No abstract

Phthalate diesters are chemicals to which humans are ubiquitously exposed. Exposure to certain phthalates during sexual differentiation causes reproductive tract malformations in male rats. In the fetal rat, exposure to the phthalates benzylbutyl phthalate (BBP), di(n)butyl phthalate (DBP), and diethylhexyl phthalate (DEHP) decreases testicular testosterone production and insulin-like 3 hormone mRNA levels. We characterized the dose-response effects of six individual phthalates (BBP, DBP, DEHP, diethyl phthalate [DEP], diisobutyl phthalate [DiBP], and dipentyl phthalate [DPP]) on gestation day (GD) 18 testicular testosterone production following exposure of Sprague-Dawley rats on GD 8-18. BBP, DBP, DEHP, and DiBP were equipotent (ED50 of 440 +/- 16 mg/kg/day), DPP was about threefold more potent (ED50 = 130 mg/kg/day) and DEP had no effect on fetal testosterone production. We hypothesized that coadministration of these five antiandrogenic phthalates would reduce testosterone production in a dose-additive fashion because they act via a common mode of toxicity. In a second study, dams were dosed at 100, 80, 60, 40, 20, 10, 5, or 0% of the mixture. The top dose contained 1300 mg of total phthalates/kg/day including BBP, DBP, DEHP, DiBP (300 mg/kg/day per chemical), and DPP (100 mg DPP/kg/day). This mixture ratio was selected such that each phthalate would contribute equally to the reduction in testosterone. As hypothesized, testosterone production was reduced in a dose-additive manner. Several of the individual phthalates and the mixture also induced fetal mortality, due to pregnancy loss. These data demonstrate that individual phthalates with a similar mechanism of action can elicit cumulative, dose additive effects on fetal testosterone production and pregnancy when administered as a mixture.

In 1991, a group of expert scientists at a Wingspread work session on endocrine-disrupting chemicals (EDCs) 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. Endocrine disruption can be profound because of the crucial role hormones play in controlling development." Since that time, there have been numerous documented examples of adverse effects of EDCs in invertebrates, fish, wildlife, domestic animals, and humans. Hormonal systems can be disrupted by numerous different anthropogenic chemicals including antiandrogens, androgens, estrogens, AhR agonists, inhibitors of steroid hormone synthesis, antithyroid substances, and retinoid agonists. In addition, pathways and targets for endocrine disruption extend beyond the traditional estrogen/androgen/thyroid receptor-mediated reproductive and developmental systems. For example, scientists have expressed concern about the potential role of EDCs in increasing trends in early puberty in girls, obesity and type II diabetes in the United States and other populations. New concerns include complex endocrine alterations induced by mixtures of chemicals, an issue broadened due to the growing awareness that EDCs present in the environment include a variety of potent human and veterinary pharmaceutical products, personal care products, nutraceuticals and phytosterols. In this review we (1) address what have we learned about the effects of EDCs on fish, wildlife, and human health, (2) discuss representative animal studies on (anti)androgens, estrogens and 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals, and (3) evaluate regulatory proposals being considered for screening and testing these chemicals.

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

1Chemicals that act as androgen receptor (AR) agonists and antagonists or inhibit fetal steroidogenesis can induce reproductive malformations in humans and laboratory animals. Several environmental chemicals disrupt development in rats and/or rabbits at fetal concentrations at, or near, exposure levels seen in some segments of the human population. In rats, fetal tissues concentrations of 1 6 2 0 p.p.m. of the DDT metabolite, p,p'-DDE, are correlated with reproductive abnormalities in male offspring. These concentrations are similar to those measured in fist-trimester human fetal tissues in the late 1960s. The pesticides vinclozolin, procymidone, linuron and DDT are AR antagonists. They reduce male rat anogenital distance, and induce areolas at relatively low dosages. Hypospadias, agenesis of the sex accessory tissues and retained nipples are seen in the middle dosages, while undescended testes and epididymal agenesis are seen in the highest doses. Phthalate esters (PE) inhibit testosterone synthesis during fetal life, but do not appear to be AR antagonists. Prenatal administration of a single low dose of dioxin (50-1000 ng TCDDkg) alters the differentiation of androgen-dependent tissues at p.p.t. concentrations, but the mechanism of action likely involves interaction with a hormone-like nuclear transcription factor, the hormone-like receptor AhR, rather than AR. p,p'-DDT and p,p'-DDE, vinclozolin and di-n-butyl phthalate affect reproductive function in rabbits when administered during prenatal and/or neonatal life. Cryptorchidism and carcinoma in situ-like (CIS) testicular lesions were seen in male rabbits treated during development with p,p'-DDT or p,p'-DDE. Extrapolation of effects from rodents to humans would be enhanced if future studies incorporate determination of tissue concentrations of the active metabolites. Knowledge of the tissue concentrations of the active toxicants also would provide an important link to in-vitro studies, which provide more useful mechanistic information when they are executed at relevant concentrations.

Antiandrogenic chemicals alter sex differentiation by several different mechanisms. Some, like flutamide, procymidone, or vinclozolin compete with androgens for the androgen receptor (AR), inhibit AR-DNA binding, and alter androgen-dependent gene expression in vivo and in vitro. Finasteride and some phthalate esters demasculinize male rats by inhibiting fetal androgen synthesis. Linuron, which is a weak competitive inhibitor of AR binding (reported Ki of 100 microM), alters sexual differentiation in an antiandrogenic manner. However, the pattern of malformations more closely resembles that produced by the phthalate esters than by vinclozolin treatment. The present study was designed to determine if linuron acted as an AR antagonist in vitro and in vivo. In vitro, we (1) confirmed the affinity of linuron for the rat AR, and found (2) that linuron binds human AR (hAR), and (3) acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR (EC(50) = 100-300 microM) and human AR (hAR) in a COS cell-binding assay (EC(50) = 20 microM). Linuron inhibited dihydrotestosterone (DHT)-hAR induced gene expression in CV-1 and MDA-MB-453-KB2 cells (EC(50) = 10 microM) at concentrations that were not cytotoxic. In short-term in vivo studies, linuron treatment reduced testosterone- and DHT-dependent tissue weights in the Hershberger assay (oral 100 mg/kg/d for 7 days, using castrate-immature-testosterone propionate-treated male rats; an assay used for decades to screen for AR agonists and antagonists) and altered the expression of androgen-regulated ventral prostate genes (oral 100 mg/kg/d for 4 days). Histological effects of in utero exposure to linuron (100 mg/kg/d, day 14-18) or DBP (500 mg/kg/d, day 14 to postnatal day 3) on the testes and epididymides also are shown here. Taken together, these results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action.

Many chemicals released into the environment display estrogenic activity including the oral contraceptive ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). EE2 is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and central nervous system (CNS) as an area of "some concern," whereas most effects were rated as of "negligible" or "minimal" concern. However, the number of robust studies in this area was limited. The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well-characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female Long-Evans rat offspring. Pregnant rats were gavaged with vehicle, EE2 (0.05-50 microg/kg/day), or BPA (2, 20, and 200 microg/kg/day) from day 7 of gestation to postnatal day (PND) 18, and the female offspring were studied. EE2 (50 microg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external genitalia (5 microg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 microg/kg) and absence of lordosis behavior (15 microg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat.

In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14-18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.