Relationship: 1880: Inhibition, 5α-reductase leads to Decrease, DHT level

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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.

Evidence Supporting this KER

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), Δ^4,5 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).

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

 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).

Quantitative Understanding of the Linkage

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).

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.

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.

Androgens can regulate gene expression of 5α-reductases (Andersson et al., 1989; Berman & Russell, 1993).

References

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

Relationship: 1935: Decrease, DHT level leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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 (Davey & Grossmann, 2016; Marks, 2004). 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’ (Davey & Grossmann, 2016). Hence, AR transcriptional function is directly dependent on the presence of ligands, with DHT being a more potent AR activator than testosterone (Grino et al, 1990). 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.

Evidence Supporting this KER

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).

The empirical support for KER1935 is considered high.

Dose concordance:

• Increasing concentrations of DHT lead to increasing AR activation in vitro in AR reporter gene assays (OECD, 2020; Williams et al., 2017).

Indirect (supporting) evidence:

• 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%.
• 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).

Other evidence:

• Male individuals with congenital 5α-reductase deficiency (absence of DHT) fail to masculinize properly (Costa et al., 2012).
• 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).

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.

Quantitative Understanding of the Linkage

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).

Upon DHT binding to the AR, a conformational change that brings the amino (N) and carboxy (C) termini into close proximity occurs with a t_1/2 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).

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.

References

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

Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

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.

Key Event Relationship Description

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.

Evidence Supporting this KER

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).

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).

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).

Quantitative Understanding of the Linkage

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).

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.

AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).

References

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 † † 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.

Relationship: 3348: Decrease, AR activation leads to nipple retention, increased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic

The KER is considered directly applicable to rats and mice, in which males normally have no nipples due to high levels of androgens during development, leading to regression of nipple anlagen. The empirical evidence supports the relevance to rats, whereas the relevance in mice is assumed based on knowledge about developmental biology in this species. Applicability may extend to most rodents. 

While NR is not directly translatable to humans, it serves as a clear indicator of diminished androgen activity causing disrupted fetal masculinisation and sexual differentiation during development - an effect considered relevant to mammals, humans (Schwartz et al., 2021), and vertebrates more broadly (Ogino et al., 2023). NR is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD 2025a; OECD 2025b, OECD 2025c) and in OECD GD 151 considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013). NR can also be used as an indicator of anti-androgenicity in mammals and vertebrates in the environment due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).

Life stage

Programming of nipple/areola regression in males occurs during a short window of sensitivity to androgens in the nipple anlagen during fetal life. This takes place in rats around embryonic days 13-15 (Imperato-McGinley et al., 1986), which is, therefore, the relevant window of exposure. The relevant timing for the investigation of NR is PND12-14 in male rat offspring when the nipples are visible in the female littermates. At this time in development, the nipples/areolas are visible through the skin without excessive fur that may interfere with the investigation (Schwartz et al., 2021). It should be mentioned that though the occurrence of nipples/areolas in male offspring is believed to be relatively stable throughout life, it may be responsive to postnatal changes. Permanent nipple/areola retention is observed in some but not all in utero exposure studies with antiandrogens inducing nipple/areola retention at PND 12-14. Most of the differences between studies seem explainable by the window of exposure, dose levels and methods for investigation used, but the responsiveness of nipple/areola retention to postnatal changes remains to be fully explored (Schwartz et al., 2021).

Sex

Data presented in this KER support that disruption of androgen action during fetal life can lead to increased nipple/areola retention in male rat offspring. Since female mice and rat offspring, in general, have 10 (mice) or 12 (rats) nipples at the relevant time of investigation, increased nipple/areola retention at that time point is not a relevant endpoint for females.

Key Event Relationship Description

This KER  links a decrease in androgen receptor (AR) activation during fetal development to increased nipple/areola retention (NR) in male rodent offspring. It should be noted that the upstream Key Event (KE) ‘decrease, androgen receptor activation’ (KE-1614 in AOP Wiki) specifically focuses on decreased activation of the AR in vivo, while most methods that can be used to measure AR activity are carried out in vitro. Indirect information about this KE may, for example, be provided from assays showing in vitro AR antagonism, decreased in vitro or in vivo testosterone production/levels, or decreased in vitro or in vivo dihydrotestosterone (DHT) production/levels.

The KER is not directly applicable to humans as both sexes have two nipples, and there is no known effect of androgens on their development (Schwartz et al., 2021). However, NR is a clear readout of reduced androgen action, fetal masculinization and sexual differentiation during development, which is relevant to humans, mammals (Schwartz et al., 2021), and vertebrates more broadly (Ogino et al., 2023). It is included as a mandatory endpoint in several rodent OECD Test Guidelines (OECD, 2025a, 2025b, 2025c) and, in OECD GD 151, is considered an adverse outcome applicable to the setting of Points of Departure for use in human health risk assessment (OECD, 2013). NR can also be used as an indicator of anti-androgenicity in mammals and vertebrates in the environment due to the conserved nature of the AR and its implication in sexual differentiation across species (Ogino et al., 2023).

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high based on the following:

- Sexual differentiation happens in fetal life. The testes are developed and start to produce testosterone that is converted in other tissues by the enzyme 5-alpha-reductase to the more potent androgen dihydrotestosterone (DHT). Both hormones bind and activate the nuclear receptor and transcription factor AR, which in turn drives the masculinization of the male fetus (Schwartz et al., 2021; Welsh et al., 2014).

- Fetal masculinization depends on the activation of androgen signalling during a critical time window, the masculinization programming window (MPW), from gestational day (GD) 16-20 in rats, 14.5-16.5 in mice and presumably gestation weeks (GWs) 8-14 in humans (Amato et al., 2022; Welsh et al., 2008).

- The fetal masculinization process involves a range of tissues and organs, including the nipple anlagen in rats and mice. In humans, both sexes have two nipples. In contrast, common laboratory mice and rats are sexually dimorphic, with females having 12 (rats) and 10 (mice) nipples and males generally having none (Mayer et al., 2008; Schwartz et al., 2021). In both male and female mouse embryos, stem cells differentiate into a mammary gland, with nipple anlagen being visible by embryonic day 11.5 (Mayer et al., 2008). In male embryos, the presence of androgen leads the nipple anlagen to regress a few days later (Kratochwil, 1977; Kratochwil & Schwartz, 1976) . The androgen responsiveness in the nipple anlagen is rather short, in mice starting late embryonic day 13, with loss of responsiveness on embryonic day 15 (Imperato-McGinley et al., 1986; Kratochwil, 1977) and thus roughly following the timing of the MPW.

- Nipple formation is inhibited in female mice and rat fetuses exposed to androgens during gestation (Goldman et al., 1976; Greene et al., 1941; Imperato-McGinley et al., 1986).

- Male Tfm-mutant mice, which are insensitive to androgens and believed to be so due to a nonfunctional androgen receptor, present with retained nipples (Kratochwil & Schwartz, 1976).   

- Multiple mechanisms of action may potentially lead to nipple retention in male mouse and rat offspring. DHT is the main androgen responsible for nipple/areola regression through interaction with AR in the nipple anlagen (Imperato-McGinley et al., 1986). Inhibition of testosterone synthesis or the conversion of testosterone to DHT, increased metabolism of androgens, or direct interference with AR activation may thus all lead to nipple/areola retention (Imperato-McGinley et al., 1986; Schwartz et al., 2021).

The empirical support from studies in animals for this KER is judged as high overall.

It should be noted that the KE decreased AR activation (KE 1614 in AOP Wiki) specifically focuses on decreased activation of the AR in vivo, with no methods currently available to measure this. Examples of assays that provide indirect information about KE 1614 are described in upstream MIE/KEs.

The empirical evidence for this KER from animal studies in vivo is based on studies using six different substances that result in decreased AR activation by different mechanisms. Flutamide, procymidone and vinclozolin bind to the AR and inhibit the receptor activity and thereby act as AR antagonists, see MIE 26. Finasteride inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT, see MIE 1617. DEHP and DBP exposure during prenatal development in rats results in reduced fetal testosterone levels, see KE-2298 and KE1690. (MIE 26, MIE 1617 and KE 1690 can be found in AOP-Wiki).

The evidence for the upstream KE is mainly based on data from in vitro assays (AR antagonism or 5-alpha-reductase inhibition in vitro), whereas the evidence for the downstream KE is based on in vivo studies, and there is generally no evidence for both KEs from the same study. However, decreased testosterone levels can be measured in vivo, and (Howdeshell et al., 2007; Martino-Andrade et al., 2009) measured the effect of developmental phthalate exposure on both testosterone levels and nipple/areola retention (see the section about “Dose concordance”).

The empirical evidence for the six substances is summarised in Table 3.

Table 3. Summary of empirical evidence for decreased androgen receptor activation, leading to decreased nipple/areola retention. References for the studies supporting the empirical evidence are found in the section “Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, and vinclozolin, finasteride, DEHP and DBP” and in Table 4 in Appendix 2 (6djoma9gmj_KER_3348_Appendix_2_.pdf).

From Table 3, it can be deduced that fetal exposure to substances known to decrease androgen receptor activation through antagonism of the AR (vinclozolin, procymidone, flutamide), inhibition of testosterone synthesis (DEHP, DBP) or inhibition of the conversion of testosterone to DHT (finasteride), results in increased nipple/areola retention in rat male offspring.

Evidence for decreased AR activation (KE 1614) by flutamide, procymidone, vinclozolin, finasteride, DEHP and DBP.

Flutamide, a pharmaceutical, binds the AR and inhibits its activity, thereby acting as an AR antagonist. It has been used as an antiandrogen for the treatment of prostate cancer and is used as a reference chemical for antiandrogenic activity in the AR transactivation assays in the OECD test guideline No 458 (Goldspiel & Kohler, 1990; Labrie, 1993; OECD, 2023; Simard et al., 1986)

Procymidone and vinclozolin are fungicides that have been shown to be AR antagonists. Procymidone binds to the AR and inhibits the agonist binding, as shown in AR binding assays using rat prostate cytosol (Hosokawa et al., 1993) or AR transfected cells (Ostby et al., 1999). Procymidone also inhibits agonist activated transcription in AR reporter assays (Hass et al., 2012; Kojima et al., 2004; Orton et al., 2011; Ostby et al., 1999; Scholze et al., 2020). Vinclozolin binds to the AR and inhibits the agonist binding, as shown in AR binding assays using rat epididymis cytosol (Kelce & Wilson, 1997) or AR transfected cells (Wong et al., 1995). Vinclozolin also inhibits agonist activated transcription in AR reporter assays (Euling, 2002; Kojima et al., 2004; Molina-Molina et al., 2006; Orton et al., 2011; Scholze et al., 2020; Shimamura et al., 2002; Wong et al., 1995).

Finasteride is a pharmaceutical that inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT. Finasteride is used to treat benign prostatic hypertrophy (Andersson & Russell, 1990; Stoner, 1990; Wood & Rittmaster, 1994).

Prenatal exposure to DEHP in rats has been shown to reduce the production of testosterone in fetal testis measured in ex vivo testis assays, and to reduce testosterone levels in testis and in fetal plasma and serum (Borch et al., 2006; Borch J et al., 2004; Culty et al., 2008; Hannas et al., 2011, 2012; Howdeshell et al., 2007; Klinefelter et al., 2012; Parks, 2000; VO et al., 2009; Wilson et al., 2004, 2007). Conversely, prenatal DEHP exposure did not result in any effects on testosterone levels in the testis at PND1 in one study by Andrade et al. (2006) (Andrade et al., 2006). Similar to DEHP, prenatal exposure to DBP has been shown to reduce the production of testosterone in fetal rat testis measured in ex vivo testis studies (Howdeshell et al., 2007; Wilson et al., 2004) and reduce testosterone levels in the fetal rat testis (Martino-Andrade et al., 2009). The precise underlying mechanism for these effects of DEHP and DPB is presently unknown.

Evidence for increased nipple/areola retention in males (AO-1786) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride, DEHP and DBP.

All datasets that were used for the weight of evidence assessment were judged as reliable without or with restriction. The majority of datasets assessed showed an increased nipple/areola retention in male offspring after gestational exposure. The conclusion was that the level of confidence was strong for all six substances. The studies are summarised in Table 4 in Appendix 2, 6djoma9gmj_KER_3348_Appendix_2_.pdf

Dose concordance

Dose concordance is challenging to assess for this KER since in vivo AR activity is currently not possible to measure, but can only be inferred indirectly by measures of upstream events. In some studies, fetal (testicular) testosterone levels during, or close to, the fetal masculinization programming window are measured in a subset of animals exposed similarly to those investigated for NR post-natally. Such information may inform on dose concordance if more doses are included.

In a rat in utero exposure study (GD13-21) with DPB and DEHP, testosterone levels in the fetal testes were investigated at GD21, and NR was investigated at PND13 (Martino-Andrade et al., 2009). For DBP, both reduced testosterone levels in fetal testes and NR were observed at 500 mg/kg/d, whereas no effect on NR and only a slight non-significant reduction of testosterone was observed at the lower dose (100 mg/kg/d). For DEHP, a slight but non-significant decrease in testosterone levels in fetal rat testis was observed after exposure to 150 mg/kg/d DEHP, with no effects on nipple/areola retention.

Such data could suggest dose concordance for this part of the KER, although the evidence for this is not strong.

Temporal concordance

Temporal concordance can only be considered from a theoretical perspective since the downstream event, increased NR, is a result of disruption to normal regression of nipple anlagen in male rodents induced during a short window of gestational development (in mice of approximately 2 days), but usually measured at PND12-14 in rats. Earlier than this, the areolae are not yet visible through the skin and later than this, the animals grow fur and need to be shaved for proper examination. This is supported by several of the studies in the empirical evidence, where the test substance was administered during a short period during gestation and nipple retention was observed postnatally.

Based on current knowledge, it is understood that the upstream event – decreased AR activation in vivo – takes place minutes to hours after exposure to an anti-androgenic substance. If a substance decreases AR activation through inhibition of the AR, the upstream event is expected to happen immediately after exposure. If a substance decreases androgen receptor activation through inhibition of testosterone synthesis, the upstream event is expected to happen minutes to hours after the exposure.
 

For DEHP and DBP, there were some inconsistencies in the empirical evidence, but they could be explained by differences in study designs and uncertainties in measurements (see Appendix 1). Some uncertainty is imposed by the poorly supported dose-concordance. However, the dose-concordance is well supported by the current understanding of biological processes.

Quantitative Understanding of the Linkage

The quantitative understanding of the linkage is low. This is a consequence of it not being possible to measure the upstream and the downstream events in the same study.

The difficulties in extrapolating potency from in vitro to in vivo studies were exemplified by a comparison of the effects of pyrifluquinazon and bisphenol C in vitro and in utero. In vitro, bisphenol C antagonized the androgen receptor with a much higher potency than pyrifluquinazon, but in vivo the potencies were reversed with pyrifluquinazon exposure leading to NR at lower exposure levels than bisphenol C (Gray et al., 2019).

AR activation operates on a time-scale of minutes. The AR is a ligand-activated nuclear receptor and transcription factor. Upon ligand binding a conformational change and subsequent dimerization of the AR takes place within 3-6 minutes (Schaufele et al., 2005). Nuclear translocation (Nightingale et al., 2003) and promoter interactions occur within 15 minutes of ligand binding, and RNA polymerase II and coactivator recruitment are then proposed to occur transiently with cycles of approximately 90 minutes (Kang et al., 2002).

For the downstream event, the time-scale for observing a measurable effect on nipple/areola retention is closer to days and weeks, depending on species. For instance, in mice the nipple anlage are responsive to androgen action at embryonic day 13-15, while a sexual dimorphism of the nipples/areolas can first be observed after birth (Imperato-McGinley et al., 1986) .

A well established modulating factor for androgen action is genetic variations in the AR, which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Chamberlain et al., 1994; Tut et al., 1997). Rat strain is another important modulating factor, with studies showing that the Long-Evans Hooded rat is less sensitive to nipple/areola retention than the Sprague-Dawley rat  (Wolf et al., 1999; You et al., 1998)

Not relevant for this KER.

References

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. https://doi.org/10.3389/fendo.2022.910964

Andersson, S., & Russell, D. W. (1990). Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha-reductases. Proceedings of the National Academy of Sciences, 87(10), 3640–3644. https://doi.org/10.1073/pnas.87.10.3640

Andrade, A. M., Grande, S. W., Talsness, C. E., Grote, K., & Chahoud, I. (2006). Developmental exposure to high and low doses of di-(2-ethylhexyl) phthalate (DEHP); Effects on male rat offspring. NAUNYN-SCHMIEDEBERGS ARCHIVES OF PHARMACOLOGY, 372, 100–101.

Barlow, N. J., McIntyre, B. S., & Foster, P. M. D. (2004). Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate. TOXICOLOGIC PATHOLOGY, 32(1), 79–90. https://doi.org/10.1080/01926230490265894

Borch, J., Axelstad, M., Vinggaard, A. M., & Dalgaard, M. (2006). Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis. Toxicology Letters, 163(3), 183–190. https://doi.org/10.1016/j.toxlet.2005.10.020

Borch J, Ladefoged O, Hass U, & Vinggaard AM. (2004). Steroidogenesis in fetal male rats is reduced by DEHP and DINP, but endocrine effects of DEHP are not modulated by DEHA in fetal, prepubertal and adult male rats. Reproductive Toxicology (Elmsford, N.Y.), 18(1), 53–61. https://doi.org/10.1016/j.reprotox.2003.10.011

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

Christiansen S, Scholze M, Dalgaard M, Vinggaard AM, Axelstad M, Kortenkamp A, & Hass U. (2009). Synergistic disruption of external male sex organ development by a mixture of four antiandrogens. Environmental Health Perspectives, 117(12), 1839–1846. https://doi.org/10.1289/ehp.0900689

Christiansen S, Boberg J, Axelstad M, Dalgaard M, Vinggaard AM, Metzdorff SB, & Hass U. (2010). Low-dose perinatal exposure to di(2-ethylhexyl) phthalate induces anti-androgenic effects in male rats. Reproductive Toxicology (Elmsford, N.Y.), 30(2), 313–321. https://doi.org/10.1016/j.reprotox.2010.04.005

Clewell, R. A., Thomas, A., Willson, G., Creasy, D. M., & Andersen, M. E. (2013). A dose response study to assess effects after dietary administration of diisononyl phthalate (DINP) in gestation and lactation on male rat sexual development. REPRODUCTIVE TOXICOLOGY, 35, 70–80. https://doi.org/10.1016/j.reprotox.2012.07.008

Culty, M., Thuillier, R., Li, W., Wang, Y., Martinez-Arguelles, D. B., Benjamin, C. G., Triantafilou, K. M., Zirkin, B. R., & Papadopoulos, V. (2008). In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat1. Biology of Reproduction, 78(6), 1018–1028. https://doi.org/10.1095/biolreprod.107.065649

Euling, S. Y. (2002). Response-Surface Modeling of the Effect of 5alpha-Dihydrotestosterone and Androgen Receptor Levels on the Response to the Androgen Antagonist Vinclozolin. Toxicological Sciences, 69(2), 332–343. https://doi.org/10.1093/toxsci/69.2.332

Fussell KC, Schneider S, Buesen R, Groeters S, Strauss V, Melching-Kollmuss S, & van Ravenzwaay B. (2015). Investigations of putative reproductive toxicity of low-dose exposures to flutamide in Wistar rats. Archives of Toxicology, 89(12), 2385–2402. https://doi.org/10.1007/s00204-015-1622-6

Goldman AS, Shapiro B, & Neumann F. (1976). Role of testosterone and its metabolites in the differentiation of the mammary gland in rats. Endocrinology, 99(6), 1490–1495. https://doi.org/10.1210/endo-99-6-1490

Goldspiel, B. R., & Kohler, D. R. (1990). Flutamide: An Antiandrogen for Advanced Prostate Cancer. DICP, 24(6), 616–623. https://doi.org/10.1177/106002809002400612

Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DN, & Parks L. (2000). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicological Sciences : An Official Journal of the Society of Toxicology, 58(2), 350–365. https://doi.org/10.1093/toxsci/58.2.350

Gray LE Jr, Barlow NJ, Howdeshell KL, Ostby JS, Furr JR, & Gray CL. (2009). Transgenerational effects of Di (2-ethylhexyl) phthalate in the male CRL:CD(SD) rat: added value of assessing multiple offspring per litter. Toxicological Sciences : An Official Journal of the Society of Toxicology, 110(2), 411–425. https://doi.org/10.1093/toxsci/kfp109

Gray, L. E., Furr, J. R., Conley, J. M., Lambright, C. S., Evans, N., Cardon, M. C., Wilson, V. S., Foster, P. M., & Hartig, P. C. (2019). A Conflicted Tale of Two Novel AR Antagonists in Vitro and in Vivo: Pyrifluquinazon Versus Bisphenol C. Toxicological Sciences, 168(2), 632–643. https://doi.org/10.1093/toxsci/kfz010

Greene, R. R., Burrill, M. W., & Ivy, A. C. (1941). Experimental intersexuality: The effects of combined estrogens and androgens on the embryonic sexual development of the rat. Journal of Experimental Zoology, 87(2), 211–232. https://doi.org/10.1002/jez.1400870203

Hannas, B. R., Furr, J., Lambright, C. S., Wilson, V. S., Foster, P. M. D., & Gray, L. E. (2011). Dipentyl Phthalate Dosing during Sexual Differentiation Disrupts Fetal Testis Function and Postnatal Development of the Male Sprague-Dawley Rat with Greater Relative Potency than Other Phthalates. Toxicological Sciences, 120(1), 184–193. https://doi.org/10.1093/toxsci/kfq386

Hannas, B. R., Lambright, C. S., Furr, J., Evans, N., Foster, P. M. D., Gray, E. L., & Wilson, V. S. (2012). Genomic Biomarkers of Phthalate-Induced Male Reproductive Developmental Toxicity: A Targeted RT-PCR Array Approach for Defining Relative Potency. Toxicological Sciences, 125(2), 544–557. https://doi.org/10.1093/toxsci/kfr315

Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M, Metzdorff SB, & Kortenkamp A. (2007). Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environmental Health Perspectives, 115, 122–128. https://doi.org/10.1289/ehp.9360

Hass U, Boberg J, Christiansen S, Jacobsen PR, Vinggaard AM, Taxvig C, Poulsen ME, Herrmann SS, Jensen BH, Petersen A, Clemmensen LH, & Axelstad M. (2012). Adverse effects on sexual development in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reproductive Toxicology (Elmsford, N.Y.), 34(2), 261–274. https://doi.org/10.1016/j.reprotox.2012.05.090

Hellwig J, van Ravenzwaay B, Mayer M, & Gembardt C. (2000). Pre- and postnatal oral toxicity of vinclozolin in Wistar and Long-Evans rats. Regulatory Toxicology and Pharmacology : RTP, 32(1), 42–50. https://doi.org/10.1006/rtph.2000.1400

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

Hosokawa S, Murakami M, Ineyama M, Yamada T, Yoshitake A, Yamada H, Miyamoto J. The affinity of procymidone to androgen receptor in rats and mice. J Toxicol Sci. 1993 May;18(2):83-93. doi: 10.2131/jts.18.83. PMID: 8331696.

Howdeshell, K. L., Furr, J., Lambright, C. R., Rider, C. V., Wilson, V. S., & Gray, L. E. (2007). Cumulative Effects of Dibutyl Phthalate and Diethylhexyl Phthalate on Male Rat Reproductive Tract Development: Altered Fetal Steroid Hormones and Genes. Toxicological Sciences, 99(1), 190–202. https://doi.org/10.1093/toxsci/kfm069

Imperato-McGinley J, Binienda Z, Gedney J, & Vaughan ED Jr. (1986). Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5 alpha-reductase: definition of a selective role for dihydrotestosterone. Endocrinology, 118(1), 132–137. https://doi.org/10.1210/endo-118-1-132

Jarfelt K, Dalgaard M, Hass U, Borch J, Jacobsen H, & Ladefoged O. (2005). Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate. Reproductive Toxicology (Elmsford, N.Y.), 19(4), 505–515. https://doi.org/10.1016/j.reprotox.2004.11.005

Kang, H.-Y., Huang, K.-E., Chang, S. Y., Ma, W.-L., Lin, W.-J., & Chang, C. (2002). Differential Modulation of Androgen Receptor-mediated Transactivation by Smad3 and Tumor Suppressor Smad4. Journal of Biological Chemistry, 277(46), 43749–43756. https://doi.org/10.1074/jbc.M205603200

Kelce, W. R., & Wilson, E. M. (1997). Environmental antiandrogens: Developmental effects, molecular mechanisms, and clinical implications. JOURNAL OF MOLECULAR MEDICINE-JMM, 75(3), 198–207. https://doi.org/10.1007/s001090050104

Klinefelter, G. R., Laskey, J. W., Winnik, W. M., Suarez, J. D., Roberts, N. L., Strader, L. F., Riffle, B. W., & Veeramachaneni, D. N. R. (2012). Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol. REPRODUCTION, 144(6), 747–761. https://doi.org/10.1530/REP-12-0266

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., & Kobayashi, K. (2004). Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environmental Health Perspectives, 112(5), 524–531. https://doi.org/10.1289/ehp.6649

Kratochwil, K. (1977). Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland. Developmental Biology, 61(2), 358–365. https://doi.org/10.1016/0012-1606(77)90305-0

Kratochwil, K., & Schwartz, P. (1976). Tissue interaction in androgen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone. Proceedings of the National Academy of Sciences, 73(11), 4041–4044. https://doi.org/10.1073/pnas.73.11.4041

Labrie, F. (1993). Mechanism of action and pure antiandrogenic properties of flutamide. Cancer, 72(12 Suppl), 3816–3827. https://doi.org/10.1002/1097-0142(19931215)72:12+<3816::aid-cncr2820721711>3.0.co;2-3

Martino-Andrade AJ, Morais RN, Botelho GG, Muller G, Grande SW, Carpentieri GB, Leão GM, & Dalsenter PR. (2009). Coadministration of active phthalates results in disruption of foetal testicular function in rats. International Journal of Andrology, 32(6), 704–712. https://doi.org/10.1111/j.1365-2605.2008.00939.x

Matsuura I, Saitoh T, Tani E, Wako Y, Iwata H, Toyota N, Ishizuka Y, Namiki M, Hoshino N, Tsuchitani M, Ikeda Y. Evaluation of a two-generation reproduction toxicity study adding endpoints to detect endocrine disrupting activity using lindane. J Toxicol Sci. (2005) Dec;30 Spec No.:135-161. doi: 10.2131/jts.30.s135. PMID: 16641539.

Mayer, J. A., Foley, J., De La Cruz, D., Chuong, C. M., & Widelitz, R. (2008). Conversion of the nipple to hair-bearing epithelia by lowering bone morphogenetic protein pathway activity at the dermal-epidermal interface. The American Journal of Pathology, 173(5), 1339–1348. https://doi.org/10.2353/AJPATH.2008.070920

McIntyre BS, Barlow NJ, & Foster PM. (2001). Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicological Sciences : An Official Journal of the Society of Toxicology, 62(2), 236–249. https://doi.org/10.1093/toxsci/62.2.236

Molina-Molina JM, Hillenweck A, Jouanin I, Zalko D, Cravedi JP, Fernández MF, Pillon A, Nicolas JC, Olea N, Balaguer P. Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicol Appl Pharmacol. 2006 Oct 1;216(1):44-54. doi: 10.1016/j.taap.2006.04.005. Epub 2006 Jun 5. PMID: 16750840.

Moore RW, Rudy TA, Lin TM, Ko K, & Peterson RE. (2001). Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer Di(2-ethylhexyl) phthalate. Environmental Health Perspectives, 109(3), 229–237. https://doi.org/10.1289/ehp.01109229

Mylchreest E, Wallace DG, Cattley RC, & Foster PM. (2000). Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation. Toxicological Sciences : An Official Journal of the Society of Toxicology, 55(1), 143–151. https://doi.org/10.1093/toxsci/55.1.143

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(4), 347–361. https://doi.org/10.1016/S1476-5586(03)80028-3

OECD (2013), Guidance Document Supporting OECD Test Guideline 443 on the Extended One-Generational Reproductive Toxicity Test, OECD Series on Testing and Assessment, No. 151, OECD Publishing, Paris, ENV/JM/MONO(2013)10

OECD (2023). 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

OECD (2025a), Test No. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264185371-en.

OECD (2025b), Test No. 421: Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264380-en.

OECD (2025c), Test No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264403-en.

Ogino, Y., Ansai, S., Watanabe, E. et al. Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish. Nat Commun 14, 1428 (2023). https://doi.org/10.1038/s41467-023-37026-6

Orton, F., Rosivatz, E., Scholze, M., & Kortenkamp, A. (2011). Widely Used Pesticides with Previously Unknown Endocrine Activity Revealed as in Vitro Antiandrogens. ENVIRONMENTAL HEALTH PERSPECTIVES, 119(6), 794–800. https://doi.org/10.1289/ehp.1002895

Ostby J, Monosson E, Kelce WR, & Gray LE Jr. (1999). Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicology and Industrial Health, 15(1), 48–64. https://doi.org/10.1177/074823379901500106

Parks, L. G. (2000). The Plasticizer Diethylhexyl Phthalate Induces Malformations by Decreasing Fetal Testosterone Synthesis during Sexual Differentiation in the Male Rat. Toxicological Sciences, 58(2), 339–349. https://doi.org/10.1093/toxsci/58.2.339

Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. In Current Research in Toxicology (Vol. 3). Elsevier B.V. https://doi.org/10.1016/j.crtox.2022.100085

Saillenfait AM, Sabaté JP, & Gallissot F. (2008). Diisobutyl phthalate impairs the androgen-dependent reproductive development of the male rat. Reproductive Toxicology (Elmsford, N.Y.), 26(2), 107–115. https://doi.org/10.1016/j.reprotox.2008.07.006

Saillenfait AM, Sabaté JP, & Gallissot F. (2009). Effects of in utero exposure to di-n-hexyl phthalate on the reproductive development of the male rat. Reproductive Toxicology (Elmsford, N.Y.), 28(4), 468–476. https://doi.org/10.1016/j.reprotox.2009.06.013

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. Proceedings of the National Academy of Sciences, 102(28), 9802–9807. https://doi.org/10.1073/pnas.0408819102

Schneider S, Kaufmann W, Strauss V, & van Ravenzwaay B. (2011). Vinclozolin: a feasibility and sensitivity study of the ILSI-HESI F1-extended one-generation rat reproduction protocol. Regulatory Toxicology and Pharmacology : RTP, 59(1), 91–100. https://doi.org/10.1016/j.yrtph.2010.09.010

Scholze, M., Taxvig, C., Kortenkamp, A., Boberg, J., Christiansen, S., Svingen, T., Lauschke, K., Frandsen, H., Ermler, S., Hermann, S. S., Pedersen, M., Lykkeberg, A. K., Axelstad, M., & Vinggaard, A. M. (2020). Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders. Environmental Health Perspectives, 128(11), 117005. https://doi.org/10.1289/EHP6774

Schwartz, C. L., Christiansen, S., Hass, U., Ramhøj, L., Axelstad, M., Löbl, N. M., & Svingen, T. (2021). On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies. In Frontiers in Toxicology (Vol. 3). Frontiers Media S.A. https://doi.org/10.3389/ftox.2021.730752

Shimamura M, Kodaira K, Kenichi H, Ishimoto Y, Tamura H, Iguchi T. Comparison of antiandrogenic activities of vinclozolin and D,L-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo. Toxicology. 2002 May 24;174(2):97-107. doi: 10.1016/s0300-483x(02)00044-6. PMID: 11985887.

Simard, J., Luthy, I., Guay, J., Bélanger, A., & Labrie, F. (1986). Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Molecular and Cellular Endocrinology, 44(3), 261–270. https://doi.org/10.1016/0303-7207(86)90132-2

Stoner, E. (1990). The clinical development of a 5α-reductase inhibitor, finasteride. The Journal of Steroid Biochemistry and Molecular Biology, 37(3), 375–378. https://doi.org/10.1016/0960-0760(90)90487-6

Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. (2023). OECD. https://doi.org/10.1787/9789264264366-en

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

Vo TT, Jung EM, Dang VH, Jung K, Baek J, Choi KC, Jeung EB. Differential effects of flutamide and di-(2-ethylhexyl) phthalate on male reproductive organs in a rat model. J Reprod Dev. 2009 Aug;55(4):400-11. doi: 10.1262/jrd.20220. Epub 2009 Apr 13. PMID: 19367084.

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

Welsh, M., Suzuki, H., & Yamada, G. (2014). The Masculinization Programming Window. In O. Hiort & S. F. Ahmed (Eds.), UNDERSTANDING DIFFERENCES AND DISORDERS OF SEX DEVELOPMENT (DSD) (Vol. 27, pp. 17–27). https://doi.org/10.1159/000363609

Wilson, V. S., Lambright, C., Furr, J., Ostby, J., Wood, C., Held, G., & Gray, L. E. (2004). Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis. Toxicology Letters, 146(3), 207–215. https://doi.org/10.1016/j.toxlet.2003.09.012

Wilson, V. S., Howdeshell, K. L., Lambright, C. S., Furr, J., & Earl Gray, L. (2007). Differential expression of the phthalate syndrome in male SpragueDawley and Wistar rats after in utero DEHP exposure. Toxicology Letters, 170(3), 177–184. https://doi.org/10.1016/j.toxlet.2007.03.004

Wolf C Jr, Lambright C, Mann P, Price M, Cooper RL, Ostby J, Gray LE Jr. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health. 1999 Jan-Mar;15(1-2):94-118. doi: 10.1177/074823379901500109. PMID: 10188194.

Wolf CJ, LeBlanc GA, Ostby JS, & Gray LE Jr. (2000). Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicological Sciences : An Official Journal of the Society of Toxicology, 55(1), 152–161. https://doi.org/10.1093/toxsci/55.1.152

Wolf CJ, LeBlanc GA, & Gray LE Jr. (2004). Interactive effects of vinclozolin and testosterone propionate on pregnancy and sexual differentiation of the male and female SD rat. Toxicological Sciences : An Official Journal of the Society of Toxicology, 78(1), 135–143. https://pubmed.ncbi.nlm.nih.gov/14736997/

Wong, C. I., Kelce, W. R., Sar, M., & Wilson, E. M. (1995). Androgen Receptor Antagonist versus Agonist Activities on the Fungicide Vinclozolin Relative to Hydroxyflutamide. Nucleic Acids, Protein Synthesis, and Molecular Genetics, 270(34), 19998–20003.

Wood, A. J. J., & Rittmaster, R. S. (1994). Finasteride. New England Journal of Medicine, 330(2), 120–125. https://doi.org/10.1056/NEJM199401133300208

You, L., Casanova, M., Archibeque-Engle, S., Sar, M., Fan, L.-Q., & Heck, A. (1998). Impaired Male Sexual Development in Perinatal Sprague-Dawley and Long-Evans Hooded Rats Exposed in Utero and Lactationally to p,p’-DDE. In TOX1COLOGICAL SCIENCES (Vol. 45). https://academic.oup.com/toxsci/article/45/2/162/1653877