international journal of andrology ISSN 0105-6263 REVIEW ARTICLE Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development Vickie S. Wilson,* Chad R. Blystone,* Andrew K. Hotchkiss,* Cynthia V. Rider* and L. Earl Gray Jr* *US Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Laboratory, Reproductive Toxicology Division, North Carolina, and North Carolina State University, Raleigh, NC, USA Summary Keywords: Scientists have identified environmental chemicals that display anti-androgenic androgen receptor, anti-androgen, linuron, activity via multiple mechanisms of action. Early studies focused on pesticides phthalate, prochloraz, reproductive acting as androgen receptor (AR) antagonists but it soon became apparent that development, vinclozolin was not the only endocrine mode by which compounds affected the androgen signalling pathway. Classes of chemicals currently known to interfere with the Correspondence: Vickie S. Wilson, US Environmental Protection androgen signalling pathway include dicarboximide fungicides (e.g. vinclozolin), Agency, ORD, National Health and organochlorine-based insecticides (e.g. p,p¢-DDT and -DDE), conazole fungi- Environmental Effects Laboratory, cides (e.g. prochloraz), plasticizers (phthalates) and urea-based herbicides (linu- Reproductive Toxicology Division, MD-72, ron). Phthalate esters (PEs) and vinclozolin appear to act primarily via a single 2525 E. Highway 54, Research Triangle Park, mechanism of action, while others such as linuron and prochloraz, appear to NC 27711, USA. display dual mechanisms of action. Exposure to PEs decreases mRNA expres- E-mail: [email protected] sion of key steroidogenic enzymes and also the peptide hormone insulin-like Received 30 October 2007; revised 13 peptide 3 (insl3) from the foetal Leydig cells. Hence, both androgen- and inls3- December 2007; accepted 18 December 2007 dependent tissues are affected. Vinclozolin and procymidone act solely through binding to the AR as antagonists thus blocking the action of androgen at the doi:10.1111/j.1365-2605.2007.00861.x cellular level but do not affect foetal testosterone synthesis or insl3 gene expres- sion. The compounds linuron and prochloraz are AR antagonists but also inhi- bit foetal testosterone synthesis, although unlike the PEs, mRNA expression of steroidogenic enzymes and insl3 are not affected. All the above chemicals dis- rupt androgen signalling in the foetal male rat and produce some malforma- tions in common, but the precise profiles of effects in the offspring are pathognomonic for each mode of action. For example, the ‘phthalate syn- drome’ vs. the ‘vinclozolin syndrome’ each displays a profile of effects which is clearly different. In summary, as more and more molecular studies with anti- androgenic compounds are conducted, the number of mechanisms by which compounds can affect the androgen signalling pathway is likely to increase. Furthermore, the effects of mixtures of these compounds are just beginning to be explored. pathologies (Jegou et al., 2001). As only a small percent- Introduction age of these lesions can be linked directly to known Concern has risen over the apparent increase in male genetic defects, developmental exposure to man-made reproductive health problems and the potential role of chemicals has been implicated in the increases in these endocrine disrupting chemicals (EDCs) in the aetiology reproductive malformations. of these conditions (Skakkebaek, 2002a). Declining sperm Compounds that disrupt endocrine signalling pathways counts and increased incidences of hypospadias, cryptor- can act via several mechanisms including binding to hor- chidism and testis cancer have all been reported. The mone receptors, modifying the production or metabolism worldwide doubling in the rate of testicular cancer over of endogenous hormones, or modifying the number of the last 40 years is one of the more robust of these hormone receptors. For example, androgen agonists bind Journal compilation ª 2008 Blackwell Publishing Ltd • International Journal of Andrology 31, 178–187 178 No claim to original US government works V. S. Wilson et al. Diverse mechanisms of anti-androgen action to androgen receptors and mimic natural hormones signalling pathway through slightly different mechanisms, which may initiate a cascade of events resulting in inap- there are both similarities and differences in androgen- propriate expression of hormone-dependent genes. Con- dependent tissue effects and the profile of malformations versely, androgen receptor (AR) antagonists compete with which are produced. In the course of this summary, endogenous androgen for the receptor and prevent down- mechanism-based groups of anti-androgens was discussed stream activation of androgen-dependent gene expression. with focus on the pathognomic effects of each class of While early research focused on the ability of environ- anti-androgen. The terms ‘low’ dose and ‘high’ dose will mental compounds to impact androgen binding to its generally be used in the discussion. The specific doses receptor, it is now clear that it is not the only mechanism which fit each of these categories are dependent on the through which compounds can interfere with the andro- potency and mechanism of action of each compound dis- gen signalling pathway. In addition, some compounds cussed and will clearly be different for each individual appear to act through more than one mechanism, each of compound. Information on the actual dose can be found which having the potential impact on the androgen sig- in the reference literature. Generally, low dose refers to nalling pathway at different points. a dose near or just above the no observed adverse effect Mammalian gonadal development and sex differentia- level for the individual compound. Additionally, we cover tion occur during a relatively narrow time window. The the differential effects between two strains of rats exposed production of testosterone and other hormones by the to the same anti-androgen. Lastly, we discuss conclusions foetal testis during this period are critical factors for and future directions for this work including the study of the proper development of the male reproductive tract complex mixtures of anti-androgens with diverse mecha- (Schardein, 1993). In utero exposure to chemicals that nisms of action. disrupt androgen signalling during this window can result in alterations that may include reduced anogenital dis- Androgen receptor antagonists tance (AGD), increase in female-like nipple retention, epi- didymal agenesis, reduction in sex accessory gland Several environmental chemicals adversely affect male weights, cryptorchidism, hypospadias and reduced fertil- development by interfering with androgen receptor signal- ity. Generally, in utero exposure to anti-androgenic chem- ling during the critical periods (in utero or peri-pubertal) icals affects male offspring, while having little effect on of sex differentiation and maturation. Depending on the female offspring. Androgenic chemicals, conversely, have timing of developmental exposure, these anti-androgens been shown to induce malformations in exposed female affect androgen-sensitive organs and processes within the offspring and have little to no effect on males. male rat leading to altered neuroendocrine development The suite of effects following in utero exposure to and behaviour, reduced reproductive organ weights and endocrine disruptors can be indicative of the mechanism malformations of external genitalia and reproductive of action of the chemical exposure. In addition to the organs. Several anti-androgenic chemicals have been iden- effects mentioned above from androgenic and anti-andro- tified as androgen receptor (AR) antagonists including: genic exposure, environmental oestrogens have been the fungicides vinclozolin (Gray et al., 1994; Kelce & Wil- found to cause infertility or shorten the reproductive life- son, 1997), procymidone (Hosokawa et al., 1993; Ostby span of in utero exposed females. Compounds such as et al., 1999), prochloraz (Vinggaard et al., 2002; Noriega 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) elicit et al., 2005), the DDT metabolite p,p¢-DDE (Kelce et al., effects in the female reproductive tract including malfor- 1995), the herbicide linuron (Gray et al., 1999a; Lam- mations of the external genitalia, reduced ovarian weight bright et al., 2000) and polybrominated diphenyl ethers and can also decrease their reproductive lifespan. In fact, flame retardants (Stoker et al., 2005). a relatively low single oral dose of 1–2 lg ⁄ kg 2,3,7,8- In utero exposure to chemicals that disrupt AR signal- TCDD given to the dam during gestation can produce ling leads to a host of typical effects in male rat offspring. frank reproductive malformations in both male and External biomarkers of prenatal androgen disruption female offspring. Lastly, foetal germ cell toxicants, such as include the anogenital distance (AGD) and the juvenile busulfan, can cause infertility in females, reduce sperm nipple ⁄ areolae number. The AGD is defined as the dis- production in males and elicit histological lesions in both tance between the genital papilla and the anus; male ovaries and testes without affecting gross morphology of rodents have AGDs that are approximately twice the the reproductive tract in either sex. length as those of females (Vandenbergh & Huggett, The focus of this selective review was to summarize 1995; Gray et al., 1999a,b). Areolae