<td>Under development: Not open for comment. Do not cite</td>
<td>Not under active development</td>
<td></td>
<td></td>
<td>Under Development</td>
</tr>
</tbody>
</table>
</div>
</div>
<!-- Abstract Section, text as generated by author -->
<div id="abstract">
<h2>Abstract</h2>
<hr>
<p style="text-align:justify">This AOP details the linkage between CYP7B inhibition and decreased sexual behavior that adversely impacts reproductive success. CYP7B is expressed in the brain and catalyzes the conversion of pregnenolone to 7α-hydroxypregnenolone, a neurosteroid that stimulates the release of dopamine in the telencephalon. When released through this pathway, dopamine binds D<sub>2</sub> receptor which is involved in induction of sexual behaviors, among other effects. Ketoconazole and other azole fungicides are potent inhibitor of cytochrome P450s, including CYP7B. They bind to the heme site of the enzyme preventing its catalytic activity. When exposed to one of these molecules, 7α-hydroxypregnenolone synthesis decreases which, in turn, reduces dopamine release in the telencephalon and limits sexual behavior. Since sexual behaviors are closely associated to reproductive success, its inhibition negatively affects the fitness of animals. </p>
<h2>Abstract</h2>
<p style="text-align:justify">This AOP details the linkage between CYP7B inhibition and decreased sexual behavior that adversely impacts reproductive success. CYP7B is expressed in the brain and catalyzes the conversion of pregnenolone to 7α-hydroxypregnenolone, a neurosteroid that stimulates the release of dopamine in the telencephalon. When released through this pathway, dopamine binds D<sub>2</sub> receptor which is involved in induction of sexual behaviors, among other effects. Ketoconazole and other azole fungicides are potent inhibitor of cytochrome P450s, including CYP7B. They bind to the heme site of the enzyme preventing its catalytic activity. When exposed to one of these molecules, 7α-hydroxypregnenolone synthesis decreases which, in turn, reduces dopamine release in the telencephalon and limits sexual behavior. Since sexual behaviors are closely associated to reproductive success, its inhibition negatively affects the fitness of animals. </p>
<p style="text-align:justify">7α-hydroxypregnenolone was recently discovered and its function and regulation remain unclear. The few studies that focused on this neurosteroid and that were used for this AOP are based on <em>in vitro</em> and <em>in vivo</em> experiments quail and newt. Since the function of this neurosteroid differs in mammals, this AOP is only applicable to non-mammalian vertebrates. It is also limited to male. </p>
<br>
</div>
<!-- Background Section, text as generated by author -->
<div id="background">
<h2>AOP Development Strategy</h2>
<div id="context">
<h3>Context</h3>
<p style="text-align:justify">This AOP shares most of its key events with AOP 218, with the exception of <em>Locomotor activity, decreased</em> (Event 1389). Due to this difference, the domain of applicability of the two AOPs differs and limits their compatibility. For that reason, two similar AOPs with different domain of applicability were created. </p>
<h3>Background</h3>
<hr>
<p style="text-align: justify;">This AOP shares most of its key events with AOP 218, with the exception of <em>Locomotor activity, decreased</em> (Event 1389). Due to this difference, the domain of applicability of the two AOPs differs and limits their compatibility. For that reason, two similar AOPs with different domain of applicability were created. </p>
<br>
</div>
</div>
<!-- AOP summary, includes summary of each of the events associated with this aop -->
<p style="text-align:justify"><u>Taxons:</u> This AOP is supported with evidence from studies conducted with newt and quail. Based on anticipated conservation of the biology associated with the KEs and KERs described, it is presumed to be applicable to all amphibian and bird. </p>
<p style="text-align:justify"><u>Taxons:</u> This AOP is supported with evidence from studies conducted with newt and quail. Based on anticipated conservation of the biology associated with the KEs and KERs described, it is presumed to be applicable to all amphibian and bird. </p>
<p style="text-align:justify">Previous evidence suggest that this AOP is not applicable to mammal. All the key events of this AOP are described or are biologically plausible in mammal, but the relationship between them might differ, as suggested by Yau et al. (2006). </p>
<p style="text-align:justify"><u>Sex:</u> This AOP is applicable to male only. </p>
<p style="text-align:justify"><u>Life Stage:</u> This AOP applies to sexually mature animals since the endpoint is related to reproduction. </p>
<h3>Essentiality of the Key Events</h3>
<p>Few studies measured multiple key events of this AOP. For this reason, the evidence for essentiality of the key events is mainly indirect and provided by a series of antagonist/exogenous supplementation experiments. The animal models used for these investigations were newt and quail. </p>
<h3>Essentiality of the Key Events</h3>
<p>Few studies measured multiple key events of this AOP. For this reason, the evidence for essentiality of the key events is mainly indirect and provided by a series of antagonist/exogenous supplementation experiments. The animal models used for these investigations were newt and quail. </p>
<p>At present, no CYP7B knock-out experiments were conducted in species of interest. However, several indirect evidences linking CYP7B inhibition to a decreased locomotor activity suggest an important correlation between the two events.</p>
<ul>
<li>CYP7B is the only enzyme able to synthesize 7α-hydroxypregnenolone and this neurosteroid is strongly related to locomotor activity <a href="http://www.genome.jp/dbget-bin/www_bget?rn:R08943">http://www.genome.jp/dbget-bin/www_bget?rn:R08943</a>. </li>
</ul>
<ul>
<li>Inhibition of CYP7B with intracranial injection of ketoconazole decreased 7α-hydroxypregnenolone synthesis and decreased sexual behavior in newt and quail (Ogura et al., 2016, Toyoda et al., 2012). Ketoconazole is a non-specific inhibitor of cytochromes P450 activity known to bind to and inhibit CYP7B both <em>in vitro</em> and <em>in vivo</em>.</li>
</ul>
</td>
</tr>
<tr>
<td>
<p><u>KE1</u></p>
<p>7α-hydroxypregnenolone, decreased</p>
</td>
<td>
<p>Strong</p>
</td>
<td>
<p>Direct evidences connecting this neurosteroid to sexual behavior were described.</p>
<ul>
<li>Intracerebroventricular injection of 7α-hydroxypregnenolone in male quail and newt induced spontaneous sexual behavior in a dose-dependent manner. The same treatment had no effect on female (Toyoda et al., 2012; Ogura et al., 2016).</li>
</ul>
</td>
</tr>
<tr>
<td>
<p><u>KE2</u></p>
<p>Dopamine release, decreased</p>
</td>
<td>
<p>Moderate</p>
</td>
<td>
<p>There is strong evidence demonstrating the involvement of dopamine in sexual behavior among all vertebrates. However, only indirect evidence relates CYP7B inhibition to a decreased dopamine release. The rational is stronger for 7α-hydroxypregnenolone in relation to dopamine release, although this neurosteroid receptor remains to be identified. </p>
<ul>
<li>Sexual behavior was stimulated in male newt with intracerebroventricular injection of 7α-hydroxypregnenolone. Newt treated with a dopamine D2-like receptor antagonist (haloperidol or sulpiride) prior to receiving 7α-hydroxypregnenolone exhibited no increase in sexual behavior (Toyoda et al., 2012).</li>
</ul>
</td>
</tr>
<tr>
<td>
<p><u>KE3</u></p>
<p>Locomotor activity, decreased</p>
</td>
<td>
<p>Strong</p>
</td>
<td>
<p>All the previous key events can decrease sexual behavior in male quail and newt. </p>
<p style="text-align:justify;">This AOP connects the CYP7B catalyzed synthesis on an important neurosteroid to a well characterized sequence of events. For instance, the involvement of dopamine in sexual behavior that in turn impacts on reproductive success is well described and undisputed (Melis et al., 1995; Hull et al., 2004). What is less characterized is the relation between 7α-hydroxypregnenolone and dopamine release. Since the neurosteroid receptor has yet to be identified, no direct interaction between 7α-hydroxypregnenolone and dopaminergic neuron has been demonstrated. It is thus possible that an intermediate event takes place in between to indirectly connect the neurosteroid to dopamine release.</p>
<p style="text-align:justify">This AOP connects the CYP7B catalyzed synthesis on an important neurosteroid to a well characterized sequence of events. For instance, the involvement of dopamine in sexual behavior that in turn impacts on reproductive success is well described and undisputed (Melis et al., 1995; Hull et al., 2004). What is less characterized is the relation between 7α-hydroxypregnenolone and dopamine release. Since the neurosteroid receptor has yet to be identified, no direct interaction between 7α-hydroxypregnenolone and dopaminergic neuron has been demonstrated. It is thus possible that an intermediate event takes place in between to indirectly connect the neurosteroid to dopamine release.</p>
<p style="text-align:justify;">In terms of structural plausibility, the brain expresses the steroidogenic enzymes required for pregnenolone synthesis, the main substrate of CYP7B. It also expresses CYP7B which synthesizes high concentration of 7α-hydroxypregnenolone in the diencephalon. This region of the brain is populated by neurons projecting into the striatum which is known to express a high quantity of D<sub>1</sub>- and D<sub>2</sub>-like dopamine receptor and control motor activity (Orgen S. et al., 1986; Mezey S. et al., 2002; Callier S. et al., 2003).</p>
<p style="text-align:justify">In terms of structural plausibility, the brain expresses the steroidogenic enzymes required for pregnenolone synthesis, the main substrate of CYP7B. It also expresses CYP7B which synthesizes high concentration of 7α-hydroxypregnenolone in the diencephalon. This region of the brain is populated by neurons projecting into the striatum which is known to express a high quantity of D<sub>1</sub>- and D<sub>2</sub>-like dopamine receptor and control motor activity (Orgen S. et al., 1986; Mezey S. et al., 2002; Callier S. et al., 2003).</p>
<p style="text-align:justify;"> </p>
<p style="text-align:justify"> </p>
<p style="text-align:justify;"><strong>Uncertainties or inconsistencie</strong></p>
<p style="text-align:justify"><strong>Uncertainties or inconsistencie</strong></p>
<p style="text-align:justify;">At present, there are no inconsistencies reported in the literature, but some gaps remain to be filled.</p>
<p style="text-align:justify">At present, there are no inconsistencies reported in the literature, but some gaps remain to be filled.</p>
<p style="text-align:justify;">The most important ones are 7α-hydroxypregnenolone receptor localization and the connection between 7α-hydroxypregnenolone and dopamine release discussed in the previous section.</p>
<p style="text-align:justify">The most important ones are 7α-hydroxypregnenolone receptor localization and the connection between 7α-hydroxypregnenolone and dopamine release discussed in the previous section.</p>
<p style="text-align:justify;">In addition, mammalian CYP7B not only catalyzes the 7α-hydroxylation of pregnenolone but also that of dehydroepiandrosterone (DHEA). Although no clear information reported this enzymatic reaction in the bird, it is plausible that CYP7B catalyzes the hydroxylation of DHEA. Thus, the phenotypic effect of CYP7B inhibition in the brain cannot be uniquely attributed to a depletion in 7α-hydroxypregnenolone. Additionally, ketoconazole is known to inhibit a variety of CYPs, which suggest that animal exposed to it are likely to have several other enzymes inhibited. It is plausible that the impacts of ketoconazole are the result of multiple CYPs inhibition that all converge towards the same phenotype. These off target effects greatly limit the investigations on 7α-hydroxypregnenolone since its concentration cannot be specifically decreased.</p>
<p style="text-align:justify">In addition, mammalian CYP7B not only catalyzes the 7α-hydroxylation of pregnenolone but also that of dehydroepiandrosterone (DHEA). Although no clear information reported this enzymatic reaction in the bird, it is plausible that CYP7B catalyzes the hydroxylation of DHEA. Thus, the phenotypic effect of CYP7B inhibition in the brain cannot be uniquely attributed to a depletion in 7α-hydroxypregnenolone. Additionally, ketoconazole is known to inhibit a variety of CYPs, which suggest that animal exposed to it are likely to have several other enzymes inhibited. It is plausible that the impacts of ketoconazole are the result of multiple CYPs inhibition that all converge towards the same phenotype. These off target effects greatly limit the investigations on 7α-hydroxypregnenolone since its concentration cannot be specifically decreased.</p>
<p style="text-align:justify;">If a CYP7B knock-out in the brain was to be performed in an animal species, 7α-hydroxyDHEA supplementation would be required to properly study 7α-hydroxypregnenolone function.</p>
<h3>Quantitative Consideration</h3>
<p>This information is not available for the moment. </p>
<p style="text-align:justify">If a CYP7B knock-out in the brain was to be performed in an animal species, 7α-hydroxyDHEA supplementation would be required to properly study 7α-hydroxypregnenolone function.</p>
<h3>Quantitative Consideration</h3>
<p>This information is not available for the moment. </p>
</div>
<!-- potential consierations, text as entered by author -->
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<!-- end of organ term -->
<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<h4>Overview for Molecular Initiating Event</h4>
<p>The binding of inhibitors to CYP7B is demonstrated <em>in vitro </em>with purified recombinant protein in presence of the inhibitor. Ligand-induced spectral changes is analyzed using spectrophotometric titration as a shift of the heme (Yantsevich et al., 2014). </p>
<p>Ketoconazole and other conazole are known to bind to CYPs preventing its enzymatic activity.</p>
<p><!--[endif]----></p>
<ul>
<li>CYP7B inhibitor (ketoconazole, 10<sup>-4</sup> M) decreased the synthesis of 7α-hydroxypregnenolone</li>
<li>CYP7B inhibitor (intracerebroventricular injection of ketoconazole) decreased the synthesis of 7α-hydroxypregnenolone in the male quail and newt brain, <em>in vivo</em> (Matsunaga et al., 2004; Rose et al., 1997; Tsutsui et al., 2008). </li>
<li>The heme prosthetic group (catalytic site) of human recombinant CYP7B thightly bound to various imidazole- and triazole-based drugs in an in vitro spectrometric titration assay. The drugs with the highest affinities were the industrial pesticides tebuconazole (0.11 μm), propiconazole (0.13 μm) and the antifungal drugs tioconazole (0.15 μm) and miconazole (0.23 μm). Voriconazole and metyrapone (non-azole compound) also interacted with CYP7B (Yantsevich et al., 2014). </li>
<p><p>It is clearly demonstrated that ketoconazole directly inhibits CYP7B (Matsunaga et al., 2004). It is expected for the other members of the conazole family to have the same effect.</p>
<p>Some other azoles such as clotrimazole can also inhibit CYP7B activity (Liu et al., 2011; Rose et al., 1997). <!--![endif]----></p>
</p>
<br>
<h4>Tebuconazole</h4>
<p><p><em>In vitro</em>, tebuconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its catalytic activity (Yantsevich et al., 2014). </p>
</p>
<br>
<h4>Propiconazole</h4>
<p><p><em>In vitro</em>, propiconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).</p>
</p>
<br>
<h4>Tioconazole</h4>
<p><p><em>In vitro</em>, tioconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).</p>
</p>
<br>
<h4>Miconazole</h4>
<p><p><em>In vitro</em>, miconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).</p>
</p>
<br>
<h4>Fluconazole</h4>
<p><p><em>In vitro</em>, fluconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).</p>
</p>
<br>
<h4>Voriconazole</h4>
<p><p><em>In vitro</em>, voriconazole was shown to bind to the catalytic site of the human recombinant CYP7B and to inhibit its activity (Yantsevich et al., 2014).</p>
</p>
<br>
<h4>Clotrimazole</h4>
<p><p>Clotrimazoles can inhibit CYP7B activity (Liu et al., 2011; Rose et al., 1997). </p>
</p>
<br>
<!-- end Evidence for Perturbation of This Event by Stressors -->
<h4>Domain of Applicability</h4>
<br>
<!-- loop to find taxonomic applicability under event -->
<p>CYP7B is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. <a href="https://www.ncbi.nlm.nih.gov/homologene/3544">https://www.ncbi.nlm.nih.gov/homologene/3544</a></p>
<div>
<p>CYP7B is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. <a href="https://www.ncbi.nlm.nih.gov/homologene/3544">https://www.ncbi.nlm.nih.gov/homologene/3544</a></p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p><strong>Site of action:</strong></p>
<h4>Key Event Description</h4>
<p><strong>Site of action:</strong></p>
<p>CYP7B is expressed in different organs including liver, prostate and brain.</p>
<p><strong>How does it work :</strong></p>
<p>CYP7B is a member of the cytochrome P450 family of enzymes. It is involved in steroidogenic pathways as well as in the synthesis of bile acids. In the brain, it is involved in neurosteroids synthesis.</p>
<p>In the brain, the reactions catalyzed by CYP7B are : </p>
<ul>
<li>Probably in all vertebrates: Pregnenolone into 7α-hydroxypregnenolone and its stereoisomer 7β-hydroxypregnenolone (bird only) (<a id="http://www.genome.jp/dbget-bin/www_bget?rn:R08943" name="http://www.genome.jp/dbget-bin/www_bget?rn:R08943">R08943</a>) (Matsunaga et al., 2004; Rose et al., 1997; Tsutsui et al., 2008)</li>
<li>Proven in mouse and human: Dehydroepiandrosterone (DHEA) to 7α-hydroxy-DHEA and its stereoisomer 7β-hydroxy-DHEA (Martin et al., 2004; Weihua et al., 2002). </li>
</ul>
<p>In the human and mouse liver, CYP7B is responsible for (Toll et al., 1994): </p>
<li>It is expressed in the chicken liver and is probably involved in the same reactions (Handschin et al., 2005). </li>
</ul>
<p>In the prostate:</p>
<ul>
<li>Proven for human and rat: Dehydroepiandrosterone (DHEA) to 7α-hydroxy-DHEA and 7β-hydroxy-DHEA (Martin et al., 2001; Martin et al., 2004). </li>
</ul>
<p>Inhibitors prevent the metabolism of pregnenolone into 7-alpha-hydroxypregnenolone, thereby decreasing the concentration of the neurosteroid. </p>
<p>To measure CYP7B activity <em>in vitro</em>, different experiments based on HPLC and GS-MS analysis can be performed.</p>
<ul>
<li>An assay in liver microsome followed by HPLC analysis of the metabolites (Souidi et al., 2000). </li>
<li>Labeled steroid conversion <em>in vitro</em> with cell or tissue extract in presence of NADPH followed by GS-MS analysis (Rose et al., 1997; Tsutsui et al., 2008). </li>
<li>CYP7B can be cloned in bacteria to produce an active protein <em>in vitro</em>. In presence of adequate precursor and cofactors, the enzymatic activity of the protein can be measured and analyzed using HPLC. </li>
<li>CYP7B can be transfected in a cell line unable to synthesize 7α-hydroxypregnenolone in order to measure with HPLC the ability of the protein to catalyze the enzymatic reaction in presence of the appropriate substrate and cofactor (Tsutsui et al., 2008)</li>
</ul>
<p><em><u>In vivo </u></em></p>
<p>Experiments may include knock-out of mice (followed by RNA, protein blotting and enzymatic activity to confirm knock-out) (Li-Hawkins et al., 2000) followed by the measurement of substrate and metabolites of CYP7B in plasma and tissues (Rose., 2001). </p>
<br>
<h4>References</h4>
<p>Dulos, J., van der Vleuten, M.A., Kavelaars, A., Heijnen, C.J., and Boots, A.M. (2005). CYP7B expression and activity in fibroblast-like synoviocytes from patients with rheumatoid arthritis: regulation by proinflammatory cytokines. Arthritis Rheum 52, 770-778.</p>
<h4>References</h4>
<p>Dulos, J., van der Vleuten, M.A., Kavelaars, A., Heijnen, C.J., and Boots, A.M. (2005). CYP7B expression and activity in fibroblast-like synoviocytes from patients with rheumatoid arthritis: regulation by proinflammatory cytokines. Arthritis Rheum 52, 770-778.</p>
<p>Handschin C., Gnerre C., Fraser DJ., Martinez-Jimenez C., Jover R., Mever UA., (2005) Species-specific mechanisms for cholesterol 7α-hydroxylase (CYP7A1) regulation by drugs and bile acids, Archives of Biochemistry and Biophysics, Vol 434-1, pp75-85</p>
<p>Haraguchi, S., Koyama, T., Hasunuma, I., Okuyama, S., Ubuka, T., Kikuyama, S., Do Rego, J.L., Vaudry, H., and Tsutsui, K. (2012). Acute stress increases the synthesis of 7alpha-hydroxypregnenolone, a new key neurosteroid stimulating locomotor activity, through corticosterone action in newts. Endocrinology 153, 794-805.</p>
<p>Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.</p>
<p>Li-Hawkins, J., Lund, E.G., Turley, S.D., and Russell, D.W. (2000). Disruption of the oxysterol 7alpha-hydroxylase gene in mice. J Biol Chem 275, 16536-16542.</p>
<p>Martin, C., Bean, R., Rose, K., Habib, F., and Seckl, J. (2001). cyp7b1 catalyses the 7alpha-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate. Biochem J 355, 509-515.</p>
<p>Martin, C., Ross, M., Chapman, K.E., Andrew, R., Bollina, P., Seckl, J.R., and Habib, F.K. (2004). CYP7B generates a selective estrogen receptor beta agonist in human prostate. J Clin Endocrinol Metab 89, 2928-2935.</p>
<p>Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.</p>
<p>Rose, K., Allan, A., Gauldie, S., Stapleton, G., Dobbie, L., Dott, K., Martin, C., Wang, L., Hedlund, E., Seckl, J.R., et al. (2001). Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation. J Biol Chem 276, 23937-23944.</p>
<p>Rose, K.A., Stapleton, G., Dott, K., Kieny, M.P., Best, R., Schwarz, M., Russell, D.W., Bjorkhem, I., Seckl, J., and Lathe, R. (1997). Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone. Proc Natl Acad Sci U S A 94, 4925-4930.</p>
<p>Souidi, M., Parquet, M., Dubrac, S., Audas, O., Becue, T., and Lutton, C. (2000). Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols. Biochim Biophys Acta 1487, 74-81.</p>
<p>Toll, A., Wikvall, K., Sudjana-Sugiaman, E., Kondo, K.H., and Bjorkhem, I. (1994). 7 alpha hydroxylation of 25-hydroxycholesterol in liver microsomes. Evidence that the enzyme involved is different from cholesterol 7 alpha-hydroxylase. Eur J Biochem 224, 309-316.</p>
<p>Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.</p>
<p>Weihua, Z., Lathe, R., Warner, M., and Gustafsson, J.A. (2002). An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci U S A 99, 13589-13594.</p>
<p>Yantsevich, A.V., Dichenko, Y.V., Mackenzie, F., Mukha, D.V., Baranovsky, A.V., Gilep, A.A., Usanov, S.A., and Strushkevich, N.V. (2014). Human steroid and oxysterol 7alpha-hydroxylase CYP7B1: substrate specificity, azole binding and misfolding of clinically relevant mutants. FEBS J 281, 1700-1713.</p>
<br>
<!-- end event text -->
</div>
<h3>List of Key Events in the AOP</h3>
<div>
<div>
<h4><a href="/events/1387">Event: 1387: 7α-hydroxypregnenolone synthesis in the brain, decreased</a><br></h4>
<h5>Short Name: 7α-hydroxypregnenolone synthesis in the brain, decreased</h5>
</div>
<h4><a href="/events/1387">Event: 1387: 7α-hydroxypregnenolone synthesis in the brain, decreased</a></h4>
<h5>Short Name: 7α-hydroxypregnenolone synthesis in the brain, decreased</h5>
<p style="text-align: justify;">The enzyme synthesizing 7α-hydroxypregnenolone is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. <a href="https://www.ncbi.nlm.nih.gov/homologene/3544">https://www.ncbi.nlm.nih.gov/homologene/3544</a></p>
<p style="text-align: justify;">The enzyme synthesizing 7α-hydroxypregnenolone is known to be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. <a href="https://www.ncbi.nlm.nih.gov/homologene/3544">https://www.ncbi.nlm.nih.gov/homologene/3544</a></p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p style="text-align: justify;">7α-hydroxypregnenolone is an active neurosteroid synthesized in the brain from pregnenolone via a reaction catalyzed by CYP7B (<a id="http://www.genome.jp/dbget-bin/www_bget?rn:R08943" name="http://www.genome.jp/dbget-bin/www_bget?rn:R08943">R08943</a>). Pregnenolone can also be synthesized in most vertebrate brain by CYP11A from cholesterol (Tsutsui and Yamazaki, 1995; do Rego et al., 2016).</p>
<h4>Key Event Description</h4>
<p style="text-align: justify;">7α-hydroxypregnenolone is an active neurosteroid synthesized in the brain from pregnenolone via a reaction catalyzed by CYP7B (<a id="http://www.genome.jp/dbget-bin/www_bget?rn:R08943" name="http://www.genome.jp/dbget-bin/www_bget?rn:R08943">R08943</a>). Pregnenolone can also be synthesized in most vertebrate brain by CYP11A from cholesterol (Tsutsui and Yamazaki, 1995; do Rego et al., 2016).</p>
<div>
<p style="text-align: justify;">Compared to other brain regions of the male quail and newt, 7α-hydroxypregnenolone concentration is higher in the diencephalon. In the brain of both salmon and newt, the peak concentrations are measured in the hypothalamus and optic tectum (Matsunaga et al., 2004; Tsutsui et al., 2008; Haraguchi et al., 2015). </p>
</div>
<p style="text-align: justify;">7α-hydroxypregnenolone synthesis in the brain is cyclic and driven by a different mechanism according to the specie.</p>
<ul>
<li style="text-align: justify;">In male quail, a diurnal animal, it is inhibited by a melatonin-receptor mechanism after melatonin secretion from the pineal gland (Tsutsui et al., 2008). </li>
<li style="text-align: justify;">In male newt, a nocturnal animal, melatonin secretion stimulates its synthesis in the brain.</li>
<li style="text-align: justify;">Another regulating mechanism is observed in male newt where 7α-hydroxypregnenolone concentration peaks during the breeding period in response to prolactin signal (Matsunaga et al., 2004). </li>
<li style="text-align: justify;">In salmon, 7α-hydroxypregnenolone stays high during homing migration (Haraguchi et al., 2015). The endogenous factor regulating its synthesis has yet to be determined.</li>
</ul>
<p style="text-align: justify;">Thus, 7α-hydroxypregnenolone synthesis is regulated by the circadian cycle and/or by seasonal factors such as breeding and migration.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Detection and quantification of 7α-hydroxypregnenolone can be performed using GC-MS and/or HPLC analysis.</p>
<h4>How it is Measured or Detected</h4>
<p style="text-align:justify">Detection and quantification of 7α-hydroxypregnenolone can be performed using GC-MS and/or HPLC analysis.</p>
<li style="text-align:justify">Cell not expressing CYP7B can be transfected with CYP7B cDNA and incubated in presence of pregnenolone and NADPH. Concentration of 7α-hydroxypregnenolone can be measured by HPLC analysis (Haraguchi et al., 2015). <!--![endif]----></li>
<li style="text-align:justify">To distinguish 7α- and 7β-hydroxypregnenolone, HPLC analysis was performed (Tsutsui et al., 2008). Brain homogenates can be incubated in presence of pregnenolone and NADPH. Concentration of 7α-hydroxypregnenolone can be measured by HPLC analysis Haraguchi et al., 2015). <!--![endif]----></li>
</ul>
<p style="text-align:justify"><em>In vivo </em></p>
<p style="text-align:justify">The extracted steroids derived from brain homogenates and plasma can be measured using GC-MS analysis (Tsutsui et a;., 2008). </p>
<p style="text-align: justify;">Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., and Tsutsui, K. (2010). Prolactin increases the synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male Newts. Endocrinology 151, 2211-2222.</p>
<h4>References</h4>
<p style="text-align: justify;">Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., and Tsutsui, K. (2010). Prolactin increases the synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male Newts. Endocrinology 151, 2211-2222.</p>
<p style="text-align: justify;">Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.</p>
<p style="text-align: justify;">Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.</p>
<p style="text-align: justify;">Petkam, R., Renaud, R.L., Freitas, A.M., Canario, A.V., Raeside, J.I., Kime, D.E., and Leatherland, J.F. (2003). In vitro metabolism of pregnenolone to 7alpha-hydroxypregnenolone by rainbow trout embryos. Gen Comp Endocrinol 131, 241-249.</p>
<p style="text-align: justify;">Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.</p>
<p style="text-align: justify;">Tsutsui, K., and Yamazaki, T. (1995). Avian neurosteroids. I. Pregnenolone biosynthesis in the quail brain. Brain Res 678, 1-9.</p>
<p style="text-align: justify;">Yau, J.L., Noble, J., Graham, M., and Seckl, J.R. (2006). Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats. J Neurosci 26, 11034-11040.</p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/1388">Event: 1388: Dopamine release in the brain, decreased</a><br></h4>
<h5>Short Name: Dopamine release in the brain, decreased</h5>
</div>
<div>
<!-- loop to find all aops that use this event -->
<p>Dopamine is used as a neurotransmitter in multicellular animals (Barron et al., 2010). Across a wide range of vertebrates, dopamine has an "activating" effect on behavior-switching and response selection, comparable to its effect in mammals. </p>
<br>
</div>
<p>Dopamine is used as a neurotransmitter in multicellular animals (Barron et al., 2010). Across a wide range of vertebrates, dopamine has an "activating" effect on behavior-switching and response selection, comparable to its effect in mammals. </p>
<!-- event text -->
<h4>Key Event Description</h4>
<p>Dopamine is a monoamine, catecholaminergic neurotransmitter synthesized in the brain and the kidney from precursor L-DOPA (Carlsson et al., 1957). It is synthesized in neuron cells, stored in vesicules nearby the synaps, and is released into the synaptic cleft after excitation of the neuron. Once released, it can bind D<sub>1</sub>-like or D<sub>2</sub>-like G protein receptor which have different effects (Stoof and Kebabia, 1984; Vallender et al., 2010).</p>
<h4>Key Event Description</h4>
<p>Dopamine is a monoamine, catecholaminergic neurotransmitter synthesized in the brain and the kidney from precursor L-DOPA (Carlsson et al., 1957). It is synthesized in neuron cells, stored in vesicules nearby the synaps, and is released into the synaptic cleft after excitation of the neuron. Once released, it can bind D<sub>1</sub>-like or D<sub>2</sub>-like G protein receptor which have different effects (Stoof and Kebabia, 1984; Vallender et al., 2010).</p>
<p>It is conserved among vertabrates and regulates neural activity, behavior and gene expression. The main impacts are related to voluntary movement, feeding, and reward. </p>
<p>In birds, fish, and other vertebrates, dopaminergic neurons located in mesencephalic region (VTA, SN) project to the telencephalon, a region of the brain rich in D<sub>1</sub> and D<sub>2</sub> receptors (Hara et al., 2007; Ball et al., 1995; Levens et al., 2000). </p>
<br>
<h4>How it is Measured or Detected</h4>
<p><em><u>In vitro</u></em></p>
<h4>How it is Measured or Detected</h4>
<p><em><u>In vitro</u></em></p>
<p>To measure the ability of a molecule to stimulate dopamine release, brain can be incubated in physiological saline in presence of a presumptive activator (e.g. 7α-hydroxypregnenolone, a neurosteroid) and dopamine concentration in saline is measured by HPLC-ECD (Matsunaga et al., 2004). </p>
<p><em><u>In vivo</u></em></p>
<p>To measure the concentration of dopamine in the brain <em>in vivo</em>, freshly collected brain can be homogenized and dopamine concentration can be analyzed using HPLC-ECD (ECD-300, Eicom).</p>
<br>
<h4>References</h4>
<p>Barron, A.B., Sovik, E., and Cornish, J.L. (2010). The roles of dopamine and related compounds in reward-seeking behavior across animal phyla. Front Behav Neurosci 4, 163.</p>
<h4>References</h4>
<p>Barron, A.B., Sovik, E., and Cornish, J.L. (2010). The roles of dopamine and related compounds in reward-seeking behavior across animal phyla. Front Behav Neurosci 4, 163.</p>
<p>Carlsson, A., Lindqvist, M., and Magnusson, T. (1957). 3,4-Dihydroxyphenylalanine and 5-hydroxytryptophan as reserpine antagonists. Nature 180, 1200.</p>
<p>Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.</p>
<p>Stoof, J.C., and Kebabian, J.W. (1984). Two dopamine receptors: biochemistry, physiology and pharmacology. Life Sci 35, 2281-2296.</p>
<p>Vallender, E.J., Xie, Z., Westmoreland, S.V., and Miller, G.M. (2010). Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs. BMC Evol Biol 10, 51.</p>
<p> </p>
<p> </p>
<br>
<!-- end event text -->
</div>
<div>
<div>
<h4><a href="/events/1390">Event: 1390: Sexual behavior, decreased</a><br></h4>
<h5>Short Name: Sexual behavior, decreased</h5>
</div>
<div>
<!-- loop to find all aops that use this event -->
<p>This key event can be applied to any animal having sexual reproduction. It does not apply to asexual animals.</p>
<br>
</div>
<p>This key event can be applied to any animal having sexual reproduction. It does not apply to asexual animals.</p>
<!-- event text -->
<h4>Key Event Description</h4>
<p>Sexual behavior in male bird is characterized by components such as crowing, strutting, and mounting, whereas the newt exhibits a tail-vibrating behavior (Hutchison, 1978). In both species, sexual behavior varies on a daily (photoperiod) and seasonal (breeding) basis. A decrease in sexual behavior is defined by a reduction in the frequency of these typical behaviors.</p>
<h4>Key Event Description</h4>
<p>Sexual behavior in male bird is characterized by components such as crowing, strutting, and mounting, whereas the newt exhibits a tail-vibrating behavior (Hutchison, 1978). In both species, sexual behavior varies on a daily (photoperiod) and seasonal (breeding) basis. A decrease in sexual behavior is defined by a reduction in the frequency of these typical behaviors.</p>
<p> </p>
<br>
<h4>How it is Measured or Detected</h4>
<p>Since sexual behavior varies along the day and the season, timing is an important component of the measurement. Light exposure, endocrine disruptors and season should all be considered in the protocol design in order to limit the bias in the measurement.</p>
<h4>How it is Measured or Detected</h4>
<p>Since sexual behavior varies along the day and the season, timing is an important component of the measurement. Light exposure, endocrine disruptors and season should all be considered in the protocol design in order to limit the bias in the measurement.</p>
<p>Sexual behavior in male is measured in presence of a sexually receptive female. To limit the risk of bias induced by differences in female receptivity, it is important to repeat the experiment later/the day after with a different female for each male (Halldin et al., 1999).In bird, the frequency of chasing, pecking, head grabbing, and mounting for a X minutes observation can be measured (Halldin et al., 1999; Ogura et al., 2016). </p>
<p><!--![endif]----></p>
<p><!--[endif]----></p>
<p>For newt, sexual behavior is characterized by a tail-vibrating behavior and can be measured by counting the frequency and incidence of this behavior during X minutes. Incidence and frequency are expressed as the percentage of animals exhibiting the behavior and the mean number of times the behavior was recorded per test animal over the test period, respectively (Toyoda et al., 1983). </p>
<p><!--![endif]----></p>
<br>
<h4>References</h4>
<p>Adkins, E. K. and N. T. Adler. 1972. Hormonal control of behavior in the Japanese quail. J. Comp. Physiol. Psychol.81:27-36.</p>
<h4>References</h4>
<p>Adkins, E. K. and N. T. Adler. 1972. Hormonal control of behavior in the Japanese quail. J. Comp. Physiol. Psychol.81:27-36.</p>
<p>Halldin, K., Berg, C., Brandt, I., and Brunstrom, B. (1999). Sexual behavior in Japanese quail as a test end point for endocrine disruption: effects of in ovo exposure to ethinylestradiol and diethylstilbestrol. Environ Health Perspect 107, 861-866.</p>
<p>Hutchison, R.E. (1978). Hormonal differentiation of sexual behavior in Japanese quail. Horm Behav 11, 363-387.</p>
<p>Ogura, Y., Haraguchi, S., Nagino, K., Ishikawa, K., Fukahori, Y., and Tsutsui, K. (2016). 7alpha-Hydroxypregnenolone regulates diurnal changes in sexual behavior of male quail. Gen Comp Endocrinol 227, 130-135.</p>
<p>Sachs, B.D. (1967). Photoperiodic control of the cloacal gland of the Japanese quail. Science 157, 201-203.</p>
<p>Toyoda, F., Ito, M., Tanaka, S., and Kikuyama, S. (1993). Hormonal induction of male courtship behavior in the Japanese newt, Cynops pyrrhogaster. Horm Behav 27, 511-522.</p>
<td><a href="/aops/23">Aop:23 - Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/25">Aop:25 - Aromatase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/29">Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/30">Aop:30 - Estrogen receptor antagonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/100">Aop:100 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of female spawning behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/122">Aop:122 - Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/123">Aop:123 - Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/101">Aop:101 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of pheromone release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/102">Aop:102 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with meiotic prophase I /metaphase I transition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/63">Aop:63 - Cyclooxygenase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/103">Aop:103 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with spindle assembly checkpoint</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/292">Aop:292 - Inhibition of tyrosinase leads to decreased population in fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/16">Aop:16 - Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/312">Aop:312 - Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/334">Aop:334 - Glucocorticoid Receptor Agonism Leading to Impaired Fin Regeneration</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/336">Aop:336 - DNA methyltransferase inhibition leading to population decline (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/337">Aop:337 - DNA methyltransferase inhibition leading to population decline (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/338">Aop:338 - DNA methyltransferase inhibition leading to population decline (3)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/339">Aop:339 - DNA methyltransferase inhibition leading to population decline (4)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/340">Aop:340 - DNA methyltransferase inhibition leading to transgenerational effects (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/341">Aop:341 - DNA methyltransferase inhibition leading to transgenerational effects (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/297">Aop:297 - Inhibition of retinaldehyde dehydrogenase leads to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/389">Aop:389 - Oxygen-evolving complex damage leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/97">Aop:97 - 5-hydroxytryptamine transporter (5-HTT; SERT) inhibition leading to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/203">Aop:203 - 5-hydroxytryptamine transporter inhibition leading to decreased reproductive success and population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/218">Aop:218 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/219">Aop:219 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased sexual behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/567">Aop:567 - Binding to plastoquinone B site leading to decreased population growth rate via photosystem II inhibition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBDPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<p>Consideration of population size and changes in population size over time is potentially relevant to all living organisms.</p>
<h4>Key Event Description</h4>
<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>
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<h4>How it is Measured or Detected</h4>
<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="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>
<h4>Regulatory Significance of the AO</h4>
<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>
<h4>References</h4>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MD, Hartig PC, Gray LE. 2003. Effects of the androgenic growth promoter 17b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ. Toxicol. Chem. 22: 1350–1360.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Cheng WY, Zhang Q, Schroeder A, Villeneuve DL, Ankley GT, Conolly R. 2016. Computational modeling of plasma vitellogenin alterations in response to aromatase inhibition in fathead minnows. Toxicol Sci 154: 78–89.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Conolly RB, Ankley GT, Cheng W-Y, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51: 4661-4672.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Etterson MA, Ankley GT. 2021. Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55: 15596-15608. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Holbech H, Kinnberg KL, Brande-Lavridsen N, Bjerregaard P, Petersen GI, Norrgren L, Orn S, Braunbeck T, Baumann L, Bomke C, Dorgerloh M, Bruns E, Ruehl-Fehlert C, Green JW, Springer TA, Gourmelon A. 2012 Comparison of zebrafish (<em>Danio rerio</em>) and fathead minnow <em>(Pimephales promelas</em>) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155: 407–415.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT. </span><span style="color:black">2011. Adverse outcome pathways and risk assessment: Bridging to population level effects. Environ. Toxicol. Chem. 30, 64-76.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">McComb B, Zuckerberg B, Vesely D, Jordan C. 2021. Monitoring Animal Populations and their Habitats: A Practitioner's Guide. Pressbooks, Oregon State University, Corvallis, OR Version 1.13, 296 pp. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Villeneuve DL, Santana Rodriguez KJ, Ankley GT. 2022. A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. </span><span style="color:black">Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada. Environmental Toxicology and Chemistry 34(7): 1623-1633.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. </span><span style="color:black">Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (<em>Pimephales promelas</em>). Environ Toxicol Chem 26: 521–527.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (<em>Pimephales promelas</em>) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59: 1–9.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murphy CA, Nisbet RM, Antczak P, Garcia-Reyero N, Gergs A, Lika K, Mathews T, Muller EB, Nacci D, Peace A, Remien CH, Schultz IR, Stevenson LM, Watanabe KH. 2018. Incorporating suborganismal processes into dynamic energy budget models for ecological risk assessment. Integrated Environmental Assessment and Management 14(5): 615–624.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murray DL, Sandercock BK (editors). 2020. Population ecology in practice. Wiley-Blackwell, Oxford UK, 448 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Jusup M, Klanjscek T, Pecquerie L. 2011. Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models. The Journal of Experimental Biology 215: 892-902.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. </span><span style="color:black">From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69: 913–926.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Perkins EJ, Ashauer R, Burgoon L, Conolly R, Landesmann B,, Mackay C, Murphy CA, Pollesch N, Wheeler JR, Zupanic A, Scholzk S. 2019. Building and applying quantitative adverse outcome pathway models for chemical hazard and risk assessment. Environmental Toxicology and Chemistry 38(9): 1850–1865. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Vandermeer JH, Goldberg DE. 2003. Population ecology: first principles. Princeton University Press, Princeton NJ, 304 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lattieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L, Whelan M. Adverse outcome pathway (AOP) development 1: Strategies and principles. Toxicol Sci. 2014: 142:312–320</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ. 2016. Predicting fecundity of fathead minnows (<em>Pimephales promelas</em>) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11: e0146594.</span></span></span></li>
</ul>
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</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<div id="evidence_supporting_links">
<hr>
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/1493">Relationship: 1493: CYP7B activity, inhibition leads to 7α-hydroxypregnenolone synthesis in the brain, decreased</a></h4>
<p>The vertebrate brain expresses all the enzymes involved in the different steroidogenic pathways (do Rego and Vaudry, 2016; Tsutsui et al., 1999).</p>
</div>
<p>The vertebrate brain expresses all the enzymes involved in the different steroidogenic pathways (do Rego and Vaudry, 2016; Tsutsui et al., 1999).</p>
<p>The physiological function of 7α-hydroxypregnenolone is more understood in birds, newts, and rats than in human. However, the direct causal effect between CYP7B inhibition and the decrease in 7α-hydroxyPREG was demonstrated in human, fish and other vertebrates (Haraguchi et al., 2015; Yantsevich et al., 2014; Yau et al., 2006). </p>
<p>Therefore, it is plausible that this KER is applicable to all vertebrates. </p>
<h4>Key Event Relationship Description</h4>
<p>Neurosteroids are steroids synthesized in the brain that interact with cell surface receptors or ligand-gated ion channels in order to modify the neuronal excitability (Paul and Purdy, 1992). They are involved in numerous biological functions including locomotor activity, memory, learning, sexually-dimorphic behaviors and anxiety.</p>
<h4>Key Event Relationship Description</h4>
<p>Neurosteroids are steroids synthesized in the brain that interact with cell surface receptors or ligand-gated ion channels in order to modify the neuronal excitability (Paul and Purdy, 1992). They are involved in numerous biological functions including locomotor activity, memory, learning, sexually-dimorphic behaviors and anxiety.</p>
<p>Neurosteroids are synthesized from pregnenolone or its derivatives by different cytochromes P450. Among these CYPs is CYP7B hydroxylase which synthesizes the neurosteroid 7α-hydroxypregnenolone. CYP7B is the only enzyme responsible for the synthesis of this neurosteroid. Therefore, its inhibition induces a decrease in 7α-hydroxypregnenolone concentration in the brain.</p>
<p>The expression of CYP7B and the synthesis of its molecular product vary cyclically on a daily and/or seasonal basis. In male quail, a diurnal animal, CYP7B expression and 7α-hydroxypregnenolone are inhibited by melatonin secretion, a hormone involved in circadian rhythm and sleep regulation. Oppositely, in a nocturnal animal such a newt, melatonin acts as an inducer of CYP7B expression and 7α-hydroxypregnenolone synthesis. These results indicate that CYP7B expression and therefore 7α-hydroxypregnenolone synthesis follow a circadian rhythm regulation. </p>
<p>In addition to this daily variation, CYP7B and its product are regulated by seasons in salmon and male newt where it peaks during homing migration (salmon) and breeding (newt) period (Haraguchi et al., 2009). It is plausible that the same seasonal variation occurs in avian.</p>
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<h4>Evidence Supporting this KER</h4>
<p> </p>
<h4>Evidence Supporting this KER</h4>
<p> </p>
<p> </p>
<p> </p>
<strong>Biological Plausibility</strong>
<p>The vertebrate brain expresses all the enzymes involved in the different steroidogenic pathways, including CYP7B (review do Rego and Vaudry, 2016; Tsutsui and Yamazaki, 1995). These enzymes in the brain are known to convert cholesterol into pregnenolone, the precursor of 7α-hydroxypregnenolone Therefore, the brain possesses both the molecular precursor and the enzyme required to synthesized 7α-hydroxypregnenolone. Since CYP7B is the only enzyme known to synthesize 7α-hydroxypregnenolone, its inhibition is assumed to decrease 7α-hydroxypregnenolone concentration in the brain.</p>
<strong>Biological Plausibility</strong>
<p>The vertebrate brain expresses all the enzymes involved in the different steroidogenic pathways, including CYP7B (review do Rego and Vaudry, 2016; Tsutsui and Yamazaki, 1995). These enzymes in the brain are known to convert cholesterol into pregnenolone, the precursor of 7α-hydroxypregnenolone Therefore, the brain possesses both the molecular precursor and the enzyme required to synthesized 7α-hydroxypregnenolone. Since CYP7B is the only enzyme known to synthesize 7α-hydroxypregnenolone, its inhibition is assumed to decrease 7α-hydroxypregnenolone concentration in the brain.</p>
<p>In the quail brain, the precise localization of CYP7B protein was explored and the results were as followed: nucleus preopticus medialis (POM), the nucleus paraventricularis magnocellularis (PVN), the nucleus ventrodedialis hypothalami (VMN), the nucleus dorsolateralis anterior thalami (DLA) and the nucleus lateralis anterior thalami (LA) (Tsutsui et al., 2008).</p>
<p>In the salmon, cells expressing CYP7B are mainly localized in the magnocellular preoptic nucleus, oculomotor nucleus, nucleus lateralis valvulae, and nucleus lateralis valvulae (Haraguchi et al., 2015). </p>
<p>In the newt brain, CYP7B cells are mainly localized in the anterior preoptic area, the magnocellular preoptic nucleus, and the tegmental area. It was also detected in the lateral and dorsal pallium, the suprachiasmatic nucleus, the ventral hypothalamic nucleus, and the tectum mesencephali (Haraguchi et al., 2010).</p>
<strong>Empirical Evidence</strong>
<ul>
<strong>Empirical Evidence</strong>
<ul>
<li>CYP7B inhibitor (ketoconazole, 10<sup>-4</sup> M) decreased the synthesis of 7α-hydroxypregnenolone <em>in vitro </em>(Tsutsui et al., 2008; Matsunaga et al., 2008; Toyoda et al., 2012).</li>
<li>CYP7B inhibitor (intracerebroventricular injection of ketoconazole, 5 μg, from 5 AM to 6 AM) decreased the synthesis of 7α-hydroxypregnenolone in the brain (quail) <em>in vivo </em>(Tsutsui et al., 2008).</li>
<li>CYP7B activity is regulated (inhibited) by melatonin in male quail. When male quail brains were injected (intracerebroventricular) with a melatonin receptor antagonist (luzindole), the production of 7α-hydroxypregnenolone significantly increased (Tsutsui et al., 2008). The opposite effect was observed on newt where melatonin stimulated 7α-hydroxypregnenolone synthesis (Koyama et al., 2009). </li>
<li>Similarly, orbital enucleation and pinealectomy performed on male quail, which abolished melatonin synthesis, induces a significant increase in 7α-hydroxypregnenolone concentration (Tsutsui et al., 2008). </li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p> </p>
<strong>Uncertainties and Inconsistencies</strong>
<p>do Rego, J.L., and Vaudry, H. (2016). Comparative aspects of neurosteroidogenesis: From fish to mammals. Gen Comp Endocrinol 227, 120-129.</p>
<h4>References</h4>
<p>do Rego, J.L., and Vaudry, H. (2016). Comparative aspects of neurosteroidogenesis: From fish to mammals. Gen Comp Endocrinol 227, 120-129.</p>
<p>Haraguchi, S., Matsunaga, M., Koyama, T., Do Rego, J.L., and Tsutsui, K. (2009). Seasonal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts. Ann N Y Acad Sci 1163, 410-413.</p>
<p>Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., and Tsutsui, K. (2010). Prolactin increases the synthesis of 7α-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male newts. <em>Endocrinology</em> 151, 2211–2222.</p>
<p>Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.</p>
<p>Koyama, T., Haraguchi, S., Vaudry, H., and Tsutsui, K. (2009). Diurnal changes in the synthesis of the neurosteroid 7alpha-hydroxypregnenolone stimulating locomotor activity in newts. Ann N Y Acad Sci 1163, 444-447.</p>
<p>Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., and Haraguchi, S. (2008). 7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J Neurosci 28, 2158-2167.</p>
<p>Tsutsui, K., Ukena, K., Takase, M., Kohchi, C., and Lea, R.W. (1999). Neurosteroid biosynthesis in vertebrate brains. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 124, 121-129.</p>
<p>Tsutsui, K., and Yamazaki, T. (1995). Avian neurosteroids. I. Pregnenolone biosynthesis in the quail brain. Brain Res 678, 1-9.</p>
<p>Yantsevich, A.V., Dichenko, Y.V., Mackenzie, F., Mukha, D.V., Baranovsky, A.V., Gilep, A.A., Usanov, S.A., and Strushkevich, N.V. (2014). Human steroid and oxysterol 7alpha-hydroxylase CYP7B1: substrate specificity, azole binding and misfolding of clinically relevant mutants. FEBS J 281, 1700-1713.</p>
<p>Yau, J.L., Noble, J., Graham, M., and Seckl, J.R. (2006). Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats. J Neurosci 26, 11034-11040.</p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/1496">Relationship: 1496: 7α-hydroxypregnenolone synthesis in the brain, decreased leads to Dopamine release in the brain, decreased</a></h4>
<p style="text-align:justify">7α-hydroxypregnenolone is synthesized in the diencephalon and the rhombencephalon (newt only) and has a paracrine effect on dopaminergic neurons that project into the telencephalon including the striatum. </p>
</div>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify">7α-hydroxypregnenolone is synthesized in the diencephalon and the rhombencephalon (newt only) and has a paracrine effect on dopaminergic neurons that project into the telencephalon including the striatum. </p>
<p style="text-align:justify"> </p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<p> </p>
<h4>Evidence Supporting this KER</h4>
<p> </p>
<p> </p>
<strong>Biological Plausibility</strong>
<p>7α-hydroxypregnenolone cannot be directly related to dopamine release since it has no known receptor and the cells that synthesize it are not in direct contact with the dopaminergic neurons. However, it was shown that 7α-hydroxypregnenolone release induces dopamine secretion in the brain.</p>
<strong>Biological Plausibility</strong>
<p>7α-hydroxypregnenolone cannot be directly related to dopamine release since it has no known receptor and the cells that synthesize it are not in direct contact with the dopaminergic neurons. However, it was shown that 7α-hydroxypregnenolone release induces dopamine secretion in the brain.</p>
<p> </p>
<strong>Empirical Evidence</strong>
<ul>
<strong>Empirical Evidence</strong>
<ul>
<li style="text-align:justify">7α-hydroxypregnenolone concentration in the brain of salmon prior to upstream migration was decreased using aminoglutethimide (AG) injection (CYP11A inhibitor) and lead to a decreased dopamine concentration in the telencephalon compared to control. This effect was rescued with intracerebroventricular injection of 7α-hydroxypregnenolone. Simultaneous measurements of hypothalamic dopamine concentration showed an absence of variation after AG-injection or AG+7 PREG (Haraguchi et al., 2015)</li>
</ul>
<ul>
<li style="text-align:justify">Tyrosine hydroxylase (TH) is a marker of dopamine neurons. Immunolabelling of CYP7B and TH revealed that these enzymes are expressed in two different cell populations in the salmon magnocellular preoptic nucleus and that they are in close proximity to each other (Haraguchi et al., 2015).</li>
</ul>
<ul>
<li style="text-align:justify">7α-hydroxypregnenolone (1 ng) was injected in non-breeding male newt and several monoamines concentration were measured using HPLC-electrochemical detection. 7α-hydroxypregnenolone increased dopamine concentration in the rostral brain region including striatum. No change was observed in the concentration of any other monoamine (Matsunaga et al., 2004).</li>
</ul>
<ul>
<li style="text-align:justify">Newt brain incubated<em> in vitro</em> with 7α-hydroxypregnenolone (0, 10<sup>−8</sup>, 10<sup>−7</sup>, or 10<sup>−6</sup> M) induced a dose-dependent increase of dopamine concentration after 10 minutes (Matsunaga et al., 2004).</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p>Since the neurosteroid receptor has yet to be identified, no direct interaction between 7α-hydroxypregnenolone and dopaminergic neuron has been demonstrated. It is thus possible that an intermediate event takes place in between to indirectly connect the neurosteroid to dopamine release.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Since the neurosteroid receptor has yet to be identified, no direct interaction between 7α-hydroxypregnenolone and dopaminergic neuron has been demonstrated. It is thus possible that an intermediate event takes place in between to indirectly connect the neurosteroid to dopamine release.</p>
<p style="text-align:justify">Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.</p>
<h4>References</h4>
<p style="text-align:justify">Haraguchi, S., Yamamoto, Y., Suzuki, Y., Hyung Chang, J., Koyama, T., Sato, M., Mita, M., Ueda, H., and Tsutsui, K. (2015). 7alpha-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon. Sci Rep 5, 12546.</p>
<p style="text-align:justify">Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.</p>
<p style="text-align:justify"> </p>
</div>
<br>
<div>
<div>
<h4><a href="/relationships/1499">Relationship: 1499: Sexual behavior, decreased leads to Decreased, Reproductive Success</a></h4>
<td><a href="/aops/218">Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/aops/219">Inhibition of CYP7B activity leads to decreased reproductive success via decreased sexual behavior</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<br>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/1498">Relationship: 1498: 7α-hydroxypregnenolone synthesis in the brain, decreased leads to Sexual behavior, decreased</a></h4>
<p>Gonadal steroid and prolactin are required to initiate a cascade of molecular events leading to sexual behavior. The cumulation of these reactions leads to the secretion of 7α-hydroxypregnenolone in the brain, a neurosteroid that acts as a trigger on sexual behavior.</p>
</div>
<h4>Key Event Relationship Description</h4>
<p>Gonadal steroid and prolactin are required to initiate a cascade of molecular events leading to sexual behavior. The cumulation of these reactions leads to the secretion of 7α-hydroxypregnenolone in the brain, a neurosteroid that acts as a trigger on sexual behavior.</p>
<p>7α-hydroxypregnenolone is synthesized by CYP7B in the telencephalon of both male and female. Its concentration fluctuates in the male brain according to season and light exposure, whereas it remains low in female. In agreement with the variation in 7α-hydroxypregnenolone concentration is the variation in sexual behavior frequency in male. Indeed, it was previously noted that 7α-hydroxypregnenolone peaked during the breeding period, increasing locomotor activity and frequency of tail-vibrating behavior (newt) or chasing, crowing, strutting, and mounting (bird).</p>
<p>Other components are required to induce sexual behavior since injection of 7α-hydroxypregnenolone in sexually immature newt has no effect on this specific parameter. However, no sexual behavior can be elicited in absence of 7α-hydroxypregnenolone.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<p> </p>
<h4>Evidence Supporting this KER</h4>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<strong>Biological Plausibility</strong>
<p style="text-align:justify">It is known that 7α-hydroxypregnenolone is secreted in the diencephalon. However, the fine-tuning mechanism of its regulation is still undetermined.</p>
<strong>Biological Plausibility</strong>
<p style="text-align:justify">It is known that 7α-hydroxypregnenolone is secreted in the diencephalon. However, the fine-tuning mechanism of its regulation is still undetermined.</p>
<p style="text-align:justify">In newt, the neurosteroid secretion is driven by melatonin and prolactin, an important circadian and breeding regulator, respectively (Matsunaga et al., 2004; Tsutsui et al., 2008). Prolactin is synthetized in the hypophysis and is one of the molecules required to initiate the cascade of molecular events leading to sexual behavior in newt (Kikuyama et al., 1980). In male newt, it induces a dose-dependent activation of sexual behavior. Immunofluorescence experiments conducted on newt brain sections revealed the presence of prolactin receptors on neurons expressing CYP7B, which could explain the direct correlation between prolactin and 7α-hydroxypregnenolone secretion in relation to sexual behavior.</p>
<p style="text-align:justify">Quail are highly sensitive to light exposure and their behavior mostly relies on circadian rhythm. Melatonin secretion, high during the night, is known to inhibit CYP7B activity in male brain, which, in turn, decreases 7α-hydroxypregnenolone concentration. Following the same pattern, sexual behavior in male quail is high during the day and significiantly lower at night.</p>
<p style="text-align:justify">A decrease in sexual behavior can be induced by steroid hormones deregulation since their involvement in the regulation of sexual behavior is prominent. Indeed, castration of male bird induces a decrease/loss of reproductive behavior, which can be rescued by a testosterone therapy (Adkins and Adler, 1972). The same effect can be induced in bird by transferring them from a 16-hr.-light 8-hr.-dark cycle to an 8-hr.- light 16-hr.-dark cycle, which demonstrates the photoperiodic regulation of sexual behavior in bird (Sach, 1967). </p>
<strong>Empirical Evidence</strong>
<ul>
<strong>Empirical Evidence</strong>
<ul>
<li>In male newt, 7α-hydroxypregnenolone concentration in the brain peaks in April and October, similarly to their sexual behavior (Haraguchi et al., 2010).</li>
</ul>
<ul>
<li>Intracranial injection of 7α-hydroxypregnenolone in male newt increased tail-vibration (the newt sexual behavior) incidence and frequency in a dose-dependent manner (Toyoda et al., 2012). In male quail, sexual behavior (frequency of chasing, pecking, head grabbing, mounting, and cloacal contact movement) was also increased in a dose-dependent manner following 7α-hydroxypregnenolone injection (Ogura et al., 2016).</li>
</ul>
<ul>
<li>Ketoconazole, a CYP7B inhibitor, prevented 7α-hydroxypregnenolone synthesis <em>in vivo</em> and abolished sexual behavior when given to male quail and newt. The behavior in ketoconazole-treated animals was successfully retrieved with 7α-hydroxypregnenolone supplementation or after ketoconazole treatment was stopped (Toyoda et al., 2012; Ogura et al., 2016).</li>
</ul>
<p>A decrease in sexual behavior can be induced by steroid hormones deregulation since their involvement in the regulation of sexual behavior is prominent. Indeed, castration of male bird induces a decrease/loss of reproductive behavior, which can be rescued by a testosterone therapy (Adkins and Adler, 1972). The same effect can be induced in bird by transferring them from a 16-hr.-light 8-hr.-dark cycle to an 8-hr.- light 16-hr.-dark cycle, which demonstrates the photoperiodic regulation of sexual behavior in bird (Sach, 1967). </p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Courtship and sexual behavior is due to the synergistic effect of multiple hormones (Iwata et al., 2000). 7α-hydroxypregnenolone is one of them and it was shown to be essential for sexual behavior, but it is not sufficient in itself to trigger sexual behavior in absence of prolactin (secreted in sexually mature animals during the breeding season). </p>
<strong>Uncertainties and Inconsistencies</strong>
<p>Courtship and sexual behavior is due to the synergistic effect of multiple hormones (Iwata et al., 2000). 7α-hydroxypregnenolone is one of them and it was shown to be essential for sexual behavior, but it is not sufficient in itself to trigger sexual behavior in absence of prolactin (secreted in sexually mature animals during the breeding season). </p>
<p>Kikuyama, S., Yamamoto, K., Seki T. (1980). Prolactin and its role in growth, metamorphosis and reproduction in amphibians, Gunma Symp. Endocrinol., 17, 3–13</p>
<h4>References</h4>
<p>Kikuyama, S., Yamamoto, K., Seki T. (1980). Prolactin and its role in growth, metamorphosis and reproduction in amphibians, Gunma Symp. Endocrinol., 17, 3–13</p>
<p>Matsunaga, M., Ukena, K., Baulieu, E.E., and Tsutsui, K. (2004). 7alpha-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc Natl Acad Sci U S A 101, 17282-17287.</p>