AOP-Wiki

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<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 571: 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</div>
Short Title: 5α-reductase inhibition leading to hypospadias
</div>
<h2>Graphical Representation</h2>
<img src="https://aopwiki.org/system/dragonfly/production/2025/09/18/98u5iyzbkb_AOP_571_Graphic.jpg" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
Emilie Elmelund; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark
Monica K. Draskau; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark
Henrik Holbech; Department of Biology, University of Southern Denmark, DK-5230, Odense M, Denmark
Terje Svingen; National Food Institute, Technical University of Denmark, Lyngby, DK-2800, Denmark
</div>
<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Under development: Not open for comment. Do not cite</td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
This AOP links in utero inhibition of 5α-reductase with hypospadias in male offspring. Hypospadias is a common reproductive disorder with a prevalence of up to ~1/125 newborn boys (Leunbach et al., 2025; Paulozzi, 1999). Developmental exposure to endocrine disrupting chemicals is suspected to contribute to some cases of hypospadias (Mattiske & Pask, 2021). Hypospadias can be indicative of fetal disruptions to male reproductive development, and is associated with short anogenital distance and cryptorchidism (Skakkebaek et al., 2016). Thus, hypospadias is included as an endpoint in OECD test guidelines (TG) for developmental and reproductive toxicity (TG 414, 416, 421/422, and 443; (OECD, 2016b, 2016a, 2018a, 2018b, 2021)), as both a measurement of adverse reproductive effects and a direct clinical adverse outcome.
5α-reductase is an enzyme that converts testosterone to dihydrotestosterone (DHT). In normal male reproductive development, DHT activates the androgen receptor (AR) in peripheral reproductive tissues to drive differentiation of the male phenotype, including development of the penis. While testosterone also acts directly at the AR, DHT is 5-10 times more potent and in peripheral tissues conversion to DHT is necessary for proper masculinization (Amato et al., 2022; Davey & Grossmann, 2016). This AOP delineates the evidence that inhibition of 5α-reductase reduces DHT levels and consequently AR activation, thereby disrupting penis development and causing hypospadias. The AOP is supported by in vitro experiments upstream of AR activation and by in vivo and human case studies downstream of AR activation. Downstream of a reduction in AR activation, the molecular mechanisms of hypospadias development are not fully delineated, highlighting a knowledge gap in this AOP. Thus, the AOP has potential for inclusion of additional KEs and elaboration of molecular causality links, once these are established. Given that hypospadias is both a clinical and toxicological endpoint, this AOP is considered highly relevant in a regulatory context.
</div>
<div id="background">
<h3>Background</h3>
This AOP is a part of an AOP network for reduced androgen receptor activation causing hypospadias in male offspring. The other AOPs in this network are AOP-477 (‘Androgen receptor antagonism leading to hypospadias in male (mammalian) offspring’), and AOP-570 (‘Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring’). The purpose of the AOP network is to organize the well-established evidence for anti-androgenic mechanisms-of-action leading to hypospadias, thus informing predictive toxicology and identifying knowledge gaps for investigation and method development.
This work received funding from the European Food and Safety Authority (EFSA) under Grant agreement no. GP/EFSA/PREV/2022/01 and from the Danish Environmental Protection Agency under the Danish Center for Endocrine Disrupters (CeHoS).
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
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<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr>
<td></td>
<td>MIE</td>
<td>1617</td>
<td><a href="/events/1617">Inhibition, 5α-reductase</a></td>
<td>Inhibition, 5α-reductase</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>1613</td>
<td><a href="/events/1613">Decrease, dihydrotestosterone (DHT) levels</a></td>
<td>Decrease, DHT level</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1614</td>
<td><a href="/events/1614">Decrease, androgen receptor activation</a></td>
<td>Decrease, AR activation</td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>286</td>
<td><a href="/events/286">Altered, Transcription of genes by the androgen receptor</a></td>
<td>Altered, Transcription of genes by the AR</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>2082</td>
<td><a href="/events/2082">Hypospadias, increased</a></td>
<td>Hypospadias</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/1880">Inhibition, 5α-reductase</a></td>
<td>adjacent</td>
<td>Decrease, dihydrotestosterone (DHT) levels</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/1935">Decrease, dihydrotestosterone (DHT) levels</a></td>
<td>adjacent</td>
<td>Decrease, androgen receptor activation</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2124">Decrease, androgen receptor activation</a></td>
<td>adjacent</td>
<td>Altered, Transcription of genes by the androgen receptor</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2828">Decrease, androgen receptor activation</a></td>
<td>non-adjacent</td>
<td>Hypospadias, increased</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h3>Stressors</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Name</th>
<th scope="col">Evidence</th>
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</thead>
<tbody class="tbody-striped">
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<td>Finasteride</td>
<td></td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
<h3>Domain of Applicability</h3>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
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<tbody class="tbody-striped">
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<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Although the upstream part of the AOPN has a broad applicability domain, the overall AOPN is considered only applicable to male mammals during fetal life, restricted by the applicability of KER-2828 (‘Decrease, AR activation leads to hypospadias’). The term hypospadias is mainly used for describing malformation of the male, and not female, external genitalia. Some studies refer to hypospadias in females, but these have not been reported to be caused by exposure to 5α-reductase inhibitors, and the mechanisms behind these malformations are likely different from the mechanisms in males (Greene, 1937; Stewart et al., 2018). The genital tubercle is programmed by androgens to differentiate into a penis in fetal life in the masculinization programming window, followed by the morphologic differentiation (Welsh et al., 2008). In humans, hypospadias is diagnosed at birth and can also often be observed in rats and mice at this time point, although the rodent penis does not finish developing until a few weeks after birth (Baskin & Ebbers, 2006; Sinclair et al., 2017). The disruption to androgen programming leading to hypospadias thus take place during fetal life, but the AO is best detected postnatally. Regarding taxonomic applicability, hypospadias has mainly been identified in rodents and humans, and the evidence in this AOP is almost exclusively from these species. It is, however, biologically plausible that the AOP is applicable to other mammals as well, given the conserved role of androgens in mammalian reproductive development, and hypospadias has been observed in many domestic animal and wildlife species, albeit not coupled to 5α-reductase inhibition.
<h3>Essentiality of the Key Events</h3>
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Event
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:200px">
Evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:200px">
Uncertainties and inconsistencies
</td>
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<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
MIE-1617
Inhibition, 5α-reductase (high)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides strong support for the essentiality of this event, as DHT (produced by 5α-reductase) is one of the primary drivers of penis development.
In utero exposure to the 5α-reductase inhibitor finasteride can cause hypospadias in male rats (Clark et al., 1993)
Human case studies of 5α-reductase deficiency support the essentiality of this KE, as mutations in 5α-reductase can cause low DHT levels and associated hypospadias in males (Robitaille & Langlois, 2020). See also table 4 in KER-2828 listing disruptions of AR activity associated with hypospadias in humans.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
In the human case studies, DHT is only measured postnatally and not in fetal life.
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
KE-1613
Decrease, DHT levels (moderate)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides strong support for the essentiality of this event, as DHT is a ligand of the AR and one of the primary drivers of penis development.
In patients with 5α-reductase deficiency, DHT levels are reduced and hypospadias are frequently observed, as listed in table 4 in KER-2828.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
In the human case studies, DHT is only measured postnatally and not in fetal life, As hypospadias is a congenital malformation, it cannot be “reversed” by postnatal DHT treatment.
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
KE-1614
Decrease, AR activation (moderate)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides strong support for the essentiality of this event, as AR activation is critical for normal penis development.
Conditional or full knockout of Ar in mice results in partly or full sex-reversal of males, including a female-like urethral opening (Willingham et al., 2006; Yucel et al., 2004; Zheng et al., 2015). Human subjects with AR mutations may also have associated hypospadias (as listed in table 4 in KER-2828).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
KE-286
Altered, transcription of genes by AR (low)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
Biological plausibility provides support for the essentiality of this event. AR is a nuclear receptor and transcription factor regulating transcription of genes, and androgens, acting through AR, are essential for normal male penis development.
Known AR-responsive genes active in normal penis development have been thoroughly reviewed  (Amato et al., 2022).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:200px">
There are currently no AR-responsive genes proved to be causally involved in hypospadias, and it is known that the AR can also signal through non-genomic actions (Leung & Sadar, 2017).
</td>
</tr>
</tbody>
</table>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:85px">
Event
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:104px">
Direct evidence
</td>
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Indirect evidence
</td>
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Contradictory evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:148px">
Overall essentiality assessment
</td>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
MIE 1617
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
***
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
High
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE 1613
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
*
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
*
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Moderate
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE 1614
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
**
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Moderate
</td>
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<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
KE 286
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:123px">
*
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:142px">
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:148px">
Low
</td>
</tr>
</tbody>
</table>
<h3>Weight of Evidence Summary</h3>
The confidence in each of the KERs comprising the AOP are judged as high, with both high biological plausibility and high confidence in the empirical evidence. The mechanistic link between KE-286 (‘altered, transcription of genes by AR’) and AO-2082 (‘hypospadias’) is not established, but given the high confidence in the KERs including the non-adjacent KER-2828 linking to the AO, the overall confidence in the AOP is judged as high.
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:169px">
KER
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:85px">
Biological Plausibility
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:80px">
Empirical Evidence
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:268px">
Rationale
</td>
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<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-1880
Inhibition, 5α-reductase leads to decrease, DHT levels
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that 5α-reductase converts testosterone to DHT.
In vitro, in vivo and human studies with 5α-reductase inhibitors have shown dose-dependent decreases in formation of DHT (Draskau et al., 2024).
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-1935
Decrease, DHT levels leads to decrease, AR activation
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that DHT activates the AR.
Direct evidence for this KER is not possible since KE-1614 can currently not be measured and is considered an in vivo effect. Indirect evidence using proxy read-outs of AR activation, either in vitro or in vivo strongly supports the relationship (Draskau et al., 2024).
</td>
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<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-2124
Decrease, AR activation leads to altered, transcription of genes by AR
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High (canonical)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that the AR regulates gene transcription.
In vivo animal studies and human genomic profiling show tissue-specific changes to gene expression upon disruption of AR (Draskau et al., 2024).
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px">
KER-2828
Decrease, AR activation leads to hypospadias
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:85px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:80px">
High
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:268px">
It is well established that AR drives penis differentiation. Numerous in vivo toxicity studies and human case studies indirectly show that decreased AR activation leads to hypospadias, with few inconsistencies. The empirical evidence moderately supports dose, temporal, and incidence concordance for the KER.
</td>
</tr>
</tbody>
</table>
<h3>Quantitative Consideration</h3>
The quantitative understanding of this AOP is judged as low.
</div>
<div id="considerations_for_potential_applicaitons">
</div>
<div id="references">
<h2>References</h2>
Amato, C. M., Yao, H. H.-C., & Zhao, F. (2022). One Tool for Many Jobs: Divergent and Conserved Actions of Androgen Signaling in Male Internal Reproductive Tract and External Genitalia. Frontiers in Endocrinology, 13, 910964. https://doi.org/10.3389/fendo.2022.910964
Baskin, L., & Ebbers, M. (2006). Hypospadias: Anatomy, etiology, and technique. Journal of Pediatric Surgery, 41(3), 463–472. https://doi.org/10.1016/j.jpedsurg.2005.11.059
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. The Journal of Clinical Endocrinology & Metabolism, 95(6), 2536–2559. https://doi.org/10.1210/jc.2009-2354
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 Research, 22(15), 3181–3186. https://doi.org/10.1093/nar/22.15.3181
Clark, R. L., Anderson, C. A., Prahalada, S., Robertson, R. T., Lochry, E. A., Leonard, Y. M., Stevens, J. L., & Hoberman, A. M. (1993). Critical Developmental Periods for Effects on Male Rat Genitalia Induced by Finasteride, a 5α-Reductase Inhibitor. Toxicology and Applied Pharmacology, 119(1), 34–40. https://doi.org/10.1006/taap.1993.1041
Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15.
Draskau, M., Rosenmai, A., Bouftas, N., Johansson, H., Panagiotou, E., Holmer, M., Elmelund, E., Zilliacus, J., Beronius, A., Damdimopoulou, P., van Duursen, M., & Svingen, T. (2024). Aop Report: An Upstream Network for Reduced Androgen Signalling Leading to Altered Gene Expression of Ar Responsive Genes in Target Tissues. Environ Toxicol Chem, In Press.
Greene, R. R. (1937). Production of Experimental Hypospadias in the Female Rat. Proceedings of the Society for Experimental Biology and Medicine, 36(4), 503–506. https://doi.org/10.3181/00379727-36-9287P
Holmer, M. L., Zilliacus, J., Draskau, M. K., 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. Reproductive Toxicology, 128, 108662. https://doi.org/10.1016/j.reprotox.2024.108662
Leunbach, T. L., Berglund, A., Ernst, A., Hvistendahl, G. M., Rawashdeh, Y. F., & Gravholt, C. H. (2025). Prevalence, Incidence, and Age at Diagnosis of Boys With Hypospadias: A Nationwide Population-Based Epidemiological Study. Journal of Urology, 213(3), 350–360. https://doi.org/10.1097/JU.0000000000004319
Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. Frontiers in Endocrinology, 8. https://doi.org/10.3389/fendo.2017.00002
Mattiske, D. M., & Pask, A. J. (2021). Endocrine disrupting chemicals in the pathogenesis of hypospadias; developmental and toxicological perspectives. Current Research in Toxicology, 2, 179–191. https://doi.org/10.1016/j.crtox.2021.03.004
OECD. (2016a). Test No. 421: Reproduction/Developmental Toxicity Screening Test. OECD. https://doi.org/10.1787/9789264264380-en
OECD. (2016b). Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. OECD. https://doi.org/10.1787/9789264264403-en
OECD. (2018a). Test No. 414: Prenatal Developmental Toxicity Study. OECD. https://doi.org/10.1787/9789264070820-en
OECD. (2018b). Test No. 443: Extended One-Generation Reproductive Toxicity Study. OECD. https://doi.org/10.1787/9789264185371-en
OECD. (2021). Test No. 416: Two-Generation Reproduction Toxicity (Section 4).
Paulozzi, L. J. (1999). International trends in rates of hypospadias and cryptorchidism.
Robitaille, J., & Langlois, V. S. (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. General and Comparative Endocrinology, 290, 113400. https://doi.org/10.1016/j.ygcen.2020.113400
Sinclair, A., Cao, M., Pask, A., Baskin, L., & Cunha, G. (2017). Flutamide-induced hypospadias in rats: A critical assessment. Differentiation; Research in Biological Diversity, 94, 37–57. https://doi.org/10.1016/j.diff.2016.12.001
Skakkebaek, N. E., Rajpert-De Meyts, E., Louis, G. M. B., Toppari, J., Andersson, A.-M., Eisenberg, M. L., Jensen, T. K., Jorgensen, N., Swan, S. H., Sapra, K. J., Ziebe, S., Priskorn, L., & Juul, A. (2016). Male Reproductive Disorders And Fertility Trends: Influences Of Environement And Genetic susceptibility. PHYSIOLOGICAL REVIEWS, 96(1), 55–97. https://doi.org/10.1152/physrev.00017.2015
Stewart, M. K., Mattiske, D. M., & Pask, A. J. (2018). In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development&dagger;. Biology of Reproduction, 99(6), 1184–1193. https://doi.org/10.1093/biolre/ioy142
Svingen, T., Villeneuve, D. L., Knapen, D., Panagiotou, E. M., Draskau, M. K., Damdimopoulou, P., & O’Brien, J. M. (2021). A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation. Toxicological Sciences, 184(2), 183–190. https://doi.org/10.1093/toxsci/kfab113
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 1. The Journal of Clinical Endocrinology & Metabolism, 82(11), 3777–3782. https://doi.org/10.1210/jcem.82.11.4385
Welsh, M., Saunders, P. T. K., Fisken, M., Scott, H. M., Hutchison, G. R., Smith, L. B., & Sharpe, R. M. (2008). Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. Journal of Clinical Investigation, 118(4), 1479–1490. https://doi.org/10.1172/JCI34241
Willingham, E., Agras, K., Souza, A. J. de, Konijeti, R., Yucel, S., Rickie, W., Cunha, G., & Baskin, L. (2006). Steroid receptors and mammalian penile development: An unexpected role for progesterone receptor? The Journal of Urology, 176(2), 728–733. https://doi.org/10.1016/j.juro.2006.03.078
Yucel, S., Liu, W., Cordero, D., Donjacour, A., Cunha, G., & Baskin, L. (2004). Anatomical studies of the fibroblast growth factor-10 mutant, Sonic Hedge Hog mutant and androgen receptor mutant mouse genital tubercle. Advances in Experimental Medicine and Biology, 545, 123–148. https://doi.org/10.1007/978-1-4419-8995-6_8
Zheng, Z., Armfield, B., & Cohn, M. (2015). Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies. Proceedings of the National Academy of Sciences of the United States of America, 112(52), E7194-203. https://doi.org/10.1073/pnas.1515981112
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/1617">Event: 1617: Inhibition, 5α-reductase</a></h4>
<h5>Short Name: Inhibition, 5α-reductase</h5>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/120">Aop:120 - Inhibition of 5α-reductase leading to Leydig cell tumors (in rat)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>eukaryotic cell</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
This KE is applicable to both sexes, across developmental stages into adulthood, in many different tissues and across mammalian taxa. 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.
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).
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).
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).
<h4>Key Event Description</h4>
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.
There are three isozymes: 5α-reductase type 1, 2, and 3. The substrates for 5α-reductases are 3-oxo (3-keto), Δ4,5 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 5α-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 5α-reductase may exhibit differential effects depending on isoforms (Azzouni et al., 2012).
<h4>How it is Measured or Detected</h4>
There is currently (as of 2023) no OECD test guideline for the measurement of 5α-reductase inhibition.
Assessing the ability of chemicals to inhibit the activity of 5α-reductase is challenging, but has been assessed using transfected cell lines. This has been demonstrated in HEK-293 cells stably transfected with human 5α-reductase type 1, 2, and 3 (Yamana et al., 2010), in CHO cells stably transfected with human 5α-reductase type 1 and 2 (Thigpens et al., 1993), and COS cells transfected with human and rat 5α-reductase with unspecified isoforms (Andersson & Russell, 1990). 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 Escherichia coli with subsequent functionality allowing for examination of enzyme inhibition (Peng et al., 2020). The availability of the stably transfected cell lines and the isolated enzymes to the scientific community is unknown.
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 (Peng et al., 2020). The use of radiolabeled steroids has historic and continued use for 5α-reductase inhibition examination (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 advanced analytical methods such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
<h4>References</h4>
Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5a-reductases. Proc. Natl. Acad. Sci. USA, 87, 3640–3644. https://www.pnas.org
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 Advances in Urology. https://doi.org/10.1155/2012/530121
Langlois VS, Zhang D, Cooke GM, Trudeau VL. (2010). Evolution of steroid-5alpha-reductases and comparison of their function with 5beta-reductase. Gen Comp Endocrinol. 166(3):489-97. doi: 10.1016/j.ygcen.2009.08.004.
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. Endocrinology (United States), 161(8), 1–11. https://doi.org/10.1210/ENDOCR/BQAA117
Robitaille, J., & Langlois, V. S. (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. In General and Comparative Endocrinology (Vol. 290). Academic Press Inc. https://doi.org/10.1016/j.ygcen.2020.113400
Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. The Journal of Biological Chemistry, 268(23), 17404–17412.
Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. The Journal of Biological Chemistry, 268(23), 17404–17412.<!--StartFragment -->
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
</p>
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. Hormone Molecular Biology and Clinical Investigation, 2(3), 293–299. https://doi.org/10.1515/HMBCI.2010.035
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/1613">Event: 1613: Decrease, dihydrotestosterone (DHT) levels</a></h4>
<h5>Short Name: Decrease, DHT level</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>hormone biosynthetic process</td>
<td>17beta-Hydroxy-2-oxa-5alpha-androstan-3-one</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/527">Aop:527 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Hypospadias, increased</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
This KE is applicable to both sexes, across developmental stages and adulthood, in many different tissues and across mammals.
In both humans and rodents, DHT is important for the in utero 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 (Azzouni et al., 2012).
In mammals, the role of DHT in females is less established (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).
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.
<h4>Key Event Description</h4>
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.
DHT is primarily synthesized from testosterone (T) via the irreversible enzymatic reaction facilitated by 5α-Reductases (5α-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.
An alternative (“backdoor”) pathway , exists for DHT formation that is independent of T and androstenedione as precursors. 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). 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).
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.).
The initial conversion of DHT into inactive steroids is primarily through 3α-hydroxysteroid dehydrogenase (3α-HSD) and 3β-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).
Disruption of any of the aforementioned processes may lead to decreased DHT levels, either systemically or at tissue level.
<h4>How it is Measured or Detected</h4>
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.
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).
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.
<h4>References</h4>
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 Advances in Urology. https://doi.org/10.1155/2012/530121
Gerald, T., & Raj, G. (2022). Testosterone and the Androgen Receptor. In Urologic Clinics of North America (Vol. 49, Issue 4, pp. 603–614). W.B. Saunders. https://doi.org/10.1016/j.ucl.2022.07.004
Hammes, S. R., & Levin, E. R. (2019). Impact of estrogens in males and androgens in females. In Journal of Clinical Investigation (Vol. 129, Issue 5, pp. 1818–1826). American Society for Clinical Investigation. https://doi.org/10.1172/JCI125755
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. Cancer Epidemiology Biomarkers and Prevention, 16(5), 1004–1008. https://doi.org/10.1158/1055-9965.EPI-06-0792
Miller, W. L., & Auchus, R. J. (2019). The “backdoor pathway” of androgen synthesis in human male sexual development. PLoS Biology, 17(4). https://doi.org/10.1371/journal.pbio.3000198
Naamneh Elzenaty, R., du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. In Best Practice and Research: Clinical Endocrinology and Metabolism (Vol. 36, Issue 4). Bailliere Tindall Ltd. https://doi.org/10.1016/j.beem.2022.101665
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.
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.
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. Clinical Chemistry, 54(11), 1855–1863. https://doi.org/10.1373/clinchem.2008.103846
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 Endocrine Reviews (Vol. 38, Issue 3, pp. 220–254). Endocrine Society. https://doi.org/10.1210/er.2016-1067
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
<h4><a href="/events/1614">Event: 1614: Decrease, androgen receptor activation</a></h4>
<h5>Short Name: Decrease, AR activation</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>androgen receptor activity</td>
<td>androgen receptor</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/288">Aop:288 - Inhibition of 17α-hydrolase/C 10,20-lyase (Cyp17A1) activity leads to birth reproductive defects (cryptorchidism) in male (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/306">Aop:306 - Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/111">Aop:111 - Decrease in androgen receptor activity leading to Leydig cell tumors (in rat)</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
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. 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.
<h4>Key Event Description</h4>
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.
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 (Davey & Grossmann, 2016; Gao et al., 2005).
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) (Davey & Grossmann, 2016; Gao et al., 2005). 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).
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) (Davey & Grossmann, 2016; Gao et al., 2005). 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).
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 (Leung & Sadar, 2017).
<h4>How it is Measured or Detected</h4>
This KE specifically focuses on decreased in vivo activation, with most methods that can be used to measure AR activity carried out in vitro. 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). Assays may in the future be developed to measure AR activation in mammalian organisms.
<h4>References</h4>
Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15.
Gao, W., Bohl, C. E., & Dalton, J. T. (2005). Chemistry and structural biology of androgen receptor. Chemical Reviews, 105(9), 3352–3370. https://doi.org/10.1021/cr020456u
Heinlein, C. A., & Chang, C. (2002). Androgen Receptor (AR) Coregulators: An Overview. https://academic.oup.com/edrv/article/23/2/175/2424160
Leung, J. K., & Sadar, M. D. (2017). Non-Genomic Actions of the Androgen Receptor in Prostate Cancer. Frontiers in Endocrinology, 8. <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>
OECD (2022). Test No. 251: Rapid Androgen Disruption Activity Reporter (RADAR) assay. Paris: OECD Publishing doi:10.1787/da264d82-en.
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. Chemosphere. 158:24-9. doi: 10.1016/j.chemosphere.2016.05.053
<table>
<tbody>
<tr>
<td colspan="1" rowspan="1">
</td>
<td colspan="1" rowspan="1">
</td>
</tr>
</tbody>
</table>
<h4><a href="/events/286">Event: 286: Altered, Transcription of genes by the androgen receptor</a></h4>
<h5>Short Name: Altered, Transcription of genes by the AR</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>regulation of gene expression</td>
<td>androgen receptor</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/19">Aop:19 - Androgen receptor antagonism leading to adverse effects in the male foetus (mammals)</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/307">Aop:307 - Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/344">Aop:344 - Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/345">Aop:345 - Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/305">Aop:305 - 5α-reductase inhibition leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/495">Aop:495 - Androgen receptor activation leading to prostate cancer</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/306">Aop:306 - Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/496">Aop:496 - Androgen receptor agonism leading to reproduction dysfunction （in zebrafish）</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/372">Aop:372 - Androgen receptor antagonism leading to testicular cancer </a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/575">Aop:575 - Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/576">Aop:576 - 5α-reductase inhibition leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Bicalutamide</td></tr>
<tr><td>Cyproterone acetate</td></tr>
<tr><td>Epoxiconazole</td></tr>
<tr><td>Flutamide</td></tr>
<tr><td>Flusilazole</td></tr>
<tr><td>Prochloraz</td></tr>
<tr><td>Propiconazole</td></tr>
<tr><td>Stressor:286 Tebuconazole</td></tr>
<tr><td>Triticonazole</td></tr>
<tr><td>Vinclozalin</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
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).
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. 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.
<h4>Key Event Description</h4>
This KE refers to transcription of genes by the androgen receptor (AR) as occurring in complex biological systems such as tissues and organs in vivo. 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.
The Androgen Receptor and its function
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). Androgens (such as dihydrotestosterone and testosterone) are AR ligands and 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).
Altered transcription of genes by the AR as a Key Event
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). 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).
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, Jin et al. 2013).
<h4>How it is Measured or Detected</h4>
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.
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).
<h4>References</h4>
Bevan C, Parker M (1999) The role of coactivators in steroid hormone action. Exp. Cell Res. 253:349–356
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
Darde, T. A., Gaudriault, P., Beranger, R., Lancien, C., Caillarec-Joly, A., Sallou, O., et al. (2018a). TOXsIgN: a cross-species repository for toxicogenomic signatures. Bioinformatics 34, 2116–2122. doi:10.1093/bioinformatics/bty040.
Darde, T. A., Chalmel, F., and Svingen, T. (2018b). 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.
Davey RA, Grossmann M (2016) Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin Biochem Rev 37:3–15
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
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
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
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.
Maclean HE, Chu S, Warne GL, Zajact JD Related Individuals with Different Androgen Receptor Gene Deletions
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
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<a name="_Hlk148352925"></a>
Rana K, Davey RA, Zajac JD (2014) Human androgen deficiency: Insights gained from androgen receptor knockout mouse models. Asian J. Androl. 16:169–177
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.
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
<h3>List of Adverse Outcomes in this AOP</h3>
<h4><a href="/events/2082">Event: 2082: Hypospadias, increased</a></h4>
<h5>Short Name: Hypospadias</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>embryonic organ development</td>
<td>penis</td>
<td>abnormal</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/477">Aop:477 - Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/527">Aop:527 - Decreased, Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII) leads to Hypospadias, increased</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/570">Aop:570 - Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/571">Aop:571 - 5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>AdverseOutcome</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Organ</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>penis</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mammals</td>
<td>mammals</td>
<td></td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Perinatal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: Numerous studies have shown an association in humans between in utero exposure to endocrine disrupting chemicals and hypospadias. In mice and rats, in utero exposure to several endocrine disrupting chemicals, in particular estrogens and antiandrogens, have been shown to cause hypospadias in male offspring at different frequencies (Mattiske & Pask, 2021). Androgen-driven development of the male external genitalia is evolutionary conserved in most mammals and, to some extent, also in other vertebrate classes (Gredler et al., 2014). Hypospadias can in principle occur in all animals that form a genital tubercle and have been observed in many domestic animal species and wildlife species.
Life stage applicability: Penis development is finished prenatally in humans, and hypospadias is diagnosed at birth (Baskin & Ebbers, 2006). In rodents, penis development is not fully completed until weeks after birth, but hypospadias may be identified in early postnatal life as well, and in some cases in late gestation (Sinclair et al., 2017).
Sex applicability: Hypospadias is primarily used in reference to malformation of the male external genitalia.
<h4>Key Event Description</h4>
Hypospadias is a malformation of the penis where the urethral opening is displaced from the tip of the glans, usually on the ventral side on the phallus. Most cases of hypospadias are milder where the urethral opening still appears on the glans proper or on the most distal part of the shaft. In more severe cases, the opening may be more proximally placed on the shaft or even as low as the scrotum or the perineum.
In addition to the misplacement of the urethral opening, hypospadias is associated with an absence of ventral prepuce, an excess of dorsal preputial tissue, and in some cases a downward curvature of the penis (chordee).  Patients with hypospadias may need surgical repairment depending on severity, with more proximal hypospadias patients in most need of surgeries to achieve optimal functional and cosmetic results (Baskin, 2000; Baskin & Ebbers, 2006; Mattiske & Pask, 2021). The incidence of hypospadias varies greatly between countries, from 1:100 to 1:500 of newborn boys (Skakkebaek et al., 2016), and the global prevalence seems to be increasing (Paulozzi, 1999; Springer et al., 2016; Yu et al., 2019).
The external genitalia arise from the biphasic genital tubercle during fetal development. Androgens (testosterone and dihydrotestosterone) drive formation of the male external genitalia. In humans, the urethra develops by fusion of two endoderm-derived urethral folds. Disruption of genital tubercle differentiation results in an incomplete urethra, i.e. hypospadias.  (Baskin, 2000; Baskin & Ebbers, 2006).
<h4>How it is Measured or Detected</h4>
In humans, hypospadias is diagnosed clinically by physical examination of the infant and is at first recognized by the absence of ventral prepuce and concurrent excess dorsal prepuce (Baskin, 2000). Hypospadias may be classified according to the location of the urethral meatus: Glandular, subcoronal, midshaft, penoscrotal, scrotal, and perineal (Baskin & Ebbers, 2006).
In mice and rats, macroscopic assessment of hypospadias may be performed postnatally, and several OECD test guidelines require macroscopic examination of genital abnormalities in in vivo toxicity studies (TG 414, 416, 421/422, 443). The guidelines do not define hypospadias or how to identify them. Fetal and neonatal identification of hypospadias may require microscopic examination for proper evaluation of the pathology. This can be done by scanning electron microscopy (Uda et al., 2004), or by histological assessment in which the presence of the urethral opening in proximal, transverse sections (for example co-occuring with the os penis or corpus cavernosum), indicates hypospadias (Mahawong et al., 2014; Sinclair et al., 2017; Vilela et al., 2007). In a semiquantitative, histologic approach, the number of transverse sections of the penis with internalization of the urethra was related to the total length of the penis, achieving a percentage of urethral internalization. In this study, ≤89% of urethral internalization was defined as indicative of mild hypospadias (Stewart et al., 2018).
<h4>Regulatory Significance of the AO</h4>
In the OECD guidelines for developmental and reproductive toxicology, several test endpoints include examination of structural abnormalities with special attention to the organs of the reproductive system. These are: Test No. 414 ‘Prenatal Developmental Toxicity Study’ (OECD, 2018a); Test No. 416 ‘Two-Generation Reproduction Toxicity’ (OECD, 2001) and Tests No. 421/422 ‘Reproduction/Developmental Toxicity Screening Test’ (OECD, 2016a, 2016b). In Test No. 443 ‘Extended One-Generation Reproductive Toxicity Study’ (OECD, 2018b), hypospadias is specifically mentioned as a genital abnormality to note.
<h4>References</h4>
Baskin, L. S. (2000). Hypospadias and urethral development. The Journal of Urology, 163(3), 951–956.
Baskin, L. S., & Ebbers, M. B. (2006). Hypospadias: Anatomy, etiology, and technique. Journal of Pediatric Surgery, 41(3), 463–472. https://doi.org/10.1016/j.jpedsurg.2005.11.059
Gredler, M. L., Larkins, C. E., Leal, F., Lewis, A. K., Herrera, A. M., Perriton, C. L., Sanger, T. J., & Cohn, M. J. (2014). Evolution of External Genitalia: Insights from Reptilian Development. Sexual Development, 8(5), 311–326. https://doi.org/10.1159/000365771
Mahawong, P., Sinclair, A., Li, Y., Schlomer, B., Rodriguez, E., Ferretti, M. M., Liu, B., Baskin, L. S., & Cunha, G. R. (2014). Prenatal diethylstilbestrol induces malformation of the external genitalia of male and female mice and persistent second-generation developmental abnormalities of the external genitalia in two mouse strains. Differentiation, 88(2–3), 51–69. https://doi.org/10.1016/j.diff.2014.09.005
Mattiske, D. M., & Pask, A. J. (2021). Endocrine disrupting chemicals in the pathogenesis of hypospadias; developmental and toxicological perspectives. Current Research in Toxicology, 2, 179–191. https://doi.org/10.1016/j.crtox.2021.03.004
OECD. (2001). Test No. 416: Two-Generation Reproduction Toxicity. In OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. https://doi.org/10.1787/9789264070868-en
OECD. (2018). Test No. 414: Prenatal Developmental Toxicity Study. In OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. https://doi.org/10.1787/9789264070820-en
OECD. (2025a). Test No. 421: Reproduction/Developmental Toxicity Screening Test. In OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. https://doi.org/doi.org/10.1787/9789264264380-en
OECD. (2025b). Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. In OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publising. https://doi.org/doi.org/10.1787/9789264264403-en
OECD. (2025c). Test No. 443: Extended One-Generation Reproductive Toxicity Study. In OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing. https://doi.org/doi.org/10.1787/9789264185371-en
Paulozzi, L. J. (1999). International Trends in Rates of Hypospadias and Cryptorchidism. Environmental Health Perspectives, 107(4), 297–302. https://doi.org/10.1289/ehp.99107297
Sinclair, A. W., Cao, M., Pask, A., Baskin, L., & Cunha, G. R. (2017). Flutamide-induced hypospadias in rats: A critical assessment. Differentiation, 94, 37–57. https://doi.org/10.1016/j.diff.2016.12.001
Skakkebaek, N. E., Rajpert-De Meyts, E., Buck Louis, G. M., Toppari, J., Andersson, A.-M., Eisenberg, M. L., Jensen, T. K., Jørgensen, N., Swan, S. H., Sapra, K. J., Ziebe, S., Priskorn, L., & Juul, A. (2016). Male Reproductive Disorders and Fertility Trends: Influences of Environment and Genetic Susceptibility. Physiological Reviews, 96(1), 55–97. https://doi.org/10.1152/physrev.00017.2015.-It
Springer, A., van den Heijkant, M., & Baumann, S. (2016). Worldwide prevalence of hypospadias. Journal of Pediatric Urology, 12(3), 152.e1-152.e7. https://doi.org/10.1016/j.jpurol.2015.12.002
Stewart, M. K., Mattiske, D. M., & Pask, A. J. (2018). In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development. Biology of Reproduction, 99(6), 1184–1193. https://doi.org/10.1093/biolre/ioy142
Uda, A., Kojima, Y., Hayashi, Y., Mizuno, K., Asai, N., & Kohri, K. (2004). Morphological features of external genitalia in hypospadiac rat model: 3-dimensional analysis. The Journal of Urology, 171(3), 1362–1366. https://doi.org/10.1097/01.JU.0000100140.42618.54
Vilela, M. L. B., Willingham, E., Buckley, J., Liu, B. C., Agras, K., Shiroyanagi, Y., & Baskin, L. S. (2007). Endocrine Disruptors and Hypospadias: Role of Genistein and the Fungicide Vinclozolin. Urology, 70(3), 618–621. https://doi.org/10.1016/j.urology.2007.05.004
Yu, X., Nassar, N., Mastroiacovo, P., Canfield, M., Groisman, B., Bermejo-Sánchez, E., Ritvanen, A., Kiuru-Kuhlefelt, S., Benavides, A., Sipek, A., Pierini, A., Bianchi, F., Källén, K., Gatt, M., Morgan, M., Tucker, D., Canessa, M. A., Gajardo, R., Mutchinick, O. M., … Agopian, A. J. (2019). Hypospadias Prevalence and Trends in International Birth Defect Surveillance Systems, 1980–2010. European Urology, 76(4), 482–490. https://doi.org/10.1016/j.eururo.2019.06.027
<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>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/289">Inhibition of 5α-reductase leading to impaired fecundity in female fish</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>High</td>
</tr>
<tr>
<td><a href="/aops/571">5α-reductase inhibition leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</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>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
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.
<h4>Key Event Relationship Description</h4>
This key event relationship (KER) links inhibition of 5α-reductase activity to decreased dihydrotestosterone (DHT) levels.
There are three isozymes of 5α-reductase: type 1, 2, and 3. 5α-reductase type 2 is mainly involved in the synthesis of 5α-DHT from testosterone (T) (Robitaille & Langlois, 2020), 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 (Miller & Auchus, 2019). 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.
The direct conversion of T to 5α-DHT mainly takes place in the target tissue (Robitaille & Langlois, 2020). In mammals, the type 1 isoform is found in the scalp and other peripheral tissues (Miller & Auchus, 2011), such as liver, skin, prostate (Azzouni et al., 2012), bone, ovaries, and adipose tissue (Nikolaou et al., 2021). The type 2 isoform is expressed mainly in male reproductive tissues (Miller & Auchus, 2011), but also in liver, scalp and skin (Nikolaou et al., 2021). The expression level of both isoforms depend on the developmental stage and the tissue.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility of this KER is considered high.
5α-reductase can catalyze the conversion of T to DHT. The substrates for 5α-reductases are 3-oxo (3-keto), Δ4,5 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.
In both humans and rodents, DHT is important for the in utero 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 (Azzouni et al., 2012).
Empirical Evidence
The empirical evidence for this KER is considered high
Dose concordance
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. These studies at large rely on conversion of radiolabeled substrate and hence serve as an indirect measurement.
Table 1: Dose concordance from selected in vitro test systems
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:614px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:94px">
Test system
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:147px">
Model description
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:91px">
Stressor
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:178px">
Effect
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:104px">
Reference
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
HEK-293 cells
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
Cells stably transfected human 5α-reductase type 1 and 2 used to measure conversion of [14C]labeled steroids
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: IC50 = 106.9 µM
Type 2: IC50 = 14.3 µM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
(Yamana et al., 2010)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">Dutasteride</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: IC50 = 8.7 µM
Type 2: IC50 = 57 µM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px"> </td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
COS cells
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
Cell homogenates from transfected cells with human and rat 5α-reductase (unknown isoform) used to measure conversion of radiolabeled testosterone
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Human:
IC50 ≈ 1 µM
Ki = 340-620 nM
Rat:
IC50 ≈ 0.1 µM
Ki = 3-5 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
(Andersson & Russell, 1990)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">4-MA</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Human:
IC50 ≈ 0.1 µM
Ki = 7-8 nM
Rat:
IC50 ≈ 0.1 µM
Ki = 5-7 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px"> </td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
CHO cells
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
Stably transfected with human 5α-reductase type 1 and 2
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: Ki = 325 nM
Type 2: Ki = 12 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
(Thigpens et al., 1993)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">4-MA</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: Ki = 8 nM
Type 2: Ki = 4 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px"> </td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px">
Isolated enzyme
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
Human 5α-reductase type 1 and 2 used to measure conversion of radiolabeled substrate of both isoforms
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: Ki =  > 200 nM
Type 2: Ki = 0.45 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px">
(Peng et al., 2020)
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px"> </td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px">Dutasteride</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px">
Type 1: Ki = 39 nM
Type 2: Ki = 1.1 nM
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:104px"> </td>
</tr>
</tbody>
</table>
These in vitro studies clearly show effects on the enzymatic reaction induced by 5α-reductases in a concentration dependent manner (Andersson & Russell, 1990; Thigpens et al., 1993; Yamana et al., 2010).
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 (Robitaille & Langlois, 2020). This has been demonstrated in humans, rats, monkeys, and mice  (Robitaille et al. 2020).
Finasteride is a specific inhibitor of 5α-reductase type 2 (Russell & Wilson, 1994). 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 (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).
Other evidence
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).
<h4>Quantitative Understanding of the Linkage</h4>
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 (Miller & Auchus, 2011).
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 (Nikolaou et al., 2021).
Response-response relationship
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.
Time-scale
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.
Known Feedforward/Feedback loops influencing this KER
Androgens can regulate gene expression of 5α-reductases (Andersson et al., 1989; Berman & Russell, 1993).
<h4>References</h4>
Alemany, M. (2022). The Roles of Androgens in Humans: Biology, Metabolic Regulation and Health. In International Journal of Molecular Sciences (Vol. 23, Issue 19). MDPI. https://doi.org/10.3390/ijms231911952
Andersson, S., Bishop, R. W., & Russell$, D. W. (1989). THE JOURNAL OF BIOLOGICAL CHEMISTRY Expression Cloning and Regulation of Steroid 5cw-Reductase, an Enzyme Essential for Male Sexual Differentiation* (Vol. 264, Issue 27).
Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5a-reductases. Proc. Natl. Acad. Sci. USA, 87, 3640–3644. https://www.pnas.org
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 Advances in Urology. https://doi.org/10.1155/2012/530121
Berman, D. M., & Russell, D. W. (1993). Cell-type-specific expression of rat steroid 5a-reductase isozymes (sexual development/androgens/prostate/stroma/epithelium). In Proc. Natl. Acad. Sci. USA (Vol. 90). https://www.pnas.org
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. Journal of Clinical Endocrinology and Metabolism, 89(5), 2179–2184. https://doi.org/10.1210/jc.2003-030330
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). The effects of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia.
Miller, W. L., & Auchus, R. J. (2011). The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews, 32(1), 81–151. https://doi.org/10.1210/er.2010-0013
Miller, W. L., & Auchus, R. J. (2019). The “backdoor pathway” of androgen synthesis in human male sexual development. PLoS Biology, 17(4). https://doi.org/10.1371/journal.pbio.3000198
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 Journal of Steroid Biochemistry and Molecular Biology (Vol. 207). Elsevier Ltd. https://doi.org/10.1016/j.jsbmb.2021.105808
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. Endocrinology (United States), 161(8), 1–11. https://doi.org/10.1210/ENDOCR/BQAA117
Robitaille, J., & Langlois, V. S. (2020). Consequences of steroid-5α-reductase deficiency and inhibition in vertebrates. In General and Comparative Endocrinology (Vol. 290). Academic Press Inc. https://doi.org/10.1016/j.ygcen.2020.113400
Russell, D. W., & Wilson, J. D. (1994). STEROID Sa-REDUCTASE: TWO GENES/TWO ENZYMES. www.annualreviews.org
Thigpens, A. E., Cala, K. M., & Russell, D. W. (1993). Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes. The Journal of Biological Chemistry, 268(23), 17404–17412.
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. Hormone Molecular Biology and Clinical Investigation, 2(3), 293–299. https://doi.org/10.1515/HMBCI.2010.035
</div>
<div>
<h4><a href="/relationships/1935">Relationship: 1935: Decrease, DHT level leads to Decrease, AR activation</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<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>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>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
KER1935 is assessed applicable to mammals, as DHT and AR activation are known to be related in mammals. 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.
Sex applicability
KER1935 is assessed applicable to both sexes, as DHT activates AR in both males and females.
Life-stage applicability
KER1935 is considered applicable to developmental and adult life stages, as DHT-mediated AR activation is relevant from the AR is expressed.
<h4>Key Event Relationship Description</h4>
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 (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>; <a href="#_ENREF_3" title="Marks, 2004 #283">Marks, 2004</a>). 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’ (<a href="#_ENREF_1" title="Davey, 2016 #250">Davey & Grossmann, 2016</a>). 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.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility of KER1935 is considered high.
The activation of AR is dependent on binding of ligands (though a few cases of ligand-independent AR activation has been shown, see uncertainties and inconsistencies), 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). AR transcriptional activity and specificity is regulated by co-activators and co-repressors in a cell-specific manner (Heinlein and Chang, 2002).
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 in vitro (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.
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).
Empirical Evidence
The empirical support for KER1935 is considered high.
Dose concordance:
<ul>
<li>Increasing concentrations of DHT lead to increasing AR activation in vitro in AR reporter gene assays (OECD, 2020; Williams et al., 2017).</li>
</ul>
Indirect (supporting) evidence:
<ul>
<li>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%. </li>
<li>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 in utero 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).</li>
</ul>
Other evidence:
<ul>
<li>Male individuals with congenital 5α-reductase deficiency (absence of DHT) fail to masculinize properly (Costa et al., 2012). </li>
<li>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).</li>
</ul>
Uncertainties and Inconsistencies
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).
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.
<h4>Quantitative Understanding of the Linkage</h4>
Response-response relationship
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 in vivo, and such a relationship will differ between biological systems (species, tissue, cell type).
Time-scale
Upon DHT binding to the AR, a conformational change that brings the amino (N) and carboxy (C) termini into close proximity occurs with a t1/2 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).
Known modulating factors
<div>
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Age</td>
<td>AR expression changes with aging</td>
<td>Tissue-specific alterations in AR activity with aging</td>
<td>(Supakar et al., 1993; Wu et al., 2009)</td>
</tr>
<tr>
<td>Genotype</td>
<td>Number of CAG repeats in the first exon of AR</td>
<td>Decreased AR activation with increased number of CAGs</td>
<td>(Chamberlain et al., 1994; Tut et al., 1997)</td>
</tr>
<tr>
<td>Androgen deficiency syndrome</td>
<td>Low circulating testosterone levels due to primary (testicular) or secondary (pituitary-hypothalamic) hypogonadism</td>
<td>Reduced levels of circulating testosterone, precurser of DHT</td>
<td>(Bhasin et al., 2010)</td>
</tr>
<tr>
<td>Castration</td>
<td>Removal of testicles</td>
<td>Reduced levels of circulating testosterone, precurser of DHT</td>
<td>(Krotkiewski et al., 1980)</td>
</tr>
</tbody>
</table>
</div>
Known Feedforward/Feedback loops influencing this KER
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). The quantitative impact of such adaptive expression changes is unknown.
<h4>References</h4>
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
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
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
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
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
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
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
Dalton, J.T., Mukherjee, A., Zhu, Z., Kirkovsky, L., Miller, D.D., 1998. Discovery of nonsteroidal androgens. Biochem. Biophys. Res. Commun. 244, 1–4. https://doi.org/10.1006/bbrc.1998.8209
Davey, R.A., Grossmann, M., 2016. Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin. Biochem. Rev. 37, 3–15.
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
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
Heinlein, C.A., Chang, C., 2002. Androgen Receptor (AR) Coregulators: An Overview. Endocr. Rev. 23, 175–200. https://doi.org/10.1210/EDRV.23.2.0460
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.
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
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
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
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
Marks, L.S., 2004. 5Alpha-Reductase: History and Clinical Importance. Rev. Urol. 6 Suppl 9, S11-21.
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
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
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
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
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
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
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
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
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
Wu, D., Lin, G., Gore, A.C., 2009. Age-related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor α in Male Rats. J. Comp. Neurol. 512, 688. https://doi.org/10.1002/CNE.21925
</div>
<div>
<h4><a href="/relationships/2124">Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/344">Androgen receptor (AR) antagonism leading to nipple retention (NR) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/345">Androgen receptor (AR) antagonism leading to decreased fertility in females</a></td>
<td>adjacent</td>
<td>High</td>
<td>Moderate</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/306">Androgen receptor (AR) antagonism leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/307">Decreased testosterone synthesis leading to short anogenital distance (AGD) in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td>Low</td>
</tr>
<tr>
<td><a href="/aops/570">Decreased testosterone synthesis leading to hypospadias 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>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>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/575">Decreased testosterone synthesis leading to increased nipple retention (NR) in male (rodent) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/477">Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>mammals</td>
<td>mammals</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>During development and at adulthood</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
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.
<h4>Key Event Relationship Description</h4>
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 (Lamont and Tindall, 2010, Roy et al. 2001). 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 in vivo and the specific effect on transcription of AR target genes will depend on species, life stage, tissue, cell type etc.
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is considered high
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 (Roy et al. 2001). 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.
Through mapping of AREs and ChIP sequencing studies, several AR target genes have been identified, mainly studied in prostate cells (Jin, Kim, and Yu 2013). Different co-regulators and ligands lead to altered expression of different sets of genes (Jin et al. 2013; Kanno et al. 2022). Alternative splicing of the AR can lead to different AR variants that also affects which genes are transcribed (Jin et al. 2013).
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 (Jin et al. 2013).
Empirical Evidence
The empirical evidence for this KER is considered high
In humans, altered gene expression profiling in individuals with androgen insensitivity syndrome (AIS) can provide supporting empirical evidence (Holterhus et al. 2003; Peng et al. 2021). 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 (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).
Exposure to known antiandrogens has been shown to alter transcriptional profiles, for example of neonatal pig ovaries (Knapczyk-Stwora et al. 2019).
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).
Uncertainties and Inconsistencies
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 (Bennesch and Picard 2015).
<h4>Quantitative Understanding of the Linkage</h4>
Response-response relationship
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).
Time-scale
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 (Kang et al. 2002). RNA polymerase II elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb/min (Maiuri et al. 2011). Therefore, depending on the cell type and the half-life of the AR target gene transcripts, changes are to be expected within hours.
Known modulating factors
<div>
<table class="table table-bordered table-fullwidth">
<thead>
<tr>
<th>Modulating Factor (MF)</th>
<th>MF Specification</th>
<th>Effect(s) on the KER</th>
<th>Reference(s)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Age</td>
<td>AR expression in aging male rats</td>
<td>Tissue-specific alterations in AR activity with aging</td>
<td>(Supakar et al. 1993; Wu, Lin, and Gore 2009)</td>
</tr>
<tr>
<td>Genotype</td>
<td>Number of CAG repeats in the first exon of AR</td>
<td>Decreased AR activation with increased number of CAGs</td>
<td>
(Tut et al. 1997; Chamberlain et al. 1994)
</td>
</tr>
</tbody>
</table>
</div>
Known Feedforward/Feedback loops influencing this KER
AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).
<h4>References</h4>
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.
Bennesch, Marcela A., and Didier Picard. 2015. “Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors.” Molecular Endocrinology 29(3):349–63.
Chamberlain, Nancy L., Erika D. Driverand, and Roger L. Miesfeldi. 1994. The Length and Location of CAG Trinucleotide Repeats in the Androgen Receptor N-Terminal Domain Affect Transactivation Function. Vol. 22.
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.” Molecular Endocrinology 20(2):321–34. doi: 10.1210/me.2005-0113.
Fan, Wuqiang, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Takashi Sato, Hirotaka Kawano, Shigeaki Kato, and Hajime Nawata. 2005. 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. Vol. 54.
Holterhus, Paul-Martin, Olaf Hiort, Janos Demeter, Patrick O. Brown, and James D. Brooks. 2003. 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. Vol. 4.
Ikeda, Yasumasa, Ken Ichi Aihara, Takashi Sato, Masashi Akaike, Masanori Yoshizumi, Yuki Suzaki, Yuki Izawa, Mitsunori Fujimura, Shunji Hashizume, Midori Kato, Shusuke Yagi, Toshiaki Tamaki, Hirotaka Kawano, Takahiro Matsumoto, Hiroyuki Azuma, Shigeaki Kato, and Toshio Matsumoto. 2005. “Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-Induced Cardiac Fibrosis.” Journal of Biological Chemistry 280(33):29661–66. doi: 10.1074/jbc.M411694200.
Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77.
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.” Journal of Biological Chemistry 277(50):48366–71. doi: 10.1074/jbc.M209074200.
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.” Experimental Cell Research 419(2). doi: 10.1016/j.yexcr.2022.113333.
Karlsson, Sara A., Erik Studer, Petronella Kettunen, and Lars Westberg. 2016. “Neural Androgen Receptors Modulate Gene Expression and Social Recognition but Not Social Investigation.” Frontiers in Behavioral Neuroscience 10(MAR). doi: 10.3389/fnbeh.2016.00041.
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.” Journal of Animal Science and Biotechnology 10(1):1–15. doi: 10.1186/s40104-019-0340-y.
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.
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 .” The FASEB Journal 22(8):2676–89. doi: 10.1096/fj.08-105726.
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.” EMBO Reports 12(12):1280–85. doi: 10.1038/embor.2011.196.
Mora, Gloria R., and Virendra B. Mahesh. 1999. Autoregulation of the Androgen Receptor at the Translational Level: Testosterone Induces Accumulation of Androgen Receptor MRNA in the Rat Ventral Prostate Polyribosomes.
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.” Frontiers in Endocrinology 12. doi: 10.3389/fendo.2021.731107.
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.” Am J Physiol Endocrinol Me-Tab 301:767–78. doi: 10.1152/ajpendo.00584.2010.-In.
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 Annals of the New York Academy of Sciences. Vol. 949. New York Academy of Sciences.
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.” Journal of Molecular Endocrinology 49(1):1–10. doi: 10.1530/JME-12-0014.
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. Premature Ovarian Failure in Androgen Receptor-Deficient Mice. Vol. 103.
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.” Journal of Biological Chemistry 268(35):26400–408. doi: 10.1016/s0021-9258(19)74328-2.
Tut, Thein G., Farid J. Ghadessy, M. A. Trifiro, L. Pinsky, and E. L. Yong. 1997. Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility*. Vol. 82.
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.” Endocrinology 147(12):5624–33. doi: 10.1210/en.2006-0138.
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.” International Journal of Andrology 35(1):25–40. doi: 10.1111/j.1365-2605.2011.01150.x.
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.” PLoS ONE 5(11). doi: 10.1371/journal.pone.0014168.
Wu, D. I., Grace Lin, and Andrea C. Gore. 2009. “Age-Related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor in Male Rats.” The Journal of Comparative Neurology 512:688–701. doi: 10.1002/cne.21925.
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.” Endocrinology 149(5):2361–68. doi: 10.1210/en.2007-0516.
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.” Prostate 72(4):437–49. doi: 10.1002/pros.21445.
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 &dagger; &dagger; George. 2006. Oligozoospermia with Normal Fertility in Male Mice Lacking the Androgen Receptor in Testis Peritubular Myoid Cells.
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).” Biology of Reproduction 84(2):400–408.
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2828">Relationship: 2828: Decrease, AR activation leads to Hypospadias</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/477">Androgen receptor (AR) antagonism leading to hypospadias in male (mammalian) offspring</a></td>
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/570">Decreased testosterone synthesis leading to hypospadias in male (mammalian) offspring</a></td>
<td>non-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>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>human</td>
<td>Homo sapiens</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>rat</td>
<td>Rattus norvegicus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>mouse</td>
<td>Mus musculus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Foetal</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Taxonomic applicability
In mammals, androgens are one of the primary drivers of penis differentiation. Hypospadias has been observed in several mammals, but most frequently reported in laboratory rodents and in humans (Chang et al., 2020; S. Wang & Zheng, 2025). In vivo studies in rats and mice show that in utero exposure to anti-androgenic chemicals can cause hypospadias in male offspring (see table 3). Many human case studies report boys born with hypospadias and associated deficiency in steroid hormone synthesis, 5α-reductase activity, or androgen receptor (AR) activity (see table 4).
The biologically plausible domain of applicability may extend beyond the empirical domain because androgen-controlled development of male external genitalia is evolutionary conserved in most mammals and, to some extent, also in other vertebrate classes (Gredler et al., 2014). Hypospadias can in principle occur in all animals that form a genital tubercle and have been observed in many domestic animal species including dog (Sonne et al., 2008; Switonski et al., 2018), cat (Nowacka-Woszuk et al., 2014), cattle (Murakami, 2008), sheep (Smith et al., 2012), and horse (De Lorenzi et al., 2010) as well as in wildlife species such as polar bear (Stamper et al., 1999), giraffe (Meuffels et al., 2020), and Tamar Wallaby (Leihy et al., 2011). The observed hypospadias in these animals is not, per se, linked to anti-androgenic exposure, which has only been sparsely investigated in other species than mice, rats, and humans. One study in monkeys did show hypospadias upon oral exposure to finasteride (Prahalada et al., 1997), and bicalutamide exposure induced hypospadias in guinea pigs (S. Wang et al., 2018). A study in rabbits exposed to procymidone did not find hypospadias in males (Inawaka et al., 2010). Another study in hyenas did also not find hypospadias in males after exposure to the anti-androgen finasteride (Drea et al., 1998), but it should be noted that the hyenas have a remarkable sexual development where penile growth occur in both females and males before androgen synthesis is initiated (Cunha et al., 2014) (the studies in hyena and rabbit were identified in our evidence collection but were judged as ‘unreliable’ and therefore not included as empirical evidence).
Sex applicability
The androgen receptor is expressed in the fetal genital tubercle of both females and males (Amato & Yao, 2021; Baskin et al., 2020), but hypospadias is primarily a term used for a malformation of the penis (Baskin & Ebbers, 2006), limiting the applicability of this KER to males.
Life stage applicability
Differentiation of the penis occurs during fetal life in the masculinization programming window (MPW) (GD 16-20 in rats, around gestational weeks 8-14 in humans), when androgen production is high (Welsh et al., 2008; C. Wolf et al., 2000a). In rats, exposure to anti-androgenic chemicals outside of, or in the late part of the MPW does not cause hypospadias or only to a low degree (Clark et al., 1993; van den Driesche et al., 2017; C. Wolf et al., 2000a), while exposure in the earlier (or full) MPW causes a higher frequency of hypospadias (depending on dose and chemical) (table 3). In humans, hypospadias can be diagnosed at birth (X. Yu et al., 2019), while in rodents, some parts of penis development occur postnatally (Schlomer et al., 2013; Sinclair et al., 2017). In these species, hypospadias may be observed at birth but is optimally diagnosed and severity classified weeks later. Given that disruptions to androgen programming takes place in fetal life, even though the AO is best detected postnatally, the life stage applicability is defined as fetal life.
<h4>Key Event Relationship Description</h4>
This non-adjacent KER describes a fetal decrease in androgen receptor (AR) activation in the genital tubercle causing hypospadias in male offspring, postnatally. During fetal development, androgens induce differentiation of the bipotential genital tubercle to a penis, including closure of the urethra. Androgens signal through AR and reduced fetal AR activation can therefore disrupt penis differentiation and lead to the genital malformation hypospadias. Reduced AR activation may happen both through reduced ligand availability (testosterone or dihydrotestosterone (DHT)) and by direct antagonism of AR (Amato et al., 2022; Mattiske & Pask, 2021).
The upstream KE ‘decrease, androgen receptor activation’ (KE 1614) refers to the in vivo event of overall reduction in AR activation. In this case, it therefore refers to a reduction in AR activation in the genital tubercle. Currently, decreased AR activation in mammals is only directly measured in vitro and not in vivo. Instead, indirect assessment of this KE may come from assays measuring AR antagonism, 5α-reductase activity (the enzyme converting testosterone to DHT), or decreased androgen levels (Draskau et al., 2024).
<h4>Evidence Supporting this KER</h4>
Biological Plausibility
The biological plausibility for this KER is judged as high. This is largely based on canonical knowledge on normal reproductive development.
The penis originates from a sexually bipotential structure, the genital tubercle, which may differentiate to either a penis or a clitoris, depending on internal cues during fetal development. In males, the fetal testes produce large amounts of testosterone, which can subsequently be converted to the more potent androgen DHT by 5α-reductase in peripheral tissues. Testosterone and DHT both signal through AR in target tissues to initiate masculinization (Amato et al., 2022; Murashima et al., 2015). The critical developmental window for androgen programming of masculinization has been identified in rats as GD16-20, and is proposed to be gestational weeks 8-14 in humans (Sharpe, 2020; Welsh et al., 2008). As part of the masculinization process orchestrated by androgens, the genital tubercle differentiates to a penis, which at this point expresses AR in both humans and rodents (Amato & Yao, 2021; Baskin et al., 2020). This includes androgen-mediated elongation of the tubercle, formation of the prepuce, and tubular internalization of urethra, which is closed at the distal tip of the glans penis (Amato et al., 2022). Failure of full closure of the urethra can result in hypospadias, in which the urethra terminates at the ventral side of the penis instead of at the tip (Baskin & Ebbers, 2006; Cohn, 2011).
The dependency of androgens for penile development has been demonstrated in mice with conditional or full knockout of Ar, which results in partly or full sex-reversal of males, including a female-like urethral opening (Willingham et al., 2006; Yucel et al., 2004; Zheng et al., 2015). Similarly, female rats and mice exposed in utero to testosterone present with varying degrees of intersexuality, including, in some cases, a penis (Greene & Ivy, 1937; Zheng et al., 2015).
Empirical Evidence
The empirical evidence for this KER is generally judged high. This includes evidence from in vivo animal studies and evidence from studies in humans. The upstream KE ‘Decreased AR activity’ refers to an in vivo effect, for which no methods for measurement of this in vivo in mammals currently exist. The effects on the upstream KE were therefore indirectly informed as described in each section.
Animal studies
Effects on the upstream KE were indirectly informed by including animal studies with stressors that are known to reduce AR activity through antagonizing the AR, lowering testosterone production, or inhibiting 5α-reductase. Six stressors, with established anti-androgenic effects, were included (more detailed evaluation of these chemicals can be found in KER-2820 (Holmer et al., 2024)). Table 3 summarizes the empirical evidence and confidence level for each chemical. Details on included evidence is presented in Table 1 in Appendix 2, <a href="https://aopwiki.org/system/dragonfly/production/2025/09/18/9prbqyba2x_Appendix_2_KER_2828.pdf">9prbqyba2x_Appendix_2_KER_2828.pdf</a>. In summary, all six substances were shown to cause hypospadias in male offspring, and the confidence level for all substances was judged as strong, as conflicting results could be explained (see the section ‘Uncertainties and inconsistencies’). Thus, antagonism or AR, inhibition of 5α-reductase, or reduction in testosterone synthesis, all lead to hypospadias.
Table 3 Summary of empirical evidence for the KER – animal studies. See Table 1 in Appendix 2 ( <a href="https://aopwiki.org/system/dragonfly/production/2025/09/18/9prbqyba2x_Appendix_2_KER_2828.pdf">9prbqyba2x_Appendix_2_KER_2828.pdf</a>) for details.
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:159px">
Chemical
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:160px">
Upstream effect
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px">
Downstream effect
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:141px">
Overall confidence
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Flutamide
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
Androgen receptor antagonist (Simard et al., 1986).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat and mouse
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Dibutyl phthalate (DBP)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
Has been shown to reduce fetal intratesticular testosterone and serum testosterone in vivo, but exact mechanism is unknown (Foster, 2006).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Vinclozolin
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
AR antagonist (Kelce et al., 1994, 1997)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat and mouse
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Di(2-ethylhexyl) phthalate (DEHP)
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
Has been shown to reduce fetal intratesticular testosterone and serum testosterone in vivo, but exact mechanism is unknown (Parks et al., 2000).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Procymidone
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
AR antagonist (Ostby et al., 1999).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:159px">
Finasteride
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:160px">
5α-reductase inhibitor, causing a reduction in DHT (Rittmaster & Wood, 1994).
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
In utero exposure causes hypospadias in rat
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:141px">
Strong
</td>
</tr>
</tbody>
</table>
Supporting human evidence
Effects on the upstream KE were indirectly informed by including studies in humans with a condition (genetic or other) that would reduce or disrupt either 1) function of AR, 2) conversion of testosterone to DHT by disrupting 5α-reductase activity, or 3) production of androgen hormones. Studies measuring low testosterone levels with no underlying cause were also included (see evidence collection strategy). Table 4 lists the studies, in which these conditions were linked to hypospadias in males.
Table 4 Supporting evidence for the KER – human studies. The table lists human studies reporting hypospadias in association with an upstream defect in AR activity, grouped according to the precise effect, and how it was diagnosed (mutation, in vitro activity, or blood hormone and metabolite profile). SRD5A2: 5α-reductase 2; HSD17B3: 17β-hydroxysteroid dehydrogenase 3; HSD3B2: 3β-hydroxysteroid dehydrogenase 2; CYP17A1: 17α-hydroxylase. See table 2 in Appendix 2  (<a href="https://aopwiki.org/system/dragonfly/production/2025/09/18/9prbqyba2x_Appendix_2_KER_2828.pdf">9prbqyba2x_Appendix_2_KER_2828.pdf</a>) for all included references.
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:614px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:.100px; vertical-align:top; width:501px">
Effect on upstream KE
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:.100px; vertical-align:top; width:113px">
Supporting studies
</td>
</tr>
<tr>
<td colspan="2" style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:6px; vertical-align:top; width:614px">
Effects on Androgen receptor
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:6px; vertical-align:top; width:501px">
AR mutations
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:6px; vertical-align:top; width:113px">
27 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:2px; vertical-align:top; width:501px">
Extended CAG repeat length in AR
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:2px; vertical-align:top; width:113px">
4 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:3px; vertical-align:top; width:501px">
Reduced AR activity (e.g. low receptor binding) in in vitro genital skin fibroblasts
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:3px; vertical-align:top; width:113px">
11 studies
</td>
</tr>
<tr>
<td colspan="2" style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:3px; vertical-align:top; width:614px">
Effects on 5α-reductase activity
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
SRD5A2 mutations
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
30 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
SRD5A2 deficiency, diagnosed by T/DHT-ratio and/or reduced in vitro 5α-reductase activity in genital skin fibroblasts
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
8 studies
</td>
</tr>
<tr>
<td colspan="2" style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:614px">
Effects on upstream steroidogenesis enzymes
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
HSD17B3 mutations
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
6 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
HSD3B2 mutations
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
5 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
CYP17A1 mutation
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
1 study
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
HSD17B3 deficiency, diagnosed by hormone and metabolite profile
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
2 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
HSD3B2 deficiency, diagnosed by hormone and metabolite profile
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
4 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
CYP17A1 deficiency, diagnosed by hormone and metabolite profile
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
5 studies
</td>
</tr>
<tr>
<td colspan="2" style="background-color:#e7e6e6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:614px">
Other upstream effects on low testosterone
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
Low testosterone due to gonadal dysgenesis or hypogonadism
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
7 studies
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:501px">
Low basal testosterone or low testosterone response to hCG stimulation. Idiopathic or rare mutations.
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:.47px; vertical-align:top; width:113px">
7 studies
</td>
</tr>
</tbody>
</table>
Six case-control studies were extracted, all of which found a correlation between lower testosterone levels (basal or hCG-stimulated) and hypospadias (Austin et al., 2002; Okuyama et al., 1981; Raboch et al., 1976; Ratan et al., 2012; Svensson et al., 1979; Yadav et al., 2011). In two of these studies, the correlation was age-dependent (Austin et al., 2002; Raboch et al., 1976)
One epidemiologic study was extracted, which investigated the association between phthalate exposure and hypospadias risk.  Western Australian women exposed through their occupation to phthalates were more likely to have sons with hypospadias (Nassar et al., 2010). It should be noted that there are reported species differences in the effects of phthalates (including DEHP and DBP) on fetal testosterone production between humans, mice, and rats, and the direct translatability of the in vivo evidence is uncertain (Sharpe, 2020).
Dose concordance
Direct information about dose concordance is not available because AR activity currently cannot be measured in vivo.
Indirect information on dose concordance can be obtained from empirical evidence. In utero exposure of rats to DBP caused a dose-dependent decrease in serum testosterone levels at PND70 with LOAEL 250 mg/kg bw/day. Hypospadias was observed at this stage with LOAEL 500 mg/kg bw/day (Jiang et al., 2007). It should be noted that fetal testosterone levels were not measured.
Temporal concordance
Direct information about temporal concordance is not available because AR activity currently cannot be measured in vivo.
Indirect information on temporal concordance can be obtained from empirical evidence. In two studies, in which rats were exposed in utero to 750 mg/kg bw/day DBP, intratesticular testosterone levels were reduced in fetal testes, while hypospadias was identified in adult males. Plasma levels of testosterone were also measured in adults, and testosterone levels in exposed males were not significantly different from control males (van den Driesche et al., 2017, 2020). This has also been shown in a study with 500 mg/kg bw/day DBP (Drake et al., 2009). These studies indicate temporal concordance. Another study with DBP-induced hypospadias in rats saw a dose-dependent reduction in serum testosterone levels at PND70 after in utero exposure to as low as 250 mg/kg bw/day from GD14-18 (Jiang et al., 2007), though fetal testosterone levels were not measured in this study.
Incidence Concordance
Direct information about dose concordance is not available because AR activity currently cannot be measured in vivo.
Indirect information on incidence concordance can be obtained from empirical evidence. In the dose-response study with DBP, the incidence of hypospadias was 6.8% for 500 mg/kg bw/day DBP and 41.3% for 750 mg/kg bw/day. When separating hypospadias males from exposed males without hypospadias, plasma testosterone levels were decreased in both groups, indicating that DBP reduced testosterone levels at higher incidence than hypospadias (Jiang et al., 2007). The same was seen in another study with DBP, in which serum testosterone levels at PND7 were reduced in both hypospadiac and non-malformed males exposed to 750 mg/kg bw/day DBP from GD14-18 (Jiang et al., 2016).
Uncertainties and Inconsistencies
The in vivo studies do not directly inform about the upstream KE, ‘decreased AR activity’. The direct concordance between the KEs can therefore not be determined from the evidence.
For flutamide, two studies reported 100% hypospadias frequencies at doses of 6.25 and 10 mg/kg bw/day (Goto et al., 2004; McIntyre et al., 2001), while another study found a frequency of 56.9% when giving 20 mg/kg bw/day (Kita et al., 2016). This might be explained by a longer exposure window in the first two studies and uncertainties in assessment of hypospadias.
For DBP, there were discrepancies in whether 250 mg/kg bw/day was LOAEL (Mylchreest et al., 1998, 1999) or NOAEL (Jiang et al., 2007) for DBP. This conflict was explained by differences in exposure windows, supported by the observation that the frequency of hypospadias at 250 mg/kg bw/day was reported as very low (Mylchreest et al., 1998, 1999).
One study with vinclozolin (Ostby J et al., 1999) and one with procymidone (Hass et al., 2012) did not find hypospadias after in utero exposure. In both cases, this was likely due to too low doses tested.
In most of the human studies of steroidogenesis deficiency, serum or plasma levels of testosterone were reduced at baseline and/or upon hCG stimulation (Al-Sinani et al., 2015; Ammini et al., 1997; Cara et al., 1985; Chen, Huang, et al., 2021; Dean et al., 1984; Galli-Tsinopoulou et al., 2018; Imperato-McGinley et al., 1979; Kaufman et al., 1983; Mendonca et al., 1987, 2000; Neocleous et al., 2012; New, 1970; Pang et al., 1983; Perrone et al., 1985; Rabbani et al., 2012; Sherbet et al., 2003), but in a few studies, testosterone levels were normal (Donadille et al., 2018; Kon et al., 2015; Luna et al., 2021). In these cases, the effect of these deficiencies on tissue AR activity is uncertain.
For AR CAG repeat length, a case-control study did not find an association with hypospadias (Radpour R et al., 2007), but this could be because the hypospadias cases included had other etiologies.
Lastly, as there are currently no universal guidelines for identification and scoring of hypospadias in rodents, there are large variations in methods of assessment, and minor cases of hypospadias may be overlooked in some studies and included in others. This poses an uncertainty in the frequency reports in the scientific evidence.
<h4>Quantitative Understanding of the Linkage</h4>
The quantitative understanding of the relationship is low. As there are currently no direct measurement methods of the upstream KE (reduced AR activity) in mammals, quantification of the relationship is difficult to assess.
Response-response relationship
A model for phthalates has been developed, aiming to predict the frequency of hypospadias in male offspring based on reductions in ex vivo testosterone production, an indirect indication of AR activity. In this model, hypospadias was induced from around a 60% reduction in testosterone levels. The model does not consider hypospadias severity and is only for phthalate chemicals (Earl Gray et al., 2024).
Time-scale
The time-scale of this KER depends on the species but is likely days to weeks.
AR activation happens within minutes, from ligand binding to nuclear translocation and promotor activation (Nightingale et al., 2003; Schaufele et al., 2005), while transcriptional and translational effects are observed minutes to hours later (Kang et al., 2002). AR programming of the genital tubercle occurs during fetal development in the Masculinization Programming Window (Sharpe, 2020). The time-scale for morphological effects in the tissue then depends on the species. In humans, penis development is completed prior to birth and hypospadias can be observed at birth. In rodents, penis development is not fully completed until weeks after birth, but hypospadias can often be observed earlier than this (table 3).
Known modulating factors
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Modulating Factors
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MF details
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Effects on the KER
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References
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AR CAG repeat length
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Extended CAG repeat length in AR is associated with reduced AR activity
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Higher risk of hypospadias development
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(Chamberlain et al., 1994)
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</table>
Known Feedforward/Feedback loops influencing this KER
There are no known feedforward/feedback loops influencing this KER.
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