<td>Under development: Not open for comment. Do not cite</td>
<td>Under Development</td>
<td>1.90</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="coaches">
<h2>Coaches</h2>
<ul>
<li class="contributor" id="coach_33">
Judy Choi
</li>
<li class="contributor" id="coach_44">
Shihori Tanabe
</li>
</ul>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p>This AOP links Androgen receptor antagonism during fetal life with short anogenital distance (AGD) in male offspring. A short AGD around birth is a marker for feminization of male fetuses and is associated with male reproductive disorders, including reduced fertility in adulthood. Although a short AGD is not necessarily ‘adverse’ from a human health perspective, it is considered an ‘adverse outcome’ in OECD test guidelines; AGD measurements are mandatory in specific tests for developmental and reproductive toxicity in chemical risk assessment (TG 443, TG 421/422, TG 414).</p>
<p>The AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species. Its main ligand is testosterone and dihydrotestosterone (DHT). Under normal physiological conditions, testosterone produced mainly by the testicles, is converted in peripheral tissues by 5α-reductase into DHT, which in turn binds AR and activates downstream target genes. AR signaling is necessary for normal masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in male fetuses. The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.</p>
<p>The key events in this pathway is antagonism of the AR in target cells of the primitive perineal region, which leads to inactivation of the AR and failure to properly masculinize the perineum/LABC complex. In this instance, the local levels of testosterone or DHT may be normal, but prevented from binding the AR.</p>
<p>Intrauterine exposure in rats can result in shorter male AGD in male offspring as reported in:</p>
<p>Bowman et al (2003), Toxicol Sci 74:393-406; doi: 10.1093/toxsci/kfg128</p>
<p>Christiansen et al (2009), Environ Health Perspect 117:1839-1846; doi: 10.1289/ehp.0900689</p>
<p>Schwartz et al (2019), Toxicol Sci 169:303-311; doi: 10.1093/toxsci/kfz046</p>
<p> </p>
<p> </p>
<h4>Flutamide</h4>
<p>Finasteride is a selective androgen receptor (AR) antagonist (Simard et al 1986) that has been shown to induce shorter male AGD in rats after in utero exposure (Foster & Harris 2005; Hass et al 2007; Kita et al 2016; McIntyre et al 2001; Mylchreest et al 1999; Scott et al 2007; Welsh et al 2007).</p>
<p> </p>
<p><strong>References:</strong></p>
<p>Foster & Harris (2005), Toxicol Sci 85:1024-1032; doi: 10.1093/toxsci/kfi159</p>
<p>Hass et al (2007), Environ Health Perspect 115(suppl 1):122-128; doi: 10.1289/ehp.0360</p>
<p>Kita et al (2016), Toxicology 368-369:152-161; doi: 10.1016/j.tox.2016.08.021</p>
<p>McIntyre et al (2001), Toxicol Sci 62:236-249; doi: 10.1093/toxsci/62.2.236</p>
<p>Mylchreest et al (1999), Toxicol Appl Pharmacol 156:81-95; doi: 10.1006/taap.1999.8643</p>
<p>Scott et al (2007), Endocrinology 148:2027-2036; doi: 10.1210/en.2006-1622</p>
<p>Simard et al (1986), Mol Cell Endocrinol 44:261-270; doi: 10.1016/0303-7207(86)90132-2</p>
<p><span style="font-family:calibri,sans-serif; font-size:11.0pt">1. Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U and <strong>Svingen T</strong> (2019), Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. <em>Arch Toxicol</em> 93: 253-272.</span></p>
<p>A large number of drugs and chemicals have been shown to antagonise the AR using various AR reporter gene assays. The AR is specifically targeted in AR-sensitive cancers, for example the use of the anti-androgenic drug flutamide in treating prostate cancer (<a href="#_ENREF_1" title="Alapi, 2006 #262">Alapi & Fischer, 2006</a>). Flutamide has also been used in several rodent in vivo studies showing anti-androgenic effects (feminization of male offspring) evident by e.g. short anogenital distance (AGD) in males (<a href="#_ENREF_4" title="Foster, 2005 #53">Foster & Harris, 2005</a>; <a href="#_ENREF_5" title="Hass, 2007 #76">Hass et al, 2007</a>; <a href="#_ENREF_8" title="Kita, 2016 #34">Kita et al, 2016</a>). QSAR models can predict AR antagonism for a wide range of chemicals, many of which have shown in vitro antagonistic potential (<a href="#_ENREF_17" title="Vinggaard, 2008 #263">Vinggaard et al, 2008</a>).</p>
<h4>Triticonazole</h4>
<p><p>Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 uM and an IC50 of 0.3 (±0.01) uM (<a href="#_ENREF_2" title="Draskau, 2019 #258">Draskau et al, 2019</a>)</p>
<p> </p>
</p>
<h4>Flusilazole</h4>
<p><p>Using hAR-EcoScreen Assay, flusilazole showed a LOEC for antagonisms of 0.8 uM and an IC50 of 2.8 (±0.1) uM (<a href="#_ENREF_2" title="Draskau, 2019 #258">Draskau et al, 2019</a>).►</p>
</p>
<h4>Epoxiconazole</h4>
<p><p>Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 uM and an IC50 of 10 uM (<a href="#_ENREF_5" title="Kjærstad, 2010 #259">Kjærstad et al, 2010</a>)</p>
</p>
<h4>Prochloraz</h4>
<p><p>Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 uM and an IC50 of 13 uM (<a href="#_ENREF_5" title="Kjærstad, 2010 #259">Kjærstad et al, 2010</a>)</p>
</p>
<h4>Propiconazole</h4>
<p><p>Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 uM and an IC50 of 18 uM (<a href="#_ENREF_5" title="Kjærstad, 2010 #259">Kjærstad et al, 2010</a>)</p>
</p>
<h4>Tebuconazole</h4>
<p><p>Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 uM and an IC50 of 8.1 uM (<a href="#_ENREF_5" title="Kjærstad, 2010 #259">Kjærstad et al, 2010</a>)</p>
</p>
<h4>Flutamide</h4>
<p><p>Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 uM (<a href="#_ENREF_11" title="Sonneveld, 2005 #260">Sonneveld et al, 2005</a>).</p>
</p>
<h4>Cyproterone acetate</h4>
<p><p>Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM (<a href="#_ENREF_11" title="Sonneveld, 2005 #260">Sonneveld et al, 2005</a>).</p>
</p>
<h4>Vinclozolin</h4>
<p><p>Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 uM (<a href="#_ENREF_11" title="Sonneveld, 2005 #260">Sonneveld et al, 2005</a>).</p>
<p>Both the DNA-binding and ligand-binding domains of the AR are highly evolutionary conserved, whereas the transactivation domain show more divergence which may affect AR-mediated gene regulation across species (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutations studies from both humans and rodents showing strong correlation for AR-dependent development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>).</p>
<p>Both the DNA-binding and ligand-binding domains of the AR are highly evolutionary conserved, whereas the transactivation domain show more divergence which may affect AR-mediated gene regulation across species (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutations studies from both humans and rodents showing strong correlation for AR-dependent development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>). </p>
<p>This KE is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across taxa</p>
<p>This KE is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across mammalian taxa. <span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">It is, however, acknowledged that this KE most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.</span></span></span></p>
<h4>Key Event Description</h4>
<p><u>The androgen receptor (AR) and its function</u></p>
<p>Development of the male reproductive system and secondary male characteristics is dependent on androgens (foremost testosterone (T) and dihydrotestosterone (DHT). T and the more biologically active DHT act by binding to the AR (<a href="#_ENREF_4" title="MacLean, 1993 #251">MacLean et al, 1993</a>; <a href="#_ENREF_5" title="MacLeod, 2010 #27">MacLeod et al, 2010</a>; <a href="#_ENREF_8" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>), with human AR mutations and mouse knock-out models having established its pivotal role in masculinization and spermatogenesis (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>). The AR is a ligand-activated transcription factor belonging to the steroid hormone nuclear receptor family (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). The AR has three domains; the N-terminal domain, the DNA-binding domain and the ligand-binding domain, with the latter being most evolutionary conserved. Apart from the essential role AR plays for male reproductive development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>), the AR is also expressed in many other tissues and organs such as bone, muscles, ovaries and the immune system (<a href="#_ENREF_7" title="Rana, 2014 #253">Rana et al, 2014</a>). </p>
<p><span style="font-size:12.0pt">The AR is a ligand-activated transcription factor belonging to the steroid hormone nuclear receptor family (</span><span style="font-size:11.0pt"><a href="https://aopwiki.org/events/26#_ENREF_1" title="Davey, 2016 #250"><span style="font-size:12.0pt"><span style="color:#337ab7">Davey & Grossmann, 2016</span></span></a></span><span style="font-size:12.0pt">). The AR has three domains: the N-terminal domain, the DNA-binding domain and the ligand-binding domain, with the latter being most evolutionary conserved. </span>Testosterone (T) and the more biologically active dihydrotestosterone (DHT) are endogenous ligands for the AR (<a href="#_ENREF_4" title="MacLean, 1993 #251">MacLean et al, 1993</a>; <a href="#_ENREF_5" title="MacLeod, 2010 #27">MacLeod et al, 2010</a>; <a href="#_ENREF_8" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>). <span style="font-size:12.0pt">In teleost fishes, 11-ketotestosterone is the second main ligand (<a href="#" title="Schuppe et al, 2020">Schuppe et al, 2020</a>).</span> Human AR mutations and mouse knock-out models have established a pivotal role for the AR in masculinization and spermatogenesis (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>). Apart from the essential role for AR in male reproductive development and function (<a href="#_ENREF_9" title="Walters, 2010 #254">Walters et al, 2010</a>), the AR is also expressed in many other tissues and organs such as bone, muscles, ovaries, and the immune system (<a href="#_ENREF_7" title="Rana, 2014 #253">Rana et al, 2014</a>). </p>
<p><u>AR antagonism as Key Event</u></p>
<p>The main function of the AR is to activate gene transcription in cells. Canonical signaling occurs by ligands (androgens) binding to AR in the cytoplasm which results in translocation to the cell nucleus, receptor dimerization and binding to specific regulatory DNA sequences (<a href="#_ENREF_2" title="Heemers, 2007 #255">Heemers & Tindall, 2007</a>). The gene targets regulated by AR activation depends on cell/tissue type and what stage of development activation occur, and is, for instance, dependent on available co-factors. Apart from the canonical signaling pathway, AR can also function through non-genomic modalities, for instance rapid change in cell function by ion transport changes (<a href="#_ENREF_3" title="Heinlein, 2002 #256">Heinlein & Chang, 2002</a>). However, with regard to this specific KE the canonical signaling pathway is what is referred to.</p>
<p>The main function of the AR is to activate gene transcription in cells. Canonical signaling occurs by ligands (androgens) binding to AR in the cytoplasm which results in translocation to the cell nucleus, receptor dimerization and binding to specific regulatory DNA sequences (<a href="#_ENREF_2" title="Heemers, 2007 #255">Heemers & Tindall, 2007</a>). The gene targets regulated by AR activation depends on cell/tissue type and what stage of development activation occur, and is, for instance, dependent on available co-factors. Apart from the canonical signaling pathway, AR can also <span style="font-size:12.0pt">initiate cytoplasmic signaling pathways with other functions than the nuclear pathway,</span> for instance rapid change in cell function by ion transport changes (<a href="#_ENREF_3" title="Heinlein, 2002 #256">Heinlein & Chang, 2002</a>) <span style="font-size:12.0pt">and association with Src kinase to activate MAPK/ERK signaling and activation of the PI3K/Akt pathway (<a href="#" title="Leung & Sadar, 2017">Leung & Sadar, 2017</a>)</span>. </p>
<h4>How it is Measured or Detected</h4>
<p>AR antagonism can be measured in vitro by transientor stable transactivation assays to evaluate nuclear receptor activation. There is already a validated assay for AR (ant)agonism adopted by the OECD, Test No. 458: <em>Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals </em>(<a href="#_ENREF_13" title="OECD, 2016 #257">OECD, 2016</a>). The stably transfected AR-EcoScreen<sup>TM</sup> cells (<a href="#_ENREF_15" title="Satoh, 2004 #280">Satoh et al, 2004</a>) should be used for the assay and is freely available for the Japanese Collection of Research Bioresources (JCRB) Cell Bank under reference number JCRB1328.</p>
<p>AR antagonism can be measured in vitro by transient or stable transactivation assays to evaluate nuclear receptor activation. There is already a validated test guideline for AR (ant)agonism adopted by the OECD, Test No. 458: <em>Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals </em>(<a href="#_ENREF_13" title="OECD, 2016 #257">OECD, 2016</a>). <span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">This test guideline contains three different methods. More information on limitations, advantages, protocols, and availability and description of cells are given in the test guideline.</span></span></span></p>
<p>Other assays include the AR-CALUX reporter gene assay that is derived from human U2-OS cells stably transfected with the human AR and an AR responsive reporter gene (<a href="#_ENREF_18" title="van der Burg, 2010 #261">van der Burg et al, 2010</a>), various transiently transfected reporter cell lines (<a href="#_ENREF_10" title="Körner, 2004 #282">Körner et al, 2004</a>), and more.</p>
<h4>References</h4>
<p><a name="_ENREF_1">Alapi EM, Fischer J (2006) Table of Selected Analogue Classes. In <em>Analogue-based Drug Discovery</em>, Fischer J, Ganellin CR (eds), p 515. Weinheim: Wiley-VCH Verlag GmbH & Co</a></p>
<p> </p>
<p><a name="_ENREF_2">Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. <em>Clinical Biochemist Reviews</em> <strong>37:</strong> 3-15</a></p>
<p> </p>
<p><a name="_ENREF_3">Draskau MK, Boberg J, Taxvig C, Pedersen M, Frandsen HL, Christiansen S, Svingen T (2019) In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. <em>Environ Pollut</em> <strong>255:</strong> 113309</a></p>
<p> </p>
<p><a name="_ENREF_4">Foster PM, Harris MW (2005) Changes in androgen-mediated reproductive development in male rat offspring following exposure to a single oral dose of flutamide at different gestational ages. <em>Toxicol Sci</em> <strong>85:</strong> 1024-1032</a></p>
<p> </p>
<p><a name="_ENREF_5">Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M, Metzdorff SB, Kortenkamp A (2007) Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. <em>Environ Health Perspect</em> <strong>115 Suppl. 1:</strong> 122-128</a></p>
<p> </p>
<p><a name="_ENREF_6">Heemers HV, Tindall DJ (2007) Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. <em>Endocr Rev</em> <strong>28:</strong> 778-808</a></p>
<p> </p>
<p><a name="_ENREF_7">Heinlein CA, Chang C (2002) The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions. <em>Mol Endocrinol</em> <strong>16:</strong> 2181-2187</a></p>
<p> </p>
<p><a name="_ENREF_8">Kita DH, Meyer KB, Venturelli AC, Adams R, Machado DL, Morais RN, Swan SH, Gennings C, Martino-Andrade AJ (2016) Manipulation of pre and postnatal androgen environments and anogenital distance in rats. <em>Toxicology</em> <strong>368-369:</strong> 152-161</a></p>
<p> </p>
<p><a name="_ENREF_9">Kjærstad MB, Taxvig C, Nellemann C, Vinggaard AM, Andersen HR (2010) Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. <em>Reprod Toxicol</em> <strong>30:</strong> 573-582</a></p>
<p>Besides these validated methods, other transiently or stably transfected reporter cell lines are available as well as yeast based systems (Campana et al, 2015; <a href="#_ENREF_10" title="Körner, 2004 #282">Körner et al, 2004</a>). <span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">AR nuclear translocation can be monitored by various assays (Campana et al 2015), for example by monitoring fluorescent rat AR movement in living cells (Tyagi et al 2020), with several human AR translocation assays being commercially available; e.g. Fluorescent AR Nuclear Translocation Assay (tGFP-hAR/HEK293) or Human Androgen NHR Cell Based Antagonist Translocation LeadHunter Assay. </span></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Additional information on AR interaction can be obtained employing competitive AR binding assays (Freyberger et al 2010, Shaw et al 2018), which can also inform on relative potency of the compounds, though not on downstream effect of the AR binding.</span></span></span></p>
<p><a name="_ENREF_10">Körner W, Vinggaard AM, Térouanne B, Ma R, Wieloch C, Schlumpf M, Sultan C, Soto AM (2004) Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals. <em>Environ Health Perspect</em> <strong>112:</strong> 695-702</a></p>
<p> </p>
<p><a name="_ENREF_11">MacLean HE, Chu S, Warne GL, Zajac JD (1993) Related individuals with different androgen receptor gene deletions. <em>J Clin Invest</em> <strong>91:</strong> 1123-1128</a></p>
<p> </p>
<p><span style="font-size:11.0pt">The recently developed AR dimerization assay provides an assay with an improved ability to measure potential stressor-mediated disruption of dimerization/activation (</span><span style="font-size:11.0pt"><a href="#_ENREF_11" title="Lee, 2021 #288">Lee et al, 2021</a></span><span style="font-size:11.0pt">).</span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Campana C, Pezzi V, Rainey WE (2015) Cell based assays for screening androgen receptor ligands. Semin Reprod Med 33: 225-234.</span></span></span></p>
<p><a name="_ENREF_12">MacLeod DJ, Sharpe RM, Welsh M, Fisken M, Scott HM, Hutchison GR, Drake AJ, van den Driesche S (2010) Androgen action in the masculinization programming window and development of male reproductive organs. <em>Int J Androl</em> <strong>33:</strong> 279-287</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_2">Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. <em>Clin Biochem Rev</em> <strong>37:</strong> 3-15</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Freyberger A, Weimer M, Tran HS, Ahr HJ. Assessment of a recombinant androgen receptor binding assay: initial steps towards validation. Reprod Toxicol. 2010 Aug;30(1):2-8. doi: 10.1016/j.reprotox.2009.10.001. Epub 2009 Oct 13. PMID: 19833195.</span></span></span></p>
<p><a name="_ENREF_13">OECD. (2016) Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. <em>OECD Guidelines for the Testing of Chemicals, Section 4</em>, Paris.</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_6">Heemers HV, Tindall DJ (2007) Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. <em>Endocr Rev</em> <strong>28:</strong> 778-808</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_7">Heinlein CA, Chang C (2002) The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions. <em>Mol Endocrinol</em> <strong>16:</strong> 2181-2187</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_10">Körner W, Vinggaard AM, Térouanne B, Ma R, Wieloch C, Schlumpf M, Sultan C, Soto AM (2004) Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals. <em>Environ Health Perspect</em> <strong>112:</strong> 695-702</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_11">Lee SH, Hong KY, Seo H, Lee HS, Park Y (2021) Mechanistic insight into human androgen receptor-mediated endocrine-disrupting potentials by a stable bioluminescence resonance energy transfer-based dimerization assay. <em>Chem Biol Interact</em> <strong>349:</strong> 109655</a></span></span></p>
<p><a name="_ENREF_15">Satoh K, Ohyama K, Aoki N, Iida M, Nagai F (2004) Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen. <em>Food Chem Toxicol</em> <strong>42:</strong> 983-993</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a id="_ENREF_23" name="_ENREF_23">Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. <em>Frontiers in Endocrinology</em>, <em>8</em>. https://doi.org/10.3389/fendo.2017.00002</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_12">MacLean HE, Chu S, Warne GL, Zajac JD (1993) Related individuals with different androgen receptor gene deletions. <em>J Clin Invest</em> <strong>91:</strong> 1123-1128</a></span></span></p>
<p><a name="_ENREF_16">Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U, Svingen T (2019) Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. <em>Arch Toxicol</em> <strong>93:</strong> 253-272</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_13">MacLeod DJ, Sharpe RM, Welsh M, Fisken M, Scott HM, Hutchison GR, Drake AJ, van den Driesche S (2010) Androgen action in the masculinization programming window and development of male reproductive organs. <em>Int J Androl</em> <strong>33:</strong> 279-287</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_14">OECD. (2016) Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. <em>OECD Guidelines for the Testing of Chemicals, Section 4</em>, Paris.</a></span></span></p>
<p><a name="_ENREF_17">Sonneveld E, Jansen HJ, Riteco JA, Brouwer A, van der Burg B (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. <em>Toxicol Sci</em> <strong>83:</strong> 136-148</a></p>
<p><a name="_ENREF_18">van der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, Witters H, van der Linden S (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. <em>Reprod Toxicol</em> <strong>30:</strong> 18-24</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_16">Satoh K, Ohyama K, Aoki N, Iida M, Nagai F (2004) Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen. <em>Food Chem Toxicol</em> <strong>42:</strong> 983-993</a></span></span></p>
<p> </p>
<p><a id="_ENREF_22" name="_ENREF_22"><span style="font-size:14px">Schuppe, E. R., Miles, M. C., and Fuxjager, M. J. (2020). Evolution of the androgen receptor: Perspectives from human health to dancing birds. Mol. Cell. Endocrinol. 499, 110577. doi:10.1016/J.MCE.2019.110577 </span></a></p>
<p><a name="_ENREF_19">Vinggaard AM, Niemelä J, Wedebye EB, Jensen GE (2008) Screening of 397 chemicals and development of a quantitative structure--activity relationship model for androgen receptor antagonism. <em>Chem Res Toxicol</em> <strong>21:</strong> 813-823</a></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_ENREF_17">Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U, Svingen T (2019) Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. <em>Arch Toxicol</em> <strong>93:</strong> 253-272</a></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Shaw J, Leveridge M, Norling C, Karén J, Molina DM, O'Neill D, Dowling JE, Davey P, Cowan S, Dabrowski M, Main M, Gianni D. Determining direct binders of the Androgen Receptor using a high-throughput Cellular Thermal Shift Assay. Sci Rep. 2018 Jan 9;8(1):163. doi: 10.1038/s41598-017-18650-x. PMID: 29317749; PMCID: PMC5760633.</span></span></span></p>
<p><a name="_ENREF_20">Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. <em>Hum Reprod Update</em> <strong>16:</strong> 543-558</a></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Tyagi RK, Lavrovsky Y, Ahn SC, Song CS, Chatterjee B, Roy AK (2000) Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol 14: 1162-1174</span></span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a id="_ENREF_21" name="_ENREF_21">Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. <em>Hum Reprod Update</em> <strong>16:</strong> 543-558</a></span></span></p>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/306">Aop:306 - Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<p><span style="font-size:11pt">This KE is considered broadly applicable across mammalian taxa as all mammals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and functions. <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">It is, however, acknowledged that this KE most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.</span></span></span></p>
<h4>Key Event Description</h4>
<p style="text-align: justify;"><span style="font-size:14px"><span style="font-family:times new roman,times,serif">Androgen receptor activation is regulated by the binding of androgens. AR activity can be decreased by either a lack of steroidal ligands (testosterone, DHT) or the presence of antagonist compounds. <sup>12</sup></span></span></p>
<p><span style="font-size:11pt">This KE refers to decreased activation of the androgen receptor (AR) as occurring in complex biological systems such as tissues and organs in vivo. It is thus considered distinct from KEs describing either blocking of AR or decreased androgen synthesis.</span></p>
<p style="text-align:justify"><span style="font-size:11pt">The AR is a nuclear transcription factor with canonical AR activation regulated by the binding of the androgens such as testosterone or dihydrotestosterone (DHT). Thus, AR activity can be decreased by reduced levels of steroidal ligands (testosterone, DHT) or the presence of compounds interfering with ligand binding to the receptor <span style="color:black">(Davey & Grossmann, 2016; Gao et al., 2005)</span>.</span></p>
<p style="text-align:justify"><span style="font-size:11pt">In the inactive state, AR is sequestered in the cytoplasm of cells by molecular chaperones. In the classical (genomic) AR signaling pathway, AR activation causes dissociation of the chaperones, AR dimerization and translocation to the nucleus to modulate gene expression. AR binds to the androgen response element (ARE) <span style="color:black">(Davey & Grossmann, 2016; Gao et al., 2005)</span>. <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Notably, for transcriptional regulation the AR is closely associated with other co-factors that may differ between cells, tissues and life stages. In this way, the functional consequence of AR activation is cell- and tissue-specific. This dependency on co-factors such as the SRC proteins also means that stressors affecting recruitment of co-activators to AR can result in decreased AR activity (Heinlein & Chang, 2002).</span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt">Ligand-bound AR may also associate with cytoplasmic and membrane-bound proteins to initiate cytoplasmic signaling pathways with other functions than the nuclear pathway. Non-genomic AR signaling includes association with Src kinase to activate MAPK/ERK signaling and activation of the PI3K/Akt pathway. Decreased AR activity may therefore be a decrease in the genomic and/or non-genomic AR signaling pathways <span style="color:black">(Leung & Sadar, 2017)</span>.</span></p>
<h4>How it is Measured or Detected</h4>
<p style="text-align: justify;"><span style="font-size:14px"><span style="font-family:times new roman,times,serif">Significance of AR signaling in fetal development can be studied through a conditional deletion of the androgen receptor using a Cre/loxP approach. The recommended animal model for reproductive study is the mouse.<sup>3</sup></span></span></p>
<p style="text-align: justify;"><span style="font-size:14px"><span style="font-family:times new roman,times,serif">Also, epidemiological case-studies following mouse or humans expressing a complete androgen insensitivity allow to directly assess the effects of a lack of AR activation on the development.<sup>4</sup></span></span></p>
<p><span style="font-size:11pt">This KE specifically focuses on decreased <em>in vivo</em> activation, with most methods that can be used to measure AR activity carried out <em>in vitro</em>. They provide indirect information about the KE and are described in lower tier MIE/KEs (see for example MIE/KE-26 for AR antagonism, KE-1690 for decreased T levels and KE-1613 for decreased dihydrotestosterone levels). In this way, this KE is a placeholder for tissue-specific responses to AR activation or inactivation that will depend on the adverse outcome (AO) for which it is included. </span></p>
<p style="text-align:justify;"><span style="font-size:14px"><span style="font-family:times new roman,times,serif">Enzyme immunoassay (ELISA) kits for in vitro quantitative measurement of AR activity are available. Androgen receptors activity can be measured using bioassay such as the (Anti-)Androgen Receptor CALUX reporter gene assay.<sup>5</sup></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">In fish, The Rapid Androgen Disruption Activity Reporter (RADAR) assay included in OECD test guideline no. 251 can be used to measure genomic AR activity (OECD, 2022). Employing a spg1-gfp construct under control of the AR-binding promoter spiggin1 in medaka fish embryos, any stressor activating or inhibiting the androgen axis will be detected. This includes for instance stressors that agonize or antagonize AR, as well as stressors that modulate androgen synthesis or metabolism. Non-genomic AR activity cannot be detected by the RADAR assay (OECD, 2022). Similar assays may in the future be developed to measure AR activity in mammalian organisms. </span></span></span></p>
<h4>References</h4>
<table>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. <em>The Clinical Biochemist. Reviews</em>, <em>37</em>(1), 3–15.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gao, W., Bohl, C. E., & Dalton, J. T. (2005). Chemistry and structural biology of androgen receptor. <em>Chemical Reviews</em>, <em>105</em>(9), 3352–3370. https://doi.org/10.1021/cr020456u</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Heinlein, C. A., & Chang, C. (2002). Androgen Receptor (AR) Coregulators: An Overview. https://academic.oup.com/edrv/article/23/2/175/2424160</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. <em>Frontiers in Endocrinology</em>, <em>8</em>. <a href="https://doi.org/10.3389/fendo.2017.00002" style="color:#0563c1; text-decoration:underline">https://doi.org/10.3389/fendo.2017.00002</a></span></span></p>
<p><sup>1</sup> Davey R.A and Grossmann M. (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clinical Biochemist Reviews, 37(1): 3-15. PCM4810760</p>
<p><sup>2 </sup>Gao W., Bohl C.E. and Dalton J.T. (2005) Chemistry and Structural Biology of Androgen Receptor. Chemical Reviews 105(9): 3352-3370<a href="https://www.google.com/url?q=https://doi.org/10.1021/cr020456u&sa=D&ust=1554891396627000">https://doi.org/10.1021/cr020456u</a> </p>
<p><sup>3</sup> Kaftanovskaya E.M., Huang Z., Barbara A.M., De Gendt K., Verhoeven G., Ivan P. Gorlov, and Agoulnik A.I. (2012) Cryptorchidism in Mice with an Androgen Receptor Ablation in Gubernaculum Testis. Molecular Endocrinology, 26(4): 598-607.<a href="https://www.google.com/url?q=https://doi.org/10.1210/me.2011-1283&sa=D&ust=1554891396628000">https://doi.org/10.1210/me.2011-1283</a> </p>
<p><sup>4</sup> Hutson J.M. (1985) A biphasic model for the hormonal control of testicular descent. Lancet, 24;2(8452): 419-21<a href="https://www.google.com/url?q=http://dx.doi.org/10.1016/S0140-6736(85)92739-4&sa=D&ust=1554891396629000">http://dx.doi.org/10.1016/S0140-6736(85)92739-4</a> </p>
<p><sup>5</sup> van der Burg B., Winter R., Man HY., Vangenechten C., Berckmans P., Weimer M., Witters M. and van der Linden S. (2010) Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reproductive Toxicology, 30(1):18-24 <a href="https://www.google.com/url?q=https://doi.org/0.1016/j.reprotox.2010.04.012&sa=D&ust=1554891396630000">https://doi.org/0.1016/j.reprotox.2010.04.012</a> </p>
<p> </p>
</td>
</tr>
</tbody>
</table>
<h4><a href="/events/1687">Event: 1687: decrease, transcription of genes by AR </a></h4>
<h5>Short Name: decrease, transcription of genes by AR </h5>
<p><p>Butylparaben has been shown to cause decreased male AGD in rats following intrauterine exposure to 500 and 1000 mg/kg bw/day (<a href="#_ENREF_1" title="Boberg, 2016 #12">Boberg et al, 2016</a>; <a href="#_ENREF_39" title="Zhang, 2014 #148">Zhang et al, 2014</a>). A separate study using 600 mg/kg bw/day did not see an effect on male AGD (<a href="#_ENREF_2" title="Boberg, 2008 #45">Boberg et al, 2008</a>).</p>
</p>
<h4>p,p'-DDE</h4>
<p><p>p,p,DDE has been shown to cause decreased male AGD in rats following intrauterine exposure to 100-200 mg/kg bw/day (<a href="#_ENREF_20" title="Loeffler, 1999 #60">Loeffler & Peterson, 1999</a>; <a href="#_ENREF_38" title="Wolf, 1999 #146">Wolf et al, 1999</a>).</p>
</p>
<h4>Bis(2-ethylhexyl) phthalate</h4>
<p><p>DEHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 300-1500 mg/kg bw/day (<a href="#_ENREF_4" title="Christiansen, 2010 #13">Christiansen et al, 2010</a>; <a href="#_ENREF_8" title="Gray, 2000 #110">Gray et al, 2000</a>; <a href="#_ENREF_13" title="Howdeshell, 2007 #111">Howdeshell et al, 2007</a>; <a href="#_ENREF_15" title="Jarfelt, 2005 #113">Jarfelt et al, 2005</a>; <a href="#_ENREF_16" title="Kita, 2016 #34">Kita et al, 2016</a>; <a href="#_ENREF_18" title="Li, 2013 #71">Li et al, 2013</a>; <a href="#_ENREF_19" title="Lin, 2009 #120">Lin et al, 2009</a>; <a href="#_ENREF_25" title="Moore, 2001 #124">Moore et al, 2001</a>; <a href="#_ENREF_27" title="Nardelli, 2017 #149">Nardelli et al, 2017</a>; <a href="#_ENREF_30" title="Saillenfait, 2009 #134">Saillenfait et al, 2009</a>; <a href="#_ENREF_38" title="Wolf, 1999 #146">Wolf et al, 1999</a>).</p>
</p>
<h4>Dexamethasone</h4>
<p><p>Dexamethasone has been shown to cause decreased male AGD in rats following intrauterine exposure to 0.1 mg/kg bw/day (<a href="#_ENREF_35" title="Van den Driesche, 2012 #144">Van den Driesche et al, 2012</a>).</p>
</p>
<h4>Fenitrothion</h4>
<p><p>Fenitrothion has been shown to cause decreased male AGD in rats following intrauterine exposure to 25 mg/kg bw/day (<a href="#_ENREF_34" title="Turner, 2002 #213">Turner et al, 2002</a>).</p>
</p>
<h4>Finasteride</h4>
<p><p>Finasteride has been shown to cause decreased male AGD in rats following intrauterine exposure to 100 mg/kg bw/day (<a href="#_ENREF_3" title="Bowman, 2003 #29">Bowman et al, 2003</a>).</p>
</p>
<h4>Flutamide</h4>
<p><p>Flutamide has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 16-100 mg/kg bw/day (<a href="#_ENREF_7" title="Foster, 2005 #53">Foster & Harris, 2005</a>; <a href="#_ENREF_11" title="Hass, 2007 #76">Hass et al, 2007</a>; <a href="#_ENREF_16" title="Kita, 2016 #34">Kita et al, 2016</a>; <a href="#_ENREF_23" title="McIntyre, 2001 #36">McIntyre et al, 2001</a>; <a href="#_ENREF_26" title="Mylchreest, 1999 #126">Mylchreest et al, 1999</a>; <a href="#_ENREF_32" title="Scott, 2007 #139">Scott et al, 2007</a>; <a href="#_ENREF_36" title="Welsh, 2007 #56">Welsh et al, 2007</a>).</p>
</p>
<h4>Ketoconazole</h4>
<p><p>Ketoconazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 50 mg/kg bw/day in one study (<a href="#_ENREF_33" title="Taxvig, 2008 #184">Taxvig et al, 2008</a>), but no effect in another study using same dose (<a href="#_ENREF_38" title="Wolf, 1999 #146">Wolf et al, 1999</a>).</p>
</p>
<h4>Linuron</h4>
<p><p>Linuron has been shown to cause decreased male AGD in rats following intrauterine exposure to 50-100 mg/kg bw/day (<a href="#_ENREF_12" title="Hotchkiss, 2004 #40">Hotchkiss et al, 2004</a>; <a href="#_ENREF_24" title="McIntyre, 2002 #38">McIntyre et al, 2002</a>; <a href="#_ENREF_38" title="Wolf, 1999 #146">Wolf et al, 1999</a>).</p>
</p>
<h4>Prochloraz</h4>
<p><p>Prochloraz has been shown to cause decreased male AGD in rats following intrauterine exposure to 150-250 mg/kg bw/day (<a href="#_ENREF_17" title="Laier, 2006 #15">Laier et al, 2006</a>; <a href="#_ENREF_28" title="Noriega, 2005 #54">Noriega et al, 2005</a>).</p>
</p>
<h4>Procymidone</h4>
<p><p style="margin-left:18.0pt">Procymidone has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-150 mg/kg bw/day (<a href="#_ENREF_10" title="Hass, 2012 #220">Hass et al, 2012</a>; <a href="#_ENREF_11" title="Hass, 2007 #76">Hass et al, 2007</a>; <a href="#_ENREF_38" title="Wolf, 1999 #146">Wolf et al, 1999</a>).</p>
</p>
<h4>Triticonazole</h4>
<p><p>Triticonazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 150 and 450 mg/kg bw/day (<a href="#_ENREF_6" title="Draskau, 2019 #258">Draskau et al, 2019</a>).</p>
</p>
<h4>Vinclozolin</h4>
<p><p>Vinclozolin has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-200 mg/kg bw/day (<a href="#_ENREF_5" title="Christiansen, 2009 #14">Christiansen et al, 2009</a>; <a href="#_ENREF_9" title="Gray, 1994 #109">Gray et al, 1994</a>; <a href="#_ENREF_11" title="Hass, 2007 #76">Hass et al, 2007</a>; <a href="#_ENREF_22" title="Matsuura, 2005 #243">Matsuura et al, 2005</a>; <a href="#_ENREF_29" title="Ostby, 1999 #78">Ostby et al, 1999</a>; <a href="#_ENREF_31" title="Schneider, 2011 #245">Schneider et al, 2011</a>; <a href="#_ENREF_37" title="Wolf, 2004 #51">Wolf et al, 2004</a>).</p>
</p>
<h4>di-n-hexyl phthalate</h4>
<p><p>DnHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-750 mg/kg bw/day (<a href="#_ENREF_35" title="Saillenfait, 2009 #133">Saillenfait et al, 2009a</a>; <a href="#_ENREF_36" title="Saillenfait, 2009 #134">Saillenfait et al, 2009b</a>).</p>
</p>
<h4>Dicyclohexyl phthalate</h4>
<p><p>DCHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 350-750 mg/kg bw/day (<a href="#_ENREF_1" title="Aydoğan Ahbab, 2015 #95">Aydoğan Ahbab & Barlas, 2015</a>; <a href="#_ENREF_13" title="Hoshino, 2005 #239">Hoshino et al, 2005</a>; <a href="#_ENREF_32" title="Saillenfait, 2009 #133">Saillenfait et al, 2009a</a>).</p>
</p>
<h4>butyl benzyl phthalate</h4>
<p><p>BBP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-1000 mg/kg bw/day (<a href="#_ENREF_8" title="Ema, 2002 #104">Ema & Miyawaki, 2002</a>; <a href="#_ENREF_10" title="Gray, 2000 #110">Gray et al, 2000</a>; <a href="#_ENREF_15" title="Hotchkiss, 2004 #40">Hotchkiss et al, 2004</a>; <a href="#_ENREF_30" title="Nagao, 2000 #128">Nagao et al, 2000</a>; <a href="#_ENREF_41" title="Tyl, 2004 #240">Tyl et al, 2004</a>).</p>
</p>
<h4>monobenzyl phthalate</h4>
<p><p>MBeP has been shown to cause decreased male AGD in rats following intrauterine exposure to 375 mg/kg bw/day (<a href="#_ENREF_9" title="Ema, 2003 #107">Ema et al, 2003</a>).</p>
</p>
<h4>di-n-heptyl phthalate</h4>
<p><p>DHPP has been shown to cause decreased male AGD in rats following intrauterine exposure to 1000 mg/kg bw/day (<a href="#_ENREF_36" title="Saillenfait, 2011 #135">Saillenfait et al, 2011</a>).</p>
<p>A short AGD in male offspring is a marker of insufficient androgen action during critical fetal developmental stages (<a href="#_ENREF_42" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>; <a href="#_ENREF_49" title="Welsh, 2008 #23">Welsh et al, 2008</a>). A short AGD is thus a sign of undervirilization, which is also associated with a series of male reproductive disorders, including genital malformations and infertility in humans (<a href="#_ENREF_21" title="Juul, 2014 #3">Juul et al, 2014</a>; <a href="#_ENREF_44" title="Skakkebaek, 2001 #9">Skakkebaek et al, 2001</a>).</p>
<p>There are numerous human epidemiological studies showing associations with intrauterine exposure to anti-androgenic chemicals and short AGD in newborn boys alongside other reproductive disorders (<a href="#_ENREF_42" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>). This underscores the human relevance of this AO. However, in reproductive toxicity studies and chemical risk assessment, rodents (rats and mice) are what is tested on. The list of chemicals inducing short male AGD in male rat offspring is extensive, as evidenced by the ‘stressor’ list and reviewed by (<a href="#_ENREF_42" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>).</p>
<h4>Key Event Description</h4>
<p>The anogenital distance (AGD) refers to the distance between anus and the external genitalia. In rodents and humans, the male AGD is approximately twice the length as the female AGD (<a href="#_ENREF_39" title="Salazar-Martinez, 2004 #8">Salazar-Martinez et al, 2004</a>; <a href="#_ENREF_41" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>). This sexual dimorphisms is a consequence of sex hormone-dependent development of secondary sexual characteristics (<a href="#_ENREF_41" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>). In males, it is believed that androgens (primarily DHT) activate AR-positive cells in non-myotic cells in the fetal perineum region to initiate differentiation of the perineal <em>levator ani</em> and <em>bulbocavernosus </em>(LABC) muscle complex (<a href="#_ENREF_18" title="Ipulan, 2014 #185">Ipulan et al, 2014</a>). This AR-dependent process occurs within a critical window of development, around gestational days 15-18 in rats (<a href="#_ENREF_26" title="MacLeod, 2010 #27">MacLeod et al, 2010</a>). In females, the absence of DHT prevents this masculinization effect from occurring.</p>
<p>The involvement of androgens in masculinization of the male fetus, including the perineum, has been known for a very long time (<a href="#_ENREF_20" title="Jost, 1953 #151">Jost, 1953</a>), and AGD has historically been used to, for instance, sex newborn kittens. It is now well established that the AGD in newborns is a proxy readout for the intrauterine sex hormone milieu the fetus was developing. Too low androgen levels in XY fetuses makes the male AGD shorter, whereas excess (ectopic) androgen levels in XX fetuses makes the female AGD longer, in humans and rodents (<a href="#_ENREF_41" title="Schwartz, 2019 #252">Schwartz et al, 2019</a>).</p>
<h4>How it is Measured or Detected</h4>
<p>The AGD is a morphometric measurement carried out by trained technicians (rodents) or medical staff (humans).</p>
<p>In rodent studies AGD is assessed as the distance between the genital papilla and the anus, and measured using a stereomicroscope with a micrometer eyepiece. The AGD index (AGDi) is often calculated by dividing AGD by the cube root of the body weight. It is important in statistical analysis to use litter as the statistical unit. This is done when more than one pup from each litter is examined. Statistical analyses is adjusted using litter as an independent, random and nested factor. AGD are analysed using body weight as covariate as recommended in Guidance Document 151 (<a href="#_ENREF_37" title="OECD, 2013 #30">OECD, 2013</a>).</p>
<p> </p>
<h4>Regulatory Significance of the AO</h4>
<p>In regulatory toxicology, the AGD is mandatory inclusions in OECD test guidelines used to test for developmental and reproductive toxicity of chemicals. Guidelines include ‘TG 443 extended one-generation study’, ‘TG 421/422 reproductive toxicity screening studies’ and ‘TG 414 developmental toxicity study’.</p>
<h4>References</h4>
<p><a name="_ENREF_1">Aydoğan Ahbab M, Barlas N (2015) Influence of in utero di-n-hexyl phthalate and dicyclohexyl phthalate on fetal testicular development in rats. <em>Toxicol Lett</em> <strong>233:</strong> 125-137</a></p>
<p><a name="_ENREF_2">Boberg J, Axelstad M, Svingen T, Mandrup K, Christiansen S, Vinggaard AM, Hass U (2016) Multiple endocrine disrupting effects in rats perinatally exposed to butylparaben. <em>Toxicol Sci</em> <strong>152:</strong> 244-256</a></p>
<p><a name="_ENREF_3">Boberg J, Metzdorff S, Wortziger R, Axelstad M, Brokken L, Vinggaard AM, Dalgaard M, Nellemann C (2008) Impact of diisobutyl phthalate and other PPAR agonists on steroidogenesis and plasma insulin and leptin levels in fetal rats. <em>Toxicology</em> <strong>250:</strong> 75-81</a></p>
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<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2130">Relationship: 2130: Antagonism, Androgen receptor leads to Decrease, AR activation</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">This KER is applicable to mammals as AR expression and activity is highly conserved (Davey & Grossmann, 2016). AR activity is important for sexual development and reproduction in both males and females (Prizant et al., 2014; Walters et al., 2010). AR function is required during development, puberty, and adulthood. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.</span></span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="text-align:justify"><span style="font-size:11pt">The androgen receptor (AR) is a ligand-activated steroid hormone nuclear receptor <span style="color:black">(Davey & Grossmann, 2016)</span>. In its inactive state, the AR locates to the cytoplasm <span style="color:black">(Roy et al., 2001)</span>. When activated, the AR translocates to the nucleus, dimerizes, and, together with co-regulators, binds to specific DNA regulatory sequences to regulate gene transcription <span style="color:black">(Davey & Grossmann, 2016)</span> (Lamont and Tindall, 2010). This is considered the canonical AR signaling pathway. The AR can also activate non-genomic signalling <span style="color:black">(Jin et al., 2013)</span>. However, this KER focuses on the canonical pathway.</span></p>
<p style="text-align:justify"><span style="font-size:11pt">The two main AR ligands are the androgens testosterone (T) and the more potent dihydrotestosterone (DHT). Androgens bind to the AR to mediate downstream androgenic responses, such as male development and masculinization <span style="color:black">(Rey, 2021; Walters et al., 2010)</span>. Antagonism of the AR would decrease AR activation and therefore the downstream AR-mediated effects. </span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-size:11pt">The biological plausibility for this KER is considered high.</span></p>
<p style="text-align:justify"><span style="font-size:11pt">The AR belongs to the steroid hormone nuclear receptor family. The AR has 3 main domains essential for its activity, the N-terminal domain, the ligand binding domain, and the DNA binding domain <span style="color:black">(Roy et al., 2001)</span>. Ligands, such as T and DHT, must bind to the ligand binding domain to activate AR allowing it to fulfill its role as a transcription factor. The binding of the ligand induces a change in AR conformation allowing it to translocate to the nucleus and congregate into a subnuclear compartment <span style="color:black">(Marcelli et al., 2006; Roy et al., 2001)</span> homodimerize and bind to the DNA target sequences and regulate transcription of target genes. Regulation of AR target genes is greatly facilitated by numerous co-factors. Active AR signaling is essential for male reproduction and sexual development and is also crucial in several other tissues and organs such as ovaries, the immune system, bones, and muscles <span style="color:black">(Ogino et al., 2011; Prizant et al., 2014; Rey, 2021; William H. Walker, 2021)</span>. </span></p>
<p style="text-align:justify"><span style="font-size:11pt">AR antagonists can compete with or prevent in different ways AR ligand binding, thereby preventing AR activation. Antagonism of the AR can prevent translocation to the nucleus, compartmentalization, dimerization and DNA binding. Consequently, AR cannot regulate transcription of target genes and androgen signalling is disrupted. This can be observed using different AR activation assays such as AR dimerization, translocation, DNA binding or transcriptional activity assays <span style="color:black">(Brown et al., 2023; <em>OECD</em>, 2020)</span><span style="color:black">.</span> </span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-size:11pt">The empirical evidence for this KER is considered high</span></p>
<p style="text-align:justify"><span style="font-size:11pt">The effects of AR antagonism have been shown in many studies <em>in vivo</em> and <em>in vitro</em>. </span></p>
<p><span style="font-size:11pt">Several stressors can act as antagonists of the AR and lead to decreased AR activation. Some of these are detailed in an AOP key event relationship report by <span style="color:black">(Pedersen et al., 2022)</span> and shown below, exhibiting evidence of dose-concordance:</span></p>
<li><span style="font-size:11pt">Cyproterone acetate: Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM <span style="color:black">(Sonneveld, 2005)</span></span></li>
<li><span style="font-size:11pt">Epoxiconazole: Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 µM and an IC50 of 10 µM <span style="color:black">(Kjærstad et al., 2010)</span>.</span></li>
<li><span style="font-size:11pt">Flutamide: Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 µM <span style="color:black">(Sonneveld, 2005).</span></span></li>
<li><span style="font-size:11pt">Flusilazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.8 µM and an IC50 of 2.8 (±0.1) µM <span style="color:#0563c1"><u><span style="color:black">(Draskau et al., 2019)</span></u></span>.</span></li>
<li><span style="font-size:11pt">Prochloraz: Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 µM and an IC50 of 13 µM <span style="color:black">(Kjærstad et al., 2010)</span>.</span></li>
<li><span style="font-size:11pt">Propiconazole: Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 µM and an IC50 of 18 µM <span style="color:black">(Kjærstad et al., 2010)</span>.</span></li>
<li><span style="font-size:11pt">Tebuconazole: Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 µM and an IC50 of 8.1 µM <span style="color:black">(Kjærstad et al., 2010)</span>.</span></li>
<li><span style="font-size:11pt">Triticonazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 µM and an IC50 of 0.3 (±0.01) µM <span style="color:black">(Draskau et al., 2019)</span>.</span></li>
<li><span style="font-size:11pt">Vinclozolin: Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 µM<span style="color:black">(Sonneveld, 2005)</span>.”<span style="color:black">(Pedersen et al., 2022)</span></span></li>
<p style="text-align:justify"><span style="font-size:11pt">Known AR antagonists are used for treatment of AR-sensitive cancers such as flutamide for prostate cancer (Mahler et al., 1998). </span></p>
<p> </p>
<p> </p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p><span style="font-size:11pt"> The quantitative relationship between AR antagonism and AR activation will depend on the type of antagonist.</span></p>
<strong>Time-scale</strong>
<p style="text-align:justify"><span style="font-size:11pt">Nuclear translocation in HeLa cells transfected with AR-GFP show a response within 2 hours after ligand exposure <span style="color:black">(Marcelli et al., 2006; Szafran et al., 2008)</span>. Another assay focusing on AR binding to promoters in LNCaP cells has shown that after ligand binding, AR is able to translocate and bind to the DNA sequences within 15min showing the speed of AR activation <span style="color:black">(Kang et al., 2002).</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p style="text-align:justify"><span style="font-size:11pt">AR antagonism can lead to increased AR transcript stability and levels as a compensatory mechanism in prostate cancer cells <span style="color:black">(Dart et al., 2020)</span>. In turn, in presence of increased AR levels, AR antagonists can exhibit agonistic activity<span style="color:black"> (Chen et al., 2003).</span> </span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Brown, E. C., Hallinger, D. R., Simmons, S. O., Puig-Castellví, F., Eilebrecht, E., Arnold, L., & Bioscience, P. A. (2023). High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites. <em>Front. Toxicol</em>, <em>5</em>, 1134783. https://doi.org/10.3389/ftox.2023.1134783</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chen, C. D., Welsbie, D. S., Tran, C., Baek, S. H., Chen, R., Vessella, R., Rosenfeld, M. G., & Sawyers, C. L. (2003). A R T I C L E S Molecular determinants of resistance to antiandrogen therapy. <em>NATURE MEDICINE</em>, <em>10</em>(1). https://doi.org/10.1038/nm972</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Dart, D. A., Ashelford, K., & Jiang, W. G. (2020). <em>AR mRNA stability is increased with AR-antagonist resistance via 3′UTR variants</em>. https://doi.org/10.1530/EC-19-0340</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. In <em>Androgen Receptor Biology Clin Biochem Rev</em> (Vol. 37, Issue 1).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Draskau, M. K., Boberg, J., Taxvig, C., Pedersen, M., Frandsen, H. L., Christiansen, S., & Svingen, T. (2019). In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. <em>Environmental Pollution</em>, <em>255</em>, 113309. https://doi.org/10.1016/j.envpol.2019.113309</span></span></p>
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<h4><a href="/relationships/2128">Relationship: 2128: Decrease, AR activation leads to decrease, transcription of genes by AR </a></h4>