<td>Under development: Not open for comment. Do not cite</td>
<td>Under Development</td>
<td>Under Review</td>
<td>1.90</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">This AOP links Androgen receptor (AR) 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 (Schwartz et al 2019). 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), with measurement guidance provided in OECD guidance documents 43 (OECD, 2008) and 151 (OECD, 2013).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">The AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species (Heemers & Tindall, 2007; Davey and Grossmann, 2016). 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 (Davey and Grossmann, 2016). AR signaling is necessary for normal masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in male fetuses (Keller et al, 1996; Robitaille and Langlois, 2020). The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">The AR is a nuclear receptor involved in the transcriptional regulation of various target genes during development and adulthood across species (Heemers & Tindall, 2007; Davey and Grossmann, 2016). 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 (Davey and Grossmann, 2016). AR signaling is necessary for normal masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in male fetuses (Keller et al, 1996; OECD, 2009; Robitaille and Langlois, 2020). The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">A central step in this pathway is antagonism of the AR in target cells of the undifferentiated 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 physiologically normal (or sufficient) levels, but prevented from binding the AR.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">A central step in this pathway is antagonism of the AR in target cells of the undifferentiated perineal region, which leads to reduced AR signaling and failure to properly masculinize the perineum/LABC complex. In this instance, the local levels of testosterone or DHT may be physiologically normal (or sufficient) levels, but prevented from binding the AR.</span></span></p>
</div>
<div id="background">
<h3>Background</h3>
<p>This AOP was developed as part of an AOP network for developmental androgen signalling-inhibition leading to short AGD in male offspring. The other AOPs in this network are AOP 305 (5α-reductase inhibition leading to short AGD) and 307 (Decreased testosterone synthesis leading to short AGD).</p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">Androgen signaling is critical for male sex differentiation during fetal life and suboptimal action during critical life stages leads to under-masculinized offspring. Androgens, primarily testosterone and dihydro-testosterone (DHT), act by binding to and activating the AR is target cells. Blocking the AR basically blocks androgen signaling and masculinization of tissues and organs that otherwise should masculinize in male fetuses. One morphometric marker for reduced fetal androgen action is a shorter than normal anogenital distance. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">The upstream part of the AOP, culminating at KE-286 (altered transcription of genes by the AR), has a broad applicability domain. It is built primarily on mammalian data and includes all life stages and both sexes. It could be extended to cover non-mammalian vertebrates by adding additional relevant knowledge, as previously discussed (Draskau et al, 2024). The overall applicability domain is limited by AO-1688 (decreased AGD). The AGD is strongly influenced by androgen action during critical fetal stages in mammals, with evidence from humans (Murashima et al, 2015; Thankamony et al, 2016), and from numerous gestational exposure studies in rats and mice to anti-androgenic chemicals (Gray et al, 2001; Schwartz et al, 2019a). The male masculinisation programming window occurs at a developmental stage included in the applicability domain of these AOPs and corresponds to around gestational day 16-20 in rats and gestation weeks 8-14 in humans (Welsh et al, 2008). Only males are included in the applicability domain since the male AGD, but not the female AGD, is shortened by decreased androgen action (Schwartz et al, 2019a).</span></span></p>
<h3>Essentiality of the Key Events</h3>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">The essentiality of each key event (KE) was evaluated, meaning that if an upstream KE is blocked or does not occur, subsequent downstream KEs or the adverse outcome (AO) are prevented or altered. Both direct and indirect evidence of essentiality were assessed according to the OECD developer’s handbook (see Supplementary Table S1, </span></span></span></span><a href="https://aopwiki.org/system/dragonfly/production/2025/08/15/4hsit0i1mm_Supplementary_Table_S1_Essentiality_table_AOPs_305_307_forwiki_R2.pdf">4hsit0i1mm_Supplementary_Table_S1_Essentiality_table_AOPs_305_307_forwiki_R2.pdf</a>)<span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">, with a summary provided in Table 1. </span></span></span></span></p>
<p><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Table 1:</span></span></strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"> Essentiality assessment of KEs of AOP 306:</span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-family:"Times New Roman",serif">Moderate</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-family:"Times New Roman",serif">*Low level of evidence (some support for essentiality), ** Intermediate level of evidence (evidence for impact on one or more downstream KEs), ***High level of evidence (evidence for impact on AO).</span></span></span> </p>
<h3>Weight of Evidence Summary</h3>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">Evidence for anti-androgenicity, by antagonizing the AR, is strong. In this AOP, most KERs are considered highly biologically plausible with strong empirical evidence in support of this assessment, both from human data and animal studies. The overall evidence assessment scores for each KER are summarized in the below Table:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that antagonism of the AR leads to decreased AR activity.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that 5α-reductase converts testosterone to DHT and that decreased 5α-reductase activity leads to decreased DHT levels.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that DHT activates the AR and that decreased DHT levels leads to decreased AR activation.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that testosterone activates the AR and that decreased testosterone levels leads to decreased AR activation.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that the AR regulates gene transcription, and that decreased AR activity leads to altered gene transcription.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is well established that decreased AR activity leads to decreased AGD in male offspring.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"Times New Roman",serif">It is highly plausible that altered gene transcription in the perineum leads to decreased AGD in male offspring.</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<h3>Quantitative Consideration</h3>
<p>The quantitative understanding of this AOP remains low.</p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM; Task Force, Endocrine Society (2010). Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. <em>J Clin Endocrinol Metab</em> 95(6):2536-59.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Chamberlain NL, Driver ED, Miesfeld RL (1994). The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. <em>Nucleic Acids Res</em> 22(15):3181-6.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Davey RA, Grossmann M (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. <em>Clin Biochem Rev</em> 37(1):3-15.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Draskau MK, Rosenmai AK, Bouftas N, Johansson HKL, Panagiotou EM, Holmer ML, Elmelund E, Zilliacus J, Beronius A, Damdimopolou P, van Duursen M, Svingen T (2024). AOP Report: An Upstream Network for Reduced Androgen Signaling Leading to Altered Gene Expression of Androgen Receptor-Responsive Genes in Target Tissues. <em>Environ Toxicol Chem</em> In Press (doi: 10.1002/etc.5972).</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Gray LE, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DN, Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L (2001). Effects of environmental antiandrogens on reproductive development in experimental animals. <em>Hum Reprod Update</em> 7(3):248-64.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">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> 28(7):778-808.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Holmer ML, Zilliacus J, Draskau MK, Hlisníková H, Beronius A, Svingen T (2024). Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance. <em>Reprod Toxicol</em> 128:108662.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Keller ET, Ershler WB, Chang C (1996). </span></span><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">The androgen receptor: a mediator of diverse responses. <em>Front Biosci</em> 1:d59-71.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Murashima A, Kishigami S, Thomson A, Yamada G (2015). Androgens and mammalian male reproductive tract development. <em>Biochim Biophys Acta</em> 1849(2):163-70.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">OECD (2008), Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment, OECD Series on Testing and Assessment, No. 43, OECD Publishing, Paris. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Times New Roman,Times,serif">OECD (2009), <em>Test No. 441: Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti)Androgenic Properties</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264076334-en">https://doi.org/10.1787/9789264076334-en</a>.</span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">OECD (2013) Guidance document in support of the test guideline on the extended one generation reproductive toxicity study no. 151. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Robitaille J, Langlois VS (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. <em>Gen Comp Endocrinol</em> 290:113400.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">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> 93(2):253-272.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-family:Times New Roman,Times,serif">Simanainen U, Brogley M, Gao YR, Jimenez M, Harwood DT, Handelsman DJ, Robins DM. Length of the human androgen receptor glutamine tract determines androgen sensitivity in vivo. Mol Cell Endocrinol. 2011 Aug 6;342(1-2):81-6. doi: 10.1016/j.mce.2011.05.011. </span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Supakar PC, Song CS, Jung MH, Slomczynska MA, Kim JM, Vellanoweth RL, Chatterjee B, Roy AK (1993). A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene. <em>J Biol Chem</em> 268(35):26400-8.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Svingen T, Villeneuve DL, Knapen D, Panagiotou EM, Draskau MK, Damdimopoulou P, O'Brien JM (2021). A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation. <em>Toxicol Sci</em> 184(2):183-190.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Thankamony A, Pasterski V, Ong KK, Acerini CL, Hughes IA (2016). Anogenital distance as a marker of androgen exposure in humans. <em>Andrology</em> 4(4):616-25.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL (1997). Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility. <em>J Clin Endocrinol Metab</em> 82(11):3777-82.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Welsh M, Saunders PT, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM (2008). Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. <em>J Clin Invest</em> 118(4):1479-90.</span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Wu D, Lin G, Gore AC (2009). Age-related changes in hypothalamic androgen receptor and estrogen receptor alpha in male rats. <em>J Comp Neurol</em> 512(5):688-701.</span></span></span></span></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 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><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 <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 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>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><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><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><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><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><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><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><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><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><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><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><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><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><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><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><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><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><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>
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<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>
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<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>
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<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) 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>
<td>KeyEvent</td>
</tr>
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<td><a href="/aops/111">Aop:111 - Decrease in androgen receptor activity leading to Leydig cell tumors (in rat)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</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><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">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:Arial,Helvetica,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), as shown for the pyrethroid cypermethrin (Wang et al., 2016).</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><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). </span><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Assays may in the future be developed to measure AR activation in mammalian organisms. </span></span></span></p>
<h4>References</h4>
<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><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Wang Q, Zhou JL, Wang H, Ju Q, Ding Z, Zhou XL, Ge X, Shi QM, Pan C, Zhang JP, Zhang MR, Yu HM, Xu LC. (2016). Inhibition effect of cypermethrin mediated by co-regulators SRC-1 and SMRT in interleukin-6-induced androgen receptor activation. <em>Chemosphere</em>. 158:24-9. doi: 10.1016/j.chemosphere.2016.05.053</span></span></p>
<table>
<tbody>
<tr>
<td colspan="1" rowspan="1">
<p> </p>
</td>
<td colspan="1" rowspan="1">
<p> </p>
</td>
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</table>
<h4><a href="/events/286">Event: 286: Altered, Transcription of genes by the androgen receptor</a></h4>
<h5>Short Name: Altered, Transcription of genes by the AR</h5>
<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 (Davey and Grossmann 2016). Despite certain inter-species differences, AR function mediated through gene expression is highly conserved, with mutation studies from both humans and rodents showing strong correlation for AR-dependent development and function (Walters et al. 2010). </p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">This KE is considered broadly applicable across mammalian taxa, sex and developmental stages, as all mammals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and function. </span><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><span style="font-size:11pt"><span style="font-size:12.0pt">This KE refers to transcription of genes by the androgen receptor (AR) as occurring in complex biological systems such as tissues and organs <em>in vivo</em>. </span><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Rather than measuring individual genes, this KE aims to capture patterns of effects at transcriptome level in specific target cells/tissues. In other words, it can be replaced by specific KEs for individual adverse outcomes as information becomes available, for example the transcriptional toxicity response in prostate tissue for AO: prostate cancer, perineum tissue for AO: reduced AGD, etc. AR regulates many genes that differ between tissues and life stages and, importantly, different gene transcripts within individual cells can go in either direction since AR can act as both transcriptional activator and suppressor. Thus, the ‘directionality’ of the KE cannot be either reduced or increased, but instead describe an altered transcriptome. </span></span></span></p>
<p><u>The Androgen Receptor and its function</u></p>
<p><span style="font-size:12.0pt">The AR belongs to the steroid hormone nuclear receptor family. It is a ligand-activated transcription factor with three domains: the N-terminal domain, the DNA-binding domain, and the ligand-binding domain with the latter being the most evolutionary conserved (Davey and Grossmann 2016). </span>Androgens <span style="font-size:12.0pt">(such as dihydrotestosterone and testosterone) are AR ligands and </span>act by binding to the AR in androgen-responsive tissues (Davey and Grossmann 2016). Human AR mutations and mouse knockout models have established a fundamental role for AR in masculinization and spermatogenesis (Maclean et al.; Walters et al. 2010; Rana et al. 2014). The AR is also expressed in many other tissues such as bone, muscles, ovaries and within the immune system (Rana et al. 2014).</p>
<p> </p>
<p><u>Altered transcription of genes by the AR as a Key Event</u></p>
<p>Upon activation by ligand-binding, the AR translocates from the cytoplasm to the cell nucleus, dimerizes, binds to androgen response elements in the DNA to modulate gene transcription (Davey and Grossmann 2016). The transcriptional targets vary between cells and tissues, as well as with developmental stages and is also dependent on available co-regulators (Bevan and Parker 1999; Heemers and Tindall 2007). <span style="font-size:12.0pt">It should also be mentioned that the AR can work in other ‘non-canonial’ ways such as non-genomic signaling, and ligand-independent activation (Davey & Grossmann, 2016; Estrada et al, 2003; Jin et al, 2013). </span></p>
<p>A large number of known, and proposed, target genes of AR canonical signaling have been identified by analysis of gene expression following treatments with AR agonists (Bolton et al. 2007; Ngan et al. 2009<span style="font-size:12.0pt">, Jin et al. 2013</span>).</p>
<h4>How it is Measured or Detected</h4>
<p>Altered transcription of genes by the AR can be measured by measuring the transcription level of known downstream target genes by RT-qPCR or other transcription analyses approaches, e.g. transcriptomics.</p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Since this KE aims to capture AR-mediated transcriptional patterns of effect, downstream bioinformatics analyses will typically be required to identify and compare effect footprints. Clusters of genes can be statistically associated with, for example, biological process terms or gene ontology terms relevant for AR-mediated signaling. Large transcriptomics data repositories can be used to compare transcriptional patterns between chemicals, tissues, and species (e.g. TOXsIgN (Darde et al, 2018a; Darde et al, 2018b), comparisons can be made to identified sets of AR ‘biomarker’ genes (e.g. as done in (Rooney et al, 2018)), and various methods can be used e.g. connectivity mapping (Keenan et al, 2019).</span></span></span></p>
<h4>References</h4>
<p>Bevan C, Parker M (1999) The role of coactivators in steroid hormone action. Exp. Cell Res. 253:349–356</p>
<p>Bolton EC, So AY, Chaivorapol C, et al (2007) Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev 21:2005–2017. doi: 10.1101/gad.1564207</p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Calibri",sans-serif">Darde, T. A., Gaudriault, P., Beranger, R., Lancien, C., Caillarec-Joly, A., Sallou, O., et al. </span><span style="font-family:"Calibri",sans-serif">(2018a). TOXsIgN: a cross-species repository for toxicogenomic signatures. Bioinformatics 34, 2116–2122. doi:10.1093/bioinformatics/bty040.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Calibri",sans-serif">Darde, T. A., Chalmel, F., and Svingen, T. (2018b). </span><span style="font-family:"Calibri",sans-serif">Exploiting advances in transcriptomics to improve on human-relevant toxicology. Curr. Opin. Toxicol. 11–12, 43–50. doi:10.1016/j.cotox.2019.02.001.</span></span></span></p>
<p>Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37:3–15</p>
<p>Estrada M, Espinosa A, Müller M, Jaimovich E (2003) Testosterone Stimulates Intracellular Calcium Release and Mitogen-Activated Protein Kinases Via a G Protein-Coupled Receptor in Skeletal Muscle Cells. Endocrinology 144:3586–3597. doi: 10.1210/en.2002-0164</p>
<p>Heemers H V., Tindall DJ (2007) Androgen receptor (AR) coregulators: A diversity of functions converging on and regulating the AR transcriptional complex. Endocr. Rev. 28:778–808</p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77. doi: 10.3978/j.issn.2223-4683.2013.09.01</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Calibri",sans-serif">Keenan, A. B., Wojciechowicz, M. L., Wang, Z., Jagodnik, K. M., Jenkins, S. L., Lachmann, A., et al. (2019). Connectivity Mapping: Methods and Applications. Annu. Rev. Biomed. Data Sci. 2, 69–92. doi:10.1146/ANNUREV-BIODATASCI-072018-021211.</span></span></span></p>
<p>Maclean HE, Chu S, Warne GL, Zajact JD Related Individuals with Different Androgen Receptor Gene Deletions</p>
<p>MacLeod DJ, Sharpe RM, Welsh M, et al (2010) Androgen action in the masculinization programming window and development of male reproductive organs. In: International Journal of Andrology. Blackwell Publishing Ltd, pp 279–287</p>
<p>Ngan S, Stronach EA, Photiou A, et al (2009) Microarray coupled to quantitative RT&ndash;PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells. Oncogene 28:2051–2063. doi: 10.1038/onc.2009.68<span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a name="_Hlk148352925"></a></span></span></p>
<p>Rana K, Davey RA, Zajac JD (2014) Human androgen deficiency: Insights gained from androgen receptor knockout mouse models. Asian J. Androl. 16:169–177</p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Calibri",sans-serif">Rooney, J. P., Chorley, B., Kleinstreuer, N., and Corton, J. C. (2018). Identification of Androgen Receptor Modulators in a Prostate Cancer Cell Line Microarray Compendium. Toxicol. Sci. 166, 146–162. doi:10.1093/TOXSCI/KFY187.</span></span></span></p>
<p>Walters KA, Simanainen U, Handelsman DJ (2010) Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16:543–558. doi: 10.1093/humupd/dmq003</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>
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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>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000">Known antiandrogenic compounds like hydroxyflutamide have been shown to act as agonists when the AR carries certain mutations, therefore contributing to uncertainties <span style="color:black">(Yeh et al., 1997)</span>. Additionally, the levels of endogenous androgens (e.g., testosterone or dihydrotestosterone) and the variability in the presence and function of AR co-activators </span></span></span><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000">may modulate the effect of AR antagonism.</span></span></span></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>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Jin, H. J., Kim, J., & Yu, J. (2013). Androgen receptor genomic regulation. In <em>Translational Andrology and Urology</em> (Vol. 2, Issue 3, pp. 158–177). AME Publishing Company. https://doi.org/10.3978/j.issn.2223-4683.2013.09.01</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kang, Z., Pirskanen, A., Jänne, O. A., & Palvimo, J. J. (2002). Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex. <em>Journal of Biological Chemistry</em>, <em>277</em>(50), 48366–48371. https://doi.org/10.1074/jbc.M209074200</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kjærstad, M. B., Taxvig, C., Nellemann, C., Vinggaard, A. M., & Andersen, H. R. (2010). Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. <em>Reproductive Toxicology</em>, <em>30</em>(4), 573–582. <a href="https://doi.org/10.1016/j.reprotox.2010.07.009" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.reprotox.2010.07.009</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mahler, C., Verhelst, J., and Denis, L. (1998). Clinical pharmacokinetics of the antiandrogens and their efficacy in prostate cancer. Clin. Pharmacokinet. 34, 405–417. doi:10.2165/00003088-199834050-00005/METRICS.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Marcelli, M., Stenoien, D. L., Szafran, A. T., Simeoni, S., Agoulnik, I. U., Weigel, N. L., Moran, T., Mikic, I., Price, J. H., & Mancini, M. A. (2006). Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. <em>Journal of Cellular Biochemistry</em>, <em>98</em>(4), 770–788. <a href="https://doi.org/10.1002/jcb.20593" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1002/jcb.20593</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">OECD (2020). Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guide. Paris: OECD Publishing doi:10.1787/9789264264366-en.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ogino, Y., Miyagawa, S., Katoh, H., Prins, G. S., Iguchi, T., & Yamada, G. (2011). Essential functions of androgen signaling emerged through the developmental analysis of vertebrate sex characteristics. <em>Evolution & Development</em>, <em>13</em>(3), 315–325. https://doi.org/10.1111/j.1525-142X.2011.00482.x</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. <em>Current Research in Toxicology</em>, <em>3</em>, 100085. https://doi.org/10.1016/j.crtox.2022.100085</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Prizant, H., Gleicher, N., & Sen, A. (2014). Androgen actions in the ovary: balance is key. <em>Journal of Endocrinology</em>, <em>222</em>(3), R141–R151. https://doi.org/10.1530/JOE-14-0296</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rey, R. A. (2021). The Role of Androgen Signaling in Male Sexual Development at Puberty. <em>Endocrinology</em>, <em>162</em>(2). https://doi.org/10.1210/endocr/bqaa215</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Roy, A. K., Tyagi, R. K., Song, C. S., Lavrovsky, Y., Ahn, S. C., Oh, T. S., & Chatterjee, B. (2001). Androgen receptor: Structural domains and functional dynamics after ligand-receptor interaction. <em>Annals of the New York Academy of Sciences</em>, <em>949</em>, 44–57. https://doi.org/10.1111/j.1749-6632.2001.tb04001.x</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sonneveld, E. (2005). Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays. <em>Toxicological Sciences</em>, <em>83</em>(1), 136–148. https://doi.org/10.1093/toxsci/kfi005</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Szafran, A. T., Szwarc, M., Marcelli, M., & Mancini, M. A. (2008). Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects. <em>PLoS ONE</em>, <em>3</em>(11), e3605. https://doi.org/10.1371/journal.pone.0003605</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Walters, K. A., Simanainen, U., & Handelsman, D. J. (2010). Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. In <em>Human Reproduction Update</em> (Vol. 16, Issue 5, pp. 543–558). Hum Reprod Update. https://doi.org/10.1093/humupd/dmq003</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">William H. Walker. (2021). Androgen Actions in the Testis and the Regulation of Spermatogenesis. In <em>Advances in Experimental Medicine and Biology: Vol. volume 1381</em> (pp. 175–203).</span></span></p>
<p><span style="font-size:medium"><span style="font-family:Aptos,sans-serif"><span style="color:#000000">Yeh, S., Miyamoto, H., & Chang, C. (1997). <em>ARA70 and androgenic activity of hydroxyflutamide Hydroxyflutamide may not always be a pure antiandrogen</em> (Vol. 349).</span></span></span></p>
</div>
<div>
<h4><a href="/relationships/2124">Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR</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 for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across mammalian taxa. 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:12pt">The androgen receptor (AR) is a ligand-dependent nuclear transcription factor that upon activation translocates to the nucleus, dimerizes, and binds androgen response elements (AREs) to modulate transcription of target genes <span style="color:black">(Lamont and Tindall, 2010, Roy et al. 2001)</span>. Decreased activation of the AR affects its transcription factor activity, therefore leading to altered AR-target gene expression. This KER refers to decreased AR activation and altered gene expression occurring in complex systems, such as <em>in vivo</em> and the specific effect on transcription of AR target genes will depend on species, life stage, tissue, cell type etc. </span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="text-align:justify"><span style="font-size:12pt">The biological plausibility for this KER is considered high</span></p>
<p style="text-align:justify"><span style="font-size:12pt">The AR is a ligand-activated transcription factor part of the steroid hormone nuclear receptor family. Non-activated AR is found in the cytoplasm as a multiprotein complex with heat-shock proteins, immunophilins and, other chaperones <span style="color:black">(Roy et al. 2001)</span>. Upon activation through ligand binding, the AR dissociates from the protein complex, translocates to the nucleus and homodimerizes. Facilitated by co-regulators, AR can bind to DNA regions containing AREs and initiate transcription of target genes, that thus will be different in e.g. different tissues, life-stages, species etc. </span></p>
<p style="text-align:justify"><span style="font-size:12pt">Through mapping of AREs and ChIP sequencing studies, several AR target genes have been identified, mainly studied in prostate cells <span style="color:black">(Jin, Kim, and Yu 2013)</span>. Different co-regulators and ligands lead to altered expression of different sets of genes <span style="color:black">(Jin et al. 2013; Kanno et al. 2022)</span>. Alternative splicing of the AR can lead to different AR variants that also affects which genes are transcribed <span style="color:black">(Jin et al. 2013)</span>.</span></p>
<p style="text-align:justify"><span style="font-size:12pt">Apart from this canonical signaling pathway, the AR can suppress gene expression, indirectly regulate miRNA transcription, and have non-genomic effects by rapid activation of second messenger pathways in either presence or absence of a ligand <span style="color:black">(Jin et al. 2013)</span>.</span></p>
<strong>Empirical Evidence</strong>
<p style="text-align:justify"><span style="font-size:12pt">The empirical evidence for this KER is considered high</span></p>
<p style="text-align:justify"><span style="font-size:12pt">In humans, altered gene expression profiling in individuals with androgen insensitivity syndrome (AIS) can provide supporting empirical evidence <span style="color:black">(Holterhus et al. 2003; Peng et al. 2021)</span>. In rodent AR knockout (KO) models, gene expression profiling studies and gene-targeted approaches have provided information on differentially expressed genes in several organ systems including male and female reproductive, endocrine, muscular, cardiovascular and nervous systems <span style="color:black">(Denolet et al. 2006; Fan et al. 2005; Holterhus et al. 2003; Ikeda et al. 2005; Karlsson et al. 2016; MacLean et al. 2008; Rana et al. 2011; Russell et al. 2012; Shiina et al. 2006; Wang et al. 2006; Welsh et al. 2012; Willems et al. 2010; Yu et al. 2008, 2012; Zhang et al. 2006; Zhou et al. 2011)</span>.</span></p>
<p style="text-align:justify"><span style="font-size:12pt">Exposure to known antiandrogens has been shown to alter transcriptional profiles, for example of neonatal pig ovaries <span style="color:black">(Knapczyk-Stwora et al. 2019)</span>. </span></p>
<p style="text-align:justify"><span style="font-size:12pt">Dose concordance has also been observed for instance in zebrafish embryos; a dose of 50 µg/L of the AR antagonist flutamide resulted in 674 differentially expressed genes at 96 h post fertilization whereas 500 µg/L flutamide resulted in 2871 differentially expressed genes (Ayobahan et al., 2023). </span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="text-align:justify"><span style="font-size:12pt">AR action has been reported to occur also without ligand binding. However, not much is known about the extent and biological implications of such non-canonical, ligand-independent AR activation <span style="color:black">(Bennesch and Picard 2015)</span>.</span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-size:12pt">There is not enough data to define a quantitative relationship between AR activation and alteration of AR target gene transcription, and such a relationship will differ between biological systems (species, tissue, cell type, life stage etc).</span></p>
<strong>Time-scale</strong>
<p style="text-align:justify"><span style="font-size:12pt">AR and promoter interactions occur within 15 minutes of ligand binding, RNA polymerase II and coactivator recruitment are proposed to occur transiently with cycles of approximately 90 minutes in LNCaP cells <span style="color:black">(Kang et al. 2002)</span>. RNA polymerase II elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb/min <span style="color:black">(Maiuri et al. 2011)</span>. Therefore, depending on the cell type and the half-life of the AR target gene transcripts, changes are to be expected within hours. </span></p>
<td><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">AR expression in aging male rats</span></span></td>
<td><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Tissue-specific alterations in AR activity with aging</span></span></td>
<td><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Supakar et al. 1993; Wu, Lin, and Gore 2009)</span></span></span></td>
</tr>
<tr>
<td>Genotype</td>
<td><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Number of CAG repeats in the first exon of AR</span></span></td>
<td><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Decreased AR activation with increased number of CAGs</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(Tut et al. 1997; Chamberlain et al. 1994)</span></span></span></p>
</td>
</tr>
</tbody>
</table>
</div>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p style="text-align:justify"><span style="font-size:12pt">AR has been hypothesized to auto-regulate its mRNA and protein levels <span style="color:black">(Mora and Mahesh 1999)</span>.</span></p>
<h4>References</h4>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Ayobahan, S. U., Alvincz, J., Reinwald, H., Strompen, J., Salinas, G., Schäfers, C., et al. (2023). Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo. Ecotoxicol. Environ. Saf. 250, 114514. doi:10.1016/J.ECOENV.2023.114514.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Bennesch, Marcela A., and Didier Picard. 2015. “Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors.” <em>Molecular Endocrinology</em> 29(3):349–63.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Chamberlain, Nancy L., Erika D. Driverand, and Roger L. Miesfeldi. 1994. <em>The Length and Location of CAG Trinucleotide Repeats in the Androgen Receptor N-Terminal Domain Affect Transactivation Function</em>. Vol. 22.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Denolet, Evi, Karel De Gendt, Joke Allemeersch, Kristof Engelen, Kathleen Marchal, Paul Van Hummelen, Karen A. L. Tan, Richard M. Sharpe, Philippa T. K. Saunders, Johannes V. Swinnen, and Guido Verhoeven. 2006. “The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice.” <em>Molecular Endocrinology</em> 20(2):321–34. doi: 10.1210/me.2005-0113.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Fan, Wuqiang, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Takashi Sato, Hirotaka Kawano, Shigeaki Kato, and Hajime Nawata. 2005. <em>Androgen Receptor Null Male Mice Develop Late-Onset Obesity Caused by Decreased Energy Expenditure and Lipolytic Activity but Show Normal Insulin Sensitivity With High Adiponectin Secretion</em>. Vol. 54.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Holterhus, Paul-Martin, Olaf Hiort, Janos Demeter, Patrick O. Brown, and James D. Brooks. 2003. <em>Differential Gene-Expression Patterns in Genital Fibroblasts of Normal Males and 46,XY Females with Androgen Insensitivity Syndrome: Evidence for Early Programming Involving the Androgen Receptor</em>. Vol. 4.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” <em>Translational Andrology and Urology</em> 2(3):158–77.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Kang, Zhigang, Asta Pirskanen, Olli A. Jänne, and Jorma J. Palvimo. 2002. “Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex.” <em>Journal of Biological Chemistry</em> 277(50):48366–71. doi: 10.1074/jbc.M209074200.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Kanno, Yuichiro, Nao Saito, Ryota Saito, Tomohiro Kosuge, Ryota Shizu, Tomofumi Yatsu, Takuomi Hosaka, Kiyomitsu Nemoto, Keisuke Kato, and Kouichi Yoshinari. 2022. “Differential DNA-Binding and Cofactor Recruitment Are Possible Determinants of the Synthetic Steroid YK11-Dependent Gene Expression by Androgen Receptor in Breast Cancer MDA-MB 453 Cells.” <em>Experimental Cell Research</em> 419(2). doi: 10.1016/j.yexcr.2022.113333.</span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">MacLean, Helen E., W. S. Maria Chiu, Amanda J. Notini, Anna-Maree Axell, Rachel A. Davey, Julie F. McManus, Cathy Ma, David R. Plant, Gordon S. Lynch, and Jeffrey D. Zajac. 2008. “ Impaired Skeletal Muscle Development and Function in Male, but Not Female, Genomic Androgen Receptor Knockout Mice .” <em>The FASEB Journal</em> 22(8):2676–89. doi: 10.1096/fj.08-105726.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Maiuri, Paolo, Anna Knezevich, Alex De Marco, Davide Mazza, Anna Kula, Jim G. McNally, and Alessandro Marcello. 2011. “Fast Transcription Rates of RNA Polymerase II in Human Cells.” <em>EMBO Reports</em> 12(12):1280–85. doi: 10.1038/embor.2011.196.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Mora, Gloria R., and Virendra B. Mahesh. 1999. <em>Autoregulation of the Androgen Receptor at the Translational Level: Testosterone Induces Accumulation of Androgen Receptor MRNA in the Rat Ventral Prostate Polyribosomes</em>.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Peng, Yajie, Hui Zhu, Bing Han, Yue Xu, Xuemeng Liu, Huaidong Song, and Jie Qiao. 2021. “Identification of Potential Genes in Pathogenesis and Diagnostic Value Analysis of Partial Androgen Insensitivity Syndrome Using Bioinformatics Analysis.” <em>Frontiers in Endocrinology</em> 12. doi: 10.3389/fendo.2021.731107.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Rana, Kesha, Barbara C. Fam, Michele V Clarke, Tammy P. S. Pang, Jeffrey D. Zajac, and Helen E. Maclean. 2011. “Increased Adiposity in DNA Binding-Dependent Androgen Receptor Knockout Male Mice Associated with Decreased Voluntary Activity and Not Insulin Resistance.” <em>Am J Physiol Endocrinol Me-Tab</em> 301:767–78. doi: 10.1152/ajpendo.00584.2010.-In.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Roy, Arun K., Rakesh K. Tyagi, Chung S. Song, Yan Lavrovsky, Soon C. Ahn, Tae Sung Oh, and Bandana Chatterjee. 2001. “Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction.” Pp. 44–57 in <em>Annals of the New York Academy of Sciences</em>. Vol. 949. New York Academy of Sciences.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Russell, Patricia K., Michele V. Clarke, Jarrod P. Skinner, Tammy P. S. Pang, Jeffrey D. Zajac, and Rachel A. Davey. 2012. “Identification of Gene Pathways Altered by Deletion of the Androgen Receptor Specifically in Mineralizing Osteoblasts and Osteocytes in Mice.” <em>Journal of Molecular Endocrinology</em> 49(1):1–10. doi: 10.1530/JME-12-0014.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Shiina, Hiroko, Takahiro Matsumoto, Takashi Sato, Katsuhide Igarashi, Junko Miyamoto, Sayuri Takemasa, Matomo Sakari, Ichiro Takada, Takashi Nakamura, Daniel Metzger, Pierre Chambon, Jun Kanno, Hiroyuki Yoshikawa, and Shigeaki Kato. 2006. <em>Premature Ovarian Failure in Androgen Receptor-Deficient Mice</em>. Vol. 103.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Supakar, P. C., C. S. Song, M. H. Jung, M. A. Slomczynska, J. M. Kim, R. L. Vellanoweth, B. Chatterjee, and A. K. Roy. 1993. “A Novel Regulatory Element Associated with Age-Dependent Expression of the Rat Androgen Receptor Gene.” <em>Journal of Biological Chemistry</em> 268(35):26400–408. doi: 10.1016/s0021-9258(19)74328-2.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Tut, Thein G., Farid J. Ghadessy, M. A. Trifiro, L. Pinsky, and E. L. Yong. 1997. <em>Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility*</em>. Vol. 82.</span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Welsh, M., L. Moffat, K. Belling, L. R. de França, T. M. Segatelli, P. T. K. Saunders, R. M. Sharpe, and L. B. Smith. 2012. “Androgen Receptor Signalling in Peritubular Myoid Cells Is Essential for Normal Differentiation and Function of Adult Leydig Cells.” <em>International Journal of Andrology</em> 35(1):25–40. doi: 10.1111/j.1365-2605.2011.01150.x.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Willems, Ariane, Sergio R. Batlouni, Arantza Esnal, Johannes V. Swinnen, Philippa T. K. Saunders, Richard M. Sharpe, Luiz R. França, Karel de Gendt, and Guido Verhoeven. 2010. “Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development.” <em>PLoS ONE</em> 5(11). doi: 10.1371/journal.pone.0014168.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Wu, D. I., Grace Lin, and Andrea C. Gore. 2009. “Age-Related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor in Male Rats.” <em>The Journal of Comparative Neurology</em> 512:688–701. doi: 10.1002/cne.21925.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yu, I. Chen, Hung Yun Lin, Ning Chun Liu, Ruey Shen Wang, Janet D. Sparks, Shuyuan Yeh, and Chawnshang Chang. 2008. “Hyperleptinemia without Obesity in Male Mice Lacking Androgen Receptor in Adipose Tissue.” <em>Endocrinology</em> 149(5):2361–68. doi: 10.1210/en.2007-0516.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Yu, Shengqiang, Chiuan Ren Yeh, Yuanjie Niu, Hong Chiang Chang, Yu Chieh Tsai, Harold L. Moses, Chih Rong Shyr, Chawnshang Chang, and Shuyuan Yeh. 2012. “Altered Prostate Epithelial Development in Mice Lacking the Androgen Receptor in Stromal Fibroblasts.” <em>Prostate</em> 72(4):437–49. doi: 10.1002/pros.21445.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Zhang, Caixia, Shuyuan Yeh, Yen-Ta Chen, Cheng-Chia Wu, Kuang-Hsiang Chuang, Hung-Yun Lin, Ruey-Sheng Wang, Yu-Jia Chang, Chamindrani Mendis-Handagama, Liquan Hu, Henry Lardy, Chawnshang Chang, and † † George. 2006. <em>Oligozoospermia with Normal Fertility in Male Mice Lacking the Androgen Receptor in Testis Peritubular Myoid Cells</em>.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Zhou, Wei, Gensheng Wang, Christopher L. Small, Zhilin Liu, Connie C. Weng, Lizhong Yang, Michael D. Griswold, and Marvin L. Meistrich. 2011. “Erratum: Gene Expression Alterations by Conditional Knockout of Androgen Receptor in Adult Sertoli Cells of Utp14bjsd/Jsd (Jsd) Mice (Biology of Reproduction (2010) 83, (759-766) DOI: 10.1095/Biolreprod.110.085472).” <em>Biology of Reproduction</em> 84(2):400–408.</span></span></p>
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2820">Relationship: 2820: Decrease, AR activation leads to AGD, decreased</a></h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Fetal masculinization including the AGD is regulated by androgens interacting with the AR in all mammals, including humans (Murashima et al., 2015; Thankamony et al., 2016), although, the size of the AGD and difference between the sexes vary between species. A large number of studies exist showing that fetal exposure to anti-androgens causes shortened AGD in male rats and mice (Schwartz et al., 2019, see also Table 2). Some epidemiological studies find associations between exposure to anti-androgenic compounds and shorter AGD in boys (Thankamony et al., 2016). However, the associations are not very clear and confidence in the data is limited by conflicting results, possibly due to differences in study design and methods for exposure measurements and analyses. Nevertheless, the KER is considered applicable to humans, based on current understanding of the role of AR activation in fetal masculinization.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Programming of the AGD occurs during the masculinization programming window in fetal life. This takes place in rats around embryonic days 15.5-19.5 (GD16-20) and likely gestation weeks 8-14 in humans (Welsh et al., 2008). It should be mentioned that though AGD is believed to be relatively stable throughout life, it can be responsive to postnatal changes in androgen levels (Schwartz et al., 2019).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Data presented in this KER support that disruption of androgen action during fetal life can lead to a short AGD in male offspring. While exposure to chemicals during fetal life can also shorten female AGD, the biological </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">significance and the mechanism driving the effect is unknown (Schwartz et al., 2019). </span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">This KER refers to a decrease in androgen receptor (AR) activation during fetal development leading to decreased anogenital distance (AGD) in male offspring.<br />
It should be noted that the upstream Key Event (KE) ‘decrease, androgen receptor activation’ (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the androgen receptor in vivo, while most methods that can be used to measure AR activity are carried out in vitro. Indirect information about this KE may for example be provided from assays showing in vitro AR antagonism, decreased in vitro or in vivo testosterone production/levels or decreased in vitro or in vivo dihydrotestosterone (DHT) production/levels.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The biological plausibility for this KER is judged to be high based on the following:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- Sexual differentiation happens in fetal life. The testes are developed and start to produce testosterone that is converted in other tissues by the enzyme 5-alpha-reductase to the more potent androgen dihydrotestosterone (DHT). Both hormones bind and activate the nuclear receptor and transcription factor AR that in turn drives masculinization of the male fetus (Welsh et al., 2014; Schwartz et. al, 2019).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- Fetal masculinization depends on activation of androgen signaling during a critical time window, the masculinization programming window (MPW), from gestational day (GD) 15.5-18.5 in rats, 14.5-16.5 in mice and presumably gestation weeks (GWs) 8-14 in humans (Welsh et al., 2008; Amato et al., 2022). The onset of AR expression in the tissues of the reproductive tract follows the timing of the MPW (Welsh et al., 2008).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- The fetal masculinization process involves a range of tissues and organs, including the perineum. Perineum length can be measured as the AGD, which is the distance between the anus and the genitalia. The AGD is approximately twice as long in male as in female newborn rodents and humans (Schwartz et al., 2019). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- Male AR knockout mice present shorter AGD than wildtype males, so short that it is indistinguishable from wildtype female littermates (Yeh et al., 2002, Sato et al., 2004). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- Male AR knockout mice present with shorter AGD than wildtype males, so short that it is indistinguishable from wildtype female littermates (</span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Notini et al., 2005; Yeh et al., 2002, Sato et al., 2004). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- In human males, mutations decreasing AR activity also lead to feminization. One example is the androgen insensitivity syndrome (AIS), where mutations in the AR lead to an impaired or abolished response to androgens, and thereby some degree of feminization of XY individuals and even XY sex reversal in individuals with complete AIS (CAIS) (Thankamony et al., 2016; Hughes et al., 2012; Crouch et al., 2011). XY individuals with CAIS present as women with internally placed testes. A study showed that the clitoral to urethral distance in these individuals was similar to a control group of women, but it is not clear whether this measurement can work as a proxy for measuring the AGD (Thankamony et al 2016, Crouch 2011). Unfortunately, it seems the AGD has not at present been measured in CAIS individuals. Another example is human males lacking 5-alpha-reductase, also presenting female-like genitalia (Batista & Mendonca, 2022).</span></span><br />
</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">- The detailed mechanism by which androgens regulate the AGD is not known but it is hypothesized that the AGD is influenced by the size of the levator-ani and bulbocavernosus (LABC) muscle complex in the perineum. The growth of this complex is stimulated by AR activation, it is sexually dimorphic and larger in males than in females and (Schwartz et al., 2019). AR is required for the development of the LABC complex as demonstrated by AR general and muscle specific knockout mice. AR is expressed in non-myocytic cells in the LABC complex, starting at E15.5 in mice, and knockout of AR in these cells results in defects in the muscle formation (Ipulan et al., 2016;). Differential gene expression profiles in the perineum of male and female rats as well as in antiandrogen-exposed male rats have been identified providing further mechanistic understanding (Schwartz et al, 2019; Draskau et al, 2022).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The empirical support from studies in animals for this KER is overall judged as high.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">It should be noted that the KE decreased androgen receptor activation (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the androgen receptor in vivo, with no methods currently available to measure this. Examples of assays that provide indirect information about KE-1614 are described in upstream MIE/KEs.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_Hlk152315924">The empirical evidence for this KER from animal studies in vivo is based on studies using five different substances that result in decreased AR activation by different mechanisms. Flutamide, procymidone and vinclozolin bind to the AR and inhibit the receptor activity and thereby act as AR antagonists, see MIE26. Finasteride inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT, see MIE1617. DEHP exposure during prenatal development in rats results in reduced fetal testosterone levels, see KE1690. (MIE26, MIE1617 and KE1690 can be found in AOP Wiki).</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The evidence for the upstream KE is mainly based on data from in vitro assays (AR antagonism or 5-alpha-reductase inhibition in vitro) whereas the evidence for the downstream KE is based on in vivo studies, and there is generally not evidence for both KEs from the same study. However, decreased testosterone levels can be measured in vivo, and Borch et al., 2004 measured the effect of developmental DEHP exposure on both testosterone levels and AGD (see section about “Dose concordance”).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The empirical animal evidence for the five substances is summarized in table 3.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Table 3. Summary of empirical evidence for decreased androgen receptor activation, leading to decreased male AGD. References for the studies supporting the empirical evidence are found in section “<u>Evidence for decreased AR activation (KE1614) by flutamide, procymidone, and vinclozolin, finasteride and DEHP” and in table 2.</u></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AR antagonism in in vitro assay receptor binding and transactivation assays</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Decreased male AGD after prenatal exposure in studies in rat</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AR antagonism in in vitro assay receptor binding and transactivation assays</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Decreased male AGD after prenatal exposure in studies in rat</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AR antagonism in in vitro assay receptor binding and transactivation assays</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Decreased male AGD after prenatal exposure in studies in rat and mouse </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Inhibition of 5-alpha-reductase enzyme in in vitro assays</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Decreased male AGD after prenatal exposure in studies in rat</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Reduced production of testosterone in fetal testis measured in ex vivo testis assays, reduced testosterone levels in testis and reduced fetal plasma or serum testosterone levels</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Decreased male AGD after prenatal exposure in studies in rat</span></span></p>
<p> </p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">From table 3, it can be deducted that fetal exposure to substances known to decrease androgen receptor activation through antagonism of the AR (vinclozolin, procymidone, flutamide), inhibition of testosterone synthesis (DEHP) or inhibition of conversion of testosterone to DHT (finasteride), results in decreased AGD in rat and mouse male offspring.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_Hlk152319119"><u>Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, vinclozolin, finasteride and DEHP</u></a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Flutamide, a pharmaceutical, binds the AR and inhibits the receptor activity, thereby acting as an AR antagonist. It has been used as an antiandrogen for treatment of prostate cancer and is used as a reference chemical for antiandrogenic activity in the AR transactivation assays in the OECD test guideline No 458 (Goldspiel & Kohler, 1990; Labrie, 1993; OECD, 2023; Simard et al., 1986). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Procymidone and vinclozolin are fungicides that have been shown to be AR antagonists. Procymidone binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat prostate cytosol (Hosokawa et al., 1993) or AR transfected COS cells (Ostby et al., 1999). Procymidone also inhibits agonist activated transcription in AR reporter assays (Hass et al., 2012; Kojima et al., 2004; Orton et al., 2011; Ostby et al., 1999; Scholze et al., 2020). Vinclozolin binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat epididymis cytosol (Kelce et al., 1997) or AR transfected COS-1 cells (Wong et al., 1995).<br />
Vinclozolin also inhibits agonist activated transcription in AR reporter assays (Euling et al, 2002; Kojima et al., 2004; Molina-Molina et al., 2006; Orton et al., 2011; Scholze et al., 2020; Shimamura et al., 2002; Wong et al., 1995). Finasteride is a pharmaceutical that inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT. Finasteride is used to treat benign prostatic hypertrophy (Andersson & Russel, 1990; Rittmaster & Wood, 1994; Stoner, 1990).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Prenatal exposure to DEHP in rats results in reduced production of testosterone in fetal testis measured in ex vivo testis assays, reduced testosterone levels in testis and reduced fetal plasma or serum testosterone levels (<a name="_Hlk155267343">Borch et al., 2004; Borch et al., 2006; Culty et al., 2008; Hannas et al., 2011; Hannas et al., 2012; Klinefelter et al., 2012; Parks et al., 2000; Wilson et al., 2004; Wilson et al., 2007; Vo et al., 2009</a>). Two studies don’t show an effect on testosterone levels in testis or fetal plasma testosterone levels, respectively (Andrade et al., 2006; Borch et al., 2006). The precise underlying mechanism is presently unknown.</span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><u>Evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP</u></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">All datasets that were used for the weight of evidence assessment were judged as reliable without or with restriction. The majority of datasets assessed showed a decreased male AGD. The conclusion was that the level of confidence was strong for all five substances. The studies are summarized in table 4.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Empirical evidence for the included substances</strong></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Table 4. Empirical evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP. *</span></span><span style="font-size:10.0pt"><span style="font-family:"Calibri",sans-serif">One dose only.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:9pt">Martinez et al., 2011</span></span></span></p>
</td>
</tr>
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</table>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_Hlk152320981"><strong>Epidemiological data on DEHP</strong></a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><a name="_Hlk164418693">The biggest relevant epidemiological dataset was identified on associations between DEHP and AGD. </a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Six prospective cohort studies and one cross-sectional study on the association between maternal DEHP metabolites and length of AGD (anopenile distance (APD) and anoscrotal distance (ASD)) in boys were assessed as reliable without or with restriction. Decreased AGD (anopenile distance (APD) and/or anoscrotal distance (ASD)) was observed in three prospective cohort studies (<a name="_Hlk155275587">Martino-Adrade et al., 2016; Swan et al., 2005 reviewed and updated in Swan 2008; Wenzel et al., 2018</a>). In contrast, no significant association was observed in three other prospective cohort studies (<a name="_Hlk155275688">Arbuckle et al., 2018</a><span style="font-size:9.0pt">; </span>Henriksen et al., 2023; Jensen et al., 2016) and the cross-sectional study (Sunman et al., 2019). This inconsistency introduces a level of uncertainty regarding the overall association. Therefore, the level of confidence was judged as weak. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Dose concordance is challenging to assess for this KER since in vivo AR activity is currently not possible to measure, but only can be informed indirectly by measures of upstream events.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">However, some studies provide useful information that support dose concordance between the KEs.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In a publication by Borch et al., rats were exposed in utero to DEHP at GD7-21. Fetal testosterone levels in testes and serum and testosterone production in fetal testes ex vivo were investigated at GD21, whereas AGD was investigated at PND3. The LOAELs for reduced testosterone production in ex vivo fetal testes and reduced testosterone levels in fetal testes were 300 mg/kg/d, whereas the LOAEL for decreased AGD in male offspring was 750 mg/kg/d (Borch et al., 2004). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In a publication by Scholze et al, AR antagonism and decreased testosterone synthesis was quantitatively assessed (IC50) in vitro for a list of substances. In addition, internal concentrations in male fetuses and effects on AGD were measured after fetal exposure to the same substances. In utero exposure to all the substances lead to reduced AGDIndex (AGDI) in the exposed male offspring. Further, for all substances except Cyprodinil, the internal exposure levels in the fetuses leading to reduced AGD exceeded the IC50 levels observed in one or both of the in vitro assays.<br />
Three different doses of linuron exposure were included. The medium exposure dose led to a higher level of internal exposure and a higher degree of AGDI reduction than the low dose. AGDI could not be determined in the highest dose due to maternal toxicity (Scholze et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Temporal concordance can only be considered from a theoretical perspective since the downstream event, decreased AGD, is usually measured at GD21, PND0 or PND1 in rats, and due to the size of the fetuses is not feasible to measure at earlier timepoints. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Considering the biology, the upstream event – decreased AR activation<em> in vivo</em> – is foreseen to happen minutes to hours after exposure. If a substance decreases AR activation through inhibition of the AR, the upstream event is expected to happen immediately after exposure. If a substance decreases androgen receptor activation through inhibition of testosterone synthesis, the upstream event is expected to happen minutes to hours after the exposure, though it is uncertain exactly when the change will be big enough to be measurable. On the other hand, the downstream event – decreased AGD - is a measurement of relative growth of the perineal tissue, which is expected to take days in the developing fetus.</span></span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">For the model substances, there were some inconsistencies in the empirical evidence, but they could be explained by differences in study designs and uncertainties in measurements, see appendix 1: </span></span><a href="https://aopwiki.org/system/dragonfly/production/2025/05/06/8dh20j155i_FINAL_Appendix_KER2820_For_Wiki.pdf">8dh20j155i_FINAL_Appendix_KER2820_For_Wiki.pdf</a><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Species differences in effects of phthalates (including DEHP and DBP) on fetal testes testosterone production have been observed between humans, mice and rats. In human fetal testes exposed to DEHP or DBP in vitro or ex vivo, no suppression of testosterone production is observed, which contrasts observations in rat fetal testes under similar conditions. Also in mice, testosterone production in the fetal testes is unaffected by treatment with DEHP or DBP in vitro or in utero (Sharpe, 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The species differences described above are specific for some phthalates and their interference with fetal testicular testosterone production. This uncertainty should not be reflected on other antiandrogenic substances, especially not those acting through other mechanisms of action.<br />
The association between exposure to DEHP and reduced AGD in humans is judged to be weak, which may further support a species difference between rodents and humans, but it may also reflect the large uncertainties inherent in the epidemiological studies.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Observational epidemiological studies face challenges in proving cause-effect relationships as they cannot control conditions like experimental animal and in vitro studies. Human studies can identify associations between variables but cannot offer conclusive proof of causation (Lanzoni et al., 2019). Various study designs and statistical methods are employed to strengthen evidence within the inherent limitations of observational research (Song & Chung, 2010; Olier et al., 2023). Inconsistencies in epidemiological data arise from various factors, such as different methodologies used in exposure and outcome measurement and also in statistical analyses. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">These differences collectively contribute to the complexity of interpreting and weighing the evidence in epidemiological research.</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The quantitative understanding of the linkage is low. This is a consequence of it not being possible to measure the upstream and the downstream event in the same study.</span></span></p>
<strong>Response-response relationship</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In one study, a quantitative model was developed to predict the decrease in AGD from in vitro AR antagonism or in vitro decreased testosterone synthesis. The authors conclude that predicting the effect on AGD in vivo based on the in vitro results is only possible on a qualitative level, but the model cannot predict AGD reductions quantitatively (Scholze et al., 2020).</span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AR activation operates on a time-scale of minutes. The AR is a ligand-activated nuclear receptor and transcription factor. Upon ligand binding a conformational change and subsequent dimerization of the AR takes place within 3-6 minutes (Schaufele et al., 2005). Nuclear translocation (Nightingale et al., 2003) and promoter interactions occur within 15 minutes of ligand binding, and RNA polymerase II and coactivator recruitment are then proposed to occur transiently with cycles of approximately 90 minutes (Kang et al., 2002). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">For the downstream event, the time-scale for observing a measurable effect on growth of a tissue (in this case the perineum) is closer to days and weeks depending on species. For instance, in humans, the masculinization programming window is presumed to start around GW 8, while a sexual dimorphism of the AGD can first be observed from around GWs 11-13 (Thankamony et al., 2016) and reaches its maximum 2-fold difference around GWs 17-20 (Sharpe, 2020). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">It has been demonstrated that exposure to flutamide for one day (Foster & Harris, 2005) or vinclozolin for two days (Wolf et al., 2000) during the sensitive window of exposure can elicit a detectable decrease in the AGD in male rat offspring.</span></span></p>
<strong>Known modulating factors</strong>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">A well established modulating factor is genetic variations in the AR which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Tut et al 1997, Chamberlain et al 1994) and a shorter AGD in adult men (Eisenberg et al., 2013). Other modulating factors being discussed in the literature is maternal age and parity (Barrett et al., 2014), but these associations are only suggestive with more studies needed to confirm the associations (Barrett et al., 2014).</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Not relevant for this KER. </span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Amato, Ciro M., Humphrey H-C. Yao, and Fei Zhao. “One Tool for Many Jobs: Divergent and Conserved Actions of Androgen Signaling in Male Internal Reproductive Tract and External Genitalia.” <em>Frontiers in Endocrinology</em> 13 (2022). <a href="https://www.frontiersin.org/articles/10.3389/fendo.2022.910964" style="color:#0563c1; text-decoration:underline">https://www.frontiersin.org/articles/10.3389/fendo.2022.910964</a>.</span></span></p>
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<h4><a href="/relationships/2127">Relationship: 2127: Altered, Transcription of genes by the AR leads to AGD, decreased</a></h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This KER applies to humans, mice, and rats based on biological plausibility. Current empirical evidence is from rat studies only. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fetal masculinization including the AGD is regulated by androgens interacting with the AR in all mammals, including humans (Murashima et al., 2015; Thankamony et al., 2016), although, the size of the AGD and difference between the sexes vary between species. A large number of studies exist showing that fetal exposure to anti-androgens causes shortened AGD in male rats and mice (Schwartz et al., 2019a). Some epidemiological studies find associations between exposure to anti-androgenic compounds and shorter AGD in boys (Thankamony et al., 2016). However, the associations are not very clear and confidence in the data is limited by conflicting results, possibly due to differences in study design and methods for exposure measurements and analyses. Nevertheless, the KER is considered applicable to humans, based on current understanding of the role of AR activation in fetal masculinization.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The length of the AGD is programmed during fetal life during the masculinization programming window. This takes place in rats around embryonic days 15.5-19.5 (GD16-20) and likely gestation weeks 8-14 in humans (Welsh et al., 2008). It should be mentioned that though AGD is believed to be relatively stable throughout life, it can be responsive to postnatal changes in androgen levels (Schwartz et al., 2019a).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">A decrease in the male AGD is a consequence of disrupted androgen action (Welsh et al 2008). While exposure to chemicals during fetal life can also shorten female AGD, the biological significance and the mechanism driving the effect is unknown (Schwartz et al 2019a).</span></span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">During male reproductive development, the androgen receptor (AR) regulates gene transcription in target tissues to induce masculinization. Target tissues include the perineum, the tissue located between the anus and the genitals. This tissue is sexually dimorphic, with males developing the levator ani-bulbocavernosus (LABC) muscle complex in response to androgen signaling. The anogenital distance (AGD) is about twice as long in newborn males than in females in many mammals such mice, rats and humans. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">A consequence of reduced androgen action during the masculinization programming window in utero, the male AGD will end up being shorter, approaching female AGD when AR signaling is almost blocked. Measuring of the AGD thus serves as a morphometric biomarker for compromised androgen action during fetal life and is used in OECD test guidelines for assessing endocrine disruption. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This KER refers to a tissue-specific, in this case the perineum, alteration in AR-mediated gene transcription during fetal development leading to a decreased AGD in male offspring.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This KER refers to a tissue-specific alteration in AR-mediated gene transcription during fetal development leading to a decreased AGD in male offspring.<span style="font-size:12px"><span style="font-family:Tahoma,Geneva,sans-serif"> </span></span></span></span><span style="font-size:12px"><span style="font-family:Verdana,Geneva,sans-serif">It should be noted that the AR‑mediated transcription operates within a broader developmental context, where timing, tissue specificity, and local signaling environments, including patterning mechanisms and morphogen gradients, jointly determine masculinization outcomes such as AGD. While such contextual influences are acknowledged, the KER remains focused on the androgen‑dependent transcriptional component that drives AGD outcomes.</span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexual differentiation initiates during fetal life when a surge in testosterone induces masculinization of a range of tissues and organs (Welsh et al). Testosterone and the more potent metabolite DHT mediate masculinization via activation of the AR; a nuclear transcription factor. Androgens thus induce masculinization via altered AR gene transcription in target tissues. This includes the perineum (Niel et al 2008; Ipulan et al 2014) which can be measured as the AGD and is approximately twice as long in newborn male rodents and humans compared to female (Schwartz et al 2019a). This is also evident in male AR knockout mice which present with an AGD that is indistinguishable from wildtype female littermates (MacLean et al 2008; Notini et al 2005).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexual differentiation initiates during fetal life when a surge in testosterone induces masculinization of a range of tissues and organs (Welsh et al). Testosterone and the more potent metabolite DHT mediate masculinization via activation of the AR; a nuclear transcription factor. Androgens thus induce masculinization via altered AR gene transcription in target tissues. This includes the perineum (Niel et al 2008; Ipulan et al 2014) which can be measured as the AGD and is approximately twice as long in newborn male rodents and humans compared to female (Schwartz et al 2019a). This is also evident in male AR knockout mice which present with an AGD that is indistinguishable from wildtype female littermates (MacLean et al 2008; Notini et al 2005). </span></span><span style="font-size:12px">This AR knockout model disrupts the second zinc finger required for DNA binding, demonstrating that genomic (DNA-binding-dependent) actions of the AR are essential for normal male AGD development.</span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Current evidence for direct transcriptional changes mediated by AR disruption in the perineum leading to shorter male AGD is limited. Two studies were identified investigating the transcriptional footprint in the perineum after anti-androgen exposure:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gestational exposure of rats to the 5α-reductase inhibitor finasteride (leading to decreased DHT levels) decreased fetal male AGD with 37% at gestational day (GD) 21. Microarray was used to compare transcriptional profiles between control males, finasteride-exposed males, and control females, revealing a sexually dimorphic transcriptional profile of the perineum, with the profile of finasteride-exposed males being intermediary to the male and female control groups (Schwartz et al 2019b).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gestational exposure of rats to the AR antagonist triticonazole induced decreased fetal male AGD at GD21 and a differentially expressed set of genes investigated by whole transcriptome sequencing in the perineum at both GD17 and GD21 (Draskau et al 2022). </span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Draskau MK, Schwartz CL, Evrard B, Lardenois A, Pask A, Chalmel F and Svingen T (2022). The anti-androgenic fungicide triticonazole induces region-specific transcriptional changes in the developing rat perineum and phallus. <em>Chemosphere</em> 308(Pt 2):136346. doi: 10.1016/j.chemosphere.2022.136346</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ipulan LA, Suzuki K, Sakamoto Y, Murashima A, Imai Y, Omori A, Nakagata N, Nishinakamura R, Valasek P and Yamada G (2014). Nonmyocytic androgen receptor regulates the sexually dimorphic development of the embryonic bulbocavernosus muscle. <em>Endocrinology</em> 155(7):2467-79. doi: 10.1210/en.2014-1008</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">MacLean HE, Chiu WS, Notini AJ, Axell AM, Davey RA, McManus JF, Ma C, Plant DR, Lynch GS and Zajac JD (2008). Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. <em>FASEB J</em> 22(8):2676-89. doi: 10.1096/fj.08-105726</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Murashima, Aki, Satoshi Kishigami, Axel Thomson, and Gen Yamada. “Androgens and Mammalian Male Reproductive Tract Development.” <em>Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms</em> 1849, no. 2 (February 2015): 163–70. <a href="https://doi.org/10.1016/j.bbagrm.2014.05.020" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1016/j.bbagrm.2014.05.020</a>.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Niel L, Willemsen KR, Volante SN and Monks DA (2008). Sexual dimorphism and androgen regulation of satellite cell population in differentiating rat levator ani muscle. <em>Dev Neurobiol</em> 68(1):115-22. doi: 10.1002/dneu.20580</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Notini AJ, Davey RA, McManus JF, Bate KL and Zajac JD (2005). Genomic actions of the androgen receptor are required for normal male sexual differentiation in a mouse model. <em>J Mol Endocrinol</em> 35(3):547-55. doi: 10.1677/jme.1.0188</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U and Svingen T (2019a). Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. <em>Arch Toxicol</em> 93(2):253-272. doi: 10.1007/s00204-018-2350-5</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schwartz CL, Vinggaard AM, Christiansen S, Darde TA, Chalmel F and Svingen T (2019b). Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses. <em>Toxicol Sci</em> 169(1):303-311. doi: 10.1093/toxsci/kfz046</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thankamony, A., V. Pasterski, K. K. Ong, C. L. Acerini, and I. A. Hughes. “Anogenital Distance as a Marker of Androgen Exposure in Humans.” <em>Andrology</em> 4, no. 4 (July 2016): 616–25. <a href="https://doi.org/10.1111/andr.12156" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1111/andr.12156</a>.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Welsh M, Saunders PT, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM. Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. <em>J Clin Invest</em> 118(4):1479-90. doi: 10.1172/JCI34241</span></span></p>