AOP ID and Title:

Short Title: 5α-reductase inhibition leading to short AGD

Authors

Monica Kam Draskau; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark

Terje Svingen; National Food Institute, Technical University of Denmark, Kongens Lyngby, 2800 Denmark

Status

Abstract

This AOP links 5α-reductase inhibition during fetal life with short anogenital distance (AGD) in male offspring. A short AGD around birth is a marker for feminization of male fetuses and is associated with male reproductive disorders, including reduced fertility in adulthood. Although a short AGD is not necessarily ‘adverse’ from a human health perspective, it is considered an ‘adverse outcome’ in OECD test guidelines; AGD measurements are mandatory in specific tests for developmental and reproductive toxicity in chemical risk assessment (TG 443, TG 421/422, TG 414).

5α-reductase is an enzyme responsible for the conversion of testosterone to DHT in target tissues. DHT is more potent agonist of the Androgen receptor (AR) than testosterone, so that DHT is necessary for proper masculinization of e.g. male external genitalia. Under normal physiological conditions, testosterone produced mainly by the testicles, is converted in peripheral tissues by 5α-reductase into DHT, which in turn binds AR and activates downstream target genes. AR signaling is necessary for masculinization of the developing fetus, including differentiation of the levator ani/bulbocavernosus (LABC) muscle complex in males. The LABC complex does not develop in the absence, or low levels of, androgen signaling, as in female fetuses.

The key events in this pathway is inhibition of 5α-reductase that converts testosterone into the more potent DHT in androgen sensitive target tissues. This includes developing perineal region, which, when DHT levels are low or absent, leads to inactivation of the AR and failure to properly masculinize the perineum/LABC complex.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Finasteride

Finasteride is a type II 5alpha-reductase inhibitor that blocks conversion of testosterone to dihydrotestosterone (Clark et al 1990; Imperato-McGinley et al 1992). Intrauterine exposure in rats can result in shorter male AGD in male offspring (Bowman et al 2003; Christiansen et al 2009; Schwartz et al 2019)

References:

Bowman et al (2003), Toxicol Sci 74:393-406; doi: 10.1093/toxsci/kfg128

Christiansen et al (2009), Environ Health Perspect 117:1839-1846; doi: 10.1289/ehp.0900689

Clark et al (1990), Teratology 42:91-100; doi: 10.1002/tera.1420420111

Imperato-McGinley (1992), J Clin Endocrinol Metab 75:1022-1026; doi: 10.1210/jcem.75.4.1400866

Schwartz et al (2019), Toxicol Sci 169:303-311; doi: 10.1093/toxsci/kfz046

Overall Assessment of the AOP

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

References

1. Schwartz CL, Christiansen S, Vinggaard AM, Axelstad M, Hass U and 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.

Appendix 1

List of MIEs in this AOP

Event: 1617: Inhibition, 5α-reductase

Short Name: Inhibition, 5α-reductase

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

This KE is applicable to both sexes, across developmental stages into adulthood, in many different tissues and across taxa.

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).

Key Event Description

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), Δ^4,5 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).

How it is Measured or Detected

There is currently (as of 2023) no OECD test guideline for the measurement of 5α-reductase inhibition.

Inhibition of 5α-reductase can be 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 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).

References

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

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.

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

List of Key Events in the AOP

Event: 1613: Decrease, dihydrotestosterone (DHT) level

Short Name: Decrease, DHT level

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

This KE is applicable to both sexes, across developmental stages and adulthood, in many different tissues and across vertebrate taxa.

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).

Key Event Description

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. 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.

How it is Measured or Detected

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).

References

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

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

Event: 1614: Decrease, androgen receptor activation

Short Name: Decrease, AR activation

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

This KE is considered broadly applicable across vertebrate taxa as all vertebrate animals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and functions.

Key Event Description

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 (Davey & Grossmann, 2016; Gao et al., 2005). AR does not, however, act alone in regulating gene transcription, but together with other co-factors that may differ between cells and tissues and life stages. In this way, the functional consequence of AR activation is cell- and tissue-dependent.

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).

How it is Measured or Detected

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 MIE/KE-26 for AR antagonism, KE-1690 for decreased T levels and KE-1613 for decreased dihydrotestosterone levels). In this way, this KE is a placeholder for tissue-specific responses to AR activation or inactivation that will depend on the adverse outcome (AO) for which it is included.

It should be mentioned that the Rapid Androgen Disruption Activity Reporter (RADAR) assay included in OECD test guideline no. 251 detects AR antagonism in vivo in fish (OECD 2022).

References

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

Hutson, J. M. (1985). A biphasic model for the hormonal control of testicular descent. The Lancet, 24, 419–421. https://doi.org/https://doi.org/10.1016/S0140-6736(85)92739-4

Kaftanovskaya, E. M., Huang, Z., Barbara, A. M., de Gendt, K., Verhoeven, G., Gorlov, I. P., & Agoulnik, A. I. (2012). Cryptorchidism in mice with an androgen receptor ablation in gubernaculum testis. Molecular Endocrinology, 26(4), 598–607. https://doi.org/10.1210/me.2011-1283

Lee, S. H., Hong, K. Y., Seo, H., Lee, H. S., & Park, Y. (2021). Mechanistic insight into human androgen receptor-mediated endocrine-disrupting potentials by a stable bioluminescence resonance energy transfer-based dimerization assay. Chemico-Biological Interactions, 349. https://doi.org/10.1016/j.cbi.2021.109655

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

OECD (2022). Test No. 251: Rapid Androgen Disruption Activity Reporter (RADAR) assay. Paris: OECD Publishing doi:10.1787/da264d82-en.

Pang, T. P. S., Clarke, M. v., Ghasem-Zadeh, A., Lee, N. K. L., Davey, R. A., & MacLean, H. E. (2012). A physiological role for androgen actions in the absence of androgen receptor DNA binding activity. Molecular and Cellular Endocrinology, 348(1), 189–197. https://doi.org/10.1016/j.mce.2011.08.017

Event: 286: Altered, Transcription of genes by the androgen receptor

Short Name: Altered, Transcription of genes by the AR

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Evidence for Perturbation by Stressor

Bicalutamide

Using analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line (Ngan et al. 2009).

Cyproterone acetate

Using analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line (Ngan et al. 2009) and using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM (Sonneveld et al. 2005).

Epoxiconazole

Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 mM and an IC50 of 10 mM (Kjærstad et al. 2010).

Flutamide

Analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line (Ngan et al. 2009) and using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 uM (Sonneveld et al. 2005).

Flusilazole

Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.8 mM and an IC50 of 2.8 (±0.1) mM (Draskau et al. 2019)

Prochloraz

Using gene expression analysis of the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I. Gene expression levels were reduced in ventral prostates of male Wistar pups at postnatal day 16 following in utero and lactational exposure from maternal perinatal dosing with prochloraz (50 and 150 mg/kg/day) from gestational day 7 to postnatal day 16 (Laier et al. 2006). Also, using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 mM and an IC50 of 13 mM (Kjærstad et al. 2010).

Propiconazole

Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 mM and an IC50 of 18 mM (Kjærstad et al. 2010).

Stressor:286 Tebuconazole

Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 mM and an IC50 of 8.1 mM (Kjærstad et al. 2010).

Triticonazole

Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 mM and an IC50 of 0.3 (±0.01) mM (Draskau et al. 2019).

Vinclozalin

Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 uM (Sonneveld et al. 2005).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

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). Likewise in fish, androgens are important for development of sexual characteristics (Ogino et al., 2014, 2023). One difference that must be mentioned is that in teleost fish, 11-ketotestosterone is the main androgen in addition to testosterone and DHT and that most teleosts have two ar ohnologs, ara and arb, with arb functioning in a similar manner to the AR in other vertebrates (Ogino et al., 2023).

This KE is considered broadly applicable across vertebrate taxa, sex and developmental stages, as all vertebrate animals express the AR in numerous cells and tissues where it regulates gene transcription required for developmental processes and function.

Key Event Description

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.

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).

How it is Measured or Detected

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.

References

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

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

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–PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells. Oncogene 28:2051–2063. doi: 10.1038/onc.2009.68

Ogino, Y., Ansai, S., Watanabe, E., Yasugi, M., Katayama, Y., Sakamoto, H., et al. (2023). Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish. Nat. Commun. 2023 141 14, 1–16. doi:10.1038/s41467-023-37026-6.

Ogino, Y., Hirakawa, I., Inohaya, K., Sumiya, E., Miyagawa, S., Denslow, N., et al. (2014). Bmp7 and Lef1 Are the Downstream Effectors of Androgen Signaling in Androgen-Induced Sex Characteristics Development in Medaka. Endocrinology 155, 449–462. doi:10.1210/EN.2013-1507.

Rana K, Davey RA, Zajac JD (2014) Human androgen deficiency: Insights gained from androgen receptor knockout mouse models. Asian J. Androl. 16:169–177

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

List of Adverse Outcomes in this AOP

Event: 1688: anogenital distance (AGD), decreased

Short Name: AGD, decreased

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Evidence for Perturbation by Stressor

Butylparaben

Butylparaben has been shown to cause decreased male AGD in rats following intrauterine exposure to 500 and 1000 mg/kg bw/day (Boberg et al, 2016; Zhang et al, 2014). A separate study using 600 mg/kg bw/day did not see an effect on male AGD (Boberg et al, 2008).

p,p'-DDE

p,p,DDE has been shown to cause decreased male AGD in rats following intrauterine exposure to 100-200 mg/kg bw/day (Loeffler & Peterson, 1999; Wolf et al, 1999).

Bis(2-ethylhexyl) phthalate

DEHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 300-1500 mg/kg bw/day (Christiansen et al, 2010; Gray et al, 2000; Howdeshell et al, 2007; Jarfelt et al, 2005; Kita et al, 2016; Li et al, 2013; Lin et al, 2009; Moore et al, 2001; Nardelli et al, 2017; Saillenfait et al, 2009; Wolf et al, 1999).

Dexamethasone

Dexamethasone has been shown to cause decreased male AGD in rats following intrauterine exposure to 0.1 mg/kg bw/day (Van den Driesche et al, 2012).

Fenitrothion

Fenitrothion has been shown to cause decreased male AGD in rats following intrauterine exposure to 25 mg/kg bw/day (Turner et al, 2002).

Finasteride

Finasteride has been shown to cause decreased male AGD in rats following intrauterine exposure to 100 mg/kg bw/day (Bowman et al, 2003).

Flutamide

Flutamide has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 16-100 mg/kg bw/day (Foster & Harris, 2005; Hass et al, 2007; Kita et al, 2016; McIntyre et al, 2001; Mylchreest et al, 1999; Scott et al, 2007; Welsh et al, 2007).

Ketoconazole

Ketoconazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 50 mg/kg bw/day in one study (Taxvig et al, 2008), but no effect in another study using same dose (Wolf et al, 1999).

Linuron

Linuron has been shown to cause decreased male AGD in rats following intrauterine exposure to 50-100 mg/kg bw/day (Hotchkiss et al, 2004; McIntyre et al, 2002; Wolf et al, 1999).

Prochloraz

Prochloraz has been shown to cause decreased male AGD in rats following intrauterine exposure to 150-250 mg/kg bw/day (Laier et al, 2006; Noriega et al, 2005).

Procymidone

Procymidone has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-150 mg/kg bw/day (Hass et al, 2012; Hass et al, 2007; Wolf et al, 1999).

Triticonazole

Triticonazole has been shown to cause decreased male AGD in rats following intrauterine exposure to 150 and 450 mg/kg bw/day (Draskau et al, 2019).

Vinclozolin

Vinclozolin has been shown to cause decreased male AGD in rats following intrauterine exposure to doses between 50-200 mg/kg bw/day (Christiansen et al, 2009; Gray et al, 1994; Hass et al, 2007; Matsuura et al, 2005; Ostby et al, 1999; Schneider et al, 2011; Wolf et al, 2004).

di-n-hexyl phthalate

DnHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-750 mg/kg bw/day (Saillenfait et al, 2009a; Saillenfait et al, 2009b).

Dicyclohexyl phthalate

DCHP has been shown to cause decreased male AGD in rats following intrauterine exposure to 350-750 mg/kg bw/day (Aydoğan Ahbab & Barlas, 2015; Hoshino et al, 2005; Saillenfait et al, 2009a).

butyl benzyl phthalate

BBP has been shown to cause decreased male AGD in rats following intrauterine exposure to 500-1000 mg/kg bw/day (Ema & Miyawaki, 2002; Gray et al, 2000; Hotchkiss et al, 2004; Nagao et al, 2000; Tyl et al, 2004).

monobenzyl phthalate

MBeP has been shown to cause decreased male AGD in rats following intrauterine exposure to 375 mg/kg bw/day (Ema et al, 2003).

di-n-heptyl phthalate

DHPP has been shown to cause decreased male AGD in rats following intrauterine exposure to 1000 mg/kg bw/day (Saillenfait et al, 2011).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

A short AGD in male offspring is a marker of insufficient androgen action during critical fetal developmental stages (Schwartz et al, 2019; Welsh et al, 2008). A short AGD is thus a sign of undervirilization, which is also associated with a series of male reproductive disorders, including genital malformations and infertility in humans (Juul et al, 2014; Skakkebaek et al, 2001).

There are numerous human epidemiological studies showing associations with intrauterine exposure to anti-androgenic chemicals and short AGD in newborn boys alongside other reproductive disorders (Schwartz et al, 2019). This underscores the human relevance of this AO. However, in reproductive toxicity studies and chemical risk assessment, rodents (rats and mice) are what is tested on. The list of chemicals inducing short male AGD in male rat offspring is extensive, as evidenced by the ‘stressor’ list and reviewed by (Schwartz et al, 2019).

Key Event Description

The anogenital distance (AGD) refers to the distance between anus and the external genitalia. In rodents and humans, the male AGD is approximately twice the length as the female AGD (Salazar-Martinez et al, 2004; Schwartz et al, 2019). This sexual dimorphisms is a consequence of sex hormone-dependent development of secondary sexual characteristics (Schwartz et al, 2019). In males, it is believed that androgens (primarily DHT) activate AR-positive cells in non-myotic cells in the fetal perineum region to initiate differentiation of the perineal levator ani and bulbocavernosus (LABC) muscle complex (Ipulan et al, 2014). This AR-dependent process occurs within a critical window of development, around gestational days 15-18 in rats (MacLeod et al, 2010). In females, the absence of DHT prevents this masculinization effect from occurring.

The involvement of androgens in masculinization of the male fetus, including the perineum, has been known for a very long time (Jost, 1953), and AGD has historically been used to, for instance, sex newborn kittens. It is now well established that the AGD in newborns is a proxy readout for the intrauterine sex hormone milieu the fetus was developing. Too low androgen levels in XY fetuses makes the male AGD shorter, whereas excess (ectopic) androgen levels in XX fetuses makes the female AGD longer, in humans and rodents (Schwartz et al, 2019).

How it is Measured or Detected

The AGD is a morphometric measurement carried out by trained technicians (rodents) or medical staff (humans).

In rodent studies AGD is assessed as the distance between the genital papilla and the anus, and measured using a stereomicroscope with a micrometer eyepiece. The AGD index (AGDi) is often calculated by dividing AGD by the cube root of the body weight.  It is important in statistical analysis to use litter as the statistical unit. This is done when more than one pup from each litter is examined. Statistical analyses is adjusted using litter as an independent, random and nested factor. AGD are analysed using body weight as covariate as recommended in Guidance Document 151 (OECD, 2013).

 

Regulatory Significance of the AO

In regulatory toxicology, the AGD is mandatory inclusions in OECD test guidelines used to test for developmental and reproductive toxicity of chemicals. Guidelines include ‘TG 443 extended one-generation study’, ‘TG 421/422 reproductive toxicity screening studies’ and ‘TG 414 developmental toxicity study’.

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Appendix 2

List of Key Event Relationships in the AOP