Relationship: 3448: Decrease, intratesticular testosterone leads to Decrease, circulating testosterone levels

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

The KER is assessed applicable to mammals, as testicular testosterone synthesis is common for all mammals. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates.

Sex applicability

This KER is only applicable to males, as testes are only found in males.

Life stage applicability

This KER is applicable to all life stages.  Once formed, the testes produce and secrete testosterone during fetal development and throughout postnatal life, although testosterone levels do vary between life stages (Vesper et al., 2015).

Key Event Relationship Description

This KE describes a decrease in intratesticular testosterone production leading to a decrease in circulating levels of testosterone. Intratesticular testosterone can be measured in whole testicular tissue samples by testing ex vivo testicular testosterone production, and circulating testosterone is measured in plasma or serum. In males, the testes produce and secrete the majority of the circulating testosterone, with only a small contribution from the adrenal gland (Naamneh Elzenaty et al., 2022). In mammals, intratesticular testosterone levels are 30- to 200-fold higher than serum testosterone levels (Coviello et al., 2004). Reducing testicular testosterone will consequently lead to a reduction in circulating levels as well.

Evidence Supporting this KER

The biological plausibility for this KER is considered high. The testes are the primary testosterone-producing organs in male mammals and the main contributors to the circulating testosterone levels in males (Naamneh Elzenaty et al., 2022). A decrease in intratesticular testosterone will therefore lead to a decrease in secretion of testosterone and consequently lower circulating levels of testosterone.  

The empirical evidence for this KER is overall judged as high.

In vivo toxicity studies in rats and mice have shown that exposure to substances that lowers intratesticular testosterone also lowers circulating testosterone levels. This includes in utero exposure and measurements in fetal males (Borch J et al., 2004; Vinggaard AM et al., 2005) as well as exposure and measurements postnatally in male rodents (Hou X et al., 2020; Ji et al., 2010; Jiang XP et al., 2017)

Supporting this evidence are castration studies in male rats and monkeys, showing a marked reduction in circulating testosterone levels when removing the testes (Gomes & Jain, 1976; Perachio et al., 1977).

Lastly, in humans, males with hypogonadism or gonadal dysgenesis present with lower circulating testosterone levels (Hirose Y et al., 2007; Jones LW et al., 1970).

Dose concordance

In vivo toxicity studies support dose concordance for this KER, as exemplified below.

In pre-pubertal/pubertal male rats, chlorocholine chloride exposure (postnatal day (PND) 23-60) in three doses reduced both intratesticular and serum testosterone levels at PND60 at all doses tested (Hou X et al., 2020).

Perinatal exposure (gestational day (GD) 10-birth) of male mice to diethylhexyl phthalate (DEHP) in three doses (100, 500, and 1000 mg/kg bw/day) reduced intratesticular testosterone at 500 and 1000 mg/kg bw/day at PND1, while only 1000 mg/kg bw/day reduced serum levels of testosterone, although this was measured later, at PND56 (Xie Q et al., 2024)

In utero exposure (GD7-21) of male rats to DEHP in doses of 300 or 750 mg/kg bw/day reduced intratesticular testosterone levels at GD21, while only the high dose also reduced plasma testosterone levels (Borch J et al., 2004).

Temporal concordance

In vivo toxicity studies moderately support temporal concordance for this KER, as exemplified below.

Several studies show that a decrease in intratesticular and circulating testosterone can be measured at the same time point (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005).

In utero exposure of male mice to DEHP from GD10 to birth reduced intratesticular testosterone levels at PND1 with LOAEL 500 mg/kg bw/day, and when measured at PND56, circulating testosterone levels were decreased, but with LOAEL 1000 mg/kg bw/day (Xie Q et al., 2024).

In Fisher JS et al., 2003, exposure of male rats from GD13-21 to 500 mg/kg bw/day dibutyl phthalate reduced intratesticular testosterone by ~90% (measured at GD19). When analyzing circulating testosterone levels at PND4, 10, 15, 25, and 90, only the testosterone levels on PND25 were decreased.

One study report conflicting results on the temporal concordance of this KER (Caceres et al., 2023). Here, male rats were exposed for 20 weeks from PND60 to a mixture of the phytoestrogens genistein and daidzein (combined dose of either 29 or 290 mg/kg bw/day). Intratesticular testosterone was measured every 4 weeks, while serum levels of testosterone were measured every second week. While the mixture caused a reduction of serum testosterone after 2 weeks of exposure, a reduction in intratesticular testosterone was not measured until after 8 weeks. The discrepancy might be explained by the multiple mechanisms of action of the phytoestrogens, as they, besides affecting testicular testosterone synthesis, may also influence peripheral aromatization of testosterone to estrogens (van Duursen et al., 2011).

Incidence concordance

Incidence concordance can not be evaluated for this KER.

There are examples of in vivo studies, in which stressors exposure have caused a reduction in intratesticular testosterone levels without a reduction in circulating testosterone levels.

Quantitative Understanding of the Linkage

The time-scale for this KER is likely minutes or hours, as testosterone is secreted into the blood from the testes after synthesis. In vivo, a decrease in intratesticular and circulating testosterone can be measured at the same time, both in fetal and postnatal studies (Borch J et al., 2004; Hou X et al., 2020; Jiang XP et al., 2017; Vinggaard AM et al., 2005). Ex vivo, chemically-induced reduction in testicular production of testosterone can be measured in culture media after 3 hours incubation (earlier time points were not measured) (Wilson et al., 2009).

Testosterone is a part of the hypothalamic-pituitary-gonadal (HPG) axis, which controls testosterone synthesis in puberty and adulthood. In this axis, gonatropin-releasing hormone (GnRH) is released from the hypothalamus and stimulates release of luteinizing hormone (LH) from the pituitary. LH acts on the testes to produce and secrete testosterone. Elevated circulating testosterone levels exerts negative feedback on the HPG axis (decreasing GnRH secretion) to keep testosterone levels in balance (Tilbrook & Clarke, 2001).

Importantly, there are species-specific differences in when the HPG axis is functional during development. In the mouse, fetal testosterone synthesis is independent of pituitary LH (O’Shaughnessy et al., 1998), whereas in humans, human chorionic gonadotropin (hCG) act similarly to LH and appear to be critical in stimulating testosterone synthesis in the fetal testis (Huhtaniemi, 2025).

References

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

Caceres, S., Crespo, B., Alonso-Diez, A., De Andrés, P. J., Millan, P., Silván, G., Illera, M. J., & Illera, J. C. (2023). Long-Term Exposure to Isoflavones Alters the Hormonal Steroid Homeostasis-Impairing Reproductive Function in Adult Male Wistar Rats. Nutrients, 15(5), 1261. https://doi.org/10.3390/nu15051261

Coviello, A. D., Bremner, W. J., Matsumoto, A. M., Herbst, K. L., Amory, J. K., Anawalt, B. D., Yan, X., Brown, T. R., Wright, W. W., Zirkin, B. R., & Jarow, J. P. (2004). Intratesticular Testosterone Concentrations Comparable With Serum Levels Are Not Sufficient to Maintain Normal Sperm Production in Men Receiving a Hormonal Contraceptive Regimen. Journal of Andrology, 25(6), 931–938. https://doi.org/10.1002/j.1939-4640.2004.tb03164.x

Fisher JS, Macpherson S, Marchetti N, & Sharpe RM. (2003). Human “testicular dysgenesis syndrome”: A possible model using in-utero exposure of the rat to dibutyl phthalate. Human Reproduction (Oxford, England), 18(7), 1383–1394. https://doi.org/10.1093/humrep/deg273

Gomes, W. R., & Jain, S. K. (1976). Effect of unilateral and bilateral castration and cryptorchidism on serum gonadotrophins in the rat. The Journal of Endocrinology, 68(02), 191–196. https://doi.org/10.1677/joe.0.0680191

Hirose Y, Sasa M, Bando Y, Hirose T, Morimoto T, Kurokawa Y, Nagao T, & Tangoku A. (2007). Bilateral male breast cancer with male potential hypogonadism. World Journal of Surgical Oncology, 5, 60. https://doi.org/10.1186/1477-7819-5-60

Hou X, Hu H, Xiagedeer B, Wang P, Kang C, Zhang Q, Meng Q, & Hao W. (2020). Effects of chlorocholine chloride on pubertal development and reproductive functions in male rats. Toxicology Letters, 319, 1–10. https://doi.org/10.1016/j.toxlet.2019.10.024

Huhtaniemi, I. T. (2025). Luteinizing hormone receptor knockout mouse: What has it taught us? Andrology, andr.70000. https://doi.org/10.1111/andr.70000

Ji, Y.-L., Wang, H., Liu, P., Wang, Q., Zhao, X.-F., Meng, X.-H., Yu, T., Zhang, H., Zhang, C., Zhang, Y., & Xu, D.-X. (2010). Pubertal cadmium exposure impairs testicular development and spermatogenesis via disrupting testicular testosterone synthesis in adult mice. Reproductive Toxicology, 29(2), 176–183. https://doi.org/10.1016/j.reprotox.2009.10.014

Jiang XP, Tang JY, Xu Z, Han P, Qin ZQ, Yang CD, Wang SQ, Tang M, Wang W, Qin C, Xu Y, Shen BX, Zhou WM, & Zhang W. (2017). Sulforaphane attenuates di-N-butylphthalate-induced reproductive damage in pubertal mice: Involvement of the Nrf2-antioxidant system. Environmental Toxicology, 32(7), 1908–1917. https://doi.org/10.1002/tox.22413

Jones LW, Isaacs H Jr, Edelbrock H, & Donnell GN. (1970). Reifenstein’s syndrome: Hereditary familial hypogonadism with hypospadias and gynecomastia. The Journal of Urology, 104(4), 608–611. https://doi.org/10.1016/s0022-5347(17)61793-2

Naamneh Elzenaty, R., Du Toit, T., & Flück, C. E. (2022). Basics of androgen synthesis and action. Best Practice & Research Clinical Endocrinology & Metabolism, 36(4), 101665. https://doi.org/10.1016/j.beem.2022.101665

O’Shaughnessy, P. J., Baker, P., Sohnius, U., Haavisto, A.-M., Charlton, H. M., & Huhtaniemi, I. (1998). Fetal Development of Leydig Cell Activity in the Mouse Is Independent of Pituitary Gonadotroph Function*. Endocrinology, 139(3), 1141–1146. https://doi.org/10.1210/endo.139.3.5788

Perachio, A. A., Alexander, M., Marr, L. D., & Collins, D. C. (1977). Diurnal variations of serum testosterone levels in intact and gonadectomized male and female rhesus monkeys. Steroids, 29(1), 21–33. https://doi.org/10.1016/0039-128X(77)90106-4

Tilbrook, A. J., & Clarke, I. J. (2001). Negative Feedback Regulation of the Secretion and Actions of Gonadotropin-Releasing Hormone in Males. Biology of Reproduction, 64(3), 735–742. https://doi.org/10.1095/biolreprod64.3.735

van Duursen, M. B. M., Nijmeijer, S. M., de Morree, E. S., de Jong, P. Chr., & van den Berg, M. (2011). Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model. Toxicology, 289(2), 67–73. https://doi.org/10.1016/j.tox.2011.07.005

Vesper, H. W., Wang, Y., Vidal, M., Botelho, J. C., & Caudill, S. P. (2015). Serum Total Testosterone Concentrations in the US Household Population from the NHANES 2011-2012 Study Population. Clinical Chemisty, 61(12), 1495–1504. https://doi.org/10.1373/clinchem.2015.245969

Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, Dalgaard M, Nellemann C, & Hass U. (2005). Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicological Sciences : An Official Journal of the Society of Toxicology, 85(2), 886–897. https://doi.org/doi.org/10.1093/toxsci/kfi150

Wilson, V. S., Lambright, C. R., Furr, J. R., Howdeshell, K. L., & Gray, L. E., Jr. (2009). The herbicide linuron reduces testosterone production from the fetal rat testis during both in utero and in vitro exposures. TOXICOLOGY LETTERS, 186(2), 73–77. https://doi.org/10.1016/j.toxlet.2008.12.017

Xie Q, Cao H, Liu H, Xia K, Gao Y, & Deng C. (2024). Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression. Translational Andrology and Urology, 13(3), 369–382. https://doi.org/10.21037/tau-23-503

Relationship: 2131: Decrease, circulating testosterone levels leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

KER2131 is assessed applicable to mammals, as T 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

KER2131 is assessed applicable to both sexes, as T activates AR in both males and females.

Life-stage applicability

KER2131 is considered applicable to developmental and adult life stages, as T-mediated AR activation is relevant from the AR is expressed.

Key Event Relationship Description

This key event relationship links decreased testosterone (T) levels to decreased androgen receptor (AR) activation. T is an endogenous steroid hormone important for, amongst other things, reproductive organ development and growth as well as muscle mass and spermatogenesis (Marks, 2004).T is, together with dihydrotestosterone (DHT), a primary ligand for the AR in mammals (Schuppe et al., 2020). Besides its genomic actions, the AR can also mediate rapid, non-genomic second messenger signaling (Davey & Grossmann, 2016). When T levels are reduced, less substrate is available for the AR, and hence, AR activation is decreased (Gao et al., 2005).

Evidence Supporting this KER

The biological plausibility for this KER is considered high

AR activation is dependent on ligand binding (though a few cases of ligand-independent AR activation has been shown, see uncertainties and inconsistencies). T is a primary ligand for the AR, and when T levels are decreased there is less substrate for the AR, and hence, AR activation is decreased. In the male, T is primarily synthesized by the testes, and in some target tissues T is irreversibly metabolized to the more potent metabolite DHT. T and DHT both bind to the AR, but DHT has a higher binding affinity (Gao et al., 2005). The lower binding affinity of T compared to DHT is due to the faster dissociation rate of T from the full-length AR, as T has less effective FXXLF motif binding to AF2 (Askew et al., 2007). Binding of T or DHT has different effects in different tissues. E.g. in the developing male, T 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). In the adult male, androgen action in the reproductive tissues is DHT dependent, whereas action in muscle and bone is DHT independent (Gao et al., 2005). In patients with male androgen deficiency syndrome (AIS), clinically low levels of T leads to reduced AR activation (either due to low T or DHT in target tissue), which manifests as both androgenic related symptoms (such as incomplete or delayed sexual development, loss of body hair, small or shrinking testes, low or zero sperm count) as well as anabolic related symptoms (such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat). All symptoms can be counteracted by treatment with T, which acts directly on the AR receptor in anabolic tissue (Bhasin et al., 2010). Similarly, removal of the testicles in weanling rats results in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980). As this demonstrates, the consequences of low T regarding AR activation will depend on tissue, life stage, species etc.

The empirical evidence for this KER is considered high

Dose concordance

There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023).

Other evidence

• In male patients with androgen deficiency, treatment with T counteracts anabolic (DHT independent) related symptoms such as height loss, low trauma fracture, low bone mineral density, reduced muscle bulk and strength, increased body fat (Bhasin et al., 2010; Katznelson et al., 1996).
• Removal of the testicles in weanling rats result in a feminized body composition and muscle metabolism, which is reversed by administration of T (Krotkiewski et al., 1980).

Ligand-independent actions of the AR have been identified. To what extent and of which biological significance is not well defined (Bennesch & Picard, 2015).

Quantitative Understanding of the Linkage

There is a positive dose-response relationship between increasing concentrations of T and AR activation (U.S. EPA., 2023). 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).

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 can upregulate and downregulate AR expression (Lee & Chang, 2003).

References

Askew, E. B., Gampe, R. T., Stanley, T. B., Faggart, J. L., & Wilson, E. M. (2007). Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone. Journal of Biological Chemistry, 282(35), 25801–25816. https://doi.org/10.1074/jbc.M703268200

Bennesch, M. A., & Picard, D. (2015). Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors. Molecular Endocrinology, 29(3), 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. The Journal of Clinical Endocrinology & Metabolism, 95(6), 2536–2559. https://doi.org/10.1210/jc.2009-2354

Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. The Clinical Biochemist. Reviews, 37(1), 3–15. http://www.ncbi.nlm.nih.gov/pubmed/27057074

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

Kang, Z., Pirskanen, A., Jänne, O. A., & Palvimo, J. J. (2002). Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex. Journal of Biological Chemistry, 277(50), 48366–48371. https://doi.org/10.1074/jbc.M209074200

Katznelson, L., Finkelstein, J. S., Schoenfeld, D. A., Rosenthal, D. I., Anderson, E. J., & Klibanski, A. (1996). Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. The Journal of Clinical Endocrinology & Metabolism, 81(12), 4358–4365. https://doi.org/10.1210/jcem.81.12.8954042

Keller, E. T., Ershler, W. B., & Chang, Chawnshang. (1996). The androgen receptor: A mediator of diverse responses. Frontiers in Bioscience, 1(4), 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 Physiologica Scandinavica, 109(3), 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. The Journal of Clinical Endocrinology & Metabolism, 88(9), 4043–4054. https://doi.org/10.1210/jc.2003-030261

Marks, L. S. (2004). 5alpha-reductase: history and clinical importance. Reviews in Urology, 6 Suppl 9(Suppl 9), S11-21. http://www.ncbi.nlm.nih.gov/pubmed/16985920

Schuppe, E. R., Miles, M. C., and Fuxjager, M. J. (2020). Evolution of the androgen receptor: Perspectives from human health to dancing birds. Mol. Cell. Endocrinol. 499, 110577. doi:10.1016/J.MCE.2019.110577.

U.S. EPA. (2023). ToxCast & Tox21 AR agonism of testosterone. Retrieved from Https://Www.Epa.Gov/Chemical-Research/Toxicity-Forecaster-Toxcasttm-Data June 23, 2023. Data Released October 2018.

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.

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Relationship: 2127: Altered, Transcription of genes by the AR leads to AGD, decreased

AOPs Referencing Relationship

Relationship: 2820: Decrease, AR activation leads to AGD, decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic

Fetal masculinization including the AGD is regulated by androgens interacting with the AR in all mammals, including humans (Murashima et al., 2015; Thankamony et al., 2016), although, the size of the AGD and difference between the sexes vary between species. A large number of studies exist showing that fetal exposure to anti-androgens causes shortened AGD in male rats and mice (Schwartz et al., 2019, see also Table 2).  Some epidemiological studies find associations between exposure to anti-androgenic compounds and shorter AGD in boys (Thankamony et al., 2016). However, the associations are not very clear and confidence in the data is limited by conflicting results, possibly due to differences in study design and methods for exposure measurements and analyses. Nevertheless, the KER is considered applicable to humans, based on current understanding of the role of AR activation in fetal masculinization.

Life stage

Programming of the AGD occurs during the masculinization programming window in fetal life. This takes place in rats around embryonic days 15.5-19.5 (GD16-20) and likely gestation weeks 8-14 in humans (Welsh et al., 2008). It should be mentioned that though AGD is believed to be relatively stable throughout life, it can be responsive to postnatal changes in androgen levels (Schwartz et al., 2019).

Sex

Data presented in this KER support that disruption of androgen action during fetal life can lead to a short AGD in male offspring. While exposure to chemicals during fetal life can also shorten female AGD, the biological significance and the mechanism driving the effect is unknown (Schwartz et al., 2019).

Key Event Relationship Description

This KER refers to a decrease in androgen receptor (AR) activation during fetal development leading to decreased anogenital distance (AGD) in male 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 androgen receptor in vivo, while most methods that can be used to measure AR activity are carried out in vitro. Indirect information about this KE may for example be provided from assays showing in vitro AR antagonism, decreased in vitro or in vivo testosterone production/levels or decreased in vitro or in vivo dihydrotestosterone (DHT) production/levels.

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 that in turn drives masculinization of the male fetus (Welsh et al., 2014; Schwartz et. al, 2019).

- Fetal masculinization depends on activation of androgen signaling during a critical time window, the masculinization programming window (MPW), from gestational day (GD) 15.5-18.5 in rats, 14.5-16.5 in mice and presumably gestation weeks (GWs) 8-14 in humans (Welsh et al., 2008; Amato et al., 2022). The onset of AR expression in the tissues of the reproductive tract follows the timing of the MPW (Welsh et al., 2008).

- The fetal masculinization process involves a range of tissues and organs, including the perineum. Perineum length can be measured as the AGD, which is the distance between the anus and the genitalia. The AGD is approximately twice as long in male as in female newborn rodents and humans (Schwartz et al., 2019).

- Male AR knockout mice present shorter AGD than wildtype males, so short that it is indistinguishable from wildtype female littermates (Yeh et al., 2002, Sato et al., 2004).

- In human males, mutations decreasing AR activity also lead to feminization. One example is the androgen insensitivity syndrome (AIS), where mutations in the AR lead to an impaired or abolished response to androgens, and thereby some degree of feminization of XY individuals and even XY sex reversal in individuals with complete AIS (CAIS) (Thankamony et al., 2016; Hughes et al., 2012; Crouch et al., 2011). XY individuals with CAIS present as women with internally placed testes. A study showed that the clitoral to urethral distance in these individuals was similar to a control group of women, but it is not clear whether this measurement can work as a proxy for measuring the AGD (Thankamony et al 2016, Crouch 2011). Unfortunately, it seems the AGD has not at present been measured in CAIS individuals. Another example is human males lacking 5-alpha-reductase, also presenting female-like genitalia (Batista & Mendonca, 2022).
 

- The detailed mechanism by which androgens regulate the AGD is not known but it is hypothesized that the AGD is influenced by the size of the levator-ani and bulbocavernosus (LABC) muscle complex in the perineum. The growth of this complex is stimulated by AR activation, it is sexually dimorphic and larger in males than in females and (Schwartz et al., 2019). AR is required for the development of the LABC complex as demonstrated by AR general and muscle specific knockout mice. AR is expressed in non-myocytic cells in the LABC complex, starting at E15.5 in mice, and knockout of AR in these cells results in defects in the muscle formation  (Ipulan et al., 2016;). Differential gene expression profiles in the perineum of male and female rats as well as in antiandrogen-exposed male rats have been identified providing further mechanistic understanding (Schwartz et al, 2019; Draskau et al, 2022).

Animal in vivo data

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

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

The empirical evidence for this KER from animal studies in vivo is based on studies using five different substances that result in decreased AR activation by different mechanisms. Flutamide, procymidone and vinclozolin bind to the AR and inhibit the receptor activity and thereby act as AR antagonists, see MIE26. Finasteride inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT, see MIE1617. DEHP exposure during prenatal development in rats results in reduced fetal testosterone levels, see KE1690. (MIE26, MIE1617 and KE1690 can be found in AOP Wiki).

The evidence for the upstream KE is mainly based on data from in vitro assays (AR antagonism or 5-alpha-reductase inhibition in vitro) whereas the evidence for the downstream KE is based on in vivo studies, and there is generally not evidence for both KEs from the same study. However, decreased testosterone levels can be measured in vivo, and Borch et al., 2004 measured the effect of developmental DEHP exposure on both testosterone levels and AGD (see section about “Dose concordance”).

The empirical animal evidence for the five substances is summarized in table 3.

Table 3. Summary of empirical evidence for decreased androgen receptor activation, leading to decreased male AGD. References for the studies supporting the empirical evidence are found in section “Evidence for decreased AR activation (KE1614) by flutamide, procymidone, and vinclozolin, finasteride and DEHP” and in table 2.

From table 3, it can be deducted that fetal exposure to substances known to decrease androgen receptor activation through antagonism of the AR (vinclozolin, procymidone, flutamide), inhibition of testosterone synthesis (DEHP) or inhibition of conversion of testosterone to DHT (finasteride), results in decreased AGD in rat and mouse male offspring.

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

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

Procymidone and vinclozolin are fungicides that have been shown to be AR antagonists. Procymidone binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat prostate cytosol (Hosokawa et al., 1993) or AR transfected COS cells (Ostby et al., 1999). Procymidone also inhibits agonist activated transcription in AR reporter assays (Hass et al., 2012; Kojima et al., 2004; Orton et al., 2011; Ostby et al., 1999; Scholze et al., 2020). Vinclozolin binds to the AR and inhibits the agonist binding as shown in AR binding assays using rat epididymis cytosol (Kelce et al., 1997) or AR transfected COS-1 cells (Wong et al., 1995).
Vinclozolin also inhibits agonist activated transcription in AR reporter assays (Euling et al, 2002; Kojima et al., 2004; Molina-Molina et al., 2006; Orton et al., 2011; Scholze et al., 2020; Shimamura et al., 2002; Wong et al., 1995). Finasteride is a pharmaceutical that inhibits the 5-alpha-reductase enzyme that converts testosterone to DHT. Finasteride is used to treat benign prostatic hypertrophy (Andersson & Russel, 1990; Rittmaster & Wood, 1994; Stoner, 1990).

Prenatal exposure to DEHP in rats results in reduced production of testosterone in fetal testis measured in ex vivo testis assays, reduced testosterone levels in testis and reduced fetal plasma or serum testosterone levels (Borch et al., 2004; Borch et al., 2006; Culty et al., 2008; Hannas et al., 2011; Hannas et al., 2012; Klinefelter et al., 2012; Parks et al., 2000; Wilson et al., 2004; Wilson et al., 2007; Vo et al., 2009). Two studies don’t show an effect on testosterone levels in testis or fetal plasma testosterone levels, respectively (Andrade et al., 2006; Borch et al., 2006). The precise underlying mechanism is presently unknown.

Evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP

All datasets that were used for the weight of evidence assessment were judged as reliable without or with restriction. The majority of datasets assessed showed a decreased male AGD. The conclusion was that the level of confidence was strong for all five substances. The studies are summarized in table 4.

Empirical evidence for the included substances

Table 4. Empirical evidence for decreased AGD in males (KE1688) by prenatal exposure to flutamide, procymidone, vinclozolin, finasteride and DEHP. *One dose only.

>>>>>TABLE 4<<<<<

Epidemiological data on DEHP

The biggest relevant epidemiological dataset was identified on associations between DEHP and AGD.  

Six prospective cohort studies and one cross-sectional study on the association between maternal DEHP metabolites and length of AGD (anopenile distance (APD) and anoscrotal distance (ASD)) in boys were assessed as reliable without or with restriction. Decreased AGD (anopenile distance (APD) and/or anoscrotal distance (ASD)) was observed in three prospective cohort studies (Martino-Adrade et al., 2016; Swan et al., 2005 reviewed and updated in Swan 2008; Wenzel et al., 2018). In contrast, no significant association was observed in three other prospective cohort studies (Arbuckle et al., 2018; Henriksen et al., 2023; Jensen et al., 2016) and the cross-sectional study (Sunman et al., 2019). This inconsistency introduces a level of uncertainty regarding the overall association. Therefore, the level of confidence was judged as weak. 

Dose concordance

Dose concordance is challenging to assess for this KER since in vivo AR activity is currently not possible to measure, but only can be informed indirectly by measures of upstream events.

However, some studies provide useful information that support dose concordance between the KEs.

In a publication by Borch et al., rats were exposed in utero to DEHP at GD7-21. Fetal testosterone levels in testes and serum and testosterone production in fetal testes ex vivo were investigated at GD21, whereas AGD was investigated at PND3. The LOAELs for reduced testosterone production in ex vivo fetal testes and reduced testosterone levels in fetal testes were 300 mg/kg/d, whereas the LOAEL for decreased AGD in male offspring was 750 mg/kg/d (Borch et al., 2004). 

In a publication by Scholze et al, AR antagonism and decreased testosterone synthesis was quantitatively assessed (IC50) in vitro for a list of substances. In addition, internal concentrations in male fetuses and effects on AGD were measured after fetal exposure to the same substances. In utero exposure to all the substances lead to reduced AGDIndex (AGDI) in the exposed male offspring. Further, for all substances except Cyprodinil, the internal exposure levels in the fetuses leading to reduced AGD exceeded the IC50 levels observed in one or both of the in vitro assays.
Three different doses of linuron exposure were included. The medium exposure dose led to a higher level of internal exposure and a higher degree of AGDI reduction than the low dose. AGDI could not be determined in the highest dose due to maternal toxicity (Scholze et al., 2020).

Temporal concordance

Temporal concordance can only be considered from a theoretical perspective since the downstream event, decreased AGD, is usually measured at GD21, PND0 or PND1 in rats, and due to the size of the fetuses is not feasible to measure at earlier timepoints.

Considering the biology, the upstream event – decreased AR activation in vivo – is foreseen to happen minutes to hours after exposure. If a substance decreases AR activation through inhibition of the AR, the upstream event is expected to happen immediately after exposure. If a substance decreases androgen receptor activation through inhibition of testosterone synthesis, the upstream event is expected to happen minutes to hours after the exposure, though it is uncertain exactly when the change will be big enough to be measurable. On the other hand, the downstream event – decreased AGD - is a measurement of relative growth of the perineal tissue, which is expected to take days in the developing fetus.

For the model substances, there were some inconsistencies in the empirical evidence, but they could be explained by differences in study designs and uncertainties in measurements, see appendix 1.

Species differences in effects of phthalates (including DEHP and DBP) on fetal testes testosterone production have been observed between humans, mice and rats. In human fetal testes exposed to DEHP or DBP in vitro or ex vivo, no suppression of testosterone production is observed, which contrasts observations in rat fetal testes under similar conditions. Also in mice, testosterone production in the fetal testes is unaffected by treatment with DEHP or DBP in vitro or in utero (Sharpe, 2020).

The species differences described above are specific for some phthalates and their interference with fetal testicular testosterone production. This uncertainty should not be reflected on other antiandrogenic substances, especially not those acting through other mechanisms of action.
The association between exposure to DEHP and reduced AGD in humans is judged to be weak, which may further support a species difference between rodents and humans, but it may also reflect the large uncertainties inherent in the epidemiological studies.

Observational epidemiological studies face challenges in proving cause-effect relationships as they cannot control conditions like experimental animal and in vitro studies. Human studies can identify associations between variables but cannot offer conclusive proof of causation (Lanzoni et al., 2019). Various study designs and statistical methods are employed to strengthen evidence within the inherent limitations of observational research (Song & Chung, 2010; Olier et al., 2023). Inconsistencies in epidemiological data arise from various factors, such as different methodologies used in exposure and outcome measurement and also in statistical analyses.

These differences collectively contribute to the complexity of interpreting and weighing the evidence in epidemiological research.

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 event in the same study.

In one study, a quantitative model was developed to predict the decrease in AGD from in vitro AR antagonism or in vitro decreased testosterone synthesis. The authors conclude that predicting the effect on AGD in vivo based on the in vitro results is only possible on a qualitative level, but the model cannot predict AGD reductions quantitatively (Scholze et al., 2020).

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 growth of a tissue (in this case the perineum) is closer to days and weeks depending on species. For instance, in humans, the masculinization programming window is presumed to start around GW 8, while a sexual dimorphism of the AGD can first be observed from around GWs 11-13 (Thankamony et al., 2016) and reaches its maximum 2-fold difference around GWs 17-20 (Sharpe, 2020). 

It has been demonstrated that exposure to flutamide for one day (Foster & Harris, 2005) or vinclozolin for two days (Wolf et al., 2000) during the sensitive window of exposure can elicit a detectable decrease in the AGD in male rat offspring.

A well established modulating factor is genetic variations in the AR which decrease the function of the receptor. For example, longer CAG repeat lengths have been associated with decreased AR activation (Tut et al 1997, Chamberlain et al 1994) and a shorter AGD in adult men (Eisenberg et al., 2013). Other modulating factors being discussed in the literature is maternal age and parity (Barrett et al., 2014), but these associations are only suggestive with more studies needed to confirm the associations (Barrett et al., 2014).

Not relevant for this KER.

References

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Relationship: 3449: Decrease, intratesticular testosterone leads to AGD, decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

Testosterone, synthesized in the testis, is essential for growth (masculinization) of the male perineum in all mammals. It is therefore biologically plausible that this KER is applicable to all mammals (Murashima et al., 2015a). The empirical evidence in this KER strongly supports the applicability to rats in particular. Given the knowledge of normal reproductive development, the KER is also considered applicable to humans.

Sex applicability

Testes are the primary male sex organs; hence, this KER is only applicable to males. The empirical evidence in this KER supports the applicability in males.

Life stage applicability

The perineum is programmed by androgens during the masculinization programming window, a fetal period during which the testes produce high levels of testosterone. The masculinization programming window is ~gestational day (GD) 16-20 in rats and suggested to be gestational weeks (GW) 8-14 in humans (Sharpe RM, 2020; Welsh M et al., 2014). Once programmed in fetal life, the AGD is believed to be relatively stable, but the perineum can in some cases be responsive to postnatal changes in androgen levels (Schwartz CL et al., 2019; Sharpe RM, 2020; Thankamony A et al., 2016). The empirical evidence in this KER supports the fetal life stage applicability.

Key Event Relationship Description

This non-adjacent KER describes a fetal decrease in testicular testosterone leading to short AGD in male offspring. In this KER, intratesticular testosterone levels can both be measured in whole testes extracts or by measuring ex vivo testosterone production from exposed testes.

In male mammals, the testes first differentiate in early fetal life and start synthesizing testosterone through the steroidogenesis pathway. Although the adrenal glands may also produce testosterone, the testes are the main site of testosterone production (Naamneh Elzenaty et al., 2022).  Testosterone is secreted to initiate male reproductive differentiation in the peripheral tissues, either directly acting on the androgen receptor (AR) or through conversion to the more potent androgen dihydrotestosterone (DHT). The androgen hormones initiate masculinization, including elongation of the perineum, which is suggested to involve the perineal muscle complex levator ani bulbocavernous (LABC). LABC expresses AR and increases in size by androgen programming (Schwartz CL et al., 2019). The perineum is programmed in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans), when testicular testosterone production is high (Sharpe RM, 2020; Welsh M et al., 2014). Thus, a decrease in testicular testosterone levels in this window may limit the AR signaling in the LABC, leading to less elongation of the perineum and a short AGD.

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high given the canonical biological knowledge on normal reproductive development.

Sexual differentiation in males, including elongation (masculinization) of the perineum, is initiated and programmed in fetal life. Around GW 8 in humans and GD16 in rats, the testes have formed and start synthesizing testosterone through the steroidogenesis pathway.  Testicular testosterone is secreted to either act directly on the AR or be converted to the more potent androgen hormone (DHT). AR activation in the perineum of males programs it to elongate, resulting in a longer AGD in males than in females (~twice the length in rats and humans) (Murashima et al., 2015b; Trost & Mulhall, 2016; Welsh M et al., 2014)

The programming of the reproductive tissues, including masculinization of the perineum happens in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans) (Welsh M et al., 2014).

Given the dependency of testosterone for elongation of the perineum, either through direct AR activation or conversion to DHT, it is highly plausible that a decrease in testicular levels of testosterone will lead to a shorter AGD in males.

The overall empirical evidence for this KER is judged as strong.  

A total of 19 data sets were collected as evidence for this KER (table 3 and appendix 2). Of these, 14 studies, all in rats, show that exposure to a stressor that decreased fetal intratesticular testosterone also caused a short AGD.

Table 3 Empirical evidence for KER 3449 LOAEL: Lowest observed adverse effect level; NOAEL: No observed adverse effect level; ITT: Intratesticular testosterone measured in whole testes; Ex vivo: Testosterone production measured by incubation of testes and collection of media. See appendix 2 for specifications.

¹NOAEL and LOAEL were, when available, based on AGDi data. For some datasets, only AGD or AGD/bw were available, see appendix 1 for details on each dataset.

Dose concordance

Overall, the empirical evidence supports dose concordance, although with some inconsistencies.

Five different studies show that in utero dibutyl phthalate exposure reduces intratesticular testosterone and AGD. Three of these studies report the same LOAEL for reduced intratesticular testosterone and short AGD, respectively (Lourenço AC et al., 2014; Pike et al., 2014; van den Driesche S et al., 2020). In one study, the LOAEL for reduced intratesticular testosterone was 500 mg/kg/day, while the LOAEL for short AGD was 100 mg/kg/day, thus not showing dose concordance (Martino-Andrade AJ et al., 2009). In contrast, another study reports 100 mg/kg/day as LOAEL for reduced intratesticular testosterone and 500 mg/kg/day as LOAEL for short AGD  (Struve MF et al., 2009).

Two studies used prochloraz as the stressor. One study showed a reduction in testosterone in the testes at a dose of 30 mg/kg bw/day, but no effect on ex vivo testosterone production or on AGD (Vinggaard AM et al., 2005). The other study tested 50 and 150 mg/kg bw/day prochloraz and found an effect on both intratesticular testosterone (both in testes and ex vivo) and on AGD in the male offspring (Laier P et al., 2006).

Of the remaining empirical evidence, most studies report the same LOAEL for reduced intratesticular testosterone and short AGD, but for many of these cases only one chemical dose was tested.

Temporal concordance

Overall, the empirical evidence supports temporal concordance.

In several of the studies, AGD was measured at a later timepoint, often postnatally, than intratesticular testosterone. For example, exposure of rats from GD14-18 to a mixture of butyl benzyl phthalate (500 mg/kg bw/day) and linuron (75 mg/kg bw/day) reduced intratesticular testosterone levels at GD18 and caused short AGD in the males, which could be measured at both PND2 and in adult rats (Hotchkiss AK et al., 2004). Exposure to only 500 mg/kg bw/day butyl benzyl phthalate (500 mg/kg bw/day) from GD14-18 also reduced intratesticular testosterone levels at GD18 and caused short AGD at PND2, but in adult males, the effect on AGD was no longer significant. Exposure to linuron alone (75 mg/kg bw/day) in the same study caused both reduced intratesticular testosterone and short AGD at PND2 and in adult males (Hotchkiss AK et al., 2004). This may indicate that the fetal effect on AGD is best detected in early postnatal life.

In ten studies, AGD was measured prenatally, either at the same time or a day after the intratesticular testosterone measurements. Three of these studies did not find an effect on short AGD when intratesticular testosterone was measured. For example, exposure to α-cypermethrin from GD13-19 in four different doses reduced ex vivo testosterone production in testes at GD19 with LOAEL of 5 mg/kg bw/day, but AGD at GD19 was not affected at this dose or at 10 mg/kg bw/day (Saillenfait AM et al., 2017). In the same study, however, exposure to diisobutyl phthalate (250 mg/kg bw/day) caused both a reduction in ex vivo testosterone and AGD on GD19, although the effect on AGD was small (Saillenfait AM et al., 2017).

Incidence concordance

Because the data mainly includes chemicals at different doses and exposure windows, and all data are continuous, they do not firmly establish incidence concordance of this KER. However, a few studies have used the same stressors at the same doses and provide some information on incidence concordance.

Five studies used the stressor dibutyl phthalate, three of them testing the same two doses, 100 and 500 mg/kg bw/day, although with slightly different exposure windows and timepoints of AGD measurement. Of these three studies, two found the LOAEL for reduced intratesticular testosterone to be 500 mg/kg bw/day (Martino-Andrade AJ et al., 2009; Pike et al., 2014), while one detected a reduction in testosterone at 100 mg/kg bw/day (Struve MF et al., 2009). Regarding AGD, one study reported 100 mg/kg bw/day as the LOAEL, i.e. lower than the LOAEL for intratesticular testosterone (Martino-Andrade AJ et al., 2009), while the others reported 500 mg/kg bw/day as the LOAEL (Pike et al., 2014; Struve MF et al., 2009). Thus, these studies are conflicting regarding incidence concordance.

There are also three studies using diisobutyl phthalate as stressor. These vary more in terms of doses, but overall they see subtle and more uncertain effects on both intratesticular testosterone and AGD with LOAELs ranging from 50 to 250 mg/kg bw/day for both measurements (Clewell et al., 2013; Saillenfait AM et al., 2017; Taxvig C et al., 2008).
 

One uncertainty in empirical data for this KER is the studies where intratesticular testosterone was measured in an ex vivo testes incubation experiment. With this method, there is an uncertainty of the direct relationship between the ex vivo secretion, as testosterone was measured in media, and the exact intratesticular testosterone levels. However, in most of the studies using this ex vivo method, intratesticular testosterone was also measured in testes homogenates (see appendix 2) with similar outcomes using both methods, suggesting that ex vivo testosterone production after incubation can be used as a proxy for intratesticular testosterone , exemplified by very identical measurements in (Borch J et al., 2004). In the three studies, only measure testosterone production ex vivo (Conley JM et al., 2021; Saillenfait AM et al., 2017), the uncertainty in this measurements should be kept in mind.

Five data sets did not measure any effect of the stressors on AGD. In two cases, this could be due to the AGD measurements either being measured too early to measure detectable differences between groups (Saillenfait AM et al., 2017) or having too high variance to gain statistical significance (Martino-Andrade AJ et al., 2009). In the three other cases, the lack of effect on AGD was likely due to only testing one dose of the stressor (Vinggaard AM et al., 2005) (dose concordance) or the doses tested were too low (Clewell et al., 2013).

Another uncertainty is the inconsistencies between studies for the stressor dibutyl phthalate. One study report the LOAEL for reduced intratesticular testosterone as 100 mg/kg/day (Struve MF et al., 2009), while others report 500 mg/kg/day (one of these only use on dose) (Lourenço AC et al., 2014; Martino-Andrade AJ et al., 2009; Pike et al., 2014). Similarly, the LOAEL for short AGD is inconsistent, with 500 mg/kg/day being reported in three studies (Lourenço AC et al., 2014; Pike et al., 2014; Struve MF et al., 2009), and 100 mg/kg/day being reported in one (Martino-Andrade AJ et al., 2009).

Finally, one study containing uncertainties is a study on diisonyl phthalate (Boberg et al., 2011). In this study, exposure from GD7-21 to 600 mg/kg/day, but not 750 or 900 mg/kg bw/day reduced intratesticular testosterone, while 900 mg/kg/day caused short AGD. However, both 750 and 900 mg/kg bw/dag diisonyl phthalate tended to decrease intratesticular testosterone levels, and the lack of statistical significance may therefore be explained by a low sample size for these measurements (n=3-4 litters, 1-2 testes per litter).

Quantitative Understanding of the Linkage

There are no direct models for reductions in intratesticular testosterone levels and AGD. A model for phthalates has been developed, aiming to predict reductions in AGD based on the reduction in ex vivo testosterone production. In this model, a 5-parameter logistic regression model, around 60% testosterone reduction can cause a decreased AGD, with a steep declining curve as testosterone production decreases. It must be emphasized that this model, however, was only developed for the phthalates and does therefore not directly evidence the same relationship for other stressors reducing testosterone levels (Earl Gray L Jr et al., 2024).

The exact timescale of this KER depends on the species, but it may take days or weeks for growth changes in the perineum to be measurable. In humans, testosterone production in the testes begin around GW8, and sexual dimorphism of the perineum between males and females can be measured by GW11-13, reaching the full 2:1 male:female ratio in length at GW17-20 (Thankamony A et al., 2016)

There are no known modulating factors for this KER.

There are no known feedback/feedforward loops for this KER

References

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Relationship: 3349: Decrease, circulating testosterone levels leads to AGD, decreased

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic applicability

Male-specific development of the fetal perineum in male mammals is strongly influenced by androgen signaling. It is therefore biologically plausible that this KER is applicable to all mammals (Murashima et al., 2015). The empirical evidence in this KER provides strong support that reduced circulating testosterone levels in fetal life can cause short AGD in rats.  The empirical evidence for this KER in humans is sparse and conflicting; however, given the known role of androgens in human male reproductive development, the KER is considered applicable to humans.

Sex applicability

The empirical evidence in this KER supports that reduced circulating testosterone in fetal life can cause reduced AGD in males. Females do have circulating testosterone, but in much lower concentrations than males (Vesper et al., 2015), and it is unlikely that further reduction can cause a short AGD in females (Schwartz CL et al., 2019). Of note is that ‘reduced AGD’ in males is not a reduction per se, but a failure to elongate in response to androgen action.

Life stage applicability

This KER is applicable to fetal life, as this is when the perineum is programmed by androgen hormones in males. The masculinization programming window is around gestational days (GD) 16-20 in rats, and suggested to be gestational weeks (GW) 8-14 in humans (Sharpe RM, 2020; Welsh M et al., 2014). Once programmed in fetal life, the AGD is believed to be relatively stable, but the perineum can in some cases be responsive to postnatal changes in androgen levels (Schwartz CL et al., 2019; Sharpe RM, 2020; Thankamony A et al., 2016). The empirical evidence in this KER supports the fetal life stage applicability.

Key Event Relationship Description

This non-adjacent KER describes a fetal decrease in circulating testosterone (often measured in serum or plasma) leading to short anogenital distance (AGD) in male offspring.

In male mammals, testosterone is one of the primary hormonal drivers of male reproductive differentiation. Produced by the fetal testes, testosterone is transported through blood to the peripheral reproductive tissues to bind the androgen receptor (AR) or be converted to the higher potency androgen hormone dihydrotestosterone (Murashima et al., 2015). The androgen hormones signal through AR to program the reproductive tissue to differentiate along the male pathway. This includes elongation of the perineum, which is suggested to involve the perineal muscle complex levator ani bulbocavernous (LABC). LABC expresses AR and increases in size by androgen programming (Schwartz CL et al., 2019). The male programming of the tissue happens during fetal life in the masculinization programming window (GD 16-20 in rats, GW 8-14 in humans), when circulating testosterone levels are high (Sharpe RM, 2020; Welsh M et al., 2014). Thus, a decrease in circulating testosterone levels in this window may limit the AR signaling in the LABC, leading to less elongation of the perineum and a short AGD.

Evidence Supporting this KER

The biological plausibility for this KER is judged to be high given the canonical biological knowledge on normal reproductive development.

Sexual differentiation in males, including elongation (masculinization) of the perineum, is initiated and programmed in fetal life. Once the testes have formed, they start producing testosterone through the steroidogenesis pathway and secrete testosterone into circulation. Testosterone is transported in the blood either as free testosterone or bound to albumin or sex-hormone binding globulin. In peripheral tissues, testosterone can be converted to the more potent androgen hormone dihydrotestosterone (DHT) by the enzyme 5α-reductase. Both DHT and testosterone bind and activate the androgen receptor (AR) to program fetal tissues to differentiate along the male pathway, including elongation of the perineum, resulting in a longer AGD in males than in females (~twice the length in rats and humans) (Murashima et al., 2015; Trost & Mulhall, 2016; Welsh M et al., 2014)

Testosterone is produced from around GD15 in fetal rats and GW8 in humans, which is also the onset of when testosterone levels can be measured in circulation. The programming of the reproductive tissues, including masculinization of the perineum happens in the masculinization programming window (GD16-20 in rats, GW8-14 in humans) (Welsh M et al., 2014).

Given the dependency of testosterone for elongation of the perineum, either through direct AR activation or conversion to DHT, it is highly plausible that a decrease in circulating levels of testosterone will lead to a shorter AGD in males

The empirical evidence from studies in animals for this KER is overall judged as strong.

From the data collection, ten data sets were extracted. The data sets included different stressors causing reduced fetal levels of testosterone, all in rats (table 3 and appendix 2). Of these ten data sets, eight showed concurrent short AGD.

Table 3 Empirical evidence for KER 3349 LOAEL: Lowest observed adverse effect level; NOAEL: No observed adverse effect level. See appendix 2 for specifications.

¹NOAEL and LOAEL were, when available, based on AGDi data. For some datasets, only AGD or AGD/bw were available, see appendix 2 for details on each dataset.

²No statistics available as samples were pooled for measurement of testosterone.

Supporting epidemiological evidence

No studies have shown a direct association between fetal circulating testosterone levels and AGD. A few epidemiologic studies can inform indirectly on the human evidence for this KER, and the current studies on this are conflicting.

As some phthalates are known to reduce testosterone production, an association between phthalate exposure and short AGD could support the KER in humans. A meta-analysis found an association between maternal urinary concentrations of some phthalate metabolites and short AGD (Zarean M et al., 2019). Moreover, in a Taiwan Maternal and Infant Cohort study, maternal urinary concentrations of some phthalate metabolites were also associated with a shorter AGD in male infants, although there was no association between cord blood testosterone levels and AGD, and the metabolites were not associated with lower cord blood testosterone levels, either  (Lu et al., 2024). Another longitudinal mother-child cohort study did not find an association between AGD in adult men with maternal serum concentrations of phthalate metabolites during pregnancy (Henriksen LS et al., 2023).

One study related cord blood testosterone levels to AGD in infants boys and did not find associations between the two (Liu C et al., 2016).

In adult men, anogenital distance was significantly associated with serum testosterone levels (Eisenberg ML et al., 2012).

Dose concordance

The in vivo rat toxicity studies for this KER moderately supports dose concordance.

One study with the stressor perfluorotridecanoic acid showed dose concordance with the LOAEL for reduced serum testosterone being 1 mg/kg bw/day, while the LOAEL for short AGD was 10 mg/kg (Li C et al., 2021).

In another study with two doses of prochloraz, the LOAEL was the same (50 mg/kg) for decreased testosterone and short AGD (Laier P et al., 2006).

Finally, in two studies with dicyclohexyl phthalate and zearalenone, respectively, AGD was shortened at lower doses than testosterone was reduced (Aydoğan Ahbab M & Barlas N, 2015; Pan P et al., 2020), and these do therefore not support dose concordance. However, in both cases the testosterone levels tended to be lower in the non-significant doses as well, and the lack of effect could therefore be due to high variation in the testosterone measurements.

Temporal concordance

Overall, the empirical evidence supports temporal concordance between the events.

Mably TA et al., 1992 followed plasma testosterone levels in male rats after in utero exposure to 1 µg/kg 2, 3, 7, 8-Tetrachlrodibenzo-p-dioxin on GD15. From GD17-21, testosterone levels steadily decreased in both control and exposed males, but the overall levels in the exposed males were lower than in control males. After birth, the testosterone levels in exposed male increased to match values in control males. At PND1, 3, and 5, plasma levels were normal in exposed male rats, while their AGD were reduced at all days.

Five of the data sets also measure circulating testosterone prenatally (GD20 or GD21) and AGD postnatally (between PND0 and PND3), and three of these observe short AGD when testosterone is reduced during fetal life (Borch J et al., 2004; Laier P et al., 2006). In one of these studies, where diethylhexyl phthalate alone or diethylhexyl phthalate in combination with diethylhydroxylamine was administered from GD7-PND17, serum testosterone levels were decreased at GD21, but not at PND22 or PD90 (Borch J et al., 2004). Similarly, PND16 serum testosterone was not altered by perinatal (GD7-PND16) prochloraz exposure which reduced serum testosterone at GD21 and AGD at PND1 (Laier P et al., 2006).

Incidence concordance

The data does not inform incidence concordance.
 

Two data sets, both from the same study (Vinggaard AM et al., 2005), showed no effect of decreased circulating testosterone levels on AGD, which may be due to too low doses of the stressors (prochloraz and a mixture). For two studies (Aydoğan Ahbab M & Barlas N, 2015; Pan P et al., 2020), the LOEAL was lower for the downstream event, short AGD, than the upstream event, reduced circulating testosterone. In both cases, lower doses of stressors tended to lower testosterone levels as well, and the inconsistency could therefore be due to high variance in testosterone measurements.

Another uncertainty is the AGD results in the study investigating di-n-hexyl phthalate exposure from GD6-19 in rats (Aydoğan Ahbab M & Barlas N, 2015). Three doses of the phthalate (20, 100, and 500 mg/kg bw/day) all reduced plasma testosterone levels, but only 20 and 500 mg/kg bw/day caused short AGD, when calculating the anogenital distance index (AGDi, AGD/bw^1/3 ). When analyzing the direct AGD, all doses of di-n-hexyl phthalate decreased AGD. In contrast, when analyzing the relative AGD (AGD/bw), only the highest dose (500 mg/kg bw/day) decreased the AGD. This study thus identified different LOAELs for AGD, depending on if and how body weight was considered, posing an uncertainty on the results.

Quantitative Understanding of the Linkage

Testosterone is secreted from around GW8 in humans (GD16 in rats), marking the beginning of the masculinization programming window and programming of the perineal tissue. Depending on the species, the time scale for observing effects on tissue growth is days or weeks. In humans, sexual dimorphism of the AGD can be measured by GW11-13, reaching the full 2:1 male:female ratio in length at GW17-20 (Thankamony A et al., 2016).

There are no known modulating factors for this KER.

There are no known feedback/feedforward loops for this KER.

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