<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 Do not cite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 5α-reductase inhibition 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">This AOP links 5α-reductase inhibition (primarily type 2) 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">5α-reductase is an enzyme responsible for the conversion of testosterone to DHT in target tissues (Azzouni et al 2012; Davey and Grossmann, 2016). DHT is more potent agonist of the Androgen receptor (AR) than testosterone, so that DHT is necessary for proper masculinization of e.g. male external genitalia. Under normal physiological conditions, testosterone produced mainly by the testes, 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 masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in males (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">5α-reductase is an enzyme responsible for the conversion of testosterone to DHT in target tissues (Azzouni et al 2012; Davey and Grossmann, 2016). DHT is more potent agonist of the Androgen receptor (AR) than testosterone, so that DHT is necessary for proper masculinization of e.g. male external genitalia. Under normal physiological conditions, testosterone produced mainly by the testes, 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 masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in males (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:11.0pt"><span style="font-family:"Aptos",sans-serif">A key step of this pathway is the inhibition of 5α-reductase, which converts testosterone into the more potent dihydrotestosterone (DHT) in androgen-sensitive tissues. In the developing perineal region, low or absent DHT levels result in inactivation of the androgen receptor (AR), leading to failure in proper masculinization of the perineum and the levator ani-bulbocavernosus (LABC) complex.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Aptos",sans-serif">A key step of this pathway is the inhibition of 5α-reductase, which converts testosterone into the more potent dihydrotestosterone (DHT) in androgen-sensitive tissues. In the developing perineal region, low or absent DHT levels result in reduced AR activation and downstream signaling, leading to failure in proper masculinization of the perineum and the levator ani-bulbocavernosus (LABC) complex.<span style="font-size:14px"> </span></span></span><span style="font-size:14px"><span style="font-family:Aptos,sans-serif">5α<span style="font-family:"Cambria Math",serif">‑</span>reductase type 2 is the critical isoform for DHT production in the developing perineum, and its inhibition - exemplified by finasteride, a selective type 2 inhibitor - is sufficient to reduce androgen receptor activation and shorten AGD in rats (Schwartz et al 2019).</span></span></p>
</div>
<div id="background">
<h3>Background</h3>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif">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 306 (</span></span>AR antagonism leading to short AGD)<span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"> and 307 (</span></span>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. Testosterone is a main androgen, but during fetal differentiation, particularly in tissues distant to the testes, the more potent androgen receptor ligand dihydro-testosterone (DHT) is critical. The formation of DHT from testosterone requires the enzyme 5α-reductase, hence the role of both this enzyme and DHT must be considered when assessing overall effects of disrupted androgen signaling on sex differentiation. </span></span></p>
<p>Finasteride is a type II 5alpha-reductase inhibitor that blocks conversion of testosterone to dihydrotestosterone (Clark et al 1990; Imperato-McGinley et al 1992). Intrauterine exposure in rats can result in shorter male AGD in male offspring (Bowman et al 2003; Christiansen et al 2009; Schwartz et al 2019)</p>
<p><strong>References:</strong></p>
<p>Bowman et al (2003), Toxicol Sci 74:393-406; doi: 10.1093/toxsci/kfg128</p>
<p>Christiansen et al (2009), Environ Health Perspect 117:1839-1846; doi: 10.1289/ehp.0900689</p>
<p>Clark et al (1990), Teratology 42:91-100; doi: 10.1002/tera.1420420111</p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",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></span></span></p>
<h3>Essentiality of the Key Events</h3>
<p style="text-align:justify"><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><a href="https://aopwiki.org/system/dragonfly/production/2025/08/15/4imfpttqsh_Supplementary_Table_S1_Essentiality_table_AOPs_305_307_forwiki_R2.pdf">4imfpttqsh_Supplementary_Table_S1_Essentiality_table_AOPs_305_307_forwiki_R2.pdf</a>)<span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">, with a summary provided in Table 1. </span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Aptos",sans-serif"><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 for AOP 305. </span></span></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>
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</table>
<p style="text-align:justify"> </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 perturbing DHT signaling through 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 is summarized in the below Table:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Aptos",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:"Aptos",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:"Aptos",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:"Aptos",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:"Aptos",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 style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">The quantitative understanding of the AOP is limited. A major challenge is that it is difficult to measure upstream and downstream events in the same study since MIE-26 and MIE-1617 are measured in vitro and KE-1614 focus on AR activation in vivo with no methods currently available to measure it. </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">Azzouni F, Godoy A, Li Y, Mohler J (2012). The 5 alpha-reductase isozyme family: a review of basic biology and their role in human diseases. <em>Adv Urol</em> 2012:530121.</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">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">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-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">OECD (2009), </span></span></span></span><span style="font-family:Times New Roman,Times,serif"><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, https://doi.org/10.1787/9789264076334-en.</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 (2011). Length of the human androgen receptor glutamine tract determines androgen sensitivity in vivo. <em>Mol Cell Endocrinol</em> 342(1-2):81-6.</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><span style="font-size:11pt">This KE is applicable to both sexes, across developmental stages into adulthood, in many different tissues and across mammalian taxa. <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>
<p><span style="font-size:11pt">Essentially the reaction performed by the isozymes is the same, but the enzyme is differentially expressed in the body. 5α-reductase type 1 is mainly linked to the production of neurosteroids, 5α-reductase type 2 is mainly involved in production of 5α-DHT, whereas 5α-reductase type 3 is involved in N-glycosylation (Robitaille & Langlois, 2020). </span></p>
<p><span style="font-size:11pt">The expression profile of the three 5α-reductase isoforms depends on the developmental stage, the tissue of interest, and the disease state of the tissue. The enzymes have been identified in, for instance, non-genital and genital skin, scalp, prostate, liver, seminal vesicle, epididymis, testis, ovary, kidney, exocrine pancreas, and brain (Azzouni, 2012, Uhlen 2015).</span></p>
<p><span style="font-size:11pt">5α-reductase is well-conserved, all primary species in Eukaryota contain all three isoforms (from plant, amoeba, yeast to vertebrates) (Azzouni, 2012) and the enzymes are expressed in both males and females (Langlois, 2010, Uhlen 2015).</span></p>
<h4>Key Event Description</h4>
<p><span style="font-size:11pt">This KE describes the inhibition of 5α-reductases (3-oxo-5α-steroid 4-dehydrogenases). These enzymes are widely expressed in tissues of both sexes and responsible for conversion of steroid hormones.</span></p>
<p><span style="font-size:11pt">There are three isozymes: 5α-reductase type 1, 2, and 3.<span style="color:black"> The substrates for 5</span><span style="color:black">α</span><span style="color:black">-reductases are 3-oxo (3-keto), </span><span style="color:black">Δ</span><sup><span style="color:black">4,5</span></sup><span style="color:black"> C19/C21 steroids such as testosterone, progesterone, androstenedione, epi-testosterone, cortisol, aldosterone, and deoxycorticosterone. The enzymatic reaction leads to an irreversible breakage of the double bond between carbon 4 and 5 and subsequent insertion of a hydride anion at carbon 5 and insertion of a proton at carbon 4. The reaction is aided by the cofactor NADPH. The substrate affinity and reaction velocity differ depending on the combination of substrate and enzyme isoform, for instance 5</span><span style="color:black">α</span><span style="color:black">-reductase type 2 has a higher substrate affinity for testosterone than the type 1 isoform of the enzyme, and the enzymatic reaction occurs at a higher velocity under optimal conditions. Likewise, inhibitors of 5</span><span style="color:black">α-reductase may exhibit differential effects depending on isoforms (Azzouni et al., 2012).</span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt">There is currently (as of 2023) no OECD test guideline for the measurement of 5α-reductase inhibition.</span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Assessing the ability of chemicals to inhibit the activity of 5α-reductase is challenging, but has been </span></span>assessed using transfected cell lines. This has been demonstrated in HEK-293 cells stably transfected with human 5α-reductase type 1, 2, and 3 <span style="color:black">(Yamana et al., 2010)</span>, in CHO cells stably transfected with human 5α-reductase type 1 and 2 <span style="color:black">(Thigpens et al., 1993)</span>, and COS cells transfected with human and rat 5α-reductase with unspecified isoforms <span style="color:black">(Andersson & Russell, 1990)</span>. The transfected cells are typically used as intact cells or cell homogenates. Further, 5α-reductase 1 and 2 has been successfully expressed and isolated from <em>Escherichia coli </em>with subsequent functionality allowing for examination of enzyme inhibition <span style="color:black">(Peng et al., 2020)</span>. <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">The availability of the stably transfected cell lines and the isolated enzymes to the scientific community is unknown.</span></span></span></p>
<p><span style="font-size:11pt">The output of the above methods could be decreased dihydrotestosterone (DHT) with increasing test chemical concentrations. Other substrates exist for the different isoforms that could be used to assess the enzymatic inhibition<span style="color:black"> (Peng et al., 2020)</span>. The use of radiolabeled steroids has historic and continued use for 5α-reductase inhibition examination <span style="color:black">(Andersson & Russell, 1990; Peng et al., 2020; Thigpens et al., 1993; Yamana et al., 2010); however, alternative methods are available, such as conventional ELISA kits or</span> advanced analytical methods such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).</span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5a-reductases. <em>Proc. Natl. Acad. Sci. </em><em>USA</em>, <em>87</em>, 3640–3644. https://www.pnas.org</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Azzouni, F., Godoy, A., Li, Y., & Mohler, J. (2012). The 5 alpha-reductase isozyme family: A review of basic biology and their role in human diseases. In <em>Advances in Urology</em>. https://doi.org/10.1155/2012/530121</span></span></p>
<p><span style="font-size:14px">Langlois VS, Zhang D, Cooke GM, Trudeau VL. (2010). Evolution of steroid-5alpha-reductases and comparison of their function with 5beta-reductase. <em>Gen Comp Endocrinol</em>. 166(3):489-97. doi: 10.1016/j.ygcen.2009.08.004. </span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Peng, H. M., Valentin-Goyco, J., Im, S. C., Han, B., Liu, J., Qiao, J., & Auchus, R. J. (2020). Expression in escherichia coli, purification, and functional reconstitution of human steroid 5α-reductases. <em>Endocrinology (United States)</em>, <em>161</em>(8), 1–11. https://doi.org/10.1210/ENDOCR/BQAA117</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Robitaille, J., & Langlois, V. S. (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. In <em>General and Comparative Endocrinology</em> (Vol. 290). Academic Press Inc. https://doi.org/10.1016/j.ygcen.2020.113400</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. <em>The Journal of Biological Chemistry</em>, <em>268</em>(23), 17404–17412.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. <em>The Journal of Biological Chemistry</em>, <em>268</em>(23), 17404–17412.</span></span><!--StartFragment --></p>
<p><span style="font-size:14px">Uhlén, M., Fagerberg, L., Hallström, B. M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, Å., Kampf, C., Sjöstedt, E., Asplund, A., Olsson, I. M., Edlund, K., Lundberg, E., Navani, S., Szigyarto, C. A. K., Odeberg, J., Djureinovic, D., Takanen, J. O., Hober, S., … Pontén, F. (2015). Tissue-based map of the human proteome. Science, 347(6220). https://doi.org/10.1126/science.1260419</span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Yamana, K., Fernand, L., Luu-The, V., & Luu-The, V. (2010). Human type 3 5</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">α</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">-reductase is expressed in peripheral tissues at higher levels than types 1 and 2 and its activity is potently inhibited by finasteride and dutasteride. <em>Hormone Molecular Biology and Clinical Investigation</em>, <em>2</em>(3), 293–299. https://doi.org/10.1515/HMBCI.2010.035</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>
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<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>KeyEvent</td>
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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/527">Aop:527 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Hypospadias, increased</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
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<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 applicable to both sexes, across developmental stages and adulthood, in many different tissues and across mammals.</span></p>
<p><span style="font-size:11pt">In both humans and rodents, DHT is important for the <em>in utero</em> differentiation and growth of the prostate and male external genitalia (Azzouni et al., 2012; Gerald & Raj, 2022). Besides its critical role in development, DHT also induces growth of facial and body hair during puberty in humans <span style="color:black">(Azzouni et al., 2012)</span>.</span></p>
<p><span style="font-size:11pt">In mammals, the role of DHT in females is less established <span style="color:black">(Swerdloff et al., 2017), however studies suggest that androgens are important in e.g. bone metabolism and growth, as well as female reproduction from follicle development to parturition (Hammes & Levin, 2019).</span></span></p>
<p><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 style="text-align:justify"><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">Dihydrotestosterone (DHT) is an endogenous steroid hormone and a potent androgen. The level of DHT in tissue or blood is dependent on several factors, such as the synthesis, uptake/release, metabolism, and elimination from the system, which again can be dependent on biological compartment and developmental stage.</span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">DHT is primarily synthesized from testosterone (T) via the irreversible enzymatic reaction facilitated by 5α</span></span><span style="background-color:white"><span style="color:black">-Reductases (5</span></span><span style="background-color:white"><span style="color:black">α-REDs) (Swerdloff et al., 2017). Different isoforms of this enzyme are differentially expressed in specific tissues (e.g. prostate, skin, liver, and hair follicles) at different developmental stages, and depending on disease status (Azzouni et al., 2012; Uhlén et al., 2015), which ultimately affects the local production of DHT. </span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">An alternative (“backdoor”) pathway , exists for DHT formation that is independent of T and androstenedione as precursors. </span></span><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">While first discovered in marsupials, the physiological importance of this pathway has now also been established in other mammals including humans (Renfree and Shaw, 2023). </span></span><span style="background-color:white"><span style="color:black">This pathway relies on the conversion of progesterone (P) or 17-OH-P to androsterone and then androstanediol through several enzymatic reactions and finally, the conversion of androstanediol into DHT probably by HSD17B6 (Miller & Auchus, 2019; Naamneh Elzenaty et al., 2022). The “backdoor” synthesis pathway is a result of an interplay between placenta, adrenal gland, and liver during fetal life (Miller & Auchus, 2019).</span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">The conversion of T to DHT by 5α-RED in peripheral tissue is mainly responsible for the circulating levels of DHT, though some tissues express enzymes needed for further metabolism of DHT consequently leading to little release and contribution to circulating levels (Swerdloff et al.). </span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">The initial conversion of DHT into inactive steroids is primarily through 3α</span></span><span style="background-color:white"><span style="color:black">-hydroxysteroid dehydrogenase (3</span></span><span style="background-color:white"><span style="color:black">α</span></span><span style="background-color:white"><span style="color:black">-HSD) and 3</span></span><span style="background-color:white"><span style="color:black">β-HSD in liver, intestine, skin, and androgen-sensitive tissues. The subsequent conjugation is mainly mediated by uridine 5´-diphospho (UDP)-glucuronyltransferase 2 (UGT2) leading to biliary and urinary elimination from the system. Conjugation also occurs locally to control levels of highly potent androgens (Swerdloff et al., 2017).</span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="color:black">Disruption of any of the aforementioned processes may lead to decreased DHT levels, either systemically or at tissue level.</span></span></span></p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:11pt"><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:black">Several methods exist for DHT identification and quantification, such as conventional immunoassay methods (ELISA or RIA) and advanced analytical methods as liquid chromatography tandem mass spectrometry (LC-MS/MS). The methods can have differences in detection and quantification limits, which should be considered depending on the DHT levels in the sample of interest. Further, the origin of the sample (e.g. cell culture, tissue, or blood) will have implications for the sample preparation. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:10.5pt"><span style="background-color:white"><span style="color:black">Conventional immunoassays have limitations in that they can overestimate the levels of DHT compared to levels determined by gas chromatography mass spectrometry and liquid chromatography tandem mass spectrometry (Hsing et al., 2007; Shiraishi et al., 2008). This overestimation may be explained by lack of specificity of the DHT antibody used in the RIA and cross-reactivity with T in samples (Swerdloff et al., 2017).</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Test guideline no. 456 (OECD 2023) uses a cell line, NCI-H295, capable of producing DHT at low levels. The test guideline is not validated for this hormone. Measurement of DHT levels in these cells require low detection and quantification limits. Any effect on DHT can be a result of many upstream molecular events that are specific for the NCI-H295 cells, and which may differ in other models for steroidogenesis.</span></span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Azzouni, F., Godoy, A., Li, Y., & Mohler, J. (2012). The 5 alpha-reductase isozyme family: A review of basic biology and their role in human diseases. In <em>Advances in Urology</em>. https://doi.org/10.1155/2012/530121</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gerald, T., & Raj, G. (2022). Testosterone and the Androgen Receptor. In <em>Urologic Clinics of North America</em> (Vol. 49, Issue 4, pp. 603–614). W.B. Saunders. https://doi.org/10.1016/j.ucl.2022.07.004</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hammes, S. R., & Levin, E. R. (2019). Impact of estrogens in males and androgens in females. In <em>Journal of Clinical Investigation</em> (Vol. 129, Issue 5, pp. 1818–1826). American Society for Clinical Investigation. https://doi.org/10.1172/JCI125755</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hsing, A. W., Stanczyk, F. Z., Bélanger, A., Schroeder, P., Chang, L., Falk, R. T., & Fears, T. R. (2007). Reproducibility of serum sex steroid assays in men by RIA and mass spectrometry. <em>Cancer Epidemiology Biomarkers and Prevention</em>, <em>16</em>(5), 1004–1008. https://doi.org/10.1158/1055-9965.EPI-06-0792</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miller, W. L., & Auchus, R. J. (2019). The “backdoor pathway” of androgen synthesis in human male sexual development. <em>PLoS Biology</em>, <em>17</em>(4). https://doi.org/10.1371/journal.pbio.3000198</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Naamneh Elzenaty, R., du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. In <em>Best Practice and Research: Clinical Endocrinology and Metabolism</em> (Vol. 36, Issue 4). Bailliere Tindall Ltd. https://doi.org/10.1016/j.beem.2022.101665</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">OECD (2023), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264122642-en.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">Renfree, M. B., and Shaw, G. (2023). The alternate pathway of androgen metabolism and window of sensitivity. J. Endocrinol., JOE-22-0296. doi:10.1530/JOE-22-0296.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shiraishi, S., Lee, P. W. N., Leung, A., Goh, V. H. H., Swerdloff, R. S., & Wang, C. (2008). Simultaneous measurement of serum testosterone and dihydrotestosterone by liquid chromatography-tandem mass spectrometry. <em>Clinical Chemistry</em>, <em>54</em>(11), 1855–1863. https://doi.org/10.1373/clinchem.2008.103846</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Swerdloff, R. S., Dudley, R. E., Page, S. T., Wang, C., & Salameh, W. A. (2017). Dihydrotestosterone: Biochemistry, physiology, and clinical implications of elevated blood levels. In <em>Endocrine Reviews</em> (Vol. 38, Issue 3, pp. 220–254). Endocrine Society. https://doi.org/10.1210/er.2016-1067</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Uhlén, M., Fagerberg, L., Hallström, B. M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, Å., Kampf, C., Sjöstedt, E., Asplund, A., Olsson, I. M., Edlund, K., Lundberg, E., Navani, S., Szigyarto, C. A. K., Odeberg, J., Djureinovic, D., Takanen, J. O., Hober, S., … Pontén, F. (2015). Tissue-based map of the human proteome. <em>Science</em>, <em>347</em>(6220). https://doi.org/10.1126/science.1260419</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>
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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>
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<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>
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<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>
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<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
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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>
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<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>KeyEvent</td>
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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>
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<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
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<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>
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<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>
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<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>
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<p><a name="_ENREF_53">Wolf CJ, LeBlanc GA, Gray LE, Jr. (2004) Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. <em>Toxicol Sci</em> <strong>78:</strong> 135-143</a></p>
<p><a name="_ENREF_54">Wolf CJJ, Lambright C, Mann P, Price M, Cooper RL, Ostby J, Gray CLJ (1999) Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. <em>Toxicol Ind Health</em> <strong>15:</strong> 94-118</a></p>
<p><a name="_ENREF_55">Zhang L, Dong L, Ding S, Qiao P, Wang C, Zhang M, Zhang L, Du Q, Li Y, Tang N, Chang B (2014) Effects of n-butylparaben on steroidogenesis and spermatogenesis through changed E₂ levels in male rat offspring. <em>Environ Toxicol Pharmacol</em> <strong>37:</strong> 705-717</a></p>
<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/1880">Relationship: 1880: Inhibition, 5α-reductase leads to Decrease, DHT level</a></h4>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">This KE 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><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">This key event relationship (KER) links inhibition of 5α-reductase activity to decreased dihydrotestosterone (DHT) levels. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">There are three isozymes of 5α-reductase: type 1, 2, and 3.<span style="color:black"> 5α-reductase type 2 is mainly involved in the synthesis of 5α-DHT from testosterone (T) <span style="font-size:11.0pt">(Robitaille & Langlois, 2020)</span>, although 5α-reductase type 1 can also facilitate this reaction, but with lower affinity for T (Nikolaou et al., 2021). The type 1 isoform is also involved in the alternative (‘backdoor’) pathway for DHT formation, facilitating the conversion of progesterone or 17OH-progesterone to dihydroprogesterone or 5α-pregnan-17α-ol-3,20-dione, respectively, whereafter several subsequent reactions will ultimately lead to the formation of DHT <span style="font-size:11.0pt">(Miller & Auchus, 2019)</span>. The quantitative importance of the alternative pathway remains unclear (Alemany, 2022). The type 1 and type 2 isoforms of 5α-reductase are the primary focus of this KER. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">The direct conversion of T to 5α-DHT mainly takes place in the target tissue <span style="color:black"><span style="font-size:11.0pt">(Robitaille & Langlois, 2020)</span></span>. In mammals, the type 1 isoform is found in the scalp and other peripheral tissues <span style="color:black"><span style="font-size:11.0pt">(Miller & Auchus, 2011)</span></span>, such as liver, skin, prostate <span style="color:black">(Azzouni et al., 2012)</span>, bone, ovaries, and adipose tissue <span style="color:black">(Nikolaou et al., 2021)</span>. The type 2 isoform is expressed mainly in male reproductive tissues <span style="color:black"><span style="font-size:11.0pt">(Miller & Auchus, 2011)</span></span>, but also in liver, scalp and skin <span style="color:black">(Nikolaou et al., 2021). The expression level of both isoforms depend on the developmental stage and the tissue.</span></span></span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:black">The biological plausibility of this KER is considered high. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:black">5α-reductase can catalyze the conversion of T to DHT. The substrates for 5α-reductases are 3-oxo (3-keto), Δ<sup>4,5</sup> C19/C21 steroids such as testosterone and progesterone. The enzymatic reaction leads to an irreversible breakage of the double bond between carbon 4 and 5 and subsequent insertion of a hydride anion at carbon 5 and insertion of a proton at carbon 4. The reaction is aided by the cofactor NADPH (Azzouni et al., 2012). By inhibiting this enzyme, the described catalyzed reaction will be inhibited leading to a decrease in DHT levels.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">In both humans and rodents, DHT is important for the <em>in utero</em> differentiation and growth of the prostate and male external genitalia. Besides its critical role during fetal development, DHT also induces growth of facial and body hair during puberty in humans <span style="color:black">(Azzouni et al., 2012)</span><em>.</em> </span></span></p>
<strong>Empirical Evidence</strong>
<p>The empirical evidence for this KER is considered high</p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Several inhibitors of 5α-reductases have been developed for pharmacological uses. Inhibition of the enzymatic conversion of radiolabeled substrate has been illustrated (Table 1) and data display dose-concordance, with increasing concentrations of inhibitor leading to lower 5α-reductase product formation. </span><span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">These studies at large rely on conversion of radiolabeled substrate and hence serve as an indirect measurement.</span></span></span></p>
<p><span style="font-size:11pt"><em><span style="font-size:12.0pt">Table 1: Dose concordance from selected in vitro test systems</span></em></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Cells stably transfected human 5α-reductase type 1 and 2 used to measure conversion of [<sup>14</sup>C]labeled steroids</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Cell homogenates from transfected cells with human and rat 5α-reductase (unknown isoform) used to measure conversion of radiolabeled testosterone</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Stably transfected with human 5α-reductase type 1 and 2</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Human 5α-reductase type 1 and 2 used to measure conversion of radiolabeled substrate of both isoforms</span></span></span></p>
<p> <span style="font-size:11pt"><span style="font-size:12.0pt">These in vitro studies clearly show effects on the enzymatic reaction induced by 5α-reductases in a concentration dependent manner <span style="color:black"><span style="font-size:11.0pt">(Andersson & Russell, 1990; Thigpens et al., 1993; Yamana et al., 2010)</span></span>.</span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">In the intact organism, when 5α-reductase type 2 activity is lacking through e.g. inhibitor treatment or knockout, this will results in decreased 5α-DHT locally in the tissues, but also in blood <span style="color:black"><span style="font-size:11.0pt">(Robitaille & Langlois, 2020)</span></span>. This has been demonstrated in humans, rats, monkeys, and mice (Robitaille et al. 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Finasteride is a specific inhibitor of 5α-reductase type 2 <span style="color:black"><span style="font-size:11.0pt">(Russell & Wilson, 1994)</span></span>. Men with androgenic alopecia were treated with increasing concentrations of finasteride and presented with decreased DHT levels in biopsies from scalp, as well as a decrease in serum DHT levels with dose dependency being most apparent in serum, up to about 70% decrease <span style="color:black">(Drake et al., 1999). Likewise, men treated with dutasteride exhibited a clear dose dependent decrease in serum DHT after 24 weeks treatment with a maximum efficacy of about 98% (Clark et al., 2004).</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">The phenotype of males with deficiency in 5α-reductases are typically born with ambiguous external genitalia. They also present with small prostate, minimal facial hair and acne, or temporal hair loss. Comparison of affected individuals to non-affected individuals in regard to T/DHT ratio, conversion of infused radioactive T, and ratios of urinary metabolites of 5α-reductase and 5β-reductase concluded that these phenotypic characteristics were due to 5α-reductase defects that resulted in less conversion of T to DHT (Okeigwe et al. 2014). Mutations in the 5α-reductase gene can result in boys being born with moderate to severe undervirilization phenotypes (Elzenaty 2022).</span></span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Inhibitors of 5α-reductase are important for the prevention and treatment of many diseases. There are several compounds that have been developed for pharmaceutical purposes and they can target the different isoforms with different affinity. Examples of inhibitors are finasteride and dutasteride. Finasteride mainly has specificity for the type 2 isoform, whereas dutasteride inhibits both type 1 and 2 isoforms <span style="color:black"><span style="font-size:11.0pt">(Miller & Auchus, 2011)</span></span>.</span></span></p>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">These differences in isoform specificity reflects in the effects on DHT serum levels, hence the broader specificity of dutasteride leads to > 90% decrease in patients with benign prostatic hyperplasia, in comparison to 70% with finasteride administration <span style="color:black">(Nikolaou et al., 2021)</span>. </span></span></p>
<strong>Response-response relationship</strong>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Enzyme inhibition can occur in different ways e.g. both competitive and noncompetitive. The inhibition model depends on the specific inhibitor and hence a generic quantitative response-response relationship is difficult to derive.</span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">An inhibition of 5α-reductases would lead to an immediate change in DHT levels at the molecular level. However, the time-scale for systemic effects on hormone levels are challenging to estimate.</span></span></p>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11pt"><span style="font-size:12.0pt">Androgens can regulate gene expression of 5α-reductases <span style="font-size:11.0pt">(Andersson et al., 1989; Berman & Russell, 1993)</span>. </span></span></p>
<h4>References</h4>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Alemany, M. (2022). The Roles of Androgens in Humans: Biology, Metabolic Regulation and Health. In <em>International Journal of Molecular Sciences</em> (Vol. 23, Issue 19). MDPI. https://doi.org/10.3390/ijms231911952</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Andersson, S., Bishop, R. W., & Russell$, D. W. (1989). <em>THE JOURNAL OF BIOLOGICAL CHEMISTRY Expression Cloning and Regulation of Steroid 5cw-Reductase, an Enzyme Essential for Male Sexual Differentiation*</em> (Vol. 264, Issue 27).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5a-reductases. <em>Proc. Natl. Acad. Sci. USA</em>, <em>87</em>, 3640–3644. https://www.pnas.org</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Azzouni, F., Godoy, A., Li, Y., & Mohler, J. (2012). The 5 alpha-reductase isozyme family: A review of basic biology and their role in human diseases. In <em>Advances in Urology</em>. https://doi.org/10.1155/2012/530121</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Berman, D. M., & Russell, D. W. (1993). Cell-type-specific expression of rat steroid 5a-reductase isozymes (sexual development/androgens/prostate/stroma/epithelium). In <em>Proc. Natl. Acad. Sci. USA</em> (Vol. 90). https://www.pnas.org</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Clark, R. V., Hermann, D. J., Cunningham, G. R., Wilson, T. H., Morrill, B. B., & Hobbs, S. (2004). Marked Suppression of Dihydrotestosterone in Men with Benign Prostatic Hyperplasia by Dutasteride, a Dual 5α-Reductase Inhibitor. <em>Journal of Clinical Endocrinology and Metabolism</em>, <em>89</em>(5), 2179–2184. https://doi.org/10.1210/jc.2003-030330</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Drake, L., Hordinsky, M., Fiedler, V., Swinehart, J., Unger, W. P., Cotterill, P. C., Thiboutot, D. M., Lowe, N., Jacobson, C., Whiting, D., Stieglitz, S., Kraus, S. J., Griffin, E. I., Weiss, D., Carrington, P., Gencheff, C., Cole, G. W., Pariser, D. M., Epstein, E. S., … City, O. (1999). <em>The effects of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia</em>.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miller, W. L., & Auchus, R. J. (2011). The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. <em>Endocrine Reviews</em>, <em>32</em>(1), 81–151. https://doi.org/10.1210/er.2010-0013</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miller, W. L., & Auchus, R. J. (2019). The “backdoor pathway” of androgen synthesis in human male sexual development. <em>PLoS Biology</em>, <em>17</em>(4). https://doi.org/10.1371/journal.pbio.3000198</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nikolaou, N., Hodson, L., & Tomlinson, J. W. (2021). The role of 5-reduction in physiology and metabolic disease: evidence from cellular, pre-clinical and human studies. In <em>Journal of Steroid Biochemistry and Molecular Biology</em> (Vol. 207). Elsevier Ltd. https://doi.org/10.1016/j.jsbmb.2021.105808</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Peng, H. M., Valentin-Goyco, J., Im, S. C., Han, B., Liu, J., Qiao, J., & Auchus, R. J. (2020). Expression in escherichia coli, purification, and functional reconstitution of human steroid 5α-reductases. <em>Endocrinology (United States)</em>, <em>161</em>(8), 1–11. https://doi.org/10.1210/ENDOCR/BQAA117</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Robitaille, J., & Langlois, V. S. (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. In <em>General and Comparative Endocrinology</em> (Vol. 290). Academic Press Inc. https://doi.org/10.1016/j.ygcen.2020.113400</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Russell, D. W., & Wilson, J. D. (1994). <em>STEROID Sa-REDUCTASE: TWO GENES/TWO ENZYMES</em>. www.annualreviews.org</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. <em>The Journal of Biological Chemistry</em>, <em>268</em>(23), 17404–17412.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yamana, K., Fernand, L., Luu-The, V., & Luu-The, V. (2010). Human type 3 5α-reductase is expressed in peripheral tissues at higher levels than types 1 and 2 and its activity is potently inhibited by finasteride and dutasteride. <em>Hormone Molecular Biology and Clinical Investigation</em>, <em>2</em>(3), 293–299. https://doi.org/10.1515/HMBCI.2010.035</span></span></p>
</div>
<div>
<h4><a href="/relationships/1935">Relationship: 1935: Decrease, DHT level leads to Decrease, AR activation</a></h4>
<td><a href="/aops/288">Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>adjacent</td>
<td>High</td>
<td>High</td>
</tr>
<tr>
<td><a href="/aops/305">5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/571">5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td></td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/576">5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td></td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<p><span style="font-size:11pt">KER1935 is assessed applicable to mammals, as DHT and AR activation are known to be related in mammals. <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">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>
<p><span style="font-size:11pt">KER1935 is considered applicable to developmental and adult life stages, as DHT-mediated AR activation is relevant from the AR is expressed.</span></p>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:11.0pt">Dihydrotestosterone (DHT) is a primary ligand for the Androgen receptor (AR), a nuclear receptor and transcription factor. DHT is an endogenous sex hormone that is synthesized from e.g. testosterone by the enzyme 5α-reductase in different tissues and organs </span><span style="font-size:11.0pt">(<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>; <a href="#_ENREF_3" title="Marks, 2004 #283">Marks, 2004</a>)</span><span style="font-size:11.0pt">. In the absence of ligand (e.g. DHT) the AR is localized in the cytoplasm in complex with molecular chaperones. Upon ligand binding, AR is activated, translocated into the nucleus, and dimerizes to carry out its ‘genomic function’ </span><span style="font-size:11.0pt">(<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>)</span><span style="font-size:11.0pt">. Hence, AR transcriptional function is directly dependent on the presence of ligands, with DHT being a more potent AR activator than testosterone (<a href="#_ENREF_2" title="Grino, 1990 #284">Grino et al, 1990</a>). Reduced levels of DHT may thus lead to reduced AR activation. Besides its genomic actions, the AR can also mediate rapid, non-genomic second messenger signaling (Davey and Grossmann, 2016). Decreased DHT levels that lead to reduced AR activation can thus entail downstream effects on both genomic and non-genomic signaling. </span></p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:11pt">The biological plausibility of KER1935 is considered high.</span></p>
<p><span style="font-size:11pt">The activation of AR is dependent on binding of ligands (though a few cases of ligand-independent AR activation has been shown, see <em>uncertainties and inconsistencies</em>), primarily testosterone and DHT in mammals (Davey and Grossmann, 2016; Schuppe et al., 2020). Without ligand activation, the AR will remain in the cytoplasm associated with heat-shock and other chaperones and not be able to carry out its canonical (‘genomic’) function. Upon androgen binding, the AR undergoes a conformational change, chaperones dissociate, and a nuclear localization signal is exposed. The androgen/AR complex can now translocate to the nucleus, dimerize and bind AR response elements to regulate target gene expression (Davey and Grossmann, 2016; Eder et al., 2001). <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">AR transcriptional activity and specificity is regulated by co-activators and co-repressors in a cell-specific manner </span><span style="font-family:"Verdana",sans-serif">(Heinlein and Chang, 2002)</span><span style="font-family:"Verdana",sans-serif">.</span></span></span></p>
<p><span style="font-size:11pt">The requirement for androgens binding to the AR for transcriptional activity has been extensively studied and proven and is generally considered textbook knowledge. The OECD test guideline no. 458 uses DHT as the reference chemical for testing androgen receptor activation <em>in vitro</em> (OECD, 2020). In the absence of DHT during development caused by 5α-reductase deficiency (i.e. still in the presence of testosterone) male fetuses fail to masculinize properly. This is evidenced by, for instance, individuals with congenital 5α-reductase deficiency conditions (Costa et al., 2012); conditions not limited to humans (Robitaille and Langlois, 2020), testifying to the importance of specifically DHT for AR activation and subsequent masculinization of certain reproductive tissues. </span></p>
<p><span style="font-size:11pt">Binding of testosterone or DHT has differential effects in different tissues. E.g. in the developing mammalian male; testosterone is required for development of the internal sex organs (epididymis, vas deferens and the seminal vesicles), whereas DHT is crucial for development of the external sex organs (Keller et al., 1996; Robitaille and Langlois, 2020). </span></p>
<strong>Empirical Evidence</strong>
<p><span style="font-size:11pt">The empirical support for KER1935 is considered high.</span></p>
<li><span style="font-size:11pt">Increasing concentrations of DHT lead to increasing AR activation <em>in vitro</em> in AR reporter gene assays (OECD, 2020; Williams et al., 2017).</span></li>
</ul>
<p>Indirect (supporting) evidence:</p>
<ul>
<li><span style="font-size:11pt">In cell lines where proliferation can be induced by androgens (such as prostate cancer cells) proliferation can be used as a readout for AR-activation. Finasteride, a 5α-reductase inhibitor, dose-dependently decreases AR-mediated prostate cancer cell line proliferation (Bologna et al., 1995). 0.001 µM finasteride decreased the growth rate with 44%, 0.1 µM decreased the growth rate with 80%. </span></li>
<li><span style="font-size:11pt">Specific events of masculinization during development are dependent on AR activation by DHT, including the development and length of the perineum which can be measured as the anogenital distance (AGD, (Schwartz et al., 2019)). E.g. a dose-dependent effect of rat <em>in utero</em> exposure to the 5α-reductase inhibitor finasteride was observed on the length of the AGD, where 0.01 mg/kg bw/day finasteride reduced the AGD measured at pup day 1 by 8%, whereas 1 mg/kg bw/day reduced the AGD by 23% (Bowman et al., 2003).</span></li>
<li><span style="font-size:11pt">Male individuals with congenital 5α-reductase deficiency (absence of DHT) fail to masculinize properly (Costa et al., 2012). </span></li>
<li><span style="font-size:11pt">A major driver of prostate cancer growth is AR activation (Davey and Grossmann, 2016; Huggins and Hodges, 1941). Androgen deprivation is used as treatment including 5α-reductase inhibitors to reduce DHT levels (Aggarwal et al., 2010).</span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:11pt">Ligand-independent actions of the AR have been identified. To what extent and of which biological consequences is not well defined (Bennesch and Picard, 2015). </span></p>
<p><span style="font-size:11pt">It should be noted, that in tissues, that are not DHT-dependent but rather respond to T, a decrease in DHT level may not influence AR activation significantly in that specific tissue. </span></p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p style="text-align:justify"><span style="font-size:11pt">There is a positive dose-response relationship between increasing concentrations of DHT and AR activation (Dalton et al., 1998; OECD, 2020). However, there is not enough data, or overview of the data, to define a quantitative linkage <em>in vivo</em>, and such a relationship will differ between biological systems (species, tissue, cell type).</span></p>
<strong>Time-scale</strong>
<p><span style="font-size:11pt">Upon DHT binding to the AR, a conformational change that brings the amino (N) and carboxy (C) termini into close proximity occurs with a t<sub>1/2</sub> of approximately 3.5 minutes, around 6 minutes later the AR dimerizes as shown in transfected HeLa cells (Schaufele et al., 2005). Addition of 5 nM DHT to the culture medium of ‘AR-resistant’ transfected prostatic cancer cells resulted in a rapid (from 15 minutes, maximal at 30 minutes) nuclear translocation of the AR with minimal residual cytosolic expression (Nightingale et al., 2003). AR 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></p>
<td><span style="font-size:11.0pt">AR expression changes with aging</span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Tissue-specific alterations in AR activity with aging</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Supakar et al., 1993; Wu et al., 2009)</span></span></span></td>
</tr>
<tr>
<td>Genotype</td>
<td><span style="font-size:11.0pt"><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:11.0pt"><span style="font-family:"Calibri",sans-serif">Decreased AR activation with increased number of CAGs</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Chamberlain et al., 1994; Tut et al., 1997)</span></span></td>
</tr>
<tr>
<td>Androgen deficiency syndrome</td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Low circulating testosterone levels due to primary (testicular) or secondary (pituitary-hypothalamic) hypogonadism</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Reduced levels of circulating testosterone, precurser of DHT</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Bhasin et al., 2010)</span></span></span></td>
</tr>
<tr>
<td>Castration</td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Removal of testicles</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Reduced levels of circulating testosterone, precurser of DHT</span></span></td>
<td><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Krotkiewski et al., 1980)</span></span></span></td>
</tr>
</tbody>
</table>
</div>
<strong>Known Feedforward/Feedback loops influencing this KER</strong>
<p><span style="font-size:11pt">Androgens have been shown to upregulate and downregulate AR expression as well as 5α-reductase expression, but for 5α-reductase, each isoform in each tissue is differently regulated by androgens and can display sexual dimorphism (Lee and Chang, 2003; Robitaille and Langlois, 2020). <span style="font-family:Aptos,sans-serif"><span style="font-family:"Verdana",sans-serif">The quantitative impact of such adaptive expression changes is unknown.</span></span></span></p>
<h4>References</h4>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Aggarwal, S., Thareja, S., Verma, A., Bhardwaj, T.R., Kumar, M., 2010. An overview on 5α-reductase inhibitors. Steroids 75, 109–153. https://doi.org/10.1016/j.steroids.2009.10.005</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bennesch, M.A., Picard, D., 2015. Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors. Mol. Endocrinol. 29, 349–363. https://doi.org/10.1210/ME.2014-1315</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bhasin, S., Cunningham, G.R., Hayes, F.J., Matsumoto, A.M., Snyder, P.J., Swerdloff, R.S., Montori, V.M., 2010. Testosterone Therapy in Men with Androgen Deficiency Syndromes: An Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab. 95, 2536–2559. https://doi.org/10.1210/JC.2009-2354</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bologna, M., Muzi, P., Biordi, L., Festuccia, C., Vicentini, C., 1995. Finasteride dose-dependently reduces the proliferation rate of the LnCap human prostatic cancer cell line in vitro. Urology 45, 282–290. https://doi.org/10.1016/0090-4295(95)80019-0</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bowman, C.J., Barlow, N.J., Turner, K.J., Wallace, D.G., Foster, P.M.D., 2003. Effects of in Utero Exposure to Finasteride on Androgen-Dependent Reproductive Development in the Male Rat. Toxicol. Sci. 74, 393–406. https://doi.org/10.1093/TOXSCI/KFG128</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chamberlain, N.L., Driver, E.D., Miesfeld, R.L., 1994. The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res. 22, 3181. https://doi.org/10.1093/NAR/22.15.3181</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Costa, E.F., Domenice, S., Sircili, M., Inacio, M., Mendonca, B., 2012. DSD due to 5α-reductase 2 deficiency - From diagnosis to long term outcome. Semin. Reprod. Med. 30, 427–431. https://doi.org/10.1055/S-0032-1324727/ID/JR00766-20/BIB</span></span></p>
<p style="margin-left:32px"><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. Clin. Biochem. Rev. 37, 3–15.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Eder, I.E., Culig, Z., Putz, T., Nessler-Menardi, C., Bartsch, G., Klocker, H., 2001. Molecular Biology of the Androgen Receptor: From Molecular Understanding to the Clinic. Eur. Urol. 40, 241–251. https://doi.org/10.1159/000049782</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Grino, P.B., Griffin, J.E., Wilson, J.D., 1990. Testosterone at High Concentrations Interacts with the Human Androgen Receptor Similarly to Dihydrotestosterone. Endocrinology 126, 1165–1172. https://doi.org/10.1210/endo-126-2-1165</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Huggins, C., Hodges, C. V., 1941. Studies on prostatic cancer: I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 1, 293–297.</span></span></p>
<p style="margin-left:32px"><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. J. Biol. Chem. 277, 48366–48371. https://doi.org/10.1074/jbc.M209074200</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Keller, E.T., Ershler, W.B., Chang, C., 1996. The androgen receptor: a mediator of diverse responses. Front. Biosci. (Landmark Ed) 1, 59–71. https://doi.org/10.2741/A116</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Krotkiewski, M., Kral, J.G., Karlsson, J., 1980. Effects of castration and testosterone substitution on body composition and muscle metabolism in rats. Acta Physiol. Scand. 109, 233–237. https://doi.org/10.1111/J.1748-1716.1980.TB06592.X</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Lee, D.K., Chang, C., 2003. Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication. J. Clin. Endocrinol. Metab. 88, 4043–4054. https://doi.org/10.1210/JC.2003-030261</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Marks, L.S., 2004. 5Alpha-Reductase: History and Clinical Importance. Rev. Urol. 6 Suppl 9, S11-21.</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nightingale, J., Chaudhary, K.S., Abel, P.D., Stubbs, A.P., Romanska, H.M., Mitchell, S.E., Stamp, G.W.H., Lalani, E.N., 2003. Ligand Activation of the Androgen Receptor Downregulates E-Cadherin-Mediated Cell Adhesion and Promotes Apoptosis of Prostatic Cancer Cells. Neoplasia 5, 347. https://doi.org/10.1016/S1476-5586(03)80028-3</span></span></p>
<p style="margin-left:32px"><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 Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.1787/9789264264366-en</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Robitaille, J., Langlois, V.S., 2020. Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. Gen. Comp. Endocrinol. 290. https://doi.org/10.1016/j.ygcen.2020.113400</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schaufele, F., Carbonell, X., Guerbadot, M., Borngraeber, S., Chapman, M.S., Ma, A.A.K., Miner, J.N., Diamond, M.I., 2005. The structural basis of androgen receptor activation: Intramolecular and intermolecular amino-carboxy interactions. Proc. Natl. Acad. Sci. U. S. A. 102, 9802–9807. https://doi.org/10.1073/pnas.0408819102</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schuppe, E.R., Miles, M.C., Fuxjager, M.J., 2020. Evolution of the androgen receptor: Perspectives from human health to dancing birds. Mol. Cell. Endocrinol. 499, 110577. https://doi.org/10.1016/J.MCE.2019.110577</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schwartz, C.L., Christiansen, S., Vinggaard, A.M., Axelstad, M., Hass, U., Svingen, T., 2019. Anogenital distance as a toxicological or clinical marker for fetal androgen action and risk for reproductive disorders. Arch. Toxicol. 93, 253–272. https://doi.org/10.1007/s00204-018-2350-5</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Supakar, P.C., Song, C.S., Jung, M.H., Slomczynska, M.A., Kim, J.M., Vellanoweth, R.L., Chatterjee, B., Roy, A.K., 1993. A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene. J. Biol. Chem. 268, 26400–26408. https://doi.org/10.1016/S0021-9258(19)74328-2</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Tut, T.G., Ghadessy, F.J., Trifiro, M.A., Pinsky, L., Yong, E.L., 1997. Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility. J. Clin. Endocrinol. Metab. 82, 3777–3782. https://doi.org/10.1210/JCEM.82.11.4385</span></span></p>
<p style="margin-left:32px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Williams, A.J., Grulke, C.M., Edwards, J., McEachran, A.D., Mansouri, K., Baker, N.C., Patlewicz, G., Shah, I., Wambaugh, J.F., Judson, R.S., Richard, A.M., 2017. The CompTox Chemistry Dashboard: a community data resource for environmental chemistry. J. Cheminform. 9, 61. https://doi.org/10.1186/s13321-017-0247-6</span></span></p>
<p style="margin-left:32px"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Wu, D., Lin, G., Gore, A.C., 2009. Age-related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">α</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> in Male Rats. J. Comp. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Neurol. 512, 688. https://doi.org/10.1002/CNE.21925</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>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Karlsson, Sara A., Erik Studer, Petronella Kettunen, and Lars Westberg. 2016. “Neural Androgen Receptors Modulate Gene Expression and Social Recognition but Not Social Investigation.” <em>Frontiers in Behavioral Neuroscience</em> 10(MAR). doi: 10.3389/fnbeh.2016.00041.</span></span></p>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Knapczyk-Stwora, Katarzyna, Anna Nynca, Renata E. Ciereszko, Lukasz Paukszto, Jan P. Jastrzebski, Elzbieta Czaja, Patrycja Witek, Marek Koziorowski, and Maria Slomczynska. 2019. “Flutamide-Induced Alterations in Transcriptional Profiling of Neonatal Porcine Ovaries.” <em>Journal of Animal Science and Biotechnology</em> 10(1):1–15. doi: 10.1186/s40104-019-0340-y.</span></span></p>
<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>
<p><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif">Wang, Ruey Sheng, Shuyuan Yeh, Lu Min Chen, Hung Yun Lin, Caixia Zhang, Jing Ni, Cheng Chia Wu, P. Anthony Di Sant’Agnese, Karen L. DeMesy-Bentley, Chii Ruey Tzeng, and Chawnshang Chang. 2006. “Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes.” <em>Endocrinology</em> 147(12):5624–33. doi: 10.1210/en.2006-0138.</span></span></p>
<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>
</tbody>
</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>
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<p> </p>
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<div>
<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>