William R. Kelce

Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat:

In humans and rodents, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of 100 or 200 mg vinclozolin (V) kg−1 day−1 during sexual differentiation in rats induces female-like anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, suprainguinal ectopic scrota/testes, a vaginal pouch, epididymal granulomas, and small to absent sex accessory glands in male offspring. Vinclozolin is metabolized to at least two active forms, M1 and M2, that display antiandrogenic activity by binding the androgen receptor (AR). Here, we present information on the reproductive effects of oral treatment with low dosage levels of V during sexual differentiation of the male rat. Vinclozolin was administered to the dam at 0, 3.125, 6.25, 12.5, 25, 50, or 100 mg kg−1 day−1 from gestational day 14 to postnatal day 3 (the period of fetal/neonatal testicular testosterone synthesis and sexual differentiation). At doses of 3.125 mg V kg−1 and above, AGD was significantly reduced in newborn male offspring and the incidence of areolas was increased. These effects were associated with permanent alterations in other androgen-dependent tissues. Ventral prostate weight in one year old male offspring was reduced in all treatment groups (significant at 6.25, 25, 50, and 100 mg kg−1 day−1), and permanent nipples were detected in males at 3.125 (1.4%), 6.25 (3.6%), 12.5 (3.9%), 25 (8.5%), 50 (91%), and 100 (100%) mg V kg−1 day−1. To date, permanent nipples have not been observed in a control male from any study in our laboratory. Vinclozolin treatment at 50 and 100 mg kg−1 day−1 induced reproductive tract malformations and reduced ejaculated sperm numbers and fertility. Even though all of the effects of V likely result from the same initial event (AR binding), the different endpoints displayed a wide variety of dose-response curves and ED50s. The dose-response data for several of the functional endpoints failed to display an obvious threshold. These data demonstrate that V produces subtle alterations in sexual differentiation of the external genitalia, ventral prostate, and nipple tissue in male rat offspring at dosage levels below the previously described no-observed-effect-level (NOEL). These effects occur at a dosage level an order of magnitude below that required to induce malformations and reduce fertility. Hence, multigenerational reproduction studies of antiandrogenic chemicals that were not conducted under the Environmental Protection Agencys new Harmonized Multigenerational Test Guidelines, which include endpoints sensitive to antiandrogens at low dosage levels, could yield a NOEL that is at least an order of magnitude too high.

The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro

Procymidone is a dicarboximide fungicide structurally related to the well-characterized fungicide vinclozolin. Vinclozolin metabolites bind to mammalian androgen receptors (AR) and act as AR antagonists, inhibiting androgen-dependent gene expression in vivo and in vitro by inhibiting AR-binding to DNA. The current study was designed to determine if procymidone acted as an AR antagonist in vitro and to describe the dosage levels of procymidone that alter sexual differentiation in vivo. In vitro, procymidone inhibited androgen from binding the human AR (hAR) in COS (monkey kidney) cells transfected with hAR at 3.16 μM. In vitro, procymidone acted as an androgen antagonist, inhibiting dihydrotestosterone (DHT)-induced transcriptional activation at 0.2 μM in CV-1 cells (cotransfected with the hAR and a MMTV-luciferase reporter gene). In vivo, maternal procymidone exposure at 0, 25, 50, 100, or 200 mg kg−1 day−1 during gestation and early lactation (gestational day 14 to postnatal day 3) altered reproductive development of male offspring at all dosage levels tested. Male offspring exhibited shortened anogenital distance (at 25 mg kg−1 day−1 and above), permanent nipples, reduced weight of several androgen-dependent tissues (levator ani and bulbocavernosus muscles, prostate, seminal vesicles, Cowpers gland and glans penis), and malformations (hypospadias, cleft phallus, exposed os penis, vaginal pouch, hydronephrosis, occasional hydroureter, epididymal granulomas, and ectopic, undescended testes). In addition, perinatal procymidone treatment had a marked effect on the histology of the lateral and ventral prostatic and seminal vesicular tissues of the offspring (at 50 mg kg−1 day−1 and above). These effects consisted of fibrosis, cellular infiltration, and epithelial hyperplasia. This constellation of effects is similar to that produced by perinatal exposure to vinclozolin. However, procymidone appears to be slightly less potent in inducing malformations than vinclozolin by a factor of about two. In summary, the antiandrogenic activity of procymidone was demonstrated in vivo and in vitro in cell lines transfected with hAR. Since the role of androgens in mammalian sexual differentiation is highly conserved, it is likely that humans would be adversely affected by procymidone in a predictable manner if the human fetus was exposed to sufficient levels during critical stages of intrauterine and neonatal life.

Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat

Vinclozolin is a well-characterized antiandrogenic fungicide. It produces adverse effects when administered during sexual differentiation, and it alters reproductive function in adult male rats by acting as an androgen-antagonist. Two active metabolites of vinclozolin, M1 and M2, compete with natural androgens for the rat and human androgen receptors (ARs), an effect that blocks androgen-induced gene expression in vivo and in vitro. In addition to their effects during perinatal life, androgens play a key role in pubertal maturation in young males. In this regard, the present study was designed to examine the effects of peripubertal oral administration of vinclozolin (0, 10, 30, or 100 mg kg−1 day−1) on morphological landmarks of puberty, hormone levels, and sex accessory gland development in male rats. In addition, as binding of the M1 and M2 to AR alter the subcellular distribution of AR by inhibiting AR-DNA binding, we examined the effects of vinclozolin on AR distribution in the target cells after in vivo treatment. We also examined serum levels of vinclozolin, M1, and M2 in the treated males so that these could be related to the effects on the reproductive tract and AR distribution. Vinclozolin treatment delayed pubertal maturation (at 30 and 100 mg kg−1 day−1) and retarded sex accessory gland and epididymal growth. Serum luteinizing hormone (LH; significant at all dosage levels) and testosterone and 5α-androstane,3α,17β-diol (at 100 mg kg−1 day−1) levels were increased. Testis size and sperm production, however, were unaffected. It was apparent that these effects were concurrent with subtle alterations in the subcellular distribution of AR. In control animals, most AR were in the high salt cell fraction, apparently bound to the natural ligand and DNA. Vinclozolin treatment reduced the amount of AR in the high salt (bound to DNA) fraction and it increased AR levels in the low salt (inactive, not bound to DNA) fraction. M1 and M2 were found in the serum of animals from the two highest dosage groups, but they were present at levels well below their K i values. In summary, these results suggest that when the vinclozolin metabolites occupy a small percentage of AR in the cell, this prevents maximal AR-DNA binding and alters in vivo androgen-dependent gene expression and protein synthesis, which in turn results in obvious alterations of morphological development and serum hormone levels. It is noteworthy that similar exposures during prenatal life result in a high incidence of malformations in male rats.

The estrogenic and antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats

This study was designed to determine if long-term exposure to high doses of methoxychlor (M) would alter pituitary or testicular endocrine functions in either an estrogenic or antiandrogenic manner. Weanling male Long-Evans hooded rats were dosed daily with M (po) at 0, 200, 300, or 400 mg kg−1 day−1 for 10 months. Methoxychlor treatment delayed puberty by as much as 10 weeks and reduced fertility and copulatory plug formation in a dose-related manner at the initial mating. During mating, M-treated males exhibited shorter latencies to mount and ejaculate versus control males, but the number of intromissions prior to ejaculation was unaffected, indicating that M enhanced the arousal level in the males in an estrogen-dependent manner. Most treated males eventually mated but time-to-pregnancy was lengthened. Very low sperm counts were associated with infertility, while prolonged delays in puberty reduced fecundity. Methoxychlor treatment with 200 to 400 mg kg−1 day−1 failed to mimic the chronic effects of a sustained (8 months) low dose of estradiol-17β (3-mm silastic implants) on pituitary or testicular hormone levels. Estradiol administration increased pituitary weight 4-fold, serum levels of luteinizing hormone (LH) were reduced by almost 50%, and serum prolactin was increased 40-fold, while M did not affect any of these measures. These data demonstrate that M affects the CNS, epididymal sperm numbers, and the accessory sex glands and delays mating without significantly affecting the secretion of LH, prolactin, or testosterone. These data indicate that M did not alter pituitary endocrine function in either an estrogenic or antiandrogenic manner. To our knowledge, these data provide the first in vivo example of such a pronounced degree of target tissue selectivity to an environmental endocrine-disrupting chemical.

Latent Effects of Pesticides and Toxic Substances On Sexual Differentiation of Rodents

In humans and rodents, exposure to hormonally active chemicals during sex differentiation can produce morphological pseudohermaphrodism (Schardein, 1993; Gray, 1992). For example, hormonally active drugs like DES (estrogenic), Danazol (androgenic), and progestins cause urogenital malformations in the reproductive tracts of humans and rodents. The current discussion will present new information on the effects of toxic chemicals and pesticides that act on reproductive development via novel mechanisms, including germ cell toxicity, antiandrogenicity, and Ah-receptor binding. Information will be presented that describes how exposure during critical stages of life to synthetic chemicals present in our environment, such as benzidine- based dyes, antiandrogenic fungicides, 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), and PCB congener 169, result in abnormal rodent sex differentiation. In rodents, perinatal exposure to fetal germ cell toxicants reduced the reproductive potential of female, and permanently reduced sperm production in male progeny. Phenotypic sex differentiation, however, was unaffected by these germ cell toxicants. In contrast, antiandrogenic drugs and fungicides induced profound alterations in phenotypic sex differentiation. Effects such as hypospadias, ectopic testes, vaginal pouches, agenesis of the ventral prostate, and nipple retention in male rats were observed commonly. Although these antiandrogens induced no permanent effects in female progeny, another class of chemicals, the Ah-receptor mediated toxicants, did reduce fertility in both male and female rat offspring. Cauda epididymal sperm numbers were reduced permanently in TCDD-exposed male rat and hamster progeny, and female progeny displayed malformations of the external genitalia. Other toxicants produced dramatic alterations of sex differentiation (uterus unicornis, agenesis of the vas and epididymis, and undescended testes), via mechanisms that have not been characterized yet. Since these adult/pubertal alterations resulted from gestational and/or neonatal exposures, future studies should include a comprehensive assessment of reproductive function after perinatal exposure because the developing animal is extremely sensitive to toxicants during sex differentiation, and many of the effects are difficult to detect until late in life.

Endocrine Modulation of Reproduction

The ability of foreign compounds to affect the functioning of various endocrine systems is currently thought responsible for a wide variety of effects. The presentations in this Symposium reviewed the evidence for and against the involvement of endocrine systems in several different aspects of reproduction. The mechanism behind the ability of a triazine herbicide to cause enhanced appearance of mammary tumors in one strain of female rats is reviewed by Stevens. The data suggest that enhanced aging, not direct mammary modulation, is responsible. Dietary phytoestrogens, the mediators of their actions, their effects in various biological systems, and the relationships between phytoestrogen producers and consumers are all provocatively and succinctly reviewed by Hughes. Kelce presents the strategy used to dissect the mode and mechanisms of action of a fungicide that opened a new awareness in reproductive toxicology: the possibility of xenobiotics being antiandrogens. Finally, to heighten our understanding of the interplay among hormonal systems in vivo, Hess reviews the data that show that androgens are not the only hormones important in the development of the male reproductive system: the pituitary is shown to play a critical role at specific stages of development. The breadth of these presentations, and the implications of their findings, should make us pause and realize how much there is still to discover about the interaction between the reproductive system and anthropogenic compounds.

Emerging issues: the effects of endocrine disrupters on reproductive development

The effects of ‘endocrine disrupters’ on the health of human and wildlife populations are currently receiving a great deal of interest from the popular press and the scientific community. At the present, scientists are grappling with research needs in this new area and the need for new test protocols to screen for endocrine effects. The impetus for this new approach began at a Work Session in July of 1991 on ‘Chemically Induced Alterations in Sexual Development: the Wildlife/Human Connection’ attended by a multidisciplinary group of experts (Colborn and Clement, 1992). A consensus statement from this Work Session (Colborn and Clement, 1992: 1–6) concluded that ‘Many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals, including fish, wildlife, and humans. The consequences of such disruption can be profound because of the crucial role hormones play in controlling development.’ In addition, one portion of the consensus statement indicated that many wildlife populations were already affected by such compounds, with effects (relevant to this chapter) including demasculinization and feminization of male fish, birds and mammals and defeminization and masculinzation of female fish and birds.

An Environmental Antiandrogen

Publisher Summary This chapter provides an overview of a study that determines the biochemical mechanism responsible for the antiandrogenic effects of vinclozolin exposure. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) are degradation products, detectable both within the soil and leaves of treated plants and in the serum of treated rats. The study suggests that some of the detrimental effects of vinclozolin exposure are likely to be mediated via the formation of the antiandrogenic vinclozolin metabolites M1 and M2. It is demonstrated in this chapter that vinclozolin, M1, or M2 was unable to alter the ability of epididymal 5α-reductase to convert testosterone to 5α-dihydrotestosterone and/or 5α-androstane-3a, 17β-diol (IC50 > 1000μM). The primary vinclozolin degradation products, M1 and M2, act as pure competitive antagonists of androgen receptor binding with Ki values of 92 and 9.7 μM, respectively. Vinclozolin, the parent compound, does not appear to be capable of androgen receptor binding at concentrations that are likely to exist in vivo.

Antiandrogenic Effects of Environmental Endocrine Disruptors

Steroid hormone receptors regulate embryonic development and sex differentiation by acting as ligand inducible transcription factors. Disrupting these processes can result in transient yet irreversible developmental defects. Several environmental chemicals have recently been identified with antiandrogen activity that have the potential to disrupt normal male sex development in utero. Detection of chemicals with the potential to disrupt normal androgen action is critical to protect human and ecological health. The molecular mechanisms by which several of these environmental chemicals act have been characterized and yielded insight into the development of in vitro and in vivo screening assays to detect chemicals with antiandrogen activity. These mechanisms and efforts to develop screening assays are discussed in the context of current endocrine disruptor screening and testing strategies.

Developmental Effects and Molecular Mechanisms of Environmental Antiandrogens

Industrial chemicals and environmental pollutants can disrupt reproductive development in wildlife and humans by altering the synthesis, transport, action, or elimination of gonadal steroid hormones. Steroid hormones control fundamental events in embryonic development and sex differentiation by binding to their cognate nuclear receptors, which act as steroid-inducible transcription factors to activate or repress transcription of target genes. The consequences of disrupting these events can be especially profound during embryonic development because the role of steroid hormones is crucial in controlling transient and irreversible developmental processes. Recent studies suggest that certain industrial pollutants and environmental pesticides have the potential to alter male sex development and reproductive processes in wildlife and human populations by acting as environmental antiandrogens (1–3). In some cases, laboratory studies have confirmed abnormalities of reproductive development observed in the field and have provided mechanisms to explain the disruptive effects of these environmental chemicals.