60-56-0PMRYVIKBURPHAH-UHFFFAOYSA-NPMRYVIKBURPHAH-UHFFFAOYSA-N
Methimazole2H-Imidazole-2-thione, 1,3-dihydro-1-methyl-
1,3-Dihydro-1-methyl-2H-imidazole-2-thione
1-Methyl-1,3-dihydroimidazole-2-thione
1-Methyl-1H-imidazole-2-thiol
1-Methyl-2-mercapto-1H-imidazole
1-Methyl-2-mercaptoimidazole
1-Methyl-4-imidazoline-2-thione
1-Methylimidazole-2(3H)-thione
1-Methylimidazole-2-thiol
1-Methylimidazole-2-thione
2-Mercapto-1-methyl-1H-imidazole
2-Mercapto-1-methylimidazole
2-Mercapto-N-methylimidazole
4-Imidazoline-2-thione, 1-methyl-
Basolan
Danantizol
Favistan
Frentirox
Imidazole-2-thiol, 1-methyl-
Mercaptazole
Mercazole
Mercazolyl
Metazolo
Methimazol
Methylmercaptoimidazole
Metothyrin
Metothyrine
Metotirin
N-Methyl-2-mercaptoimidazole
N-Methylimidazolethiol
NSC 38608
Strumazol
Tapazole
Thacapzol
Thiamazol
thiamazole
Thycapzol
Thymidazol
Thymidazole
tiamazol
DTXSID4020820149-30-4YXIWHUQXZSMYRE-UHFFFAOYSA-NYXIWHUQXZSMYRE-UHFFFAOYSA-N
2-Mercaptobenzothiazole(2(3H)-Benzothiazolethione)
2(3H)-Benzothiazolethione
1,3-Benzothiazole-2-thiol
1,3-Benzothiazole-2-thione
2,3-Dihydrobenzothiazole-2-thione
2-Benzothiazolethiol
2-Benzothiazolinethione
2-BENZOTHIAZOLTHIOL
2-Benzothiazolyl mercaptan
2-Mercapthobenzothiazole Technical
2-Mercapto-1H-benzothiazole
2-Mercaptobenzthiazole
2-Sulfanylbenzothiazole
Accel M
Accelerator M
Aero Promoter 412
Benz-1,3-thiazolidine-2-thione
Benzo[d]thiazole-2-thiol
Benzothiazol-2-thiol
BENZOTHIAZOLE, 2-MERCAPTO-
Benzothiazole-2-thiol
Benzothiazole-2-thione
Benzothiazolethiol
benzotiazol-2-tiol
Dermacid
Ekagom G
Kaptaks
Mebetizol
Mebetizole
Mebithizol
MERCAPTOBENZOTHIAZOLE
Mercaptobenzthiazole
Nocceler M
Nocceler M-P
Nonflex NB
NSC 2041
Perkacit MBT
Pneumax MBT
Royal MBT
Sanceler M
Sanceler M-G
Soxinol M
Thiotax
Vulkacit M
Vulkacit Mercapto
Vulkacit Mercapto MG/C
Vulkacit Mercapto/C
Vulkacit Mercapto/MG
Vulkafil ZN 94TT01
Wobezit M
DTXSID102080751-52-5KNAHARQHSZJURB-UHFFFAOYSA-NKNAHARQHSZJURB-UHFFFAOYSA-N
6-Propyl-2-thiouracil6-Propyl-2 thiouracil (PTU)
4(1H)-Pyrimidinone, 2,3-dihydro-6-propyl-2-thioxo-
2,3-Dihydro-6-propyl-2-thioxo-4(1H)-pyrimidinone
2-Mercapto-4-hydroxy-6-n-propylpyrimidine
2-Mercapto-4-hydroxy-6-propylpyrimidine
2-Mercapto-6-propylpyrimidin-4-ol
2-Thio-4-oxo-6-propyl-1,3-pyrimidine
2-Thio-6-propyl-1,3-pyrimidin-4-one
6-n-Propyl-2-thiouracil
6-n-Propylthiouracil
6-Propyl-2-thio-2,4(1H,3H)pyrimidinedione
6-Propylthiouracil
NSC 6498
NSC 70461
Procasil
Propacil
propiltiouracilo
Propycil
Propyl-Thiorist
Propylthiorit
propylthiouracil
Propylthiouracile
Propyl-Thyracil
Prothiucil
Prothiurone
Prothycil
Prothyran
Protiural
Thiuragyl
Thyreostat II
URACIL, 4-PROPYL-2-THIO-
Uracil, 6-propyl-2-thio-
DTXSID502120996-45-7PDQAZBWRQCGBEV-UHFFFAOYSA-NPDQAZBWRQCGBEV-UHFFFAOYSA-N
Ethylene thioureaETU
2-Imidazolidinethione
1,3-Ethylenethiourea
2-Imidazoline-2-thiol
2-Mercapto-2-imidazoline
2-Mercapto-4,5-dihydroimidazole
2-MERCAPTOIMIDAZOLINE
2-Thioimidazolidine
4,5-Dihydro-2-mercaptoimidazole
Ethylenethiocarbamide
ETHYLENETHIOUREA
Imidazolidin-2-thion
imidazolidina-2-tiona
Imidazolidine-2-thione
Imidazolidinethione
Imidazoline-2(3H)-thione
Imidazoline-2-thiol
Mercaptoimidazoline
Mercazin I
N,N'-AETHYLENTHIOHARNSTOFF
N,N'-ETHYLENETHIOUREA
Nocceler 22
Pennac CRA
Rhenogran ETU
Rhodanin S 62
Sanceler 22
Sanceler 22C
Sanceler 22S
Tetrahydro-2H-imidazole-2-thione
Thiourea, N,N'-1,2-ethanediyl-
Vulkacit NPV/C
Warecure C
DTXSID5020601108-46-3GHMLBKRAJCXXBS-UHFFFAOYSA-NGHMLBKRAJCXXBS-UHFFFAOYSA-N
Resorcinol1,3-Benzenediol
1,3-DIHYDROXYBENZENE
3-Hydroxyphenol
C.I. Developer 4
C.I. Oxidation Base 31
Developer O
Developer R
Developer RS
Durafur Developer G
Fouramine RS
Fourrine 79
Fourrine EW
m-Benzenediol
m-Dihydroxybenzene
m-Hydroquinone
m-Hydroxyphenol
m-Phenylenediol
Nako TGG
NSC 1571
Oxidation Base 31
Pelagol Grey RS
Pelagol RS
PHENOL, 3-HYDROXY-
Redimix 401RAP60
Resorcin
Resorcinol 80
Rezorsine
Rodol RS
UN 2876
DTXSID202123857583-54-7OWICEWMBIBPFAH-UHFFFAOYSA-NOWICEWMBIBPFAH-UHFFFAOYSA-N
Resorcinol bis(diphenyl phosphate)DTXSID8069197141-90-2ZEMGGZBWXRYJHK-UHFFFAOYSA-NZEMGGZBWXRYJHK-UHFFFAOYSA-N
Thiouracil4(1H)-Pyrimidinone, 2,3-dihydro-2-thioxo-
2,3-Dihydro-2-thioxo-4(1H)-pyrimidinone
2-Mercapto-4-hydroxypyrimidine
2-Mercapto-4-pyrimidinol
2-Mercapto-4-pyrimidinone
2-thiouracil
2-Thiouracile
2-tiouracilo
4-Hydroxy-2-mercaptopyrimidine
4-Hydroxy-2-pyrimidinethiol
6-Hydroxy-2-mercaptopyrimidine
Antagothyroil
Deracil
Nobilen
NSC 19473
NSC 290412
NSC 290413
NSC 290414
URACIL, 2-THIO-
DTXSID402134761-82-5KLSJWNVTNUYHDU-UHFFFAOYSA-NKLSJWNVTNUYHDU-UHFFFAOYSA-N
Amitrole1H-1,2,4-Triazol-3-amine
3-Amino-1,2,4-triazole
3-Aminotriazole
1H-1,2,4-Triazol-3-ylamine
1H-1,2,4-Triazol-5-amine
1H-1,2,4-Triazolamine
1H-1,2,4-Triazole, 3-amino-
2,3,5,6-Tetraazabicyclo[2.1.1]hex-1-ene
2-Amino-1,3,4-triazole
3-Amino-1H-1,2,4-triazole
3-Amino-2H-1,2,4-triazole
3-Amino-s-triazole
5-Amino-1,2,4-triazole
5-Amino-1H-1,2,4-triazole
Amitrol
Amitrol T
Azaplant
Cytrole
Herbicide, Amino-1H-1,2,4-triazole, 3-
Herbidal total
Herbizole
NSC 34809
NSC 7243
s-Triazole, 3-amino-
TRIAZOLE (1,2,4), 3-AMINO-4H
Weedazol
DTXSID0020076131-55-5WXNRYSGJLQFHBR-UHFFFAOYSA-NWXNRYSGJLQFHBR-UHFFFAOYSA-N
2,2',4,4'-TetrahydroxybenzophenoneMethanone, bis(2,4-dihydroxyphenyl)-
2,2',4,4'-tetrahidroxibenzofenona
2,2',4,4'-Tetrahydroxy diphenyl ketone
2,2',4,4'-Tetrahydroxybenzophenon
2,4,2',4'-Tetrahydroxybenzophenone
Benzophenone 2
BENZOPHENONE, 2,2',4,4'-TETRAHYDROXY-
BENZOPHENONE-2
Bis(2,4-dihydroxyphenyl)methanone
Dainsorb P 6
NSC 38556
Seesorb 106
Sumisorb 150
Uvinul 3050
Uvinul D 50
DTXSID5041306486-66-8ZQSIJRDFPHDXIC-UHFFFAOYSA-NZQSIJRDFPHDXIC-UHFFFAOYSA-N
Daidzein4',7-Dihydroxyisoflavone
4H-1-Benzopyran-4-one, 7-hydroxy-3-(4-hydroxyphenyl)-
7,4'-Dihydroxyisoflavone
7-hidroxi-3-(4-hidroxifenil)-4-benzopirona
7-Hydroxy-3-(4-hydroxyphenyl)-4-benzopyron
7-hydroxy-3-(4-hydroxyphenyl)-4-benzopyrone
7-Hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one
Daidzeol
Isoaurostatin
Isoflavone, 4',7-dihydroxy-
DTXSID9022310446-72-0TZBJGXHYKVUXJN-UHFFFAOYSA-NTZBJGXHYKVUXJN-UHFFFAOYSA-N
GenisteinGEN
4H-1-Benzopyran-4-one, 5,7-dihydroxy-3-(4-hydroxyphenyl)-
5,7,4'-Trihydroxyisoflavone
5,7-dihidroxi-3-(4-hidroxifenil)-4-benzopirona
5,7-Dihydroxy-3-(4-hydroxyphenyl)-4-benzopyron
5,7-Dihydroxy-3-(4-hydroxyphenyl)-4-benzopyrone
Baichanin A
Bonistein
Genisteol
Genisterin
Isoflavone, 4',5,7-trihydroxy-
NSC 36586
Prunetol
Sophoricol
4',5,7-Trihydroxyisoflavone
DTXSID5022308104-40-5IGFHQQFPSIBGKE-UHFFFAOYSA-NIGFHQQFPSIBGKE-UHFFFAOYSA-N
4-Nonylphenolp-Nonylphenol
p-NP
Phenol, 4-nonyl-
4-n-Nonyl phenol
Nonyl phenol
Phenol, p-nonyl-
p-n-Nonylphenol
p-nonilfenol
DTXSID503383657-68-1ASWVTGNCAZCNNR-UHFFFAOYSA-NASWVTGNCAZCNNR-UHFFFAOYSA-N
SulfamethazineBenzenesulfonamide, 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)-
2-(4-Aminobenzenesulfonamido)-4,6-dimethylpyrimidine
2-(4-Aminobenzenesulfonylamino)-4,6-dimethylpyrimidine
2-(p-Aminobenzenesulfonamido)-4,6-dimethylpyrimidine
2-Sulfanilamido-4,6-dimethylpyrimidine
4,6-Dimethyl-2-sulfanilamidopyrimidine
4-Amino-N-(2,6-dimethyl-4-pyrimidinyl)benzenesulfonamide
4-Amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide
Azolmetazin
Calfspan
Calfspan Tablets
Cremomethazine
DiazilSulfadine
Dimezathine
Dimidin R
Kelametazine
Mermeth
N-(4,6-Dimethyl-2-pyrimidyl)sulfanilamide
N1-(4,6-Dimethyl-2-pyrimidinyl)sulfanilamide
N1-(4,6-Dimethyl-2-pyrimidyl)sulfanilamide
Neasina
Neazina
NSC 67457
NSC 683529
Panazin
Pirmazin
S-Dimidine
Spanbolet
Sulfadimerazine
Sulfadimesin
Sulfadimesine
Sulfadimethyldiazine
Sulfadimethylpyrimidine
Sulfadimezin
Sulfadimezine
Sulfadimidin
sulfadimidina
Sulfadimidine
Sulfadine
SULFAMETHAZINE BASE
Sulfamethiazine
Sulfanilamide, N1-(4,6-dimethyl-2-pyrimidinyl)-
SulfaSURE SR Bolus
Sulfodimesin
Sulfodimezine
Sulka K Boluses
Sulphadimethylpyrimidine
Sulphadimidine
Sulphamethasine
Sulphamethazine
Sulphamezathine
Sulphamidine
Sulphodimezine
Superseptil
Superseptyl
Sustain III
Vertolan
DTXSID602129014797-73-0VLTRZXGMWDSKGL-UHFFFAOYSA-MVLTRZXGMWDSKGL-UHFFFAOYSA-M
PerchloratePerchlorate ion
Perchlorate ion (ClO41-)
Perchlorate ion(1-)
Perchlorate(1-)
Perchloric acid, ion(1-)
DTXSID6024252PR:000016584thyroid peroxidasePCO:0000001population of organismsCHEBI:60311thyroid hormoneCHEBI:30660thyroxineUBERON:2001277anterior chamber swim bladderCHEBI:287743,3',5'-triiodothyronineGO:0004447iodide peroxidase activityPCO:0000008population growth rateGO:0006590thyroid hormone generationMP:0005475abnormal circulating thyroxine levelGO:0048798swim bladder inflationNBO:0000371aquatic locomotionMP:0005473decreased triiodothyronine levelD009026mortality2decreased1increasedMethimazole2016-11-29T18:42:192016-11-29T18:42:19Mercaptobenzothiazole2016-11-29T18:42:172016-11-29T18:42:17Propylthiouracil2016-11-29T18:42:222016-11-29T18:42:222(3H)-Benzothiazolethione2016-11-29T18:42:082016-11-29T18:42:082-mercaptobenzothiazole2016-11-29T18:42:082016-11-29T18:42:08Ethylene thiourea2016-11-29T18:42:172016-11-29T18:42:17Resorcinol2016-11-29T18:42:222016-11-29T18:42:22Thiouracil2016-11-29T18:42:232016-11-29T18:42:23Ethylenethiourea2016-11-29T18:42:232016-11-29T18:42:23Amitrole2016-11-29T18:42:262016-11-29T18:42:26131-55-52016-11-29T18:42:262016-11-29T18:42:262,2',4,4'-Tetrahydroxybenzophenone2016-11-29T18:42:262016-11-29T18:42:26Daidzein2016-11-29T18:42:262016-11-29T18:42:26Genistein2016-11-29T18:42:262016-11-29T18:42:264-Nonylphenol2016-11-29T18:42:262016-11-29T18:42:264-propoxyphenol2016-11-29T18:42:262016-11-29T18:42:26Sulfamethazine2016-11-29T18:42:262016-11-29T18:42:26Perchlorate2016-11-29T18:42:262016-11-29T18:42:2610116ratWCS_9606humans9823pigsWCS_8355Xenopus laevisWCS_9031chickenWCS_7955zebrafishWCS_90988fathead minnow10090mouseWikiUser_22all speciesWCS_9606human9823Sus scrofa70862teleost fishWCS_8355African clawed frogThyroperoxidase, InhibitionThyroperoxidase, InhibitionMolecular<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis. TPO catalyzes several reactions in the thyroid gland, including: the oxidation of iodide; nonspecific iodination of tyrosyl residues of thyroglobulin (Tg); and the coupling of iodotyrosyls to produce Tg-bound monoiodotyrosine (MIT) and diiodotyrosine (DIT) (Divi et al., 1997; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996). The outcome of TPO inhibition is decreased synthesis of thyroxine (T4) and triiodothyronine (T3), a decrease in release of these hormones from the gland into circulation, and unless compensated, a consequent decrease in systemic concentrations of T4, and possibly T3. The primary product of TPO-catalyzed TH synthesis is T4 (Taurog et al., 1996; Zoeller et al., 2007) that would be peripherally or centrally deiodinated to T3.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">It is important to note that TPO is a complex enzyme that has two catalytic cycles and is capable of iodinating multiple species (Divi et al., 1997). Alterations in all of these events are not covered by some of the commonly used assays that measure “TPO inhibition” (e.g., guaiacol and AmplexUltraRed, see below). Ususally just the first step of this series of events is covered by assays that measure TPO inhibition. Therefore, in the context of this AOP we are using TPO inhibition not in the classical sense, but instead to refer to the empirical data derived from the assays commonly used to investigate environmental chemicals. Therefore, in the context of this AOP we are using TPO inhibition </span><span style="color:black">not in the classical sense, but instead to refer to the empirical data derived from the assays commonly used to investigate environmental chemicals.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Figure 1 </span> <span style="color:black">illustrates the enzymatic and nonenzymatic reactions mediated by TPO that result in the synthesis of thyroxine (T4) .</span></span></span></span></p>
<p style="text-align:start"><a href="https://aopwiki.org/system/dragonfly/production/2017/04/16/9jyfbzvr2o_Synthesis_figure.jpg"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2017/04/16/9jyfbzvr2o_Synthesis_figure.jpg" style="height:330px; width:604px" /></a></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Inhibition of TPO can be reversible, with transient interaction between the enzyme and the chemical, or irreversible, whereby suicide substrates permanently inactivate the enzyme. Reversible and irreversible (isoflavones such as genistein) TPO inhibition may be determined by the chemical structure, may be concentration dependent, or may be influenced by other conditions, including the availability of iodine (Doerge and Chang, 2002).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The ontogeny of TPO has been determined using both direct and indirect evidence in <strong>mammals</strong>. Available evidence suggests the 11</span><sup><span style="font-size:9pt"><span style="color:black">th</span></span></sup><span style="color:black"> to 12</span><sup><span style="font-size:9pt"><span style="color:black">th </span></span></sup><span style="color:black">fetal week as the beginning of functional TPO in humans. In rodents, TPO function begins late in the second fetal week, with the first evidence of T4 secretion on gestational day 17 (Remy et al., 1980). Thyroid-specific genes appear in the thyroid gland according to a specific temporal pattern; thyroglobulin (<em>Tg</em>), TPO (<em>Tpo</em>), and TSH receptor (<em>Tshr</em>) genes are expressed by gestational day 14 in rats, and the sodium iodide symporter, NIS (<em>Nis</em>), is expressed by gestational day 16 in rats. Maturation to adult function is thought to occur within a few weeks after parturition in rats and mice, and within the first few months in neonatal humans (Santisteban and Bernal, 2005). Tg is first detected in human fetuses starting at 5</span><sup><span style="font-size:9pt"><span style="color:black">th</span></span></sup><span style="color:black"> week of gestation and rises throughout gestation (Thorpe-Beeston et al., 1992), but iodine trapping and T4 production does not occur until around 10-12 weeks. Also, the dimerization of Tg, a characteristic of adult TH storage, is not found until much later in human gestation (Pintar, 2000). In rats, Tg immunoreactivity does not appear until day 15 of gestation (Fukiishi et al., 1982; Brown et al., 2000). The vast majority of research and knowledge on Tg is from mammals, although genomic orthologs are known for a variety of other species (Holzer et al., 2016). It is important to note that prior to the onset of fetal thyroid function, TH</span><span style="color:black">s</span><span style="color:black"> are still required by the developing fetus which until that time relies solely on maternal sources. Chemical-induced TPO inhibition can affect synthesis in the maternal gland and in the fetal gland.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The components of the TH system responsible for TH synthesis are highly conserved across vertebrates. In fish and amphibians TPO and NIS inhibition result in an expected decrease of TH synthesis (Hornung et al., 2010; Tietge et al., 2013; Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020) like in mammals. Although the </span><span style="color:black">TH</span> <span style="color:black">system is highly conserved across vertebrates, there are some taxon-specific considerations.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zebrafish and fathead minnows are oviparous <strong>fish </strong>species in which maternal </span><span style="color:black">THs </span><span style="color:black">are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic </span><span style="color:black">TH </span><span style="color:black">synthesis is initiated. Maternal transfer of </span><span style="color:black">THs</span> <span style="color:black">to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Inhibition of </span><span style="color:black">TPO</span> <span style="color:black">can only occur after activation of embryonic TH synthesis mediated by </span><span style="color:black">TPO</span><span style="color:black">. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for </span><span style="color:black">TPO </span><span style="color:black">is maternally transferred in relatively high amounts with subsequent mRNA degradation followed by initiation of embryonic transcription around hatching (Vergauwen et al., 2018).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">There are no approved OECD or EPA guideline study protocols for measurement of TPO inhibition. However, there is an OECD scoping document on identification of chemicals that modulate TH signaling that provides details on a TPO assay (OECD, 2017). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">From the early 1960's, microsomal fractions prepared from porcine thyroid glands and isolated porcine follicles were used as a source of TPO for inhibition experiments (Taurog, 2005). Microsomes from human goiter samples (Vickers et al., 2012) and rat thyroid glands (Paul et al., 2013; 2014; Paul-Friedman et al., 2016) have also been used as a source of TPO.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">TPO activity has been measured for decades via indirect assessment by kinetic measurement of the oxidation of guaiacol (Chang & Doerge 2000; Hornung et al., 2010; Schmutzler et al., 2007). This method is a low-throughput assay due to the very rapid kinetics of the guaiacol oxidation reaction. More recently, higher-throughput methods using commercial fluorescent and luminescent substrates with rodent, porcine, and human microsomal TPO have been developed (Vickers et al., 2012; Paul et al., 2013; 2014; Kaczur et al., 1997). This assay substitutes a pre-fluorescent substrate (Amplex UltraRed) for guaiacol, that when incubated with a source of peroxidase and excess hydrogen peroxidase, results in a stable fluorescent product proportional to TPO activity (Vickers et al., 2012). The stability of the fluorescent reaction product allows this assay to be used in a higher throughput format (Paul-Friedman et al., 2016). This approach is appropriate for high-throughput screening but does not elucidate the specific mechanism by which a chemical may inhibit TPO (Paul-Friedman et al., 2016), and as with most </span><em><span style="color:black">in vitro</span></em><span style="color:black"> assays, is subject to various sources of assay interference (Thorne et al., 2010). Recombinant sources of TPO have also been used (e.g. Schmutzler et al., 2007; Dong et al., 2020)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">HPLC has been used to measure the activity of TPO via formation of the precursors monoiodotyrosine (MIT), diiodotyrosine (DIT), and both T3 and T4, in a reaction mixture containing TPO, or a surrogate enzyme such as lactoperoxidase (Divi & Doerge 1994). The tools and reagents for this method are all available. However, HPLC or other analytical chemistry techniques make this a low throughput assay, depending on the level of automation. A primary advantage of this </span><em><span style="color:black">in vitro</span></em><span style="color:black"> method is that it directly informs hypotheses regarding the specific mechanism by which a chemical may impact </span><span style="color:black">TH</span><span style="color:black"> synthesis in vitro. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In fish, increases of TPO mRNA levels are often used as indirect evidence of TPO inhibition in </span><em><span style="color:black">in vivo</span></em><span style="color:black">experiments (Baumann et al., 2016; Nelson et al., 2016; Wang et al., 2020).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This KE is plausibly applicable across vertebrates. TPO inhibition is a MIE conserved across taxa, with supporting data from experimental models and human clinical testing. This conservation is likely a function of the high degree of protein sequence similarity in the catalytic domain of mammalian peroxidases (Taurog, 1999). Ample data available for human, rat, and porcine TPO inhibition demonstrate qualitative concordance across these species (Schmultzer et al., 2007; Paul et al., 2013; Hornung et al., 2010). A comparison of rat TPO and pig TPO, bovine lactoperoxidase, and human TPO inhibition by genistein demonstrated good qualitative and quantitative (40–66%) inhibition across species, as indicated by quantification of monoiodotyrosine (MIT) and diiodotyrosine (DIT) production (Doerge and Chang, 2002). Ealey et al. (1984) demonstrated peroxidase activity in guinea pig thyroid tissue using 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a substrate that is oxidized by the peroxidase to form a brown insoluble reaction product. Formation of this reaction product was inhibited by 3-amino-1,2,4-triazole and the TPO inhibitor, methimazole (MMI). A comparative analysis of this action of MMI between rat- and human-derived TPO indicates concordance of qualitative response. Data also suggest an increased quantitative sensitivity to MMI in rats</span><span style="color:black"> compared to human</span><span style="color:black">s</span><span style="color:black"> (Vickers et al., 2012). Paul et al. (2013) tested 12 chemicals using the guaiacol assay using both porcine and rat thyroid microsomes. The authors concluded that there was an excellent qualitative concordance between rat and porcine TPO inhibition, as all chemicals that inhibited TPO in porcine thyroid microsomes also inhibited TPO in rat thyroid microsomes when tested within the same concentration range. In addition, these authors noted a qualitative concordance that ranged from 1.5 to 50-fold differences estimated by relative potency. </span><span style="color:black">Similarly</span><span style="color:black">, Takayama et al. (1986) found a very large species difference in potency for sulfamonomethoxine between cynomologus monkeys and rats.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Applicability to certain life stages may depend on the species and their dependence on maternally transferred </span><span style="color:black">THs</span> <span style="color:black">during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. Evidence supporting this hypothesis is obtained from a zebrafish TPO knockout line. In homozygous individuals TPO is inhibited from the embryonic developmental stage onwards, resulting in an abolished T4 production in thyroid follicles with phenotypical abnormalities such as reduced swim bladder inflation and growth retardation appearing at 20 dpf but not before 10 dpf (Fang et al., 2022). In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf and not at 24 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body TH</span> <span style="color:black">levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TPO inhibition.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This KE is plausibly applicable to both sexes. The molecular components responsible for </span><span style="color:black">TH</span> <span style="color:black">synthesis, including </span><span style="color:black">TPO</span><span style="color:black">, are identical in both sexes. Therefore inhibition of </span><span style="color:black">TPO </span><span style="color:black">is not expected to be sex-specific.</span></span></span></span></p>
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<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Wang JX, Shi GH, Yao JZ, Sheng N, Cui RN, Su ZB, Guo Y, Dai JY. 2020. Perfluoropolyether carboxylic acids (novel alternatives to pfoa) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International. 134.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zoeller, R. T., Tan, S. W., & Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology, 37(1-2), 11–53.</span></span></span></span></p>
2016-11-29T18:41:232022-11-04T09:24:35Decrease, Population growth rateDecrease, Population growth ratePopulation<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">A population can be defined as a group of interbreeding organisms, all of the same species, occupying a specific space during a specific time (Vandermeer and Goldberg 2003, Gotelli 2008). As the population is the biological level of organization that is often the focus of ecological risk</span> <span style="color:black">assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">If N is the size of the population and t is time, then the population growth rate (dN/dt) is proportional to the instantaneous rate of increase, r, which measures the per capita rate of population increase over a short time interval. Therefore, r, is a difference between the instantaneous birth rate (number of births per individual per unit of time; b) and the instantaneous death rate (number of deaths per individual per unit of time; d) [Equation 1]. Because r is an instantaneous rate, its units can be changed via division. For example, as there are 24 hours in a day, an r of 24 individuals/(individual x day) is equal to an r of 1 individual/(individual/hour) (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="margin-left:144px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Equation 1: r = b - d</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Examining r in the context of population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The smaller the value of r below 1, the faster the population will decrease to zero. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The larger the value that r exceeds 1, the faster the population can increase over time </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will neither increase or decrease when the population growth rate equals 0 (either due to N = 0, or if the per capita birth and death rates are exactly balanced). For example, the per capita birth and death rates could become exactly balanced due to density dependence and/or to the effect of a stressor that reduces survival and/or reproduction (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Effects incurred on a population from a chemical or non-chemical stressor could have an impact directly upon birth rate (reproduction) and/or death rate (survival), thereby causing a decline in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of direct effect on r: Exposure to 17b-trenbolone reduced reproduction (i.e., reduced b) in the fathead minnow over 21 days at water concentrations ranging from 0.0015 to about 41 mg/L (Ankley et al. 2001; Miller and Ankley 2004). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Alternatively, a stressor could indirectly impact survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of indirect effect on r: Exposure of non-sexually differentiated early life stage fathead minnow to the fungicide prochloraz has been shown to produce male-biased sex ratios based on gonad differentiation, and resulted in projected change in population growth rate (decrease in reproduction due to a decrease in females and thus recruitment) using a population model. (Holbech et al., 2012; Miller et al. 2022)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Density dependence can be an important consideration:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The effect of density dependence depends upon the quantity of resources present within a landscape. A change in available resources could increase or decrease the effect of density dependence and therefore cause a change in population growth rate via indirectly impacting survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● This concept could be thought of in terms of community level interactions whereby one species is not impacted but a competitor species is impacted by a chemical stressor resulting in a greater availability of resources for the unimpacted species. In this scenario, the impacted species would experience a decline in population growth rate. The unimpacted species would experience an increase in population growth rate (due to a smaller density dependent effect upon population growth rate for that species). </span> </span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Closed versus open systems:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The above discussion relates to closed systems (there is no movement of individuals between population sites) and thus a declining population growth rate cannot be augmented by immigration. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● When individuals depart (emigrate out of a population) the loss will diminish population growth rate. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate applies to all organisms, both sexes, and all life stages.</span></span></span></span></p>
<p> </p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate (instantaneous growth rate) can be measured by sampling a population over an interval of time (i.e. from time t = 0 to time t = 1). The interval of time should be selected to correspond to the life history of the species of interest (i.e. will be different for rapidly growing versus slow growing populations). The population growth rate, r, can be determined by taking the difference (subtracting) between the initial population size, N</span><sub><span style="font-size:9pt"><span style="color:black">t=0 </span></span></sub><span style="color:black">(population size at time t=0), and the population size at the end of the interval, N</span><sub><span style="font-size:9pt"><span style="color:black">t=1 </span></span></sub><span style="color:black">(population size at time t = 1), and then subsequently dividing by the initial population size. </span></span></span></span></p>
<p style="margin-left:96px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Equation 2: r = (N</span><sub><span style="font-size:9pt"><span style="color:black">t=1 </span></span></sub><span style="color:black">- N</span><sub><span style="font-size:9pt"><span style="color:black">t=0</span></span></sub><span style="color:black">) / N</span><sub><span style="font-size:9pt"><span style="color:black">t=0</span></span></sub></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The diversity of forms, sizes, and life histories among species has led to the development of a vast number of field techniques for estimation of population size and thus population growth over time (Bookhout 1994, McComb et al. 2021). </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For stationary species an observational strategy may involve dividing a habitat into units. After setting up the units, samples are performed throughout the habitat at a select number of units (determined using a statistical sampling design) over a time interval (at time t = 0 and again at time t = 1), and the total number of organisms within each unit are counted. The numbers recorded are assumed to be representative for the habitat overall, and can be used to estimate the population growth rate within the entire habitat over the time interval. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For species that are mobile throughout a large range, a strategy such as using a mark-recapture method may be employed (i.e. tags, bands, transmitters) to determine a count over a time interval (at time = 0 and again at time =1). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate can also be estimated using mathematical model constructs (for example, ranging from simple differential equations to complex age or stage structured matrix projection models and individual based modeling approaches), and may assume a linear or nonlinear population increase over time (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). The AOP framework can be used to support the translation of pathway-specific mechanistic data into responses relevant to population models and output from the population models, such as changing (declining) population growth rate, can be used to assess and manage risks of chemicals (Kramer et al. 2011). As such, this translational capability can increase the capacity and efficiency of safety assessments both for single chemicals and chemical mixtures (Kramer et al. 2011). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Some examples of modeling constructs used to investigate population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A modeling construct could be based upon laboratory toxicity tests to determine effect(s) that are then linked to the population model and used to estimate decline in population growth rate. Miller et al. (2007) used concentration–response data from short term reproductive assays with fathead minnow (<em>Pimephales promelas</em>) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A model construct could be based upon a combination of effects-based monitoring at field sites (informed by an AOP) and a population model. Miller et al. (2015) applied a population model informed by an AOP to project declines in population growth rate for white suckers (Catostomus commersoni) using observed changes in sex steroid synthesis in fish exposed to a complex pulp and paper mill effluent in Jackfish Bay, Ontario, Canada. Furthermore, a model construct could be comprised of a series of quantitative models using KERs that culminates in the estimation of change (decline) in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A quantitative adverse outcome pathway (qAOP) has been defined as a mathematical construct that models the dose–response or response–response relationships of all KERs described in an AOP (Conolly et al. 2017, Perkins et al. 2019). Conolly et al. (2017) developed a qAOP using data generated with the aromatase inhibitor fadrozole as a stressor and then used it to predict potential population‐level impacts (including decline in population growth rate). The qAOP modeled aromatase inhibition (the molecular initiating event) leading to reproductive dysfunction in fathead minnow (Pimephales promelas) using 3 computational models: a hypothalamus–pituitary–gonadal axis model (based on ordinary differential equations) of aromatase inhibition leading to decreased vitellogenin production (Cheng et al. 2016), a stochastic model of oocyte growth dynamics relating vitellogenin levels to clutch size and spawning intervals (Watanabe et al. 2016), and a population model (Miller et al. 2007).</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Dynamic energy budget (DEB) models offer a methodology that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources (Nisbet et al. 2000, Nisbet et al. 2011). Murphy et al. (2018) developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates.</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Endogenous Lifecycle Models (ELMs), capture the endogenous lifecycle processes of growth, development, survival, and reproduction and integrate these to estimate and predict expected fitness (Etterson and Ankley, 2021). AOPs can be used to inform ELMs of effects of chemical stressors on the vital rates that determine fitness, and to decide what hierarchical models of endogenous systems should be included within an ELM (Etterson and Ankley, 2021).</span></span></span></span></p>
<p> </p>
<p>Consideration of population size and changes in population size over time is potentially relevant to all living organisms.</p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHigh<ul>
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2016-11-29T18:41:242023-01-03T09:09:06Thyroid hormone synthesis, DecreasedTH synthesis, DecreasedCellular<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The thyroid hormones (TH), triiodothyronine (T3) and thyroxine (T4) are thyrosine</span><span style="color:black">-</span><span style="color:black">based hormones. Synthesis of TH</span><span style="color:black">s is regulated by thyroid-stimulating hormone (TSH) binding to its receptor and thyroidal availability of iodine via the sodium iodide symporter (NIS). Other proteins contributing to TH production in the thyroid gland, including thyroperoxidase (TPO), dual oxidase enzymes (DUOX), and the transport protein pendrin are also necessary for iodothyronine production (Zoeller et al., 2007).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The production of THs in the thyroid gland and resulting serum concentrations are controlled by a negatively regulated feedback mechanism. Decreased T4 and T3 serum concentrations activates the hypothalamus-pituitary-thyroid (HPT) axis which upregulates thyroid-stimulating hormone (TSH) that acts to increase production of additional THs (Zoeller and Tan, 2007). This regulatory system includes: 1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH); 2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary; 3) hormonal transport by the plasma binding proteins; 4) cellular uptake mechanisms at the tissue level; 5) intracellular control of TH concentration</span><span style="color:black">s</span><span style="color:black"> by deiodinating mechanisms; 6) transcriptional function of the nuclear TH receptor; and 7) in the fetus, the transplacental passage of T4 and T3 (Zoeller et al., 2007).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">TRH and the TSH primarily regulate the production of T4, often considered a “pro-hormone,” and to a lesser extent of T3, the transcriptionally active TH. Most of the hormone released from the thyroid gland into circulation is in the form of T4, while peripheral deiodination of T4 is responsible for the majority of circulating T3. Outer ring deiodination of T4 to T3 is catalyzed by the deiodinases 1 and 2 (DIO1 and DIO2), with DIO1 expressed mainly in liver and kidney, and DIO2 expressed in several tissues including the brain (Bianco et al., 2006). Conversion of T4 to T3 takes place mainly in </span><span style="color:black">the </span><span style="color:black">liver and kidney, but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). </span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">In <strong>mammals</strong>, most evidence for the ontogeny of TH synthesis comes from measurements of serum hormone concentrations. And, importantly, the impact of xenobiotics on fetal hormones must include the influence of the maternal compartment since a majority of fetal THs are derived from maternal blood early in fetal life, with a transition during mid-late gestation to fetal production of THs that is still supplemented by maternal THs. In humans, THs can be found in the fetus as early as gestational weeks 10-12, and concentations rise continuously until birth. At term, fetal T4 is similar to maternal levels, but T3 remains 2-3 fold lower than maternal levels. In rats, THs can be detected in the fetus as early as the second gestational week, but fetal synthesis does not start until gestational day 17 with birth at gestational day 22-23. Maternal THs continue to supplement fetal production until parturition. (see Howdeshell, 2002; Santisteban and Bernal, 2005 for review). Due to the maternal factor, the life stage specific impact of TPO inhibition after exposure to environmental chemicals is complex (Ramhoj et al., 2022).</span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Decreased TH synthesis in the thyroid gland may result from several possible molecular-initiating events (MIEs) including: 1) Disruption of key catalytic enzymes or cofactors needed for TH synthesis, including TPO, NIS, or dietary iodine insufficiency. Theoretically, decreased synthesis of Tg could also affect TH production (Kessler et al., 2008; Yi et al., 1997). Mutations in genes that encode requisite proteins in the thyroid may also lead to impaired TH synthesis, including mutations in pendrin associated with Pendred Syndrome (Dossena et al., 2011), mutations in TPO and Tg (Huang and Jap 2015), and mutations in NIS (Spitzweg and Morris, 2010). 2) Decreased TH synthesis in cases of clinical hypothyroidism may be due to Hashimoto's thyroiditis or other forms of thyroiditis, or physical destruction of the thyroid gland as in radioablation or surgical treatment of thyroid lymphoma. 3) It is possible that TH synthesis may also be reduced subsequent to disruption of the negative feedback mechanism governing TH homeostasis, e.g. pituitary gland dysfunction may result in a decreased TSH signal with concomitant T3 and T4 decreases. 4) More rarely, hypothalamic dysfunction can result in decreased TH synthesis. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased fetal </span><span style="color:black">TH</span> <span style="color:black">levels are also possible. Maternal Graves disease, which results in fetal thyrotoxicosis (hyperthyroidism and increased serum T4 levels), has been successfully treated by maternal administration of TPO inhibitors (c.f., Sato et al., 2014). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">It should be noted that different species and different life</span> <span style="color:black">stages store different amounts of TH precursor</span><span style="color:black">s</span><span style="color:black"> and iodine within the thyroid gland. Thus, decreased TH synthesis via transient iodine insufficiency or inhibition of TPO may not affect TH release from the thyroid gland until depletion of stored iodinated Tg. Adult humans may store sufficient Tg-DIT residues to serve for several months to a year of TH demand (Greer et al., 2002; Zoeller, 2004). Neonates and infants have a much more limited supply of less than a week.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">While the </span><span style="color:black">TH</span> <span style="color:black">system is highly conserved across vertebrates, there are some taxon-specific considerations.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zebrafish and fathead minnows are oviparous <strong>fish</strong> species in which maternal </span><span style="color:black">THs </span><span style="color:black">are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic </span><span style="color:black">TH </span><span style="color:black">synthesis is initiated. Maternal transfer of </span><span style="color:black">THs </span><span style="color:black">to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. The components of the TH system responsible for TH synthesis are highly conserved across vertebrates and therefore interference with the same molecular targets compared to mammals can lead to decreased TH synthesis (TPO, NIS, etc.) in fish. Endogenous transcription profiles of thyroid-related genes in zebrafish and fathead minnow showed that mRNA coding for these genes is also maternally transferred and increasing expression of most transcripts during hatching and embryo-larval transition indicates a fully functional HPT axis in larvae (Vergauwen et al., 2018). Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus </span><span style="color:black">TH</span> <span style="color:black">synthesis compared to TSH-releasing hormone (TRH). Also, in most fish species thyroid follicles are more diffusely located in the pharyngeal region rather than encapsulated in a gland.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Decreased TH synthesis is often implied by measurement of TPO and NIS inhibition measured clinically and in laboratory models as these enzymes are essential for TH synthesis. Rarely is decreased TH synthesis measured directly, but rather the impact of chemicals on the quantity of T4 produced in the thyroid gland, or the amount of T4 present in serum is used as a marker of decreased T4 release from the thyroid gland (e.g., Romaldini et al., 1988). Methods used to assess TH synthesis include, incorporation of </span><span style="color:black">radiolabeled </span><span style="color:black">tracer compounds, radioimmunoassay, ELISA, and analytical detection. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Recently, amphibian thyroid explant cultures have been used to demonstrate direct effects of chemicals on TH synthesis, as this model contains all necessary synthesis enzymes including TPO and NIS (Hornung et al., 2010). For this work THs was measured by HPLC/ICP-mass spectometry. Decreased TH synthesis and release, using T4 release as the endpoint, has been shown for thiouracil antihyperthyroidism drugs including MMI, PTU, and the NIS inhibitor perchlorate (Hornung et al., 2010).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Techniques for </span><em><span style="color:black">in vivo</span></em><span style="color:black"> analysis of </span><span style="color:black">TH</span> <span style="color:black">system disruption among other drug-related effects in fish were reviewed by Raldua and Piña (2014). TIQDT (Thyroxine-immunofluorescence quantitative disruption test) is a method that provides an immunofluorescent based estimate of thyroxine in the gland of zebrafish (Raldua and Babin, 2009; Thienpont et al., 2011; Jomaa et al., 2014; Rehberger et al., 2018). Thienpont used this method with ~25 xenobiotics (e.g., amitrole, perchlorate, methimazole, PTU, DDT, PCBs). The method detected changes for all chemicals known to directly impact TH synthesis in the thyroid gland (e.g., NIS and TPO </span><span style="color:black">inhibitors</span><span style="color:black">), but not those that upregulate hepatic catabolism of T4. Rehberger et al. (2018) updated the method to enable simultaneous semi-quantitative visualization of intrafollicular T3 and T4 levels. Most often, whole body </span><span style="color:black">TH</span><span style="color:black"> level measurements in fish early life stages are used as indirect evidence of decreased </span><span style="color:black">TH</span> <span style="color:black">synthesis (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). Analytical determination of </span><span style="color:black">TH </span><span style="color:black">levels by LC-MS is becoming increasingly available (Hornung et al., 2015).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">More recently, transgenic zebrafish with fluorescent thyroid follicles are being used to visualize the compensatory proliferation of the thyroid follicles following inhibition of </span><span style="color:black">TH</span> <span style="color:black">synthesis</span><span style="color:black"> among others</span><span style="color:black"> (Opitz et al., 2012).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: This KE is plausibly applicable across vertebrates. Decreased TH synthesis resulting from TPO or NIS inhibition is conserved across vertebrate taxa, with <em>in vivo</em> evidence from humans, rats, amphibians, some fish speci</span><span style="color:black">es, and birds, and <em>in vitro</em> evidence from rat and porcine microsomes. Indeed, TPO and NIS mutations result in congenital hypothyroidism in humans (Bakker et al., 2000; Spitzweg and Morris, 2010), demonstrating the essentiality of TPO and NIS function toward maintaining euthyroid status. Though decreased serum T4 is used as a surrogate measure to indicate chemical-mediated decreases in TH synthesis, clinical and veterinary management of hyperthyroidism and Graves’ disease using propylthiouracil and methimazole, known to decrease TH synthesis, indicates strong evidence for chemical inhibition of TPO (Zoeller and Crofton, 2005).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Applicability to certain life stages may depend on the species and their dependence on maternally transferred </span><span style="color:black">THs</span> <span style="color:black">during the earliest phases of development. The earliest life stages of teleost fish (e.g., fathead minnow, zebrafish) rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). In externally developing fish species, decreases in TH synthesis can only occur after initiation of embryonic TH synthesis. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. TPO inhibition in a homozygous knockout line abolished the T4 production in thyroid follicles of mutant zebrafish with phenotypic abnormalities occurring from 20 dpf onwards but not before 10 dpf (Fang et al., 2022). Therefore, it is still uncertain when exactly embryonic TH synthesis is activated and thus when exactly this process becomes sensitive to disruption. In fathead minnows, a significant increase of whole body TH</span> <span style="color:black">levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It currently remains unclear when exactly embryonic </span><span style="color:black">TH </span><span style="color:black">production is initiated in zebrafish.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. </span><span style="color:black">THs </span><span style="color:black">are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of </span><span style="color:black">TH</span><span style="color:black"> levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in </span><span style="color:black">TH</span> <span style="color:black">levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
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2016-11-29T18:41:232022-11-04T09:25:39 Thyroxine (T4) in serum, DecreasedT4 in serum, DecreasedTissue<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">All iodothyronines are derived from the modification of tyrosine molecules (Taurog, 2000). There are two biologically active thyroid hormones (THs) in serum, triiodothyronine (T3) and T4, and a few less active iodothyronines, reverse T3 (rT3), and 3,3'-Diiodothyronine (3,5-T2). T4 is the predominant TH in circulation, comprising approximately 80% of the TH excreted from the thyroid gland in mammals and is the pool from which the majority of T3 in serum is generated (Zoeller et al., 2007). As such, serum T4 changes usually precede changes in other serum THs. Decreased thyroxine (T4) in serum results from one or more MIEs upstream and is considered a key biomarker of altered TH homeostasis (DeVito et al., 1999).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Serum T4 is used as a biomarker of TH status because the circulatory system serves as the major transport and delivery system for TH delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In serum, it is the unbound, or ‘free’ form of the hormone that is thought to be available for transport into tissues. Free hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. There are major species differences in the predominant binding proteins and their affinities for THs (see below). However, there is broad agreement that changes in serum concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis across vertebrates (DeVito et al., 1999; Miller et al., 2009; Zoeller et al., 2007; Carr and Patiño, 2011).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Normal serum T4 reference ranges can be species and lifestage specific. In <strong>rodents</strong>, serum THs are low in the fetal circulation, increasing as the fetal thyroid gland becomes functional on gestational day 17, just a few days prior to birth. After birth serum hormones increase steadily, peaking at two weeks, and falling slightly to adult levels by postnatal day 21 (Walker et al., 1980; Harris et al., 1978; Goldey et al., 1995; Lau et al., 2003). Similarly, in <strong>humans</strong>, adult reference ranges for THs do not reflect the normal ranges for children at different developmental stages, with TH concentrations highest in infants, still increased in childhood, prior to a decline to adult levels coincident with pubertal development (Corcoran et al. 1977; Kapelari et al., 2008).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In some <strong>frog </strong>species, there is an analogous peak in </span><span style="color:black">THs </span><span style="color:black">in tadpoles that starts around embryonic NF stage 56, peaks at </span><span style="color:black">s</span><span style="color:black">tage 62 and the declines to lower levels by </span><span style="color:black">s</span><span style="color:black">tage 56 (Sternberg et al., 2011; Leloup and Buscaglia, 1977). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Additionally, ample evidence is available from studies investigating responses to inhibitors of </span><span style="color:black">TH </span><span style="color:black">synthesis in <strong>fish</strong>. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Serum T3 and T4 can be measured as free (unbound) or total (bound + unbound). Free hormone concentrations are clinically considered more direct indicators of T4 and T3 activities in the body, but in animal studies, total T3 and T4 are typically measured. Historically, the most widely used method in toxicology is the radioimmunoassay (RIA). The method is routinely used in rodent endocrine and toxicity studies. The ELISA method is commonly used as a human clinical test method. Analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates, through methods employing HPLC, liquid chromatography, immuno luminescence, and mass spectrometry are less common, but are becoming increasingly available (Hornung et al., 2015; DeVito et al., 1999; Baret and Fert, 1989; Spencer, 2013; Samanidou V.F et al., 2000; Rathmann D. et al., 2015 ). In fish early life stages most evidence for the ontogeny of thyroid hormone synthesis comes from measurements of whole body thyroid hormone levels using LC-MS techniques (Hornung et al., 2015) which are increasingly used to accurately quantify whole body thyroid hormone levels as a proxy for serum thyroid hormone levels (Nelson et al., 2016; Stinckens et al., 2016; Stinckens et al., 2020). It is important to note that thyroid hormones concentrations can be influenced by a number of intrinsic and extrinsic factors (e.g., circadian rhythms, stress, food intake, housing, noise) (see for example, Döhler et al., 1979).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Any of these measurements should be evaluated for the relationship to the actual endpoint of interest, repeatability, reproducibility, and lower limits of quantification using a fit-for-purpose approach. This is of particular significance when assessing the very low levels of TH present in fetal serum. Detection limits of the assay must be compatible with the levels in the biological sample. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by an indirect methodology, whereas analytical determination is the most direct measurement available. All these methods, particularly RIA, are repeatable and reproducible.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: This KE is plausibly applicable across vertebrates and the overall evidence supporting taxonomic applicability is strong. THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in zebrafish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002), and amphibian and lamprey metamorphoses is well established (Manzon and Youson, 1997; Yaoita and Brown, 1990; Furlow and Neff, 2006). </span><span style="color:black">T</span><span style="color:black">heir role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH</span><span style="color:black">s</span><span style="color:black"> in the different species depends on the expression and function of specific proteins (e.g receptors or enzymes) under TH control and may vary across species and tissues. As such</span><span style="color:black">,</span><span style="color:black"> extrapolation regarding TH action across species and developmental stages should be done with caution.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">With few exceptions, vertebrate species have circulating T4 (and T3) that are bound to transport proteins in blood. Clear species differences exist in serum transport proteins (Dohler et al., 1979; Yamauchi and Isihara, 2009). There are three major transport proteins in mammals; thyroid binding globulin (TBG), transthyretin (TTR), and albumin. In adult humans, the percent bound to these proteins is about 75, 15 and 10 percent, respectively (Schussler 2000). In contrast, in adult rats the majority of THs are bound to TTR. Thyroid</span><span style="color:black">-</span> <span style="color:black">binding proteins are developmentally regulated in rats. TBG is expressed in rats until approximately postnatal day (PND) 60, with peak expression occurring during weaning (Savu et al., 1989). However, low levels of TBG persist into adult ages in rats and can be experimentally induced by hypothyroidism, malnutrition, or caloric restriction (Rouaze-Romet et al., 1992). While these species differences impact TH half-life (Capen, 1997) and possibly regulatory feedback mechanisms, there is little information on quantitative dose-response relationships of binding proteins and serum hormones during development across different species. Serum THs are still regarded as the most robust measurable key event causally linked to downstream adverse outcomes.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body </span><span style="color:black">TH </span><span style="color:black">levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH </span><span style="color:black">system </span><span style="color:black">disruptors.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. </span><span style="color:black">THs</span> <span style="color:black">are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of </span><span style="color:black">TH</span> <span style="color:black">levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in </span><span style="color:black">TH</span> <span style="color:black">levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
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2016-11-29T18:41:232022-10-10T08:52:30Reduced, Anterior swim bladder inflationReduced, Anterior swim bladder inflationOrgan<p>The swim bladder of bony fish is evolutionary homologous to the lung (Zheng et al., 2011). The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96 h post fertilization (hpf) which is 2 days post hatch, and the anterior chamber inflates around 21 dpf. In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodeling of organs including the lateral line, nervous system, gut and kidneys (McMenamin and Parichy, 2013).</p>
<p>The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007). Dumbarton et al. (2010) showed that the anterior chamber of zebrafish has particularly closely packed and highly organized bundles of muscle fibres, suggesting that contraction of these muscles would reduce swim bladder volume. While it had previously been suggested that the posterior chamber had a more important role as a hydrostatic organ, this implies high importance of the anterior chamber for buoyancy. The anterior chamber has an additional role in hearing (Bang et al., 2002). Weberian ossicles (the Weberian apparatus) connect the anterior chamber to the inner ear resulting in an amplification of sound waves. Reduced inflation of the anterior chamber may manifest itself as either a complete failure to inflate the chamber or reduced size of the chamber. Reduced size is often associated with a deviating morphology.</p>
<p>In several fish species, inflation of the anterior chamber can be observed using a stereomicroscope because the larvae are still transparent during the larval stage. This is for example true for zebrafish and fathead minnow. Anterior chamber size can then be measured based on photographs with a calibrator.</p>
<p><strong>Taxonomic</strong>: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass, medaka) and physostomous (e.g., zebrafish and fathead minnow). Physostomous fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2016; Nelson et al., 2016; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020). While zebrafish and fathead minnows are physostomous fish with a two-chambered swim bladder, the Japanese rice fish or medaka (<em>Oryzias latipes</em>) is a physoclistous fish with a single chambered swim bladder that inflates during early development. The key event 'reduced anterior chamber inflation' is not applicable to such fish species. Therefore, the current key event is plausibly applicable to physostomous fish in general.</p>
<p><strong>Life stage</strong>: The anterior chamber inflates during a specific developmental time frame. In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KE is only applicable to the larval life stage.</p>
<p><strong>Sex</strong>: This KE/KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KE/KER. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 21 days post fertilization in zebrafish, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.</p>
UBERON:0006860swim bladderModerateUnspecificHighLarvaeHighHigh<p>Bang, P.I., Yelick, P.C., Malicko, J.J., Sewell, W.F. 2002. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Journal of Neuroscience Methods. 118, 177-187.</p>
<p>Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.</p>
<p>Dumbarton, T.C., Stoyek, M., Croll, R.P., Smith, F.M., 2010. Adrenergic control of swimbladder deflation in the zebrafish (Danio rerio). J. Exp. Biol. 213,2536–2546, http://dx.doi.org/10.1242/jeb.039792.</p>
<p>Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.</p>
<p>McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.</p>
<p>Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.</p>
<p>Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC, Saari T, Stinckens E, Vergauwen L, Villeneuve DL. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole – Part I: fathead minnow. Aquatic Toxicology 173: 192-203.</p>
<p>Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.</p>
<p>Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007. Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.</p>
<p>Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</p>
<p>Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.</p>
<p>van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.</p>
<p>Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture 2, 181-190.</p>
<p>Zheng, W., Wang, Z., Collins, J.E., Andrews, R.M., Stemple, D., Gong, Z. 2011. Comparative transcriptome analyses indicate molecular homology of zebrafish swim bladder and mammalian lung. PLoS One 6, http://dx.doi.org/10.1371/</p>
2016-11-29T18:41:282022-10-07T09:36:16Reduced, Swimming performanceReduced, Swimming performanceIndividual<p>Adequate swimming performance in fish is essential for behaviour such as foraging, predator avoidance and reproduction.</p>
<p>For fish larvae, automated observation and tracking systems are commercially available and increasingly used for measuring swimming performance including distance travelled, duration of movements, swimming speed, etc. This kind of measurements is often included in publications describing effects of chemicals in zebrafish larvae (Hagenaars et al., 2014; Stinckens et al., 2016; Vergauwen et al., 2015).</p>
<p>For juvenile and adult fish, measurements of swim performance vary. However, in some circumstances, swim tunnels have been used to measure various data (Fu et al., 2013).</p>
<p>Little and Finger (1990) discussed swimming behavior as an indicator of sublethal toxicity in fish.</p>
<p><strong>Taxonomic</strong>: Importance of swimming performance for natural behaviour is generally applicable to fish and tho other taxa that rely on swimming to support vital behaviours.</p>
<p><strong>Life stage</strong>: Importance of swimming performance for natural behaviour is generally applicable across all free-swimming life stages, i.e., post-embryonic life stages.</p>
<p><strong>Sex</strong>: Importance of swimming performance for natural behaviour is generally applicable across sexes.</p>
ModerateUnspecificModerateLarvaeModerateJuvenileModerateAdultHighHighHigh<p>Fu C, Cao ZD, Fu SJ. 2013. The effects of caudal fin loss and regeneration on the swimming performance of three cyprinid fish species with different swimming capactities. The Journal of Experimental Biology 216:3164-3174. doi:10.1242/jeb.084244</p>
<p>Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOS affects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235.</p>
<p>Little EE, Finger SE. 1990. Swimming behavior as an indicator of sublethal toxicity in fish. Environmental Toxicology and Chemistry. 9(1):13-19.</p>
<p>Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.</p>
<p>Vergauwen, Lucia; Nørgaard Schmidt, Stine; Maho, Walid; Stickens, Evelyn; Hagenaars, An; Blust, Ronny; Mayer, Philipp; Covaci, Adrian; Knapen, Dries. 2014. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere. 139: 9-17.</p>
2016-11-29T18:41:282021-09-08T06:12:30Decreased, Triiodothyronine (T3)Decreased, Triiodothyronine (T3)Tissue<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">There are two biologically active thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), and a few less active iodothyronines (rT3, 3,5-T2), which are all derived from the modification of tyrosine molecules (Hulbert, 2000). However, the plasma concentrations of the other iodothyronines are significantly lower than those of T3 and T4. The different iodothyronines are formed by the sequential outer or inner ring monodeiodination of T4 and T3 by the deiodinating enzymes, Dio1, Dio2, and Dio3 (Gereben et al., 2008). Deiodinase structure is considered to be unique, as THs are the only molecules in the body that incorporate iodide.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The circulatory system serves as the major transport and delivery system for THs from synthesis in the gland to delivery to tissues. The majority of THs in the blood are bound to transport proteins (Bartalena and Robbins, 1993). In humans, the major transport proteins are TBG (thyroxine binding globulin), TTR (transthyretin) and albumin. The percent bound to these proteins in adult humans is about 75, 15 and 10 percent, respectively (Schussler 2000). Unbound (free) hormones are approximately 0.03 and 0.3 percent for T4 and T3, respectively. In serum, it is the free form of the hormone that is active.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">There are major species differences in the predominant binding proteins and their affinities for THs (see section below on Taxonomic applicability). However, there is broad agreement that changes in concentrations of THs is diagnostic of thyroid disease or chemical-induced disruption of thyroid homeostasis (Zoeller et al., 2007).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">It is notable that the changes measured in the free TH concentration reflect mainly the changes in the serum transport proteins rather than changes in the thyroid status. These thyroid-binding proteins serve as hormonal storage which ensures their even and constant distribution in the different tissues, while they protect the most sensitive ones in the case of severe changes in thyroid availability, like in thyroidectomies (Obregon et al., 1981). Initially, it was believed that all of the effects of TH were mediated by the binding of T3 to the thyroid nuclear receptors (TRa and TRb), a notion which is now questionable due to the increasing evidence that support the non-genomic action of TH (Davis et al., 2010, Moeller et al., 2006). Many non-nuclear TH binding sites have been identified to date and they usually lead to rapid cellular response in TH-effects (Bassett et al., 2003). Four types of thyroid hormone signaling have been defined (Anyetei-Anum et al., 2018): type 1 is the canonical pathway in which liganded TR binds directly to DNA; type 2 describes liganded TR tethered to chromatin-associated proteins, but not bound to DNA directly; type 3 suggests that liganded TR can exert its function without recruitment to chromatin in either the nucleus or cytoplasm; and type 4 proposes that thyroid hormone acts at the plasma membrane or in the cytoplasm without binding TR, a mechanism of action that is emerging as a key component of thyroid hormone signaling.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The production of THs in the thyroid gland and the circulation levels in the bloodstream are self-controlled by an efficiently regulated feedback mechanism across the Hypothalamus-Pituitary-Thyroid (HPT) axis. TH levels are regulated, not only in the plasma level, but also in the individual cell level, to maintain homeostasis. This is succeeded by the efficient regulatory mechanism of the thyroid hormone axis which consists of the following: (1) the hypothalamic secretion of the thyrotropin-releasing hormone (TRH), (2) the thyroid-stimulating hormone (TSH) secretion from the anterior pituitary, (3) hormonal transport by the plasma binding proteins, (4) cellular uptake mechanisms in the cell level, (5) intracellular control of TH concentration by the deiodinating mechanism (6) transcriptional function of the nuclear thyroid hormone receptor and (7) in the fetus, the transplacental passage of T4 and T3 (Cheng et al., 2010).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In regards to the brain, the TH concentration involves also an additional level of regulation, namely the hormonal transport through the Blood Brain Barrier (BBB) (Williams, 2008). The TRH and the TSH regulate the production of thyroid hormones. Less T3 (the biologically more active TH) than T4 is produced by the thyroid gland. The rest of the required amount of T3 is produced by outer ring deiodination of T4 by the deiodinating enzymes D1 and D2 (Bianco et al., 2006), a process which takes place mainly in liver and kidneys but also in other target organs such as in the brain, the anterior pituitary, brown adipose tissue, thyroid and skeletal muscle (Gereben et al., 2008; Larsen, 2009). Both hormones exert their action in almost all tissues of mammals and they are acting intracellularly, and thus the uptake of T3 and T4 by the target cells is a crucial step of the overall pathway. The trans-membrane transport of TH is performed mainly through transporters that differ depending on the cell type (Hennemann et al., 2001; Friesema et al., 2005; Visser et al., 2008). Many transporter proteins have been identified to date. The monocarboxylate transporters (Mct8, Mct10) and the anion-transporting polypeptide (OATP1c1) show the highest degree of affinity towards TH (Jansen et al., 2005) and mutations in these genes have pathophysiological effects in humans (Bernal et al., 2015). Unlike humans with an MCT8 deficiency, MCT8 knockout mice do not have neurological impairment. One explanation for this discrepancy could be differences in expression of the T4 transporter OATP1C1 in the blood–brain barrier. This shows that cross-species differences in the importance of specific transporters may occur.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">T3 and T4 have significant effects on normal development, neural differentiation, growth rate and metabolism (Yen, 2001; Brent, 2012; Williams, 2008), with the most prominent ones to occur during the fetal development and early childhood. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs. The thyroidal actions though are not only restricted to mammals, as their high significance has been identified also for other vertebrates, with the most well-studied to be the amphibian metamorphosis (Furlow and Neff, 2006). The importance of the thyroid-regulated pathways becomes more apparent in iodine deficient areas of the world, where a higher rate of cretinism and growth retardation has been observed and linked to decreased TH levels (Gilbert et al., 2012). Another very common cause of severe hypothyroidism in human is the congenital hypothyroidism, but the manifestation of these effects is only detectable in the lack of adequate treatment and is mainly related to neurological impairment and growth retardation (Glinoer, 2001), emphasizing the role of TH in neurodevelopment in all above cases. In adults, the thyroid-related effects are mainly linked to metabolic activities, such as deficiencies in oxygen consumption, and in the metabolism of the vitamin, proteins, lipids and carbohydrates, but these defects are subtle and reversible (Oetting and Yen, 2007). Blood tests to detect the amount of thyroid hormone (T4) and thyroid stimulating hormone (TSH) are routinely done for newborn babies for the diagnosis of congenital hypothyroidism at the earliest stage possible.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Although the components of the thyroid hormone system as well as thyroid hormone synthesis and action are highly conserved across vertebrates, there are some taxon-specific considerations.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Although the HPT axis is highly conserved, there are some differences between fish and mammals (Blanton and Specker, 2007; Deal and Volkoff, 2020). For example, in fish, corticotropin releasing hormone (CRH) often plays a more important role in regulating thyrotropin (TSH) secretion by the pituitary and thus thyroid hormone synthesis compared to TSH-releasing hormone (TRH). TTRs from fish have low sequence identity with human TTR, for example seabream TTR has 54% sequence identity with human TTR but the only amino acid difference within the thyroxine-binding site is the conservative substitution of Ser117 in human TTR to Thr117 in seabream TTR (Santos and Power, 1999; Yamauchi et al., 1999; Eneqvist et al., 2004). In vitro binding experiments showed that TH system disrupting chemicals bind with equal or weaker affinity to seabream TTR than to the human TTR with polar TH disrupting chemicals, in particular, showing a more than 500-fold lower affinity for seabream TTR compared to human TTR (Zhang et al., 2018).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zebrafish and fathead minnow are oviparous fish species in which maternal thyroid hormones are transferred to the eggs and regulate early embryonic developmental processes during external (versus intra-uterine in mammals) development (Power et al., 2001; Campinho et al., 2014; Ruuskanen and Hsu, 2018) until embryonic thyroid hormone synthesis is initiated. Maternal transfer of thyroid hormones, both T4 and T3, to the eggs has been demonstrated in zebrafish (Walpita et al., 2007; Chang et al., 2012) and fathead minnows (Crane et al., 2004; Nelson et al., 2016).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">T3 and T4 can be measured as free (unbound) or total (bound + unbound) in serum, or in tissues. Free hormones are considered more direct indicators of T4 and T3 activities in the body. The majority of T3 and T4 measurements are made using either RIA or ELISA kits. In animal studies, total T3 and T4 are typically measured as the concentrations of free hormone are very low and difficult to detect.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Historically, the most widely used method in toxicology is RIA. The method is routinely used in rodent endocrine and toxicity studies. The ELISA method has become more routine in rodent studies. The ELISA method is commonly used as a human clinical test method.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Recently, analytical determination of iodothyronines (T3, T4, rT3, T2) and their conjugates through methods employing HPLC and mass spectrometry have become more common (DeVito et al., 1999; Miller et al., 2009; Hornung et al., 2015; Nelson et al., 2016; Stinckens et al., 2016).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Any of these measurements should be evaluated for fit-for-purpose, relationship to the actual endpoint of interest, repeatability, and reproducibility. All three of the methods summarized above would be fit-for-purpose, depending on the number of samples to be evaluated and the associated costs of each method. Both RIA and ELISA measure THs by a an indirect methodology, whereas analytical determination is the most direct measurement available. All of these methods, particularly RIA, are repeatable and reproducible.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In fish early life stages most evidence for the ontogeny of TH synthesis comes from measurements of whole-body TH levels and using LC-MS techniques (Hornung et al., 2015) are increasingly used to accurately quantify whole-body TH levels (Nelson et al., 2016; Stinckens et al., 2016, 2020).</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Taxonomic: </span></strong><span style="color:black">The overall evidence supporting taxonomic applicability is strong. With few exceptions vertebrate species have T3 and T4 that are mostly bound to transport proteins in blood as well as T3 and T4 in tissues. Therefore, the current key event is plausibly applicable to vertebrates in general. Clear species differences exist in transport proteins (Yamauchi and Isihara, 2009). Specifically, the majority of supporting data for TH decreases come from rat studies and have been measured mostly in serum. The predominant iodothyronine binding protein in rat serum is transthyretin (TTR). TTR demonstrates a reduced binding affinity for T4 when compared with thyroxine binding globulin (TBG), the predominant serum binding protein for T4 in humans. This difference in serum binding protein affinity for THs is thought to modulate serum half-life for T4; the half-life of T4 in rats is 12-24 hr, whereas the half-life in humans is 5-9 days (Capen, 1997). While these species differences impact hormone half-life, possibly regulatory feedback mechanisms, and quantitative dose-response relationships, measurement of decreased THs is still regarded as a measurable key event causatively linked to downstream adverse outcomes.</span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Several studies have reported evidence of T3 decreases after exposure to TPO inhibitors and deiodinase inhibitors in early life stages of zebrafish (Stinckens et al., 2016; Stinckens et al., 2020; Wang et al., 2020) and fathead minnow (Nelson et al., 2016; Cavallin et al., 2017). Such measurements in fish early life stages are usually based on whole animal samples and do not allow for distinguishing between systemic and tissue TH alterations.</span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">THs are evolutionarily conserved molecules present in all vertebrate species (Hulbert, 2000; Yen, 2001). Moreover, their crucial role in amphibian and lamprey metamorphoses (Manzon and Youson, 1997; Yaoita and Brown, 1990) as well as fish development, embryo-to-larval transition and larval-to-juvenile transition (Thienpont et al., 2011; Liu and Chan, 2002) is well established. Their role as environmental messenger via exogenous routes in echinoderms confirms the hypothesis that these molecules are widely distributed among the living organisms (Heyland and Hodin, 2004). However, the role of TH in the different species may differ depending on the expression or function of specific proteins (e.g., receptors or enzymes) that are related to TH function, and therefore extrapolation between species should be done with caution.</span></span></span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage: </span></strong><span style="color:black">THs are essential in all life stages, but decreases of TH levels are not applicable to all developmental phases. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, T4 levels are not expected to decrease in response to exposure to inhibitors of TH synthesis during these earliest stages of development. However, T3 levels are expected to decrease upon exposure to deiodinase inhibitors in any life stage, since maternal T4 needs to be activated to T3 by deiodinases similar to embryonically synthesized T4.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex: </span></strong><span style="color:black">The KE is plausibly applicable to both sexes. THs are essential in both sexes and the components of the HPT- axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of TH levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in TH levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
<p> </p>
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<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G. (1981). Presence of l-thyroxine and 3,5,3-triiodo-l- thyronine in tissues from thyroidectomised rats. Endocrinology 109:908-913.</span></span></span></p>
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<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Oetting A, Yen PM. (2007). New insights into thyroid hormone action. Best Pract Res Clin Endocrinol Metab. 21:193–208.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Power DM, Llewellyn L, Faustino M, Nowell MA, Bjornsson BT, Einarsdottir IE, Canario AV, Sweeney GE. 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol. 130(4):447-459.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Ruuskanen S, Hsu BY. 2018. Maternal thyroid hormones: An unexplored mechanism underlying maternal effects in an ecological framework. Physiological and Biochemical Zoology. 91(3):904-916.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Santos CRA, Power DM. 1999. Identification of transthyretin in fish (sparus aurata): Cdna cloning and characterisation. Endocrinology. 140(5):2430-2433.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Schussler, G.C. (2000). The thyroxine-binding proteins. Thyroid 10:141–149.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2- mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Thienpont B, Tingaud-Sequeira A, Prats E, Barata C, Babin PJ, Raldua D. 2011. Zebrafish eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. Environmental Science & Technology. 45(17):7525-7532</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Visser WE, Friesema EC, Jansen J, Visser TJ. (2008). Thyroid hormone transport in and out of cells. Trends Endocrinol Metab. 19:50-56.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Walpita CN, Van der Geyten S, Rurangwa E, Darras VM. 2007. The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen Comp Endocrinol. 152(2-3):206-214.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Wang JX, Shi GH, Yao JZ, Sheng N, Cui RN, Su ZB, Guo Y, Dai JY. 2020. Perfluoropolyether carboxylic acids (novel alternatives to pfoa) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International. 134.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Williams GR. (2008). Neurodevelopmental and neurophysiological actions of thyroid hormone. J Neuroendocrinol. 20:784–794.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yamauchi K, Nakajima J, Hayashi H, Hara A. 1999. Purification and characterization of thyroid-hormone-binding protein from masu salmon serum - a homolog of higher-vertebrate transthyretin. European Journal of Biochemistry. 265(3):944-949.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yamauchi K1, Ishihara A. Evolutionary changes to transthyretin: developmentally regulated and tissue-specific gene expression. FEBS J. 2009 Oct; 276(19):5357-66.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yaoita Y, Brown DD. (1990). A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev. 4:1917-1924.</span></span></span></p>
<p><span style="color:#000000"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yen PM. (2001). Physiological and molecular basis of thyroid hormone action. Physiol Rev. 81:1097-1142.</span></span></span></p>
<p style="text-align:start"><span style="color:#000000"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Zhang J, Grundstrom C, Brannstrom K, Iakovleva I, Lindberg M, Olofsson A, Andersson PL, Sauer-Eriksson AE. 2018. Interspecies variation between fish and human transthyretins in their binding of thyroid-disrupting chemicals. Environmental Science & Technology. 52(20):11865-11874.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Crit Rev Toxicol. 2007 Jan-Feb;37(1-2):11-53</span></span></span></span></p>
2016-11-29T18:41:282022-10-07T08:26:15Increased MortalityIncreased MortalityPopulation<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.</span></span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Depending on the species and the study setup, mortality can be measured:</span></span></span></span></span></span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the lab by recording mortality during exposure experiments</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</span></span></span></span></span></li>
</ul>
<p>All living things are susceptible to mortality.</p>
ModerateUnspecificHighAll life stagesHigh2016-11-29T18:41:242022-07-08T07:32:26b70ffda9-114a-41b3-b8d5-8aedd63956614a1dd514-9205-4ead-b4b3-a84f409c1b2d<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thyroperoxidase (TPO) is a heme-containing apical membrane protein within the follicular lumen of thyrocytes that acts as the enzymatic catalyst for thyroid hormone (TH) synthesis (Taurog, 2005) across vertebrates. Two commonly used reference chemicals, propylthiouracil (PTU) and methimazole (MMI), are drugs that inhibit the ability of TPO to: a) activate iodine and transfer it to thyroglobulin (Tg) (Davidson et al., 1978); and, b) couple thyroglobulin (Tg)-bound iodotyrosyls to produce Tg-bound thyroxine (T4) and triiodothyronine (T3) (Taurog, 2005).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The weight of evidence supporting a direct linkage between the MIE, TPO inhibition, and the KE of decreased TH synthesis, is strong and supported by more than three decades of research in animals, including humans (Cooper et al., 1982; Cooper et al.,1983; Divi and Doerge, 1994; Fang et al., 2022).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The biological plausibility for this KER is rated Strong. TPO is the only enzyme capable of de novo </span><span style="color:black">synthesis</span><span style="color:black"> of TH. TPO catalyzes several reactions, including the oxidation of iodide, nonspecific iodination of tyrosyl residues of thyroglobulin (Tg) to form monoiodotyrosyl (MIT) or diiodotyrosyl (DIT) residues, and the coupling of these Tg-bound iodotyrosyls to produce Tg-bound T3 and T4 (Divi and Doerge, 1994; Kessler et al., 2008; Ruf et al., 2006; Taurog et al., 1996, 2005). Therefore, inhibition of TPO activity is widely accepted to directly impact TH synthesis.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Empirical support for this KER is strong. There are several papers that have measured alterations in TPO and subsequent effects on TH synthesis across vertebrates. Taurog et al. (1996) showed decreased guicaol activity, decreased bound I</span><sup><span style="font-size:9pt"><span style="color:black">125</span></span></sup><span style="color:black">, and subsequent decreases in newly formed T3 and T4 per molecule of Tg, following exposure to PTU, MMI and some antibiotics. There is important evidence in <strong>mammals</strong>. Following </span><em><span style="color:black">in vivo</span></em><span style="color:black"> exposure to PTU in rats (Cooper et al., 1982; 1983), there are concentration and time-dependent decreases in thyroid protein bound iodine and serum T4 and T3 that recovered one month after cessation of PTU exposure. In addition, measures of thyroidal iodine content were highly correlated with intra-thyroidal PTU concentration. Vickers et al. (2012) demonstrated dose- and time- dependent inhibition of TPO activity in both human and rat thyroid homogenates exposed to MMI. Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of </span><span style="color:black">THs</span> <span style="color:black">in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Tietge et al (2010) showed decreases in thyroidal T4 following MMI exposure in <strong>Xenopus</strong>. Also in Xenopus, Haselman et al (2020) showed decreases in thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3) following exposure to MMI. Doerge et al (1998) showed that a tryphenylmethane dye, malachite green, inhibited TPO and lowered thyroxine production. A recent paper used a series of benzothiazoles and showed TPO inhibition (guicaol assay) and inhibition of TSH stimulated thyroxine release from Xenopus thyroid gland explant cultures (Hornung et al., 2015). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Additionally, evidence is available from studies investigating responses to TPO inhibitors in <strong>fish</strong>. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). Using a TPO knockout line Fang et al. (2022) showed that TPO inhibition abolished the T4 synthesis in 7 dpf zebrafish mutants. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Temporal Evidence:</span></em><span style="color:black"> In <strong>mammals</strong>, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The impact of decreased TPO activity on </span><span style="color:black">TH</span> <span style="color:black">synthesis is similar across all ages in mammals. Good evidence for the temporal relationship of the KER comes from thyroid system modeling (e.g., Degon et al., 2008; Fisher et al., 2013) using data from studies of iodine deficiency and chemicals that inhibit NIS. In addition, there is ample evidence of the temporal impacts of TPO inhibition on TH synthesis, using </span><em><span style="color:black">ex vivo</span></em><span style="color:black"> and </span><em><span style="color:black">in vitro</span></em><span style="color:black"> measures that demonstrate the time course of inhibition following chemical exposures, including some data from human thyroid microsomes and </span><em><span style="color:black">ex vivo</span></em><span style="color:black"> thyroid slices (Vickers et al., 2012). Future work is needed that measures both TPO inhibition and TH production during development. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In oviparous <strong>fish </strong>such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal </span><span style="color:black">THs</span> <span style="color:black">transferred to the eggs. Embryonic </span><span style="color:black">TH</span> <span style="color:black">synthesis is activated later during embryo-larval development. (See Domain of applicability)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Dose-Response Evidence:</span></em><span style="color:black"> Dose-response data is available from a number of studies in <strong>mammals </strong>that correlate TPO inhibition with decreased TH production measured using a variety of endpoints including iodine organification (e.g., Taurog et al., 1996), inhibition of guicaol oxidation in thyroid microsomes (e.g., Doerge and Chang, 2002), and direct measure of thyroid gland T4 concentrations (e.g., Hornung et al., 2015). However, there is a lack of dose-response data from developmental studies showing direct linkages from TPO inhibition to thyroidal TH synthesis.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">While it is clear that TPO inhibition will lead to altered </span><span style="color:black">TH</span> <span style="color:black">synthesis, there is a need for data that will inform quantitative modeling of the relationship between TPO inhibition and the magnitude of effects on </span><span style="color:black">TH</span> <span style="color:black">synthesis.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Data from studies on genistein highlight this uncertainty. Doerge and colleagues have demonstrated that for this compound up to 80% TPO inhibition did not result in decreased serum T4 in rats (Doerge and Chang, 2002). This is not consistent with other prototypical TPO inhibitors (e.g., PTU, MMI). Genistein is however a well-known phytoestrogen and the observed inconsistency may be the result of feedback mechanisms resulting from its estrogenic effect.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In <em>Xenopus laevis</em>, Haselman et al.(2020) demonstrated temporal profiles of thyroidal iodotyrosines (MIT/DIT) and iodothyronines (T4/T3), the products of TPO activity, following exposure to three different model TPO inhibitors (MMI, PTU, MBT) at multiple concentrations. This study established that, in <em>Xenopus</em>, measurable decreases in the products of TPO activity can occur as early as 2 days of exposure during pro-metamorphosis. However, despite consistent profiles of some iodo-species across chemicals, other iodo-species showed inconsistent profiles across chemicals. This highlights the multiple mechanisms of TPO (iodination and coupling) and differential </span><span style="color:black">susceptibility</span> <span style="color:black">to inhibition of those mechanisms depending on the chemical's type of interaction with TPO. The most consistent concentration-response relationship across chemicals and over time was demonstrated by thyroidal T4, which is the most relevant product to subsequent key events. At the highest concentrations tested for each </span><span style="color:black">chemical</span><span style="color:black">, thyroidal T4 was below detection by 7 days of exposure across all three TPO inhibitors. Keeping in mind that the thyroid gland has follicular lumen space where thyroglobulin/T4 is stored until proteolysis and release to the blood, full inhibition of TPO would result in a delayed </span><span style="color:black">measurable</span> <span style="color:black">response due to the time it takes to deplete stored hormone</span><span style="color:black">s</span><span style="color:black">. Regardless of the delay, the results from this study imply full inhibition of TPO by each of these three chemicals at the highest test concentrations, but would require chemical residue analysis and/or toxicokinetic modeling to relate cellular/tissue concentrations at the site of TPO catalysis to levels of inhibition via Michaelis-Menten kinetic descriptions.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Profiles of thyroidal iodinated species demonstrated by Haselman et al. (2020) across three different TPO inhibitors suggests that a high level of TPO inhibition must occur in order to elicit responses in subsequent key events. Although the level of TPO inhibition is not directly quantifiable from this study, these data suggest that at least 90-100% inhibition was occurring since circulating T4 was not detectable at 10 days of exposure to the highest concentrations of MMI and MBT. However, additional efforts would be necessary to determine the minimum level of TPO inhibition that leads to a measurable decrease in thyroidal T4 and subsequently circulating T4. Furthermore, Hassan et al. (2017, 2020) and Handa et al. (2021) predicted the level of </span><span style="color:black">TH</span><span style="color:black">s in serum after treatment with PTU and MMI in rats. They developed a quantitative model by comparing dose- response data. </span></span></span></span></p>
HighMaleHighFemaleHighAll life stagesHighHighHighHighLow<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: This KER is plausibly applicable across vertebrates. Inhibition of TPO activity is widely accepted to directly impact TH synthesis. This is true for both rats and humans, as well as some fishes, frogs and birds. Most of the data supporting a causative relationship between TPO inhibition and altered TH synthesis is derived from animal studies,</span><em><span style="color:black"> in vitro </span></em><span style="color:black">thyroid microsomes from rats or pigs, and a limited number of human </span><em><span style="color:black">ex vivo</span></em><span style="color:black"> (Nagasaka and Hidaka, 1976; Vickers et al., 2012) and clinical studies. There are data to support that gene mutations in TPO result in congenital hypothyroidism, underscoring the essential role of TPO in human </span><span style="color:black">TH</span> <span style="color:black">synthesis.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Applicability to certain life stages may depend on the species and their dependence on maternally transferred </span><span style="color:black">THs</span> <span style="color:black">during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. Evidence supporting this hypothesis is obtained from a zebrafish TPO knockout line. In homozygous individuals TPO is inhibited from the embryonic developmental stage onwards, resulting in an abolished T4 production in thyroid follicles with phenotypical abnormalities such as reduced swim bladder inflation and growth retardation appearing at 20 dpf but not before 10 dpf (Fang et al., 2022). In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnow, a significant increase of whole body </span><span style="color:black">TH</span> <span style="color:black">levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH</span><span style="color:black"> system</span><span style="color:black"> disruptors.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Chang HC, Doerge DR. Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect. Toxicol Appl Pharmacol 168:244–252 (2000).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in Thyroid Hormone Levels during Zebrafish Development. Zoological Science 29, 181-184.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Cooper DS, Kieffer JD, Halpern R, Saxe V, Mover H, Maloof F, Ridgway EC (1983) Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113:921-928.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Cooper DS, Saxe VC, Meskell M, Maloof F, Ridgway EC. Acute effects of propylthiouracil (PTU) on thyroidal iodide organification and peripheral iodothyronine deiodination: correlation with serum PTU levels measured by radioimmunoassay. J Clin Endocrinol Metab. 1982 54(1):101-7.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Crane HM, Pickford DB, Hutchinson TH, Brown JA. 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences. 93(2):278-285.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Davidson, B., Soodak, M., Neary, J.T., Strout, H.V., and Kieffer, J.D. (1978). The irreversible inactivation of thyroid peroxidase by methylmercaptoimidazole, thiouracil, and propylthiouracil in vitro and its relationship to in vivo findings. Endocrinology 103:871–882.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Divi, R. L., and Doerge, D. R. (1994). Mechanism-based inactivation of lactoperoxidase and thyroid peroxidase by resorcinol derivatives. Biochemistry 33(32), 9668-74.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Doerge DR, Chang HC, Divi RL, Churchwell Mechanism for inhibition of thyroid peroxidase by leucomalachite green. Chem Res Toxicol. 1998 11(9):1098-104.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Doerge DR, Chang HC. Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Sep 25;777(1-2):269-79</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Fang, Y., Wan, J. P., Zhang, R. J., Sun, F., Yang, L., Zhao, S. X., Dong, M., & Song, H. D. (2022). Tpo knockout in zebrafish partially recapitulates clinical manifestations of congenital hypothyroidism and reveals the involvement of TH in proper development of glucose homeostasis. General and Comparative Endocrinology, 323–324. https://doi.org/10.1016/j.ygcen.2022.114033</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Handa S, Hassan I, Gilbert M, El-Masri H. 2021. Mechanistic Computational Model for Extrapolating In Vitro Thyroid Peroxidase (TPO) Inhibition Data to Predict Serum Thyroid Hormone Levels in Rats. Toxicological Sciences 183(1):36-48.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Haselman, J.T., Olker, J.H., Kosian, P.A., Korte, J.J., Swintek, J.A., Denny, J.S., Nichols, J.W., Tietge, J.E., Hornung, M.W. and Degitz, S.J., 2020. Targeted pathway-based in vivo testing using thyroperoxidase inhibition to evaluate plasma thyroxine as a surrogate metric of metamorphic success in model amphibian Xenopus laevis. Toxicological Sciences, 175(2), pp.236-250.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Hassan I, El-Masri H, Ford J, Brennan A, Handa S, Friedman KP, Gilbert ME. 2020. Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat. Toxicological Sciences 173(2):280-292.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Hassan I, El-Masri H, Kosian PA, Ford J, Degitz SJ, Gilbert ME. 2017. Neurodevelopment and Thyroid Hormone Synthesis Inhibition in the Rat: Quantitative Understanding Within the Adverse Outcome Pathway Framework. Toxicological Sciences 160(1):57-73.</span></span></span></span></p>
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<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Kessler, J., Obinger, C., and Eales, G. (2008). Factors influencing the study of peroxidase-generated iodine species and implications for thyroglobulin synthesis. Thyroid 18(7), 769-74, 10.1089/thy.2007.0310.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Liu CS, Zhang XW, Deng J, Hecker M, Al-Khedhairy A, Giesy JP, Zhou BS. 2011. Effects of prochloraz or propylthiouracil on the cross-talk between the hpg, hpa, and hpt axes in zebrafish. Environmental Science & Technology. 45(2):769-775.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Nagasaka, A., and Hidaka, H. (1976). Effect of antithyroid agents 6-propyl-2-thiouracil and 1-mehtyl-2-mercaptoimidazole on human thyroid iodine peroxidase. J. Clin. Endocrinol. Metab. 43:152–158.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Nelson K, Schroeder A, Ankley G, Blackwell B, Blanksma C, Degitz S, Flynn K, Jensen K, Johnson R, Kahl M et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part i: Fathead minnow. Aquatic Toxicology. 173:192-203.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Opitz, R., Maquet, E., Zoenen, M., Dadhich, R., Costagliola, S., 2011. TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish. Molecular Endocrinology 25, 1579-1599.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Raldua, D., Babin, P.J., 2009. Simple, Rapid Zebrafish Larva Bioassay for Assessing the Potential of Chemical Pollutants and Drugs to Disrupt Thyroid Gland Function. Environmental Science & Technology 43, 6844-6850.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Rehberger, K., Baumann, L., Hecker, M., Braunbeck, T., 2018. Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption. Fish Physiology and Biochemistry 44, 997-1010.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Ruf, J., and Carayon, P. (2006). Structural and functional aspects of thyroid peroxidase. Archives of biochemistry and biophysics 445(2), 269-77, 10.1016/j.abb.2005.06.023.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stinckens E, Vergauwen L, Blackwell BR, Anldey GT, Villeneuve DL, Knapen D. 2020. Effect of thyroperoxidase and deiodinase inhibition on anterior swim bladder inflation in the zebrafish. Environmental Science & Technology. 54(10):6213-6223.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stinckens E, Vergauwen L, Schroeder A, Maho W, Blackwell B, Witters H, Blust R, Ankley G, Covaci A, Villeneuve D et al. 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part ii: Zebrafish. Aquatic Toxicology. 173:204-217.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Taurog A. 2005. Hormone synthesis. In: Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (Braverman LE, Utiger RD, eds). Philadelphia:Lippincott, Williams and Wilkins, 47–81.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Taurog, A., Dorris, M. L., and Doerge, D. R. (1996). Mechanism of simultaneous iodination and coupling catalyzed by thyroid peroxidase. Archives of biochemistry and biophysics 330(1), 24-32,</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thienpont, B., Tingaud-Sequeira, A., Prats, E., Barata, C., Babin, P.J., Raldua, D., 2011. Zebrafish Eleutheroembryos Provide a Suitable Vertebrate Model for Screening Chemicals that Impair Thyroid Hormone Synthesis. Environmental Science & Technology 45, 7525-7532.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Tietge JE, Butterworth BC, Haselman JT, Holcombe GW, Hornung MW, Korte JJ, Kosian PA, Wolfe M, Degitz SJ. Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis. </span><span style="color:black">Aquat Toxicol. 2010 98(1):44-50</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">van der Ven LTM, van den Brandhof EJ, Vos JH, Power DM, Wester PW. 2006. </span><span style="color:black">Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish. Environmental Science & Technology. 40(1):74-81.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Vickers AE, Heale J, Sinclair JR, Morris S, Rowe JM, Fisher RL. Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways. Toxicol Appl Pharmacol. 2012 260(1):81-8.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Walter, K.M., Miller, G.W., Chen, X.P., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). General and Comparative Endocrinology 272, 20-32.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.</span></span></span></span></p>
<p style="text-align:start"> </p>
<p> </p>
2016-11-29T18:41:332022-11-04T09:27:294a1dd514-9205-4ead-b4b3-a84f409c1b2d4683c179-3444-4ba0-a0fa-1d70a42171e2<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) are synthesized by NIS and TPO in the thyroid gland as iodinated thyroglobulin (Tg) and stored in the colloid of thyroid follicles across vertebrates. Secretion from the follicle into serum is a multi-step process. The first involves thyroid stimulating hormone (TSH) stimulation of the separation of the peptide linkage between Tg and TH. The next steps involve endocytosis of colloid, fusion of the endosome with the basolateral membrane of the thyrocyte, and finally release of TH into blood. More detailed descriptions of this process can be found in reviews by Braverman and Utiger (2012) and Zoeller et al. (2007).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The weight of evidence linking these two KEs of decreased TH synthesis and decreased T4 in serum is strong. It is commonly accepted dogma that decreased synthesis in the thyroid gland will result in decreased circulating TH (serum T4).</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The biological relationship between two KEs in this KER is well understood and documented fact within the scientific community.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">It is widely accepted that TPO inhibition leads to declines in serum T4 levels in adult <strong>mammals</strong>. This is due to the fact that the sole source for circulating T4 derives from hormone synthesis in the thyroid gland. Indeed, it has been known for decades that insufficient dietary iodine will lead to decreased serum TH concentrations due to inadequate synthesis. Strong qualitative and quantitative relationships exist between reduced TH synthesis and reduced serum T4 (Ekerot et al., 2013; Degon et al., 2008; Cooper et al., 1982; 1983; Leonard et al., 2016; Zoeller and Tan, 2007). There is more limited evidence supporting the relationship between decreased TH synthesis and lowered circulating hormone levels during development. Lu and Anderson (1994) followed the time course of TH synthesis, measured as thyroxine secretion rate, in non-treated pregnant rats and correlated it with serum T4 levels. Modeling of TH in the rat fetus demonstrates the quantitative relationship between TH synthesis and serum T4 concentrations (Hassan et al., 2017, 2020; Handa et al., 2021). Furthermore, a wide variety of drugs and chemicals that inhibit TPO are known to result in decreased release of TH from the thyroid gland, as well as decreased circulating TH concentrations. This is evidenced by a very large number of studies that employed a wide variety of techniques, including thyroid gland explant cultures, tracing organification of 131-I and </span><em><span style="color:black">in vivo</span></em><span style="color:black"> treatment of a variety of animal species with known TPO inhibitors (King and May,1984; Atterwill et al., 1990; Brown et al., 1986; Brucker-Davis, 1998; Haselman et al., 2020; Hornung et al., 2010; Hurley et al., 1998; Kohrle, 2008; Tietge et al., 2010).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Additionally, evidence is available from studies investigating responses to TPO inhibitors in <strong>fish</strong>. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. Several other studies have also shown that chemically induced Inhibition of TPO results in reduced TH synthesis in zebrafish (Van der Ven et al., 2006; Raldua and Babin, 2009; Liu et al., 2011; Thienpont et al., 2011; Rehberger et al., 2018). A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Temporal Evidence:</span></em><span style="color:black"> In <strong>mammals</strong>, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). There are currently no studies that measured both TPO synthesis and TH production during development. However, the impact of decreased TH synthesis on serum hormones is similar across all ages in mammals. Good evidence for the temporal relationship comes from thyroid system modeling of the impacts of iodine deficiency and NIS inhibition (e.g., Degon et al., 2008; Fisher et al., 2013). In addition, recovery experiments have demonstrated that serum thyroid hormones recovered in athyroid mice following grafting of in-vitro derived follicles (Antonica et al., 2012).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In <strong><em>Xenopus</em></strong>, it has been shown that depression of TH synthesis in the thyroid gland precedes depression of circulating TH within 7 days of exposure during pro-metamorphosis (Haselman et al., 2020). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In oviparous <strong>fish</strong> such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal </span><span style="color:black">TH</span><span style="color:black">s transferred to the eggs. Embryonic </span><span style="color:black">TH</span> <span style="color:black">synthesis is activated later during embryo-larval development. (See Domain of applicability)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Dose-response Evidence</span></em><span style="color:black">: Dose-response data is lacking from studies that include concurrent measures of both TH synthesis and serum TH concentrations. However, data is available demonstrating correlations between thyroidal TH and serum TH concentrations during gestation and lactation during development (Gilbert et al., 2013). This data was used to develop a rat quantitative biologically-based dose-response model for iodine deficiency (Fisher et al., 2013). In <em>Xenopus</em>, dose-responses were demonstrated in both thyroidal T4 and circulating T4 following exposure to three TPO inhibitors (Haselman et al., 2020).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">There are no inconsistencies in this KER, but there are some uncertainties. The first uncertainty stems from the paucity of data for quantitative modeling of the relationship between the degree of synthesis decrease and resulting changes in circulating T4 concentrations. In addition, most of the data supporting this KER comes from inhibition of TPO, and there are a number of other processes (e.g., endocytosis, lysosomal fusion, basolateral fusion and release) that are not as well studied.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">For example, Kim et al. (2015) investigated the adverse effects of Triphenyl phosphate (TPP), a substance that disrupts the thyroid system. Therefore,<strong> Rat pituitary</strong> (GH3) and <strong>thyroid follicular cell lines</strong> (FRTL-5) were studied. In the GH3 cells, TPP led to an upregulation of the expression of important thyroid genes (tsh</span><span style="color:black">, tr </span><span style="color:black">alpha</span> <span style="color:black">and tr </span><span style="color:black">beta</span><span style="color:black">) while T3, a positive control, downregulated the expression of these genes. In FRTL-5 cells, the expression of nis and tpo genes was significantly upregulated, suggesting that TPP stimulates </span><span style="color:black">TH</span> <span style="color:black">synthesis in the thyroid gland.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In <strong>zebrafish larvae </strong>at the age of 7 days post-fertilisation (dpf), TPP exposure resulted in a significant <strong>increase in T3 and T4</strong> concentrations and the expression of genes involved in thyroid hormone synthesis. Exposure to TPP also significantly regulated the expression of genes involved in the metabolism (dio1), transport (ttr) and excretion (ugt1ab) of </span><span style="color:black">TH</span><span style="color:black">s. The down-regulation of the crh and tsh genes in the zebrafish larvae suggests the activation of a central regulatory feedback mechanism that is triggered by the increased T3 levels in vivo. Taken together, these observations indicate that TPP increases </span><span style="color:black">TH</span> <span style="color:black">concentrations in early life stages of zebrafish by disrupting central regulatory and hormone synthesis pathways.</span></span></span></span></p>
<p> </p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In rats, Hassan et al. (2020) demonstrated</span><em><span style="color:black"> in vitro: ex vivo</span></em><span style="color:black"> correlations of TPO inhibition using PTU and MMI and constructed a quantitative model relating level of TPO inhibition with changes in circulating T4 levels. They determined that 30% inhibition of TPO was sufficient to decrease circulating T4 levels by 20%. This is further supported by studies of Hassan et al. (2017) and Handa et al. (2021)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In <em>Xenopus</em>, Haselman et al. (2020) collected temporal and dose-response data for both thyroidal and circulating T4 which showed strong qualitative concordance of the response-response relationship. A quantitative relationship exists there in, but is yet to be demonstrated mathematically in this species. </span></span></span></span></p>
HighMaleHighFemaleHighAll life stagesHighHighHighHighLowLow<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: This KER is plausibly applicable across vertebrates. While a majority of the empirical evidence comes from work with laboratory rodents, there is a large amount of supporting data from humans (with anti-hyperthyroidism drugs including propylthiouracil and methimazole), some amphibian species (e.g., frog), fish species (e.g., zebrafish and fathead minnow), and some avian species (e.g, chicken). The following are samples from a large literature that supports this concept: Cooper et al. </span><span style="color:black">(1982; 1983); Hornung et al. (2010); Van Herck et al. (2013); Paul et al. (2013); Nelson et al. (2016); Alexander et al. (2017); Stinckens et al. </span><span style="color:black">(2020).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones </span><span style="color:black">(TH) </span><span style="color:black">during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body </span><span style="color:black">TH</span> <span style="color:black"> levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). It is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH </span><span style="color:black">system </span><span style="color:black">disruptors.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Laurberg P, Lazarus JH, Mandel SJ, Peeters RP, Sullivan S. 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017 Mar;27(3):315-389.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Antonica F, Kasprzyk DF, Opitz R, Iacovino M, Liao XH, Dumitrescu AM, Refetoff S, Peremans K, Manto M, Kyba M, Costagliola S. Generation of functional thyroid from embryonic stem cells. Nature. 2012 491(7422):66-71.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Atterwill CK, Fowler KF. A comparison of cultured rat FRTL-5 and porcine thyroid cells for predicting the thyroid toxicity of xenobiotics. Toxicol In Vitro. 1990. 4(4-5):369-74.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Braverman, L.E. and Utiger, R.D. (2012). Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text (10 ed.). Philadelphia, PA: Lippincott Williams & Wilkins. pp. 775-786. ISBN 978-1451120639.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Brown CG, Fowler KL, Nicholls PJ, Atterwill C. Assessment of thyrotoxicity using in vitro cell culture systems. Food Chem Toxicol. 1986 24(6-7):557-62.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Brucker-Davis F. Effects of environmental synthetic chemicals on thyroid function. Thyroid. 1998 8(9):827-56.</span></span></span></span></p>
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<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">van der Ven LTM, van den Brandhof EJ, Vos JH, Power DM, Wester PW. 2006. Effects of the antithyroid agent propylthiouracil in a partial life cycle assay with zebrafish. Environmental Science & Technology. 40(1):74-81.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Van Herck SL, Geysens S, Delbaere J, Darras VM. Regulators of thyroid hormone availability and action in embryonic chicken brain development. Gen Comp Endocrinol. 2013. 190:96-104.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Wabukebunoti MAN, Firling CE. 1983. The prehatching development of the thyroid-gland of the fathead minnow, pimephales-promelas (rafinesque). General and Comparative Endocrinology. 49(2):320-331.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Walter KM, Miller GW, Chen XP, Yaghoobi B, Puschner B, Lein PJ. 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (danio rerio). General and Comparative Endocrinology. 272:20-32.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zoeller, R. T., Tan, S. W., and Tyl, R. W. (2007). General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical reviews in toxicology 37(1-2), 11-53.</span></span></span></span></p>
2016-11-29T18:41:332022-10-10T08:56:384683c179-3444-4ba0-a0fa-1d70a42171e2816f9e81-06db-4e7b-ad0f-06e9dfb09c5d<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">When serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). While some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Since in fish early life stages TH are typically measured on a whole-body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level. Pending more dedicated studies, whole-body TH levels are </span><span style="color:black">often </span><span style="color:black">considered a proxy for serum TH levels.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">When serum thyroxine (T4) levels are decreased, less T4 is available for conversion to the more biologically active triiodothyronine (T3). It is plausible to assume that while some thyroid hormone (TH) disrupting mechanisms can immediately affect T3 levels, including deiodinase inhibition, other mechanisms reduce T4 levels, for example through inhibition of TH synthesis, leading to decreased T3 levels.</span></span></span></span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">A decrease in whole-body T4 and T3 was observed in zebrafish exposed to methimazole from fertilization until the age of 21 and 32 days and to propylthiouracil until the age of 14, 21 and 32 days (Stinckens et al., 2020). Additionally, a strong correlation was observed between T4 and T3 levels. Both compounds are TPO inhibitors expected to inhibit TH synthesis.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">A dose-dependent decrease in whole-body T4 and T3 was observed in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the TH system remain uncertain.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">While T4 measurements could not be acquired in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole, a TPO inhibitor, for 14 days, a significant decrease in T3 was observed (Nelson et al., 2016). The decreased T3 levels were likely the result of reduced T4 synthesis.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Besson et al. (2020) showed both decreased T4 levels and decreased T3 levels in metamorphosing convict surgeonfish exposed to chlorpyrifos.</span></span></li>
</ul>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Since in fish early life stages THs are typically measured on a whole body level, it is currently uncertain whether TH level changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. Examples of studies showing reduced T4 levels in the absence of reduced T3 levels:</span></span>
<ul style="list-style-type:circle">
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Zebrafish exposed to 0.35 mg/L 2-mercaptobenzothiazole, a TPO inhibitor, through 32 dpf showed decreased whole-body T4, but T3 levels showed particularly large variation and overall were not significantly decreased (Stinckens et al., 2016).</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Although T4 content of 28 dpf larval fathead minnows exposed to 32 or 100 µg/l methimazole, a TPO inhibitor, was reduced, these fish showed no change in whole body T3 content (Crane et al., 2006). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole</span></span></li>
</ul>
</li>
</ul>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stinckens et al. (2020, supplementary information) showed a significant linear relationship between whole body T3 and T4 concentrations at 21 and 32 days post fertilization after continuous exposure of zebrafish to methimazole and propylthiouracil, two inhibitors of TH synthesis.</span></span></span></span></p>
ModerateUnspecificModerateJuvenileModerateLarvaeHighModerate<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: Thyroid follicles mainly produce T4 and to a lesser extent T3 across vertebrates. When serum T4 levels are decreased, less T4 is available for conversion to the more biologically active T3. This key event relationship is not always evident. This could be due to feedback/compensatory mechanisms that in some cases seem to be able to maintain T3 levels even though T4 levels are reduced, for example through increased conversion of T4 to T3 by deiodinases. These feedback mechanisms can also differ across species. Therefore, although this KER is plausibly applicable across vertebrates, variation can be expected. In zebrafish and fathead minnow, several studies reported</span> <span style="color:black">evidence for a relationship between </span><span style="color:black">whole body </span><span style="color:black">T4 and T3 levels (Nelson et al., 2016; Stinckens et al., 2020, Wang et al., 2020).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: This key event relationship is applicable to late larvae and juveniles rather than to embryos, because of the presence of maternal TH in embryos.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Uncertainties during embryonic life</span> <span style="color:black">stage:</span></span></span></span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">A decrease in whole body T4 was observed in fathead minnows exposed to 1 mg/L 2-mercaptobenzothiazole (MBT), a TPO inhibitor, until 6 dpf (Nelson et al., 2016). In contrast, there was no observed effect on T3 in fathead minnows exposed to MBT until 6 dpf. Comparably, zebrafish exposed to 0.4 or 0.7 mg/L MBT thruntilough 120 hpf showed decreased whole body T4 but not T3 (Stinckens et al., 2016). During this early larval life stage, T3 may have been derived from maternal T4. In addition, it could be produced from further depletion of any T4 still produced by the thyroid gland (as TPO may not have been fully inhibited at the tested exposure concentrations).</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Since exposure to PFAS did result in decreased whole-body T4 and T3 in 5 day old zebrafish, the life-stage specificity possibly depends on the mechanism that lies at the basis of the TH changes (Wang et al., 2020). The exact mechanisms by which PFAS disrupt the TH system remain uncertain. Compounds that directly reduce T3 levels (e.g., deiodinase inhibitors) in addition to reducing T4 levels via another mechanism can be expected to result in decreased T4 and T3 levels.</span></span></li>
</ul>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex: </span></strong><span style="color:black">The KE is plausibly applicable to both sexes. Thyroid hormones are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of thyroid hormone levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in thyroid hormone levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Besson, M., Feeney, W. E., Moniz, I., François, L., Brooker, R. M., Holzer, G., Metian, M., Roux, N., Laudet, V., & Lecchini, D. (2020). Anthropogenic stressors impact fish sensory development and survival via thyroid disruption. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17450-8</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2006. The effects of methimazole on development of the fathead minnow, pimephales promelas, from embryo to adult. Toxicological Sciences 93, 278-285.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Hernandez-Mariano JA, Torres-Sanchez L, Bassol-Mayagoitia S, Escamilla-Nunez M, Cebrian ME, Villeda-Gutierrez EA, Lopez-Rodriguez G, Felix-Arellano EE, Blanco-Munoz J. 2017. Effect of exposure to p,p '-dde during the first half of pregnancy in the maternal thyroid profile of female residents in a mexican floriculture area. Environmental Research. 156:597-604.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Liu XS, Cai Y, Wang Y, Xu SH, Ji K, Choi K. 2019. Effects of tris(1,3-dichloro-2-propyl) phosphate (tdcpp) and triphenyl phosphate (tpp) on sex-dependent alterations of thyroid hormones in adult zebrafish. Ecotoxicology and Environmental Safety. 170:25-32.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Wang, J.X., Shi, G.H., Yao, J.Z., Sheng, N., Cui, R.N., Su, Z.B., Guo, Y., Dai, J.Y., 2020. Perfluoropolyether carboxylic acids (novel alternatives to PFOA) impair zebrafish posterior swim bladder development via thyroid hormone disruption. Environment International 134.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Webster GM, Venners SA, Mattman A, Martin JW. 2014. Associations between perfluoroalkyl acids (pfass) and maternal thyroid hormones in early pregnancy: A population-based cohort study. Environmental Research. 133:338-347.</span></span></span></span></p>
2020-02-19T09:39:462022-10-10T09:02:24816f9e81-06db-4e7b-ad0f-06e9dfb09c5de94bcd3a-949c-4f3e-81a9-7706f62cb58b<p>Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition and larval-to-juvenile transition, including anterior chamber inflation in fish. Reduced T3 levels prohibit local TH action in the target tissues. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired anterior chamber inflation.</p>
<p>There is convincing evidence that decreased T3 levels result in impaired anterior chamber inflation, but the underlying mechanisms are not completely understood. A very convincing linear quantitative relationship between reduced T3 levels and reduced anterior chamber volume was shown in zebrafish across exposure to a limited set of three compounds. Therefore the evidence supporting this KER can be considered moderate.</p>
<p>Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. Inflation of the anterior swim bladder chamber is part of the larval-to-juvenile transition in fish, together with the development of adult fins and fin rays, ossification of the axial skeleton, formation of an adult pigmentation pattern, scale formation, maturation and remodelling of organs including the lateral line, nervous system, gut and kidneys (Brown, 1997; Liu and Chan, 2002; McMenamin and Parichy, 2013).</p>
<p>Dedicated studies with two different experimental setups have been conducted to investigate the link between reduced T3 levels and reduced anterior chamber inflation:</p>
<p>1. Studies applying larval exposures initiated after posterior chamber inflation</p>
<ul>
<li>In a study in which larval fathead minnows (<em>Pimephales promelas</em>) were exposed to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole (MBT), T3 concentrations measured at 14dpf were reduced at the same concentration (1 mg/L) that significantly reduced anterior swim bladder inflation at the same time-point (Nelson et al. 2016).</li>
<li>In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed pronounced decreases of whole-body T3 concentrations and increases of whole-body T4 concentrations, together with impaired inflation of the anterior swim bladder chamber. More specifically, inflation was delayed and the size of the swim bladder chamber was reduced until the end of the exposure experiment.</li>
</ul>
<p>Since exposures were started after inflation of the posterior chamber, these studies show that DIO inhibition can directly affect anterior chamber inflation.</p>
<p>2. Studies applying continuous exposure initiated immediately after fertilization and thus including both posterior and anterior chamber inflation</p>
<ul>
<li>In the study of Stinckens et al. (2020) exposure concentrations were chosen where the posterior chamber inflates. A strong correlation between reduced T3 levels and reduced anterior chamber inflation was observed in zebrafish exposed to iopanoic acid, a deiodinase inhibitor, as well as methimazole and propylthiouracil, both thyroperoxidase inhibitors, from fertilization until the age of 32 days. Anterior chamber inflation was delayed and a number of larvae did not manage to inflate the anterior chamber by the end of the 32 day exposure period. Additionally, exposed fish that had inflated the swim bladder had reduced anterior chamber sizes.</li>
</ul>
<ul>
<li>Since in fish early life stages THs are typically measured on a whole-body level, it is currently uncertain whether TH levels changes occur at the serum and/or tissue level.</li>
<li>The mechanism underlying the link between reduced T3 and reduced anterior chamber inflation remains unclear, but several hypotheses exist (Stinckens et al., 2020). For example, altered gas distribution between chambers could be the result of impaired development of smooth muscle fibers, delayed and/or impaired evagination of the anterior chamber, impaired anterior budding through altered Wnt and hedgehog signalling, etc. Reinwald et al. (2021) showed that T3 and propylthiouracil treatment of zebrafish embryos altered expression of genes involved in muscle contraction and functioning in an opposing fashion. The authors suggested impaired muscle function as an additional key event between decreased T3 levels and reduced swim bladder inflation.</li>
<li>Increased T3 levels also seem to result in reduced swim bladder inflation. For example, Li et al. (2011) reported impairment of swim bladder inflation in Chinese rare minnows (Gobiocypris rarus) exposed to exogenous T3.</li>
</ul>
<p>Stinckens et al. (2020, supplementary information) established a very convincing linear quantitative relationship between reduced T3 levels and reduced anterior chamber volume (measured as surface in 2D images) in 32 day old zebrafish across three compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis on the one hand, and iopanoic acid, a deiodinase inhibitor, on the other hand.</p>
ModerateUnspecificHighLarvaeModerateHigh<p><strong>Taxonomic</strong>: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass, medaka) and physostomous (e.g., zebrafish and fathead minnow). Physostomous fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2016; Nelson et al., 2016; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020). While zebrafish and fathead minnows are physostomous fish with a two-chambered swim bladder, the Japanese rice fish or medaka (Oryzias latipes) is a physoclistous fish with a single chambered swim bladder that inflates during early development. This KER is not applicable to such fish species. Therefore, the current key event is plausibly applicable to physostomous fish in general.</p>
<p><strong>Life stage</strong>: The anterior chamber inflates during a specific developmental time frame. In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KER is only applicable to the larval life stage.</p>
<p><strong>Sex</strong>: This KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KER. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 21 days post fertilization in zebrafish, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current KER.</p>
<p>Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.</p>
<p>Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.</p>
<p>Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.</p>
<p>Li W, Zha J, Yang L, Li Z, Wang Z. Regulation of thyroid hormone related genes mRNA expression by exogenous T₃ in larvae and adult Chinese rare minnow (Gobiocypris rarus). Environ Toxicol Pharmacol. 2011 Jan;31(1):189-97. doi: 10.1016/j.etap.2010.10.007.</p>
<p>Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.</p>
<p>McMenamin, S.K., Parichy, D.M., 2013. Metamorphosis in Teleosts. Animal Metamorphosis 103, 127-165.</p>
<p>Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.</p>
<p>Nelson KR, Schroeder AL, Ankley GT, Blackwell BR, Blanksma C, Degitz SJ, Flynn KM, Jensen KM, Johnson RD, Kahl MD, Knapen D, Kosian PA, Milsk RY, Randolph EC,Saari T, Stinckens E, Vergauwen L, Villeneuve DL. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquat Toxicol. 2016 Apr;173:192-203. doi: 10.1016/j.aquatox.2015.12.024.</p>
<p>Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.</p>
<p>Reinwald H, Konig A, Ayobahan SU, Alvincz J, Sipos L, Gockener B, Bohle G, Shomroni O, Hollert H, Salinas G et al. 2021. Toxicogenomic fin(ger)prints for thyroid disruption aop refinement and biomarker identification in zebrafish embryos. Science of the Total Environment. 760.</p>
<p>Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</p>
<p>Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.</p>
<p>Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2002. Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. Journal of Experimental Biology 205, 711-718.</p>
<p>van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.Zeng FX, Sherry JP, Bols NC. 2016. Evaluating the toxic potential of benzothiazoles with the rainbow trout cell lines, rtgill-w1 and rtl-w1. Chemosphere. 155:308-318.</p>
2016-11-29T18:41:362022-10-07T09:44:35e94bcd3a-949c-4f3e-81a9-7706f62cb58badebe552-5f5f-4c64-889b-543acb4dbb97<p>Effects on swim bladder inflation can alter swimming performance and buoyancy of fish, which is essential for predator avoidance, energy sparing, migration, reproduction and feeding behaviour, resulting in increased mortality.</p>
<p>The weight of evidence supporting a direct linkage between these two KEs, i.e. reduced anterior swim bladder inflation and reduced swimming performance, is weak.</p>
<p>The anterior chamber of the swim bladder has a function in regulating the buoyancy of fish, by altering the volume of the swim bladder (Roberston et al., 2007). Fish rely on the lipid and gas content in their body to regulate their position within the water column, with the latter being more efficient at increasing body buoyancy. Therefore, fish with functional swim bladders have no problem supporting their body (Brix 2002), while it is highly likely that impaired inflation severely impacts swimming performance. Fish with no functional swim bladder can survive, but are severely disadvantaged, making the likelihood of surviving smaller.</p>
<p>Several studies in zebrafish and fathead minnow showed that a smaller AC was associated with a larger posterior chamber (Nelson et al., 2016; Stinckens et al., 2016; Cavallin et al., 2017, Stinckens et al., 2020) suggesting a possible compensatory mechanism. As shown by Stoyek et al. (2011) however, the AC volume is highly dynamic under normal conditions due to a series of regular corrugations running along the chamber wall, and is in fact the main driver for adjusting buoyancy while the basic PC volume remains largely invariable. Therefore, it is plausible to assume that functionality of the swim bladder is affected when AC inflation is incomplete, even when the PC appears to fully compensate the gas volume of the swim bladder.</p>
<ul>
<li>Lindsey et al. (2010) showed that zebrafish started swimming deeper down in the water column upon inflation of the anterior chamber, confirming a role of the anterior chamber in supporting swimming performance.</li>
<li>After exposure to 2-mercaptobenzothiazole, a TPO inhibitor, from 0 to 32 days post fertilization (dpf) in zebrafish, the swimming activity of fish was impacted starting at 26 dpf if the inflation of the anterior chamber of the swim bladder was impaired or had no normal structure/size (Stinckens et al., 2016).</li>
<li>Methimazole (MMI) and propylthiouracil (PTU), two thyroperoxidase inhibitors, and iopanoic acid (IOP), a deiodinase inhibitor, each reduced both anterior chamber inflation and swimming distance in zebrafish exposed from fertlization until the age of 32 days (Stinckens et al., 2020). Stinckens et al. (2020) showed a specific, direct link between reduced anterior chamber inflation and reduced swimming performance.
<ul>
<li>First, after 21 d of exposure to 111 mg/L propylthiouracil around 30% of anterior chambers were not inflated and swimming distance was reduced, while by 32 days post fertilization all larvae had inflated their anterior chamber (although chamber surface was still smaller) and the effect on swimming distance had disappeared.</li>
<li>The most direct way to assess the role of anterior chamber inflation in swimming performance, however, is to compare larvae with and without inflated anterior chamber at the same time point and within the same experimental treatment. Both in the propylthiouracil exposure at 21 days post fertilization and in the iopanoic acid exposure at 21 and 32 days post fertilization, swimming distance was clearly reduced in larvae lacking an inflated anterior chamber, while the swimming distance of larvae with inflated anterior chamber was equal to that of controls.</li>
<li>Exposure concentrations were selected where the posterior chamber inflates. Even though the posterior chamber was generally larger when anterior chamber inflation was reduced, this did not remove the effect on swimming performance, confirming a direct link between proper anterior chamber inflation and swimming performance.</li>
<li>No morphological effects were observed, but in some treatments reduced length and/or condition factor was observed. However, reduced swimming performance after 32 days of IOP exposure to medium concentrations was not accompanied by reduced length or condition factor. Therefore, at least in this study no evidence was found that the effect on swimming performance was an indirect consequence of effects other than reduced swim bladder inflation.</li>
</ul>
</li>
<li>It has also been reported that larvae that fail to inflate their swim bladder use additional energy to maintain buoyancy (Lindsey et al., 2010, Goodsell et al. 1996), possibly contributing to reduced swimming activity. </li>
</ul>
<p>After exposure to 100 mg/L methimazole, 95% of the zebrafish larvae failed to inflate their anterior chamber at 32 dpf and swimming distance was reduced (Stinckens et al., 2020). On the other hand, there was no effect of impaired anterior chamber inflation on swimming distance in the methimazole exposure of 50 mg/L. Also, inflated but smaller anterior chambers did not result in a decreased swimming performance in this study. A similar result, where non-inflated anterior chambers did not consistently lead to reduced swimming performance, was previously found after exposure to 2-mercaptobenzothiazole (Stinckens et al., 2016). In summary, the precise relationship between these two KEs is not easy to determine and may be different for different chemicals. This is in part due to the complexity of the swim bladder system and the difficulty of distinguishing effects resulting from altered anterior chamber inflation from those resulting from altered posterior chamber inflation. Additionally, swimming capacity can be affected via other processes which may or may not depend on the HPT axis, such as general malformations, decreased cardiorespiratory function, energy metabolism and growth.</p>
<p>As Robertson et al., (2007) reported, the swim bladder only starts regulating buoyancy actively from 32 dpf onward in zebrafish, possibly explaining the lack of effect on swimming capacity in some cases.</p>
<p>The anterior chamber is also important for producing and transducing sound through the Weberian Apparatus (Popper, 1974; Lechner and Ladich, 2008). It is highly plausible that impaired inflation or size of the anterior swim bladder could lead to increased mortality as hearing loss would affect their ability to respond to their surrounding environment, thus impacting ecological relevant endpoints such as predator avoidance or prey seeking (Wisenden et al., 2008; Fay, 2009).</p>
<p> </p>
<p> </p>
<p>The direct quantitative evidence supporting the connection between anterior chamber impairment and swimming capacity is lacking.</p>
ModerateUnspecificHighLarvaeHighLow<p><strong>Taxonomic</strong>: Importance of proper functioning of the swim bladder for supporting natural swimming behaviour can be plausibly assumed to be generally applicable to fish possessing an anterior chamber. Evidence exists for the role of the posterior chamber in swimming performance comes from a wide variety of freshwater and marine fish species. Evidence for the specific role of the anterior chamber is however less abundant.</p>
<p><strong>Life stage</strong>: In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KER is only applicable to the larval life stage. To what extent fish can survive and swim with partly inflated swim bladders during later life stages is unknown.</p>
<p><strong>Sex</strong>: This KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KER. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 21 days post fertilization in zebrafish, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current KER.</p>
<p>Brix O (2002) The physiology of living in water. In: Hart PJ, Reynolds J (eds) Handbook of Fish Biology and Fisheries, Vol. 1, pp. 70–96. Blackwell Publishing, Malden, USA.</p>
<p>Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.</p>
<p>Czesny, S.J., Graeb, B.D.S., Dettmersn, J.M., 2005. Ecological consequences of swimbladder noninflation for larval yellow perch. Trans. Am. Fish. Soc. 134,1011–1020, http://dx.doi.org/10.1577/T04-016.1.</p>
<p>Fay, R., 2009. Soundscapes and the sense of hearing of fishes. Integrative Zool. 4,26–32.</p>
<p>Goodsell, D.S., Morris, G.M., Olsen, A.J. 1996. Automated docking of fleixble ligands. Applications of Autodock. J. Mol. Recogonition, 9:1-5.</p>
<p>Lechner, W., Ladich, F., 2008. Size matters: diversity in swimbladders andWeberian ossicles affects hearing in catfishes. J. Exp. Biol. 211, 1681–1689.</p>
<p>Lindsey, B.W., Smith, F.M., Croll, R.P., 2010. From inflation to flotation: contribution of the swimbladder to whole-body density and swimming depth during development of the zebrafish (Danio rerio). Zebrafish 7, 85–96, http://dx.doi.org/10.1089/zeb.2009.0616.</p>
<p>Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.</p>
<p>Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.</p>
<p>Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.</p>
<p>Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.</p>
<p>Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007.Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.</p>
<p>Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</p>
<p>Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.</p>
<p>Stoyek, M.R., Smith, F.M., Croll, R.P., 2011. Effects of altered ambient pressure on the volume and distribution of gas within the swimbladder of the adult zebrafish, Danio rerio. Journal of Experimental Biology 214, 2962-2972.</p>
<p>van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.</p>
<p>Wisenden, B.D., Pogatschnik, J., Gibson, D., Bonacci, L., Schumacher, A., Willet, A.,2008. Sound the alarm: learned association of predation risk with novelauditory stimuli by fathead minnows (Pimephales promelas) and glowlighttetras (Hemigrammus erythrozonus) after single simultaneous pairings withconspecific chemical alarm cues. Environ. Biol. Fish 81, 141–147.</p>
<p>Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to theculture of marine finfish larvae. Rev. Aquac. 2, 181–190, http://dx.doi.org/10.1111/j.1753-5131.2010.01035.x.</p>
2016-11-29T18:41:362022-10-07T09:48:25adebe552-5f5f-4c64-889b-543acb4dbb976f971661-f4a8-4ebe-96f4-7e0ba97f17f6<p>Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction in taxa that rely on swimming to support these vital behaviours. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.</p>
<p>A direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (<a href="https://aopwiki.org/relationships/1041">https://aopwiki.org/relationships/2213</a>), which can be plausibly assumed to be related to reduced swimming performance.</p>
<p>For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.</p>
<p>Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.</p>
<p>A direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (see non-adjacent KER 1041), which can be plausibly assumed to be related to reduced swimming performance.</p>
<p>For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.</p>
<p>A direct relationship between reduced swimming performance and reduced survival is difficult to establish in a laboratory environment where food is abundant and there are no predators.</p>
<p>Quantitative understanding of this linkage is currently limited.</p>
ModerateUnspecificModerateAdultModerateJuvenileModerateLarvaeModerateModerate<p>Importance of swimming performance on survival is generally applicable to all hatched fish across life stages and sexes and to other taxa that rely on swimming to support vital behaviours.</p>
<p><span style="font-family:calibri,sans-serif; font-size:11.0pt">Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</span></p>
2020-11-30T06:19:022021-09-08T10:05:176f971661-f4a8-4ebe-96f4-7e0ba97f17f680e93738-bca7-472d-a2fd-a3980b8388b7<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased mortality in the reproductive population may lead to a declining population. This depends on the excess mortality due to the applied stressor and the environmental parameters such as food availability and predation rate. Most fish species are r-strategist, meaning they produce a lot of offspring instead of investing in parental care. This results in natural high larval mortality causing only a small percentage of the larvae to survive to maturity. If the excess larval mortality due to a stressor is small, the population dynamics might result in constant population size. Should the larval excess be more significant, or last on the long-term, this will affect the population. To calculate the long-term persistence of the population, population dynamic models should be used.</span></span></span></span></p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Survival rate is an obvious determinant of population size and is therefore included in population modeling (e.g., Miller et al., 2020).</span></span></span></span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Survival to reproductive maturity is a parameter of demographic significance. Assuming resource availability (i.e., food, habitat, etc.) is not limiting to the extant population, sufficient mortality in the reproductive population may ultimately lead to declining population trajectories.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Under some conditions, reduced larval survival may be compensated by reduced predation and increased food availability, and therefore not result in population decline (Stige et al., 2019).</span></span></li>
</ul>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">According to empirical data, combined with population dynamic models, feeding larvae are the crucial life stage in zebrafish (and other r-strategists) for the regulation of the population. (Schäfers et al., 1993)</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">In fathead minnow, natural survival of early life stages has been found to be highly variable and influential on population growth (Miller and Ankley, 2004)</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Rearick et al. (2018) used data from behavioural assays linked to survival trials and applied a modelling approach to quantify changes in antipredator escape performance of larval fathead minnows in order to predict changes in population abundance. This work was done in the context of exposure to an environmental oestrogen. Exposed fish had delayed response times and slower escape speeds, and were more susceptible to predation. Population modelling showed that this can result in population decline.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">In the context of fishing and fisheries, ample evidence of a link between increased mortality and a decrease of population size has been given. Important insights can result from the investigation of optimum modes of fishing that allow for maintaining a population (Alekseeva and Rudenko, 2018). Jacobsen and Essington (2018) showed the impact of varying predation mortality on forage fish populations.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Boreman (1997) reviewed methods for comparing the population-level effects of mortality in fish populations induced by pollution or fishing.</span></span></li>
</ul>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">The extent to which larval mortality affects population size could depend on the fraction of surplus mortality compared to a natural situation.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">There are scenarios in which individual mortality may not lead to declining population size. These include instances where populations are limited by the availability of habitat and food resources, which can be replenished through immigration. Effects of mortality in the larvae can be compensated by reduced competition for resources (Stige et al., 2019).</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">The direct impact of pesticides on migration behavior can be difficult to track in the field, and documentation of mortality during migration is likely underestimated (Eng 2017).</span></span></span></li>
</ul>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Assuming other relevant demographic parameters are available, the effect of increased mortality rates on population status can be quantitatively predicted using standard population modeling approaches.</span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Stage population matrix models (Caswell, 2000) simulate population growth rates based on age-specific parameters and can be adapted to a range of species (Pinceel et al., 2016). For zebrafish, individually based models (IBM) have been developed to link responses at the individual level to the population level (Beaudouin et al., 2015). However, authors agree that survival is one of the most uncertain parameters in the model and more research on the topic is needed.</span></span></li>
</ul>
ModerateUnspecificHighAll life stagesHighHigh<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: All organisms must survive to reproductive age in order to reproduce and sustain populations. The additional considerations related to survival made above are applicable to other fish species in addition to zebrafish and fathead minnows with the same reproductive strategy (r-strategist as described in the theory of MaxArthur and Wilson (1967). The impact of reduced survival on population size is even greater for k-strategists that invest more energy in a lower number of offspring.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Density dependent effects start to play a role in the larval stage of fish when free-feeding starts (Hazlerigg et al., 2014).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: This linkage is independent of sex.</span></span></span></span></p>
<p> </p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Alekseeva SM, Rudenko AI. 2018. Modeling of optimum fishing population. Marine Intellectual Technologies. 3(4):142-146.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Beaudouin, R., Goussen, B., Piccini, B., Augustine, S., Devillers, J., Brion, F., Pery, A.R., 2015. An individual-based model of zebrafish population dynamics accounting for energy dynamics. PloS one 10, e0125841.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Boreman J. 1997. Methods for comparing the impacts of pollution and fishing on fish populations. Transactions of the American Fisheries Society. 126(3):506-513.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Caswell, H., 2000. Matrix population models. Sinauer Sunderland, MA, USA.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Eng, M.L., Stutchbury, B.J.M. & Morrissey, C.A. Imidacloprid and chlorpyrifos insecticides impair migratory ability in a seed-eating songbird. Sci Rep 7, 15176 (2017)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Hazlerigg, C.R., Lorenzen, K., Thorbek, P., Wheeler, J.R., Tyler, C.R., 2012. Density-dependent processes in the life history of fishes: evidence from laboratory populations of zebrafish Danio rerio. PLoS One 7, e37550.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Jacobsen NS, Essington TE. 2018. Natural mortality augments population fluctuations of forage fish. Fish and Fisheries. 19(5):791-797.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">MacArthur, R., Wilson, E., 1967. The Theory of Island Biogeography. Princeton: Princeton Univ. Press. 203 p.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Miller, D.H., Ankley, G.T., 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17β-trenbolone as a case study. Ecotoxicology and Environmental Safety 59, 1-9.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Miller, D.H., Clark, B.W., Nacci, D.E. 2020. A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations. Ecotoxicology and environmental safety 201, 110786</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Pinceel, T., Vanschoenwinkel, B., Brendonck, L., Buschke, F., 2016. Modelling the sensitivity of life history traits to climate change in a temporary pool crustacean. Scientific reports 6, 29451.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Rearick, D.C., Ward, J., Venturelli, P., Schoenfuss, H., 2018. Environmental oestrogens cause predation-induced population decline in a freshwater fish. Royal Society open science 5, 181065.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Schäfers, C., Oertel, D., Nagel, R., 1993. Effects of 3, 4-dichloroaniline on fish populations with differing strategies of reproduction. In: Braunbeck, T. , Hanke, W and Segner, H. (eds) Ecotoxicology and Ecophysiology, VCH, Weinheim, 133-146.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, Ø., Durant, J.M., 2019. Density‐and size‐dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.Hazlerigg, C.R.E., Tyler, C.R., Lorenzen, K., Wheeler, J.R., Thorbek, P., 2014. Population relevance of toxicant mediated changes in sex ratio in fish: An assessment using an individual-based zebrafish (Danio rerio) model. Ecological Modelling 280, 76-88.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, O., Durant, J.M., 2019. Density- and size-dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.</span></span></span></span></p>
<p> </p>
2019-12-20T16:07:122022-07-08T08:29:35b70ffda9-114a-41b3-b8d5-8aedd63956614683c179-3444-4ba0-a0fa-1d70a42171e2<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thyroperoxidase (TPO) is the enzyme that catalyzes iodine organification of thyroglobulin to produce thyroglobulin (Tg)-bound T3 and T4 in the lumen of thyroid follicles. Tg-bound THs are endocytosed across the apical lumen-follicular cell membrane, undergo thyroglobulin proteolysis, followed by hormone section into the blood stream (see Taurog, 2005 for review). This indirect KER describes the relationship of TPO inhibition to reduced circulating levels of thyroid hormone (TH) in the serum.</span></span></span></span></p>
<p>The weight of evidence linking thyroperoxidase inhibition to reductions in circulating serum TH is strong. Many studies support this basic linkage. There is no inconsistent data.</p>
<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">It is a well-accepted fact that inhibition of the only enzyme capable of synthesizing THs, TPO, results in subsequent decrease in serum TH concentrations. A large amount of evidence from clinical and animal studies clearly support the commonly accepted dogma that inhibition of TPO leads to decreased serum THs. </span></span></span></span><span style="font-size:14px"> </span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The majority of research in support of this KER involve</span><span style="color:black">s</span><span style="color:black"> exposure to known TPO inhibitors and measurement of serum hormones. There are many in vivo studies that link decreases in serum TH concentrations with exposure to xenobiotics that inhibit thyroperoxidase (TPO) in <strong>mammals </strong>(Brucker-Davis, 1998; Hurley, 1998; Boas et al., 2006; Crofton, 2008; Kohrle, 2008; Pearce and Braverman, 2009; Murk et al., 2013). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">While these studies support the connection between exposure to a known TPO inhibitor and decreased TH, many of these studies do not empirically measure TPO inhibition or decreased TH synthesis. Thus, many studies support the indirect linkage between TPO inhibition (for chemicals identified as TPO inhibitors in in vivo or ex vivo studies) and decreased TH, with the well accepted theory that these proceed via decreased TH synthesis. That exposure to TPO inhibitors leads to decreased serum TH concentrations, via decreased TH synthesis is strongly supported by decades of mechanistic research in a variety of species.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This indirect relationship is also evidenced by the use of clinically-relevant anti-hyperthyroidism drugs, MMI and PTU (Laurberg & Anderson, 2014; Sundaresh et al., 2013). These drugs are both recognized TPO inhibitors and are part of a standard drug-based regimen of care for clinically hyperthyroid patients including those with Grave's disease. Serum THs are measured as the bioindicator of successful treatment with anti-hyperthyroidism drugs; the actual decrease in TH synthesis in the thyroid gland is implied in the efficacious use of these drugs (Trepanier, 2006).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In <strong>rats</strong>, MMI and PTU are often used as control chemicals to decrease serum THs to study biological phenomena related to disruption of TH homeostasis (many examples, including Zoeller and Crofton, 2005; Morreale de Escobar et al, 2004; Schwartz et al., 1997; Herwig et al., 2014; Wu et al., 2013; Pathak et al., 2011). Further, MMI is recommended as a positive control for use in the <strong>Amphibian </strong>Metamorphosis (Frog) Assay within Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program (US EPA, 2009; Coady et al., 2010), an assay used to evaluate the potential for chemicals to disrupt TH homeostasis. PTU has been suggested as positive control chemical in the guidance for the Comparative Developmental Thyroid Assay (US EPA, 2005), a non-guideline assay used to evaluate the potential for chemicals to disrupt TH homeostasis during gestation and early neonatal development.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Additionally, evidence is available from studies investigating responses to TPO inhibitors in <strong>fish</strong>. For example, Stinckens et al. (2020) showed reduced whole body T4 concentrations in zebrafish larvae exposed to 50 or 100 mg/L methimazole, a potent TPO inhibitor, from immediately after fertilization until 21 or 32 days of age. Exposure to 37 or 111 mg/L propylthiouracil also reduced T4 levels after exposure up to 14, 21 and 32 days in the same study. Walter et al. (2019) showed that propylthiouracil had no effect on T4 levels in 24h old zebrafish, but decreased T4 levels of 72h old zebrafish. This difference is probably due to the onset of embryonic TH production between the age of 24 and 72 hours (Opitz et al., 2011). Stinckens et al. (2016) showed that exposure to 2-mercaptobenzothiazole (MBT), an environmentally relevant TPO inhibitor, decreased whole body T4 levels in continuously exposed 5 and 32 day old zebrafish larvae. A high concentration of MBT also decreased whole body T4 levels in 6 day old fathead minnows, but recovery was observed at the age of 21 days although the fish were kept in the exposure medium (Nelson et al., 2016). Crane et al. (2006) showed decreased T4 levels in 28 day old fathead minnows continuously exposed to 32 or 100 µg/L methimazole.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Thus, an indirect key event relationship between TPO inhibition and decreased serum THs is strongly supported by a large database of clinical medicine and investigative research with whole animals (with a great deal of supporting evidence in rats and frogs).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Temporal Evidence:</span></em><span style="color:black"> In <strong>mammals</strong>, the temporal nature of this KER is applicable to all life stages, including development (Seed et al., 2005). The qualitative impact of </span><span style="color:black">TPO</span> <span style="color:black">inhibition on serum hormones is similar across all ages in mammals. The temporal nature of the impact on serum THs by TPO inhibitors in developmental exposure studies is evidenced by the duration of exposure and developmental age (Goldey et al., 1995; Ahmed et al., 2010; Tietge et al., 2010), as well as recovery after cessation of exposure (Cooke et al., 1993; Goldey et al., 1995; Sawin et al., 1998; Axelstad et al., 2008; Shibutani et al., 2009; Lasley and Gilbert, 2011). The temporal relationship between TPO inhibitor exposure duration and serum hormone decreases in adult organisms has been widely demonstrated (e.g., Hood et al., 1999; Mannisto et al., 1979). In addition, MMI and PTU induced decreases in serum T4 are alleviated by </span><span style="color:black">TH</span> <span style="color:black"> replacement in both fetal and postnatal age rats (Calvo et al., 1990; Sack et al., 1995; Goldey and Crofton, 1998). Computational modeling of the thyroid also provides evidence for the indirect temporal relationship between these two KEs (e.g., Degon et al., 2008; Fisher et a., 2013).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">In oviparous <strong>fish </strong>such as zebrafish and fathead minnow, the nature of this KER depends on the life stage since the earliest stages of embryonic development rely on maternal thyroid hormones transferred to the eggs. Embryonic thyroid hormone synthesis is activated later during embryo-larval development. (See Domain of applicability)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><em><span style="color:black">Dose-Response Evidence: </span></em><span style="color:black">Empirical data is available from enough studies in animals treated with TPO inhibitors during development to make it readily accepted dogma that a dose-response relationship exists between TPO inhibition and serum TH concentrations. Again, these studies do not empirically measure TPO inhibition or decreased TH synthesis, but rely on the strong support of decades of mechanistic research in a variety of species of the causative relationship between these KEs. Examples of dose-responsive changes in TH concentrations following developmental exposure to TPO inhibitors include studies a variety of species, including: rodents (Blake and Henning, 1985; Goldey et al., 1995; Sawin et al., 1998); frogs (Tietge et al., 2013); fish tissue levels (Elsalini and Rohr, 2003.); and, chickens (Wishe et al., 1979). Computational modeling of the thyroid also provides evidence for the indirect dose-response relationship between these two KEs (e.g., Leonard et al., 2016; Fisher et a., 2013).</span></span></span></span></p>
<p>There are no inconsistencies in this KER, but there are some uncertainties. The predominant uncertainty regarding the indirect key event relationship between inhibition of TPO activity and decreased serum T4 is the quantitative nature of this relationship, i.e., to what degree must TPO be inhibited in order to decrease serum T4 by a certain magnitude. Many animal (rat) studies typically employ relatively high exposures of TPO-inhibiting chemicals that result in hypothyroidism (severe decrements in T4 and T3). Thus, a dose-response relationship between TPO inhibition and decreased serum T4 is not typically defined. However, there are numerous publications demonstrating clear dose- and duration- dependent relationships between TPO inhibitors dose and reduced serum T3 and T4 in rodent models (see for example: Cooper et al., 1983; Hood et al., 1999; Goldey et al., 2005; Gilbert, 2011). The relationship between maternal and fetal levels of hormone following chemically-induced TPO inhibiton has not been well characterized and may differ based on kinetics. Reductions in serum TH in the fetus, in rats and humans is derived from a chemical's effect on the maternal thyroid gland as well as the fetal thyroid gland.</p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Hassan et al. (2017, 2020) and Handa et al. (2021) worked out a quantitative model to predict serum T4 levels based on TPO inhibition in rats.</span></span></span></span></p>
HighMaleHighFemaleHighAll life stagesHighHighModerateHighHighModerate<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Taxonomic</span></strong><span style="color:black">: Use of TPO inhibitors as anti-hyperthyroidism drugs in humans and pets (Emiliano et al., 2010; Trepanier, 2006) and effects of these drugs on serum TH concentrations in rats (US EPA, 2005), amphibian, fish and avian species (Coady et al., 2010; Grommen et al., 2011; Nelson et al., 2016; Rosebrough et al., 2006; Stinckens et al.; 2020; Tietge et al., 2012), strongly supports a causative linkage between inhibition of TPO and decreased serum T4 across species. Therefore, this KER is plausibly applicable across vertebrate species. Therefore, this KER is plausibly applicable across vertebrates.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Life stage</span></strong><span style="color:black">: Applicability to certain life stages may depend on the species and their dependence on maternally transferred thyroid hormones during the earliest phases of development. The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, TPO inhibition is not expected to decrease TH synthesis during these earliest stages of development. In zebrafish, Opitz et al. (2011) showed the formation of a first thyroid follicle at 55 hours post fertilization (hpf), Chang et al. (2012) showed a first significant TH increase at 120 hpf and Walter et al. (2019) showed clear TH production already at 72 hpf but did not analyse time points between 24 and 72 hpf. In fathead minnows, a significant increase of whole body </span><span style="color:black">TH</span> <span style="color:black">levels was already observed between 1 and 2 dpf, which corresponds to the appearance of the thyroid anlage at 35 hpf prior to the first observation of thyroid follicles at 58 hpf (Wabuke-Bunoti and Firling, 1983). Therefore, iIt is still uncertain when exactly embryonic TH synthesis is activated and how this determines sensitivity to TH </span><span style="color:black">system </span><span style="color:black">disruptors.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><strong><span style="color:black">Sex</span></strong><span style="color:black">: The KE is plausibly applicable to both sexes. T</span><span style="color:black">Hs</span> <span style="color:black">are essential in both sexes and the components of the HPT-axis are identical in both sexes. There can however be sex-dependent differences in the sensitivity to the disruption of </span><span style="color:black">TH</span> <span style="color:black">levels and the magnitude of the response. In humans, females appear more susceptible to hypothyroidism compared to males when exposed to certain halogenated chemicals (Hernandez‐Mariano et al., 2017; Webster et al., 2014). In adult zebrafish, Liu et al. (2019) showed sex-dependent changes in </span><span style="color:black">TH</span><span style="color:black"> levels and mRNA expression of regulatory genes including corticotropin releasing hormone (crh), thyroid stimulating hormone (tsh) and deiodinase 2 after exposure to organophosphate flame retardants. The underlying mechanism of any sex-related differences remains unclear.</span></span></span></span></p>
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<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Zoeller RT, Crofton KM (2005) Mode of action: developmental thyroid hormone insufficiency--neurological abnormalities resulting from exposure to propylthiouracil. Crit Rev Toxicol 35:771-781.</span></span></span></span></p>
<p style="text-align:start"> </p>
2016-11-29T18:41:332022-07-08T08:33:454683c179-3444-4ba0-a0fa-1d70a42171e2e94bcd3a-949c-4f3e-81a9-7706f62cb58b<p>Reduced T4 levels in serum prohibit local production of active T3 hormone by deiodinases expressed in the target tissues. There is evidence suggesting that anterior swim bladder inflation relies on increased thyroid hormone levels at this specific developmental time point.</p>
<p>There is convincing evidence that decreased T4 levels result in impaired anterior chamber inflation, but the underlying mechanisms are not completely understood. A convincing linear quantitative relationship between reduced T4 levels and reduced anterior chamber volume was shown in zebrafish across exposure to a limited set of three compounds. Therefore the evidence supporting this KER can be considered moderate.</p>
<p>Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. The formation of the anterior chamber coincides with the second transition phase (Winata et al., 2009) and with a peak in T4 synthesis (Chang et al., 2012) suggesting that anterior inflation is under thyroid hormone regulation. Since most of the more biologically active T3 originates from the conversion of T4, decreased circulatory T4 levels are plausibly linked to reduced anterior chamber inflation.</p>
<ul>
<li>Chang et al. (2012) observed an increase of whole body T4 concentrations in zebrafish larvae at 21 days post fertilization, corresponding to the timing of anterior swim bladder inflation.</li>
<li>Nelson et al. (2016) showed reduced whole body T4 levels at 6 days post fertilization and delayed anterior inflation (lower proportion of inflation at 14 dpf compared to controls) after exposure of fathead minnow embryos to 2-mercaptobenzothiazole. All anterior chambers eventually inflated but their size was reduced and morphology deviated.</li>
<li>Stinckens et al. (2016) showed reduced whole body T4 levels both at 5 (before anterior inflation) and 32 days post fertilization (after anterior inflation) when zebrafish were exposed to 2-mercaptobenzothiazole (a thyroperoxidase inhibitor) from 0 to 32 days post fertilization. A large percentage of MBT-exposed fish had an uninflated anterior swim bladder, although some recovery was observed over time.</li>
<li>Stinckens et al. (2016) further showed a significant correlation between whole body T4 levels and anterior chamber volume, with reduced T4 levels leading to smaller anterior chambers.</li>
<li>Stinckens et al. (2020) established a significant correlation between reduced whole body T4 levels and reduced anterior chamber volume in 32 day old zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis. Godfrey et al. (2017) also observed impaored anterior chamber inflation after exposure of zebrafish to Methimazole. Stinckens et al. (2020) continued the follow-up until 32 dpf and observed no recovery.</li>
<li>Chopra et al. (2019) found that a nonsense mutation of the duox gene, coding for the enzyme dual oxidase, another enzyme that is important for TH synthesis since it provides hydrogen peroxide for iodide oxidation, resulted in decreased intrafollicular T4 levels and impaired anterior chamber inflation until at least 54 dpf in zebrafish. It should be noted that dual oxidase is not only involved in thyroid hormone synthesis, but also in the production of reactive oxygen species (ROS) (Flores et al. 2010; Niethammer et al. 2009). In zebrafish, ROS can also be induced e.g. by copper (Zhou et al. 2016), which has also been shown to impair swim bladder development (Xu et al. 2017). Impaired production of ROS after dual oxidase knockdown may contribute to an impairment of swim bladder development.</li>
</ul>
<p>Reduced anterior chamber inflation upon disruption of the thyroid hormone system is in most cases, but not always, accompanied by reduced whole body T3 levels. Stinckens et al. (2016) found a consistent relationship between reduced whole body T4 levels, but not T3 levels, and reduced anterior chamber inflation after exposure to 2-mercaptobenzothiazole (MBT). Possibly, local T4 levels in the swim bladder tissue were too low to allow for enough local activation to T3. This relates to the general uncertainty on serum versus tissue TH levels. Alternatively, differences in timing between T3/T4 measurements (at 120hpf and 32dpf), the moment when there is a need for T3 to inflate the swim bladder (unknown but probably in between 120hpf and 32dpf) and the observation of the phenotype (32dpf), could lead to the hypothesis that T3 concentration was reduced in between the two measurements. There is also a possibility that the effect of MBT on anterior chamber inflation is not directly caused by decreased thyroid hormone levels, but rather by another mechanism such as oxidative stress. MBT is known to elevate the production of reactive oxygen species (ROS) levels in fish cells (Zeng et al., 2016). In general, chemicals may have multiple modes of action and effects on autophagy, ROS, cardiac function may impact swim bladder inflation.</p>
<p>The mechanism through which reduced T4 hormone concentrations in serum result in anterior chamber inflation impairment is not yet understood. The anterior chamber is formed by evagination from the cranial end of the posterior chamber (Robertson et al., 2007, Winata et al., 2009). Several hypotheses could explain effects on anterior chamber inflation due to reduced T4 levels:</p>
<ul>
<li>Evagination from the posterior chamber could be impaired. Villeneuve et al. (unpublished results) showed that although the anterior bud was present after exposure to a deiodinase 2 inhibitor, the anterior chamber did not inflate.</li>
<li>The formation of the tissue layers of the anterior swim bladder could be affected, although Villeneuve et al. (unpublished results) observed intact tissue layers of the anterior swim bladder after exposure to a deiodinase 2 inhibitor.</li>
<li>The anterior chamber is inflated with gas from the posterior chamber through the communicating duct. Impaired gas exchange between the two chambers could be at the basis of impaired anterior inflation. Both Nelson et al. (2016) and Stinckens et al. (2016) found that posterior chambers were larger when anterior chambers were smaller or not inflated at all. The sum of the areas of the posterior and anterior chambers remained constant independent of inflation of the anterior chamber (Stinkens et al., 2016). These results suggest retention of the gas in the posterior chamber.</li>
<li>Since gas exchange relies on a functional communicating duct between the posterior and anterior chamber, and the communicating duct is known to progressively narrow and eventually close during development, a dysfunctional communicating duct or a closure prior to anterior inflation could inhibit inflation. However, Villeneuve et al. (unpublished results) showed that the communicating duct was anatomically intact and open after exposure to iopanoic acid (a deiodinase 2 inhibitor), still leading to impaired anterior inflation.</li>
<li>Lactic acid production which is essential for producing gas to fill the swim bladder could be affected, although the observation that the total amount of gas in both chambers is not affected when anterior inflation is impaired seems to contradict this (Stinckens et al., 2016).</li>
<li>Possibly there is an effect on the production of surfactant, which is crucial to maintain the surface tension necessary for swim bladder inflation.</li>
<li>Reinwald et al. (2021) showed that T3 and propylthiouracil treatment of zebrafish embryos altered expression of genes involved in muscle contraction and functioning in an opposing fashion. The authors suggested impaired muscle function as an additional key event between decreased T3 levels and reduced swim bladder inflation.</li>
</ul>
<p>In some cases indirect effects may play a role in the impact of chemical exposure or genetic knockdown/knockout on swim bladder inflation. For example, dual oxidase also plays a role in oxidative stress.</p>
<p>Stinckens et al. (2016) showed a significant linear quantitative relationship between whole body T4 levels and anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish, with reduced T4 levels leading to smaller anterior chambers after continuous exposure to 2-mercaptobenzothiazole, a thyroid hormone synthesis inhibitor.</p>
<p>Stinckens et al. (2020, supplementary information) established a significant linear quantitative relationship between reduced T4 levels and reduced anterior chamber volume (measured as surface in 2D images) in 32 day old juvenile zebrafish across two compound exposures. This includes methimazole and propylthiouracil, two inhibitors of TH synthesis.</p>
ModerateUnspecificHighLarvaeNot SpecifiedNot Specified<p><strong>Taxonomic</strong>: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass) and physostomus (e.g., zebrafish and fathead minnow). Physostomus fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). The evidence for impaired inflation of the anterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2016; Nelson et al., 2016; Cavallin et al., 2017; Godfrey et al., 2017; Stinckens et al., 2020). While zebrafish and fathead minnows are physostomous fish with a two-chambered swim bladder, the Japanese rice fish (Oryzias latipes) is a physoclistous fish with a single chambered swim bladder that inflates during early development. This KER is not applicable to such fish species. Therefore, the current key event is plausibly applicable to physostomous fish in general.</p>
<p><strong>Life stage</strong>: The anterior chamber inflates during a specific developmental time frame. In zebrafish, the anterior chamber inflates around 21 days post fertilization (dpf) which is during the larval stage. In the fathead minnow, the anterior chamber inflates around 14 dpf, also during the larval stage. Therefore this KER is only applicable to the larval life stage.</p>
<p><strong>Sex</strong>: This KE/KER plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KE/KER. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 21 days post fertilization in zebrafish, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.</p>
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<p>Stinckens, E., Vergauwen, L., Ankley, G.T., Blust, R., Darras, V.M., Villeneuve, D.L., Witters, H., Volz, D.C., Knapen, D., 2018. An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish. Aquatic Toxicology 200, 1-12.</p>
<p>Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</p>
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<p>Woolley LD, Qin JG. 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture. 2(4):181-190.</p>
<p>Xu JP, Zhang RT, Zhang T, Zhao G, Huang Y, Wang HL, Liu JX. 2017. Copper impairs zebrafish swimbladder development by down-regulating wnt signaling. Aquatic Toxicology. 192:155-164.</p>
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2016-11-29T18:41:362022-10-10T09:13:15Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflationTPOi anterior swim bladder<p>Lucia Vergauwen [1], [lucia.vergauwen(at)uantwerpen.be]</p>
<p>Evelyn Stinckens [1], [evelyn.stinckens(at)uantwerpen.be]</p>
<p>Dan Villeneuve [2], [villeneuve.dan(at)epa.gov]</p>
<p>Dries Knapen [1], [dries.knapen (at)uantwerpen.be]</p>
<p>[1] Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium</p>
<p>[2] United States Environmental Protection Agency, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN, USA.</p>
Under Development: Contributions and Comments WelcomeWPHA/WNT EndorsedIncluded in OECD Work Plan1.35<p>This AOP describes the sequence of events leading from thyroperoxidase inhibition to increased mortality via reduced anterior swim bladder inflation. The enzyme thyroperoxidase (TPO) is essential for the synthesis of thyroxine (T4) and triiodothyronine (T3) in the thyroid follicles. Inhibition of TPO reduces thyroid hormone (TH) levels. Thyroid hormones are critical in regulating developmental processes and thyroid hormone disruption can interfere with normal development. Swim bladder inflation is known to be under TH control (Brown et al., 1988; Liu and Chan, 2002). Many fish species have a swim bladder which is a gas-filled organ that typically consists of two chambers (Robertson et al., 2007). The posterior chamber inflates during early development in the embryonic phase, while the anterior chamber inflates during late development in the larval phase. Both the posterior and the anterior chamber have an important role in regulating buoyancy, and the anterior chamber has an additional role in hearing (Robertson et al., 2007).</p>
<p>This AOP describes how inhibition of TPO results in decreased synthesis of THs in the thyroid follicles. This reduces the availability of T4 for conversion to the more biologically active T3. Reduced T3 levels prohibit normal inflation of the anterior swim bladder chamber. Due to its role in regulating buoyancy, this results in reduced swimming performance. Since reduced swimming performance results in a decreased ability to forage and avoid predators, this reduces chances of survival. The final adverse outcome is a decrease of the population growth rate. Since many AOPs eventually lead to this more general adverse outcome at the population level, the more specific and informative adverse outcome at the organismal level, increased mortality, is used in the AOP title. Support for this AOP is mainly based on chemical exposures in zebrafish and fathead minnows (Nelson et al., 2016; Godfrey et al., 2017; Stinckens et al., 2016, 2020). Convincing evidence comes from a TPO knockout study in zebrafish confirming that anterior inflation was reduced (Fang et al. 2022). Additional evidence of a link between reduced TH synthesis and reduced anterior chamber inflation is available from a study where a mutation was inserted in the gene coding for dual oxidase, another enzyme that is important for TH synthesis since it provides hydrogen peroxide for iodide oxidation (Chopra et al., 2019).</p>
<p>This AOP is part of a larger AOP network describing how decreased synthesis and/or decreased biological activation of THs leads to incomplete or improper inflation of the swim bladder, leading to reduced swimming performance, increased mortality and decreased population trajectory (Knapen et al., 2018; Knapen et al., 2020; Villeneuve et al., 2018). Apart from the upstream part, the current AOP is identical to the corresponding AOPs leading from DIO1 and DIO2 inhibition to increased mortality via anterior swim bladder inflation (<a href="https://aopwiki.org/aops/156">https://aopwiki.org/aops/156</a>, <a href="https://aopwiki.org/aops/158">https://aopwiki.org/aops/158</a>).</p>
<p>The larger AOP network describing the effect of deiodinase and thyroperoxidase inhibition on swim bladder inflation consists of 5 AOPs:</p>
<ul>
<li>Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation: <a href="https://aopwiki.org/aops/155">https://aopwiki.org/aops/155</a></li>
<li>Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation: <a href="https://aopwiki.org/aops/156">https://aopwiki.org/aops/156</a></li>
<li>Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation : <a href="https://aopwiki.org/aops/157">https://aopwiki.org/aops/157</a></li>
<li>Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation : <a href="https://aopwiki.org/aops/158">https://aopwiki.org/aops/158</a></li>
<li>Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation: <a href="https://aopwiki.org/aops/159">https://aopwiki.org/aops/159</a></li>
</ul>
<p>The development of these AOPs was mainly based on a series of dedicated experiments (using a set of reference chemicals as prototypical stressors) in zebrafish and fathead minnow that form the core of the empirical evidence. Specific literature searches were used to add evidence from other studies, mainly in zebrafish and fathead minnow. No systematic review approach was applied.</p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">There is a wealth of information on the inhibition of TPO by drugs such as MMI and PTU, as well as environmental xenobiotics. In the landmark paper on </span><span style="color:black">TH system</span><span style="color:black"> disruption by environmental chemicals, Brucker-Davis (1998) identified environmental chemicals that depressed TH synthesis by inhibiting TPO. Hurley (1998) listed TPO as a major target for thyroid tumor inducing pesticides. More recent work has tested over 1000 chemicals using a high-throughput screening assay (Paul-Friedman et al., 2016).</span></span></span></span></p>
<p>Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.</p>
<p>Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.</p>
adjacentLowHighadjacentLowModerateadjacentModerateModerateadjacentModerateModerateadjacentLowModerateadjacentLowModerateadjacentModerateModeratenon-adjacentLowHighnon-adjacentModerateModerate<p>Overall, the confidence in the supporting data for essentiality of KEs within the AOP is moderate. There is indirect evidence that reduced thyroid hormone synthesis causes reduced anterior swim bladder inflation from a study where a similar MIE was targeted: Chopra et al. (2019) showed that knockdown of dual oxidase, another enzyme that is important for TH synthesis since it provides hydrogen peroxide for iodide oxidation, reduced anterior swim bladder inflation. It should be noted that dual oxidase also plays a role in oxidative stress. Additionally, there is indirect evidence from deiodinase knockdowns supporting the downstream part of the AOP linking decreased T3 levels to reduced swim bladder inflation (targeted at posterior chamber inflation, not specifically at anterior chamber inflation, see AOPs 155-158). There is also evidence that alleviation of the effect on anterior chamber inflation reduces the effect on swimming performance.</p>
<p> </p>
ModerateUnspecificHighLarvaeHighHigh<p>The attached document includes:</p>
<ul>
<li>Support for biological plausibility of KERs</li>
<li>Support for essentiality of KEs</li>
<li>Empirical support for KERs</li>
<li>Dose and temporal concordance table covering the larger AOP network</li>
</ul>
<p>Overall, the weight of evidence for the sequence of key events laid out in the AOP is moderate to high. Nonetheless, the exact underlying mechanism of TH disruption leading to impaired swim bladder inflation is not understood.</p>
<p><strong>Taxonomic</strong>: Organogenesis of the swim bladder begins with an evagination from the gut. In physostomous fish, a connection between the swim bladder and the gut is retained. In physoclystous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Woolley and Qin, 2010). This AOP is currently mainly based on experimental evidence from studies on zebrafish and fathead minnows, physostomous fish with a two-chambered swim bladder. This AOP is not applicable to fish that do not have a second swim bladder chamber that inflates during larval development, e.g., the Japanese rice fish or medaka (<em>Oryzias latipes</em>).</p>
<p><strong>Life stage</strong>: The current AOP is applicable to the larval life stage, the period in which the anterior chamber of the swim bladder inflates (21 days post fertilization in zebrafish).</p>
<p><strong>Sex</strong>: All key events in this AOP are plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. For zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes across the sequence of events in this AOP. Different fish species have different sex determination and differentiation strategies. Zebrafish do not have identifiable heteromorphic sex chromosomes and sex is determined by multiple genes and influenced by the environment (Nagabhushana and Mishra, 2016). Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the anterior chamber inflates around 21 days post fertilization in zebrafish, sex differences are expected to play a minor role in the current AOP. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the anterior chamber inflates around 14 days post fertilization (9 dph) in fathead minnows, sex differences are expected to play a minor role in the current AOP.</p>
<p>Overall, the confidence in the supporting data for essentiality of KEs within the AOP is moderate. There is indirect evidence that reduced thyroid hormone synthesis causes reduced anterior swim bladder inflation from a study where a similar MIE was targeted: Chopra et al. (2019) showed that knockdown of dual oxidase, another enzyme that is important for TH synthesis since it provides hydrogen peroxide for iodide oxidation, reduced anterior swim bladder inflation. It should be noted that dual oxidase also plays a role in oxidative stress. Additionally, there is indirect evidence from deiodinase knockdowns supporting the downstream part of the AOP linking decreased T3 levels to reduced swim bladder inflation (targeted at posterior chamber inflation, not specifically at anterior chamber inflation, see AOPs 155-158). There is also evidence that alleviation of the effect on anterior chamber inflation reduces the effect on swimming performance.</p>
<p> </p>
<p><strong>Biological plausibility</strong>: see Table. Overall, the weight of evidence for the biological plausibility of the KERs in the AOP is moderate since there is empirical support for an association between the sets of KEs and the KERs are plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established.</p>
<p><strong>Empirical support</strong>: see Table. Overall, the empirical support for the KERs in the AOP is moderate since dependent changes in sets of KEs following exposure to several specific stressors has been demonstrated, with limited evidence for dose and temporal concordance and some uncertainties.</p>
<p>There is some level of quantitative understanding that can form the basis for development of a quantitative AOP. Quantitative relationships between reduced T4 and reduced T3, and between reduced T3 and reduced anterior chamber inflation were established. The latter is particularly critical for linking impaired swim bladder inflation to TH disruption.</p>
<p>A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. Villeneuve et al. (2014) discussed the relevance of swim bladder inflation as a potential key event and endpoint of interest in fish tests. Knapen et al. (2020) provide an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early-life stage tests, and fish tests in general. While the AOP is only applicable to fish, some of the upstream KEs are relevant across vertebrates. The taxonomic domain of applicability call of the KEs can be found on the respective pages. A suite of assays covering all the essential biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption in fish, using the levels of assessment of the OECD’s Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide. Specifically, for this AOP, thyroperoxidase inhibition can be assessed using an in chemico assay, measurements of T4 and T3 levels could be added to the Fish Embryo Acute Toxicity (FET) test (OECD TG 236), the Fish Early Life Stage Toxicity (FELS) Test (OECD TG210) and the Fish Sexual Development Test (FSDT), and assessments of anterior chamber inflation and swimming performance could be added to the FELS Test and FSDT.</p>
<p>Thyroid hormone system disruption causes multiple unspecific effects. Addition of TH measurements could aid in increasing the diagnostic capacity of a battery of endpoints since they are specific to the TH system. A battery of endpoints would ideally include the MIE, the AO and TH levels as the causal link. It is also in this philosophy that TH measurements are currently being considered as one of the endpoints in project 2.64 of the OECD TG work plan, “Inclusion of thyroid endpoints in OECD fish Test Guidelines”. While thyroid hormone measurements showed low levels of variation and were highly predictive of downstream effects in dedicated experiments to support this AOP, more variability may be present in other studies. Because of the rapid development in fish, it is important to compare thyroid hormone levels within specific developmental stages. For example, clear changes in thyroid hormone levels have been observed in zebrafish at 5, 14, 21 and 32 dpf (Stinckens et al., 2016; Stinckens et al., 2020) and in fathead minnows at 4, 6, 10, 14, 18 and 21 dpf (Nelson et al., 2016; Cavallin et al., 2017) using liquid chromatography tandem mass spectrometry (LC−MS/MS).</p>
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<p>Chopra, K., Ishibashi, S., Amaya, E., 2019. Zebrafish duox mutations provide a model for human congenital hypothyroidism. Biology Open 8.</p>
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<p>Godfrey, A., Hooser, B., Abdelmoneim, A., Horzmann, K.A., Freemanc, J.L., Sepulveda, M.S., 2017. Thyroid disrupting effects of halogenated and next generation chemicals on the swim bladder development of zebrafish. Aquatic Toxicology 193, 228-235.</p>
<p>Knapen, D., Angrish, M.M., Fortin, M.C., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J.M., Pollesch, N., Smith, L.C., Zhang, X.W., Villeneuve, D.L., 2018. Adverse outcome pathway networks I: Development and applications. Environmental Toxicology and Chemistry 37, 1723-1733.</p>
<p>Knapen, D., Stinckens, E., Cavallin, J.E., Ankley, G.T., Holbech, H., Villeneuve, D.L., Vergauwen, L., 2020. Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption. Environmental Science & Technology 54, 8491-8499.</p>
<p>Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.</p>
<p>Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.</p>
<p>Nagabhushana A, Mishra RK. 2016. Finding clues to the riddle of sex determination in zebrafish. Journal of Biosciences. 41(1):145-155.</p>
<p>Nelson, K., Schroeder, A., Ankley, G., Blackwell, B., Blanksma, C., Degitz, S., Flynn, K., Jensen, K., Johnson, R., Kahl, M., Knapen, D., Kosian, P., Milsk, R., Randolph, E., Saari, T., Stinckens, E., Vergauwen, L., Villeneuve, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow. Aquatic Toxicology 173, 192-203.</p>
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<p>Olmstead AW, Villeneuve DL, Ankley GT, Cavallin JE, Lindberg-Livingston A, Wehmas LC, Degitz SJ. 2011. A method for the determination of genetic sex in the fathead minnow, pimephales promelas, to support testing of endocrine-active chemicals. Environmental Science & Technology. 45(7):3090-3095.</p>
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<p>Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.</p>
<p>Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquatic Toxicology 173, 204-217.</p>
<p>van Aerle R, Runnalls TJ, Tyler CR. 2004. Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology. 64(2):355-369.</p>
<p>Villeneuve, D., Angrish, M., Fortin, M., Katsiadaki, I., Leonard, M., Margiotta-Casaluci, L., Munn, S., O'Brien, J., Pollesch, N., Smith, L., Zhang, X., Knapen, D., 2018. Adverse Outcome Pathway Networks II: Network Analytics. Environ Toxicol Chem doi: 10.1002/etc.4124.</p>
<p>Villeneuve, D., Volz, D.C., Embry, M.R., Ankley, G.T., Belanger, S.E., Leonard, M., Schirmer, K., Tanguay, R., Truong, L., Wehmas, L., 2014. Investigating alternatives to the fish early-life stage test: a strategy for discovering and annotating adverse outcome pathways for early fish development. Environmental Toxicology and Chemistry 33, 158-169.</p>
<p>Woolley, L.D., Qin, J.G., 2010. Swimbladder inflation and its implication to the culture of marine finfish larvae. Reviews in Aquaculture 2, 181-190.</p>
<p>Xu JP, Zhang RT, Zhang T, Zhao G, Huang Y, Wang HL, Liu JX. 2017. Copper impairs zebrafish swimbladder development by down-regulating wnt signaling. Aquatic Toxicology. 192:155-164.</p>
<p>Zhou XY, Zhang T, Ren L, Wu JJ, Wang WM, Liu JX. 2016. Copper elevated embryonic hemoglobin through reactive oxygen species during zebrafish erythrogenesis. Aquatic Toxicology. 175:1-11.</p>
2016-11-29T18:41:172023-04-29T13:02:13