment of learning and memory</span></span></span></strong></span></span></p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td colspan="2" style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p style="margin-right:-7px"> </p>
</td>
<td colspan="2" style="background-color:#b4c6e7; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
<td style="background-color:#d9e2f3; border-bottom:1px solid white; border-left:none; border-right:1px solid white; border-top:none; vertical-align:top; width:199px">
<p> </p>
</td>
</tr>
</tbody>
</table>
<ul>
<li>Contribution to the network of KEs/AOPs on Developmental Neurotoxicity (DNT)</li>
<li>Generating quantitative data by measuring all KEs in a single model after repeated/long term exposure to a wide concentration range of the chemical initiators to facilitate the development of computational predictive approaches</li>
</ul>
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<p>Agrawal, S., P. Bhatnagar and S. J. Flora (2015). "Changes in tissue oxidative stress, brain biogenic amines and acetylcholinesterase following co-exposure to lead, arsenic and mercury in rats." Food Chem Toxicol 86: 208-216.</p>
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<p>Antunes Dos Santos, A., M. Appel Hort, M. Culbreth, C. Lopez-Granero, M. Farina, J. B. Rocha and M. Aschner (2016). "Methylmercury and brain development: A review of recent literature." <u>J Trace Elem Med Biol</u> <strong>38</strong>: 99-107.</p>
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<p>Balazs, R. (2006). "Trophic effect of glutamate." <u>Curr Top Med Chem</u> <strong>6</strong>(10): 961-968.</p>
<p>Boron, WF and Boulpaep, EL (2005) Medical Physiology. Elsevier. Philadelphia.</p>
<p>Branco, V., J. Canario, J. Lu, A. Holmgren and C. Carvalho (2012). "Mercury and selenium interaction in vivo: effects on thioredoxin reductase and glutathione peroxidase." Free Radic Biol Med 52(4): 781-793.</p>
<p>Branco, V., L. Coppo, S. Sola, J. Lu, C. M. P. Rodrigues, A. Holmgren and C. Carvalho (2017). "Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury." Redox Biol 13: 278-287.</p>
<p>Bridges, K., Venables, B., Roberts, A., 2017. Effects of dietary methylmercury on the dopaminergic system of adult fathead minnows and their offspring. Environ Toxicol Chem 36, 1077-1084.</p>
<p>Brown, G. C. and A. Bal-Price (2003). "Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria." <u>Mol Neurobiol</u> <strong>27</strong>(3): 325-355.</p>
<p>Brzozowski, M. J., P. Jenner and S. Rose (2015). "Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity." <u>J Neural Transm</u> <strong>122</strong>(6): 779-788.</p>
<p>Cagiano, R., et al. (1990). "Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats." Neurotoxicol Teratol <strong>12</strong>(1): 23-28.</p>
<p>Carvalho, C.M. et al. (2008) Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity. J Biol Chem 283, 11913-11923.</p>
<p>Carvalho, C.M.L. et al. (2011), Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: Implications for treatment of mercury poisoning(. <em>FASEB Journal</em>, 25 (1), pp. 370-381.</p>
<p>Castoldi, A. F., C. Johansson, N. Onishchenko, T. Coccini, E. Roda, M. Vahter, S. Ceccatelli and L. Manzo (2008a). "Human developmental neurotoxicity of methylmercury: impact of variables and risk modifiers." <u>Regul Toxicol Pharmacol</u> <strong>51</strong>(2): 201-214.</p>
<p>Castoldi, A. F., N. Onishchenko, C. Johansson, T. Coccini, E. Roda, M. Vahter, S. Ceccatelli and L. Manzo (2008b). "Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment." <u>Regul Toxicol Pharmacol</u> <strong>51</strong>(2): 215-229.</p>
<p>Ceccatelli, S., R. Bose, K. Edoff, N. Onishchenko and S. Spulber (2013). "Long-lasting neurotoxic effects of exposure to methylmercury during development." <u>J Intern Med</u> <strong>273</strong>(5): 490-497.</p>
<p>Charleston, J. S., R. L. Body, R. P. Bolender, N. K. Mottet, M. E. Vahter and T. M. Burbacher (1996). "Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure." <u>NeuroToxicology</u> <strong>17</strong>: 127-138.</p>
<p>Chao, C. C., S. Hu and P. K. Peterson (1995). "Glia, cytokines, and neurotoxicity." <u>Crit.Rev.Neurobiol.</u> <strong>9</strong>: 189-205.</p>
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