7440-61-1JFALSRSLKYAFGM-UHFFFAOYSA-NJFALSRSLKYAFGM-UHFFFAOYSA-N
UraniumUranium, isotope of mass 238
238U Element
UN 2979 (DOT)
Uranium I
DTXSID10425227440-43-9BDOSMKKIYDKNTQ-UHFFFAOYSA-NBDOSMKKIYDKNTQ-UHFFFAOYSA-N
CadmiumCadimium
CADMIUM BLUE
CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER
DTXSID1023940FMA:61796Nicotinic acetylcholine receptorGO:0005739mitochondrionCHEBI:39124calcium ionPR:000004978calmodulinGO:0015464acetylcholine receptor activityGO:0007612learningGO:0007613memoryGO:0060756foraging behaviorD056631colony collapseGO:0005488bindingGO:0023052signalingGO:0005516calmodulin binding1increased7functional change2decreased4abnormal2-Imidazolidinimine, 1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-, (2E)-2016-11-29T18:42:272016-11-29T18:42:27Uranium2021-08-05T14:28:502021-08-05T14:28:50Nanoparticles and Micrometer Particles2022-02-04T13:43:432022-02-04T13:43:43Cadmium2017-10-25T08:33:122017-10-25T08:33:12WCS_9606human10090mouse10116ratWCS_7227fruit flyWCS_7955zebrafishWCS_160004gastropodsWikiUser_2Honey beeActivation, Nicotinic acetylcholine receptorActivation, Nicotinic acetylcholine receptorMolecular<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"Nicotinic acetylcholine receptors belong to the<br />
cys-loop superfamily of ligand-gated ion channels, responsible for<br />
rapid neurotransmission (Karlin, 2002). In insects nAChR have signaling<br />
roles in nervous systems and neuromuscular junctions and other cells<br />
(Jones and Sattelle, 2010; Lindstrom, 2003). Under normal conditions<br />
the endogenous neurotransmitter, acetylcholine (ACh), attaches to the<br />
ligand binding domains on the extracellular region of the pentameric<br />
nAChR. This initiates a conformation change that promotes the influx<br />
and efflux of calcium (Ca2+) and extracellular sodium and intracellular<br />
potassiumions, respectively, to create the action potential necessary for<br />
synaptic signaling (Jones and Sattelle, 2010). Activation of the nAChR,<br />
by natural or synthetic agonists, and subsequent involvement in neurotransmission<br />
is well established. Although the nAChR is conserved<br />
across vertebrates and invertebrates, the diverse composition and assembly<br />
of α-(containing two adjacent cysteine residues important in<br />
ACh binding) and non α-(lacking the cysteine residues) subunits confer<br />
diverse functional architecture and, therefore, toxicological responses<br />
(Jones and Sattelle, 2010)."</p>
<p>Text fromTable 2 of LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"• Radiolabeled nAChR agonists, (e.g., [3H] imidacloprid) or nAChR subunit specific antibodies to detect location and subunit<br />
composition of nAChR<br />
• Ligand competition studies evaluating [3H] agonist displacement to determine ligand affinities to the nAChR<br />
• Whole-cell voltage clamp electrophysiological measurements with agonists to measure nAChR activation"</p>
CL:0000540neuron<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Karlin, A., 2002. Emerging structure of the nicotinic acetylcholine receptors. Nat. Rev.<br />
Neurosci. 3 (2), 102–114.</p>
<p>Jones, A.K., Sattelle, D.B., 2010. Diversity of insect nicotinic acetylcholine receptor subunits.<br />
Adv. Exp. Med. Biol. 683, 25–43.</p>
<p>Lindstrom, J.M., 2003. Nicotinic acetylcholine receptors of muscles and nerves. Ann. N. Y.<br />
Acad. Sci. 998 (1), 41–52.</p>
<p>Tomizawa,M., Casida, J.E., 2003. Selective toxicity of neonictinoids attributable to specificity<br />
of insect and mammalian nicotinic receptors. Annu. Rev. Entomol. 48, 339–364.</p>
<p>Dani, J.A., Bertrand, D.D., 2007. Nicotinic acetylcholine receptors and nicotinic cholinergic<br />
mechanisms of the central nervous system.Annu. Rev. Pharmacol. Toxicol. 47, 699–729.</p>
<p>Matsuda, K., Kanaoka, S., Akamatsu,M., Sattelle, D.B., 2009. Diverse actions and target-site<br />
selectivity of neonicotinoids: structural insights. Mol. Pharmacol. 76 (1), 1–10.</p>
<p>LaLone, C.A., Villeneuve, D.L., Lyons, D., Helgen, H.W., Robinson, S.L., Swintek, J.A., Saari,<br />
T.W., Ankley, G.T., 2016. Sequence alignment to predict across species susceptibility<br />
(SeqAPASS): a web-based tool for addressing the challenges of cross-species extrapolation<br />
of chemical toxicity. Toxicol. Sci. 153 (2), 228–245.</p>
2016-11-29T18:41:252018-06-07T09:33:27N/A, Mitochondrial dysfunction 1N/A, Mitochondrial dysfunction 1Cellular<p>Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.</p>
<p>Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).</p>
<p>Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.</p>
<p>A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM), are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (mPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).</p>
<p><strong>Summing up:</strong> Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.</p>
<p>Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.</p>
<p>I. Mitochondrial dysfunction assays assessing a loss-of function.</p>
<p>1. Cellular oxygen consumption.</p>
<p>See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O<sub>2</sub> consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).</p>
<p>2. Mitochondrial membrane potential (Δψm ).</p>
<p>The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).</p>
<p>3. Enzymatic activity of the electron transport system (ETS).</p>
<p>Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).</p>
<p>4. ATP content.</p>
<p>For the evaluation of ATP levels, various commercially-available ATP assay kits are offered based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).</p>
<p><br />
II. Mitochondrial dysfunction assays assessing a gain-of function.</p>
<p><br />
1. Mitochondrial permeability transition pore opening (PTP).</p>
<p>The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).</p>
<p>2. mtDNA damage as a biomarker of mitochondrial dysfunction.</p>
<p>Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).</p>
<p>3. Generation of ROS and resultant oxidative stress.</p>
<p>a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.</p>
<p>b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential). GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.</p>
<p>c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.</p>
<p><br />
d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (<a class="external free" href="http://www.biotek.com/resources/articles/reactive-oxygen-species.html" rel="nofollow" target="_blank">http://www.biotek.com/resources/articles/reactive-oxygen-species.html</a>). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in the absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at site of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.</p>
<p>e. Detection of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H<sub>2</sub>O<sub>2</sub> form increasing amounts of fluorescent product (Tarpley et al., 2004).</p>
<p>Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (mPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion) (Promega assay, TB369; Radkowsky et al., 1986) • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent. All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE <a href="/wiki/index.php/Event:209" title="Event:209"> Oxidative Stress, Increase</a></p>
<table border="1" cellpadding="1" cellspacing="1">
<tbody>
<tr>
<td>
<p><strong>Assay Type & Measured Content</strong></p>
</td>
<td><strong>Description</strong></td>
<td><strong>Dose Range Studied</strong></td>
<td>
<p><strong>Assay Characteristics</strong></p>
<p><strong>(Length/Ease of use/Accuracy)</strong></p>
</td>
</tr>
<tr>
<td>
<p><strong>Rhodamine 123 Assay</strong></p>
<p>Measuring Mitochondrial membrane potential (MMP) and its collapse </p>
<p>(Shaki et al., 2012)</p>
</td>
<td>
<p>Mitochondrial uptake of cationic fluorescent dye, rhodamine 123, is used for estimation of mitochondrial membrane potential. The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively.</p>
</td>
<td>50, 100 and 500 μM of uranyl acetate</td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TMRE fluorescence Assay</strong></p>
<p>Measuring Mitochondrial permeability transition pore (mPTP) opening</p>
<p>(Huser et al., 1998)</p>
</td>
<td>Laser scanning confocal microscopy in combination with the potentiometric fluorescence dye tetramethylrhodamine ethyl ester to monitor relative changes in membrane potential in single isolated cardiac mitochondria. The cationic dye distributes across the membrane in a voltage-dependent manner. Therefore, the large potential gradient across the inner mitochondrial membrane results in the accumulation of the fluorescent dye within the matrix compartment. Rapid depolarizations are caused by the opening of the transition pore.</td>
<td>1 µM cyclosporin A</td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>GSH / GSSG Determination Assay</strong></p>
<p>Measuring cellular glutathione (GSH) status; ratio of GSH/GSSG</p>
<p>(Owen & Butterfield, 2010; Shaki et al., 2013)</p>
</td>
<td>GSH and GSSG levels are determinted biochemically with DTNB (Ellman’s reagent). The developed yellow color was read at 412 nm on a spectrophotometer.</td>
<td>100 µM uranyl acetate</td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>TBARS Assay</strong></p>
<p>Quantification of lipid peroxidation</p>
<p>(Yuan et al., 2016)</p>
</td>
<td>MDA content, a product of lipid peroxidation, was measured using a thiobarbituric acid reactive substances (TBARS) assay. Briefly, the kidney cells were collected in 1 ml PBS buffer solution (pH 7.4) and sonicated. MDA reacts with thiobarbituric acid forming a colored product which can be measured at an absorbance of 532 nm.</td>
<td>200, 400, 800 µM uranyl acetate</td>
<td>
<p>Medium / medium</p>
<p>High accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Aequorin-based bioluminescence assay</strong></p>
<p>Increase in mitochondrial Ca<sup>2+</sup> influx</p>
<p>(Pozzan & Rudolf, 2009)</p>
</td>
<td>Together with GFP, the aequorin moiety acts as Ca<sup>2+</sup> sensor <em>in vivo</em>, which delivers emission energy to the GFP acceptor molecule in a BRET (Bioluminescence Resonance Energy Transfer) process; the Ca2+ can then be visualized with fluorescence microscopy.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Western blot & immunostaining analyses</strong></p>
<p>Measuring cytochrome c release</p>
(Chen et al., 2000)</td>
<td>Examining the redistribution of Cyto c in cytosolic and mitochondrial cellular fractions. Cells are homogenized and centrifuged, then prepared for immunoblots. Cellular fractions were washed in PBS and lysed in 1% NP-40 buffer. Cellular proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes, probed using immunoblot analyses with antibodies specific to cyto c (6581A for Western and 65971A for immunostaining; Pharmingen)</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Quantikine Rat/Mouse Cytochrome c Immunoassay</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Shaki et al., 2012)</p>
</td>
<td>Cytochrome C release was measured a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing mono- clonal antibody specific for cytochrome c conjugated to horseradish peroxidase). After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm.</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Low accurancy</p>
</td>
</tr>
<tr>
<td>
<p><strong>Membrane potential and cell viability – Flow Cytometry</strong></p>
<p>Measuring cytochrome c release</p>
<p>(Kruidering et al., 1997)</p>
</td>
<td>“Dc and viability were determined by analyzing the R123 and propidium iodide fluorescence intensity with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser, with the Lysis software program (Becton Dickinson). R123 is a cationic dye that accumulates in the negatively charged inner side of the mitochondria. When the potential drops, less R123 accumulates in the mitochondria, which results in a lower fluorescence signal. The potential was measured as follows: at the indicated times, a 500-ml sample of the cell suspension was taken and transferred to an Eppendorf minivial. To this sample, 100 ml of 6 mM R123 in buffer D was added. After incubation for 10 min at 37°C, the cell suspension was centrifuged for 5 min at 80 3 <em>g</em>. The cell pellet was resuspended in 200 ml of buffer D, containing 0.2 mM R123 and 10 mM propidium iodide, to prevent loss of R123 and to stain nonviable cells, respectively. The samples were transferred to FACScan tubes and analyzed immediately. Analysis was performed at a flow rate of<br />
60 ml/min. R123 fluorescence was detected by the FL1 detector with an emission detection limit below 560 nm. Propidium iodide fluorescence was detected by the FL3 detector, with emission detection above 620 nm. Per sample 3,000 to 5,000 cells were counted (Van de Water <em>et al.</em>, 1993)”</td>
<td> </td>
<td>
<p>Short / easy</p>
<p>Medium accurancy</p>
</td>
</tr>
</tbody>
</table>
<p>Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al., 2010) as well as in humans (Winklhofer and Haass, 2010).</p>
CL:0000255eukaryotic cellNot SpecifiedUnspecificNot SpecifiedAll life stagesHighHighHigh<p> </p>
<p>Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149.</p>
<p>Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464.</p>
<p>Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80.</p>
<p>Belyaeva, E. A., Sokolova, T. V., Emelyanova, L. V., & Zakharova, I. O. (2012). Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063</p>
<p>Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312.</p>
<p>Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.</p>
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2016-11-29T18:41:232022-03-07T07:12:30Impairment, Learning and memoryImpairment, Learning and memoryIndividual<p> </p>
<p>Learning can be defined as the process by which new information is acquired to establish knowledge by systematic study or by trial and error (Ono, 2009). Two types of learning are considered in neurobehavioral studies: a) associative learning and b) non-associative learning. Associative learning is based on making associations between different events. In associative learning, a subject learns the relationship among two different stimuli or between the stimulus and the subject’s behaviour. On the other hand, non-associative learning can be defined as an alteration in the behavioural response that occurs over time in response to a single type of stimulus. Habituation and sensitization are some examples of non-associative learning.</p>
<p>The memory formation requires acquisition, retention and retrieval of information in the brain, which is characterised by the non-conscious recall of information (Ono, 2009). There are three main categories of memory, including sensory memory, short-term or working memory (up to a few hours) and long-term memory (up to several days or even much longer).</p>
<p>Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. For example, the hippocampus has been shown to be critical for spatial-temporal memory, visio-spatial memory, verbal and narrative memory, and episodic and autobiographical memory (Burgess et al., 2000; Vorhees and Williams, 2014). However, there is substantial evidence that fundamental learning and memory functions are not mediated by the hippocampus alone but require a network that includes, in addition to the hippocampus, anterior thalamic nuclei, mammillary bodies cortex, cerebellum and basal ganglia (Aggleton and Brown, 1999; Doya, 2000; Mitchell et al., 2002, Toscano and Guilarte, 2005; Gilbert et al., 2006, 2016). Thus, damage to variety of brain structures can potentially lead to impairment of learning and memory. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990).While the prefrontal cortex and frontostriatal neuronal circuits have been identified as the primary sites of higher-order cognition in vertebrates, invertebrates utilize paired mushroom bodies, shown to contain ~300,000 neurons in honey bees (Menzel, 2012; Puig et al., 2014).</p>
<p>For the purposes of this KE (AO), impaired learning and memory is defined as an organism’s inability to establish new associative or non-associative relationships, or sensory, short-term or long-term memories which can be measured using different behavioural tests described below.</p>
<p><strong>In laboratory animals:</strong> in rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, <span style="color:#3498db">Hebb-Williams maze</span>, passive avoidance and Spontaneous alternation and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows.</p>
<p>1) RAM, Barnes, MWM, <span style="color:#3498db">Hebb-Williams maze </span>are examples of spatial tasks, animals are required to learn the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze), or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). The <span style="color:#3498db">Hebb-Williams maze measures an animal’s problem solving abilities by providing no spatial cues to find the target (Pritchett & Mulder, 2004).</span></p>
<p>2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention – I have seen one of these objects before, but not this one (Cohen and Stackman, 2015).</p>
<p>3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009).</p>
<p>4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2001).</p>
<p><span style="color:#3498db">5) Operant Responding. Performance on operant responding reflects the cortex’ ability to organize processes (Rabin et al., 2002). </span></p>
<p><strong>In humans:</strong> A variety of standardized learning and memory tests have been developed for human neuropsychological testing, including children (Rohlman et al., 2008). These include episodic autobiographical memory, perceptual motor tests, short and long term memory tests, working memory tasks, word pair recognition memory; object location recognition memory. Some have been incorporated in general tests of intelligence (IQ) such as the Wechsler Adult Intelligence Scale (WAIS) and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include:</p>
<p>1) Rey Osterieth Complex Figure test (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006).</p>
<p>2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1994; Talley, 1986).</p>
<p>3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994).</p>
<p>4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986).</p>
<p>5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2011).</p>
<p>6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).</p>
<p><span style="color:#3498db">7) Attentional set-shifting (ATSET) task. Measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015).</span></p>
<p><strong>In Honey Bees:</strong> For over 50 years an assay for evaluating olfactory conditioning of the proboscis extension reflex (PER) has been used as a reliable method for evaluating appetitive learning and memory in honey bees (Guirfa and Sandoz, 2012; LaLone et al., 2017). These experiments pair a conditioned stimulus (e.g., an odor) with an unconditioned stimulus (e.g., sucrose) provided immediately afterward, which elicits the proboscis extension (Menzel, 2012). After conditioning, the odor alone will lead to the conditioned PER. This methodology has aided in the elucidation of five types of olfactory memory phases in honey bee, which include early short-term memory, late short-term memory, mid-term memory, early long-term memory, and late long-term memory (Guirfa and Sandoz, 2012). These phases are dependent on the type of conditioned stimulus, the intensity of the unconditioned stimulus, the number of conditioning trials, and the time between trials. Where formation of short-term memory occurs minutes after conditioning and decays within minutes, memory consolidation or stabilization of a memory trace after initial acquisition leads to mid-term memory, which lasts 1 d and is characterized by activity of the cAMP-dependent PKA (Guirfa and Sandoz, 2012). Multiple conditioning trials increase the duration of the memory after learning and coincide with increased Ca2+-calmodulin-dependent PKC activity (Guirfa and Sandoz, 2012). Early long-term memory, where a conditioned response can be evoked days to weeks after conditioning requires translation of existing mRNA, whereas late long-term memory requires de novo gene transcription and can last for weeks (Guirfa andSandoz, 2012)."</p>
<p>Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans. Recently, larval zebrafish has also been suggested as a model for the study of learning and memory (Roberts et al., 2013).</p>
<p><span style="color:#3498db"><strong>Life stage applicability: </strong>This key event is applicable to various life stages such as during brain development and maturity (Hladik & Tapio, 2016). </span></p>
<p><span style="color:#3498db"><strong>Sex applicability:</strong> This key event is not sex specific (Cekanaviciute et al., 2018), although sex-dependent cognitive outcomes have been recently ; Parihar et al., 2020). </span></p>
<p><span style="color:#3498db"><strong>Evidence for perturbation by a prototypic stressor: </strong>Current literature provides ample evidence of impaired learning and memory being induced by ionizing radiation (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). </span></p>
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<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
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<p>Menzel, R., 2012. The honeybee as a model for understanding the basis of cognition. Nat. Rev. Neurosci. 13 (11), 758–768.</p>
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<p>Vorhees CV, Williams MT (2014) Assessing spatial learning and memory in rodents. ILAR J 55:310-332.</p>
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<p> </p>
2016-11-29T18:41:242023-03-22T16:30:39Abnormal, Foraging activity and behaviorAbnormal, Foraging activity and behaviorIndividual<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"As eusocial insects, honey bees rely on theworker bee caste to forage<br />
for nectar, pollen, andwater. Foraged water can be used for evaporative<br />
cooling of the hive during warm weather (as reviewed by Jones and<br />
Oldroyd, 2006). Nectar and pollen collected by the foragers are the<br />
sole food source for the colony, with nectar providing carbohydrates<br />
and pollen providing lipids, protein, vitamins, and essential minerals<br />
(Brodschneider and Crailsheim, 2010). Upon returning to the hive, forager<br />
bees identify non-foraging, food-storing hive bees and deliver their<br />
collection by regurgitating nectar carried back in their honey stomach<br />
(i.e., foregut of proventriculus; Free, 1959). The hive bees place the nectar<br />
in wax cells for processing into honey. Hive bees also aid foragers in<br />
unloading pollen from the pollen baskets (corbicula) on the forager's<br />
hind legs and place it in cells where it is mixed with nectar to form<br />
bee bread, which is stored for consumption by the colony (Winston,<br />
1987). Foragers consume only small amounts of the food they collect.<br />
Hive bees consume the food they receive in order to produce proteinrich<br />
royal jelly and brood food, which they use to nourish both the<br />
queen and the developing brood (Winston, 1987). During winter, the<br />
colony survives on the pollen and nectar that was stored as bee bread<br />
and honey over the spring, summer, and fall seasons (Seeley and<br />
Visscher, 1985).<br />
The act of foraging is a perilous and metabolically challenging task<br />
that is typically carried out by worker bees in the later stages of life<br />
(Woyciechowski and Moroń, 2009). However, the timing of the role<br />
change from hive bee to forager can vary depending on the needs of<br />
the colony. There are environmental, hormonal, and social cues that determine<br />
when and how often foragers search for food and fluid, includingweather,<br />
abundance or scarcity of food resources, magnitude of food<br />
stockpiled in the hive, health of the colony, and size of the brood<br />
(Dreller and Tarpy, 2000). Such cues initiate physiological changes involved<br />
in the transition of a worker bee to foraging, which include<br />
changes to flight muscles andmetabolic rate. These changes accommodate<br />
the reported 70-fold increase in oxygen consumption needed to<br />
sustain physical and cognitive activities of the forager bee (Kammer<br />
and Heinrich, 1978). It has been documented that the volume of<br />
neuropil in mushroom bodies is increased by approximately 15%, and<br />
the somata of the Kenyon cells decreased by approximately 29% in foragers<br />
compared to day-old bees (Withers et al., 1995). Change in lipid<br />
stores also occurs in forager bees prior to foraging, whereby their abdominal<br />
lipid is reduced to approximately half that of nurse bees<br />
(Chang et al., 2015; Toth and Robinson, 2005). Further, there is lowprotein<br />
content in the forager's fat body cells, and vitellogenin (Vtg; egg</p>
<p>yolk) protein production is significantly reduced, while juvenile hormone<br />
levels are significantly increased (Toth and Robinson, 2005). Another<br />
change which occurs at the stage where worker bees become<br />
foragers is that their flight muscle fiber thickness decreases and diameter<br />
of the myofibrils, which contain the contractile filaments, increases<br />
in preparation for prolonged flight during foraging (Correa-Fernandez<br />
and Cruz-Landim, 2010)."</p>
<p>Text from table 2 in LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"• Radio-frequency identification tagging technology to track the frequency and duration of individual foraging events, flight time,<br />
foragers homing ability, duration of time spent at a feeder, and duration between feeding<br />
• Video tracking software for measures of total distance traveled and time spent in social interaction<br />
• Weigh bee-collected pollen from hive entrance trap<br />
• Pollen load can also be assessed by scoring the size of amount of pollen in the forager’s corbiculae (pollen basket) relative to the<br />
size of the worker bee<br />
• Nectar loads from individual forager bees can be measured with a pocket refractometer after inducing regurgitation<br />
• Video foragers returning to hive and measure waggle dance circuits performed<br />
• Food storage can be measured by visual inspection or digital imaging of the combs with the objective to estimate the percent of<br />
cells filled with nectar (uncapped), honey (capped), or pollen"</p>
<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Brodschneider, R., Crailsheim, K., 2010. Nutrition and health in honey bees. Apidologie 41<br />
(3), 278–294.</p>
<p>Jones, J.C., Oldroyd, B.P., 2006. Nest thermoregulation in social insects. Adv. Insect Physiol.<br />
33, 153–191.</p>
<p>Free, J.B., 1959. The transfer of food between the adult members of a honeybee community.<br />
Bee World 40 (8), 193–201.</p>
<p>Winston, M.L., 1987. The Biology of the Honey Bee. Harvard University Press.</p>
<p>Seeley, T.D., Visscher, P.K., 1985. Survival of honeybees in cold climates: the critical timing<br />
of colony growth and reproduction. Ecol. Entomol. 10 (1), 81–88.</p>
<p>Woyciechowski, M., Moroń, D., 2009. Life expectancy and onset of foraging in the honeybee<br />
(Apis mellifera). Insect. Soc. 56 (2), 193–201.</p>
<p>Dreller, C., Tarpy, D.R., 2000. Perception of the pollen need by foragers in a honeybee colony.<br />
Anim. Behav. 59 (1), 91–96.</p>
<p>Kammer, A.E., Heinrich, B., 1978. Insect flight metabolism. Adv. Insect Physiol. 13,<br />
133–228.</p>
<p>Withers, G.S., Fahrbach, S.E., Robinson, G.E., 1995. Effects of experience and juvenile hormone<br />
on the organization of the mushroom bodies of honey bees. J. Neurobiol. 26<br />
(1), 130–144.</p>
<p>Chang, L.H., Barron, A.B., Cheng, K., 2015. Effects of the juvenile hormone analogue<br />
methoprene on rate of behavioural development, foraging performance and navigation<br />
in honey bees (Apis mellifera). J. Exp. Biol. 218 (11), 1715–1724.</p>
<p>Toth, A.L., Robinson, G.E., 2005. Worker nutrition and division of labour in honeybees.<br />
Anim. Behav. 69, 427–435.</p>
<p>Correa-Fernandez, F., Cruz-Landim, C., 2010. Differential flight muscle development in<br />
workers, queens and males of the eusocial bees, Apis mellifera and Scaptotrigona<br />
postica. J. Insect Sci. 10, 85.</p>
2016-11-29T18:41:252018-06-07T10:51:36Death/Failure, ColonyDeath/Failure, ColonyPopulation<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"Colony death/failure is defined as demise of a functional colony. Dramatic losses in the number of managed honey bee colonies have been reported across the globe (Potts et al., 2010) and efforts have been undertaken to survey and identify trends in losses over time, particularly in the US and European Union. Most recent survey results collected in the US have shown that managed honey bee colony losses are significantly higher than those deemed acceptable by beekeepers (Seitz et al., 2015). From surveying commercial (>300 colonies), sideline (25–300 colonies), and small scale <25 colonies) beekeepers, average annual colony losses (both<br />
summer and winter losses) per operation in the US during 2014–2015 were 49%, compared to 18.7% that has been identified by beekeepers as an acceptable loss rate (Seitz et al., 2015). Starvation, poor over-winter survival, and weak colonies, were among the most common perceived causes of loss reported by bee keepers (Seitz et al., 2015). Commercial beekeepers, managing thousands of colonies, self-reported colony collapse disorder and pesticides as third and fourth leading reasons for colony loss, respectively (Seitz et al., 2015)."</p>
<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Potts, S.G., Biesmeijer, J.C., Kremen, C., Neumann, P., Schweiger, O., Kunin, W.E., 2010.<br />
Global pollinator declines: trends, impacts and drivers. Trends Ecol. Evol. 25 (6),<br />
345–353.</p>
<p>Seitz, N., Traynor, K.S., Steinhauer, N., Rennich, K., Wilson, M.E., Ellis, D., Rose, R., Tarpy,<br />
D.R., Sagili, R.R., Caron, D.M., Delaplane, K.S., Rangel, J., Lee, K., Baylis, K., Wilkes, J.T.,<br />
Skinner, J.A., Pettis, J.S., vanEngelsdorp, D., 2015. A national survey of managed<br />
honey bee 2014–2015 annual colony losses in the USA. J. Apic. Res. 54 (4), 1–12.</p>
<p> </p>
2016-11-29T18:41:252018-06-07T11:15:11Weakened, ColonyWeakened, ColonyPopulation<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"The characteristics evaluated to determine the strength/health of honey bee colonies, include adequate numbers of adult bees, presence of sealed and open brood, adequate amounts of stored pollen, nectar and sealed honey, the absence of pests and disease, and the presence of a queen that lays eggs in consistent and tight patterns, with limited eggless cells (Sagili and Burgett, 2011). If the colony is weakened by any one (or a combination) of these factors for an extended period, a critical point can be reached<br />
that will lead to colony failure. Through honey bee population dynamics models, it has been demonstrated that loss of foragers leading to precocious foraging of young bees may restore the overall foraging capacity, but the brood rearing capacity of the colony might be reduced (Khoury et al., 2011). Further, as noted above, precocious foragers are less effective and resilient, causing the forager death rate to increase. The model predicts that sustained forager losses that reduce the force by two-thirds would place a colony at risk for failure (Khoury et al., 2011). Additionally, proper brood rearing is essential to the development of healthy adult bees."</p>
<p>Text from Table 2 in LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"• Count number of adult bees, presence of sealed and open brood, assess amount of food stores by visual method or by weighing,<br />
assess presence/absence of pests and disease, evaluate egg laying patterns of queen<br />
• Brood care behavior can be evaluated by filming the brood nest and then recording nursing frequency, total nursing period per<br />
hour, and average duration of nursing episodes for individual cells<br />
• Cannibalism of brood can be detected by mapping eggs, larvae and pupae present on brood frames and noting developmental<br />
stages for each individual, then inspecting daily for missing larvae<br />
• Assess health of bee: dry weight, muscle development, protein content"</p>
<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Sagili, R.R., Burgett, D.M., 2011. Evaluating honey bee colonies for pollination: a guide for<br />
commercial growers and beekeepers. A Pacific Northwest Extension Publication. vol.<br />
623, pp. 1–8.</p>
<p>Khoury, D.S.,Myerscough,M.R., Barron, A.B., 2011. A quantitativemodel of honey bee colony<br />
population dynamics. PLoS One 6 (4), e18491.</p>
<p> </p>
2016-11-29T18:41:292018-06-07T11:04:32Altered, Ca2+-calmodulin activated signal transductionAltered, Ca2+-calmodulin activated signal transductionCellular<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"Some neuronal nAChR subunit combinations are highly permeable<br />
to Ca2+, which acts as a messenger relaying extracellular information<br />
to intracellular regions and to the nucleus (Uteshev, 2012). Upon influx<br />
of Ca2+ into neurons via nAChR, Ca2+ binds to calmodulin (CaM). This<br />
complex either activates adenylyl cyclase (AC) to catalyze the conversion<br />
of ATP to 3′5′-adenosine monophosphate (cAMP),which then activates<br />
PKA, or interacts with Ca2+-CaM kinase II (CaMKII) (e.g.,<br />
Dajas-Bailador andWonnacott, 2004; Sweatt, 2001). Regardless of signaling<br />
through PKA or CaMKII, both kinases activate the phosphorylation<br />
cascade via extracellular signal-related protein kinase/mitogenactivated<br />
protein kinase (ERK/MAPK), stimulating transcription of<br />
cAMP response element (CRE) binding protein (CREB) mediated<br />
genes (Impey et al., 1999). In neurons, these signaling cascades lead to<br />
the production of proteins that direct synaptic plasticity (i.e., changes<br />
in synaptic strength in response to signaling activity),which is essential<br />
to learning and memory."</p>
<p>Text from Table 2 in LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"• Fluorescent Ca2+ imaging in cells expressing nAChR for evaluation of Ca2+ levels entering individual nAChR-mediated ion<br />
channels<br />
• Western blotting and kinase assays can be used to evaluate ERK1/2 phosphorylation and activity<br />
• Activation of CREB/CRE transcription<br />
• Pharmacological inhibition of pathway"</p>
<p>LaLone, C.A., Villeneuve, D.L., Wu-Smart, J., Milsk, R.Y., Sappington, K., Garber, K.V., Housenger, J. and Ankley, G.T., 2017. Weight of evidence evaluation of a network of adverse outcome pathways linking activation of the nicotinic acetylcholine receptor in honey bees to colony death.<em> </em>STOTEN. 584-585, 751-775.</p>
<p>Uteshev, V.V., 2012. alpha7 nicotinic ACh receptors as a ligand-gated source of Ca(2+)<br />
ions: the search for a Ca(2+) optimum. Adv. Exp. Med. Biol. 740, 603–638.</p>
<p>Dajas-Bailador, F., Wonnacott, S., 2004. Nicotinic acetylcholine receptors and the regulation<br />
of neuronal signalling. Trends Pharmacol. Sci. 25 (6), 317–324.</p>
<p>Sweatt, J.D., 2001. The neuronalmap kinase cascade: a biochemical signal integration system<br />
subserving synaptic plasticity and memory. J. Neurochem. 76, 1–10.</p>
<p>Impey, S., Obrietan, K., Storm, D.R., 1999. Making new connections: role of ERK/MAP kinase<br />
signaling in neuronal plasticity. Neuron 23, 11–14.</p>
2016-11-29T18:41:312018-06-07T09:46:42e2457478-9004-4b80-8ca0-a72aced9180050b2923d-73a0-42b1-bb4d-7a354ec60b862016-11-29T18:41:342016-12-03T16:37:58da2197aa-6f4b-4b4f-952f-a533b6169d2f1ca80d57-e643-430b-9607-d8c08e61ab9f2016-11-29T18:41:342016-12-03T16:37:581ca80d57-e643-430b-9607-d8c08e61ab9f2a8dccb3-497f-433e-9442-4ea130310fa62016-11-29T18:41:362016-12-03T16:38:042a8dccb3-497f-433e-9442-4ea130310fa6dbf70e3c-93f3-4896-8210-ed6de466c06e2016-11-29T18:41:362016-12-03T16:38:04e2457478-9004-4b80-8ca0-a72aced91800da2197aa-6f4b-4b4f-952f-a533b6169d2f2016-11-29T18:41:362016-12-03T16:38:0450b2923d-73a0-42b1-bb4d-7a354ec60b8643254f8c-2bf9-4bd5-83da-d987cc335b0e2016-11-29T18:41:372016-12-03T16:38:0743254f8c-2bf9-4bd5-83da-d987cc335b0eda2197aa-6f4b-4b4f-952f-a533b6169d2f2016-11-29T18:41:372016-12-03T16:38:07Nicotinic acetylcholine receptor activation contributes to abnormal foraging and leads to colony death/failure 1nAChR activation - colony death 1<p>Carlie A. LaLone, U.S. Environmental Protection Agency (LaLone.Carlie@epa.gov)</p>
Open for comment. Do not citeUnder Development1.29<p>Text from LaLone et al. (2017) Weight of evidence evaluation of a network of adverse outcome pathways linking activaiton of the nicotinic acetylcholine receptor in honey bees to colony death. <em>Science of the Total Environment</em> 584-585, 751-775:</p>
<p>"The nicotinoids and neonicotinoids are both agonists of the nAChR<br />
(Tomizawa and Casida, 2003); however, neonicotinoids are the primary<br />
chemicals considered in the AOPs relevant to bees.<br />
The potency of a nAChR agonist is dependent on the receptor subunit<br />
composition, structurally important amino acid residues at the<br />
binding site, and the ionization status of the chemical at physiological<br />
pH (Tomizawa and Casida, 2003; Dani and Bertrand, 2007). For example,<br />
nicotine is a classical vertebrate nAChR agonist; however, it has relatively<br />
low affinity (and insecticidal activity) for the invertebrate<br />
nAChR. Due to ionization, nicotine is poor at passing though the ion-impermeable<br />
barrier surrounding the insect central nervous system(CNS;<br />
Tomizawa and Casida, 2003). Conversely, non-ionizable neonicotinoids<br />
readily translocate into the insect CNS and have high affinity for the<br />
nAChR (e.g., Drosophila nAChR IC50 4.6 nM imidacloprid), with limited<br />
or no binding activity to vertebrate nAChR (Tomizawa and Casida,<br />
2003). Various studies have demonstrated that similarities and differences<br />
in key amino acid residues in the ligand binding domain across<br />
species can lead to structural and binding site differences that dictate<br />
chemical interaction with the receptor (Dani and Bertrand, 2007;<br />
Matsuda et al., 2009; Tomizawa and Casida, 2009; Jones and Sattelle,<br />
2010; LaLone et al., 2016). Due to the intended insecticidal action of<br />
neonicotinoids, a growing number of studies have been conducted to</p>
<p>evaluate potential adverse effects in non-target species such as honey<br />
bees exposed to neonicotinoids, particularly imidacloprid, clothianidin,<br />
and thiamethoxam. Some of the results of these studies are included<br />
in subsequent AOP descriptions."</p>
adjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot SpecifiedadjacentNot SpecifiedNot Specifiednon-adjacentNot SpecifiedNot SpecifiedNot Specified2016-11-29T18:41:162023-04-29T13:02:11