L.E. Gray

A Mixture of Five Phthalate Esters Inhibits Fetal Testicular Testosterone Production in the Sprague-Dawley Rat in a Cumulative, Dose-Additive Manner

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.

Xenoendocrine disrupters: laboratory studies on male reproductive effects

Wildlife from ecosystems contaminated with endocrine disrupting chemicals (EDCs) display a variety of reproductive alterations including cryptorchidism in the Florida panther, small baculum in young male otters, small penises in alligators, sex reversal in fish, and altered social behavior in birds. It has been proposed that EDCs also may have contributed to increases in testicular cancer and hypospadias and the reported decline in human sperm counts. Combined in vivo and in vitro studies are necessary to fully characterize EDC induced alterations of reproduction. We have identified several pesticides (vinclozolin, procymidone, p,p-DDE) which bind rat and human androgen receptors, block androgen-induced gene expression in vitro and in vivo, delay puberty, reduce sex accessory gland size and alter sex differentiation in the male rat. Some of the phthalates, which are estrogenic in vitro but not in vivo, cause malformations in male rats that appear to result from antagonism of androgens in utero. In contrast, xenoestrogens affect male offspring but they are not malformed or infertile. Prenatal administration of an Ah receptor agonist (2,3,7,8-TCDD or PCB 169) produces a different spectrum of effects including reduced ejaculated sperm numbers in male rats.

Cumulative effects of in utero administration of mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of toxicity.

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act required the US Environmental Protection Agency to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures of chemicals to elucidate mechanisms of joint action at the systemic level with the goal of providing a framework for assessing the cumulative effects of reproductive toxicants. Previous mixture studies conducted with antiandrogenic chemicals are reviewed briefly and two new studies are described. In all binary mixture studies, rats were dosed during pregnancy with chemicals, singly or in pairs, at dosage levels equivalent to approximately one-half of the ED50 for hypospadias or epididymal agenesis. The binary mixtures included androgen receptor (AR) antagonists (vinclozolin plus procymidone), phthalate esters [di(n-butyl) phthalate (DBP) plus benzyl n-butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) plus DBP], a phthalate ester plus an AR antagonist (DBP plus procymidone), a mixed mechanism androgen signalling disruptor (linuron) plus BBP, and two chemicals which disrupt epididymal differentiation through entirely different toxicity pathways: DBP (AR pathway) plus 2,3,7,8 TCDD (AhR pathway). We also conducted multi-component mixture studies combining several antiandrogens. In the first study, seven chemicals (four pesticides and three phthalates) that elicit antiandrogenic effects at two different sites in the androgen signalling pathway (i.e. AR antagonist or inhibition of androgen synthesis) were combined. In the second study, three additional phthalates were added to make a 10 chemical mixture. In both the binary mixture studies and the multi-component mixture studies, chemicals that targeted male reproductive tract development displayed cumulative effects that exceeded predictions based on a response-addition model and most often were in accordance with predictions based on dose-addition models. In summary, our results indicate that compounds that act by disparate mechanisms of toxicity to disrupt the dynamic interactions among the interconnected signalling pathways in differentiating tissues produce cumulative dose-additive effects, regardless of the mechanism or mode of action of the individual mixture component.

An environmental antiandrogen, vinclozolin, alters the organization of play behavior.

During mammalian sexual differentiation, the androgens, testosterone and dihydrotestosterone are critical for the organization of the male phenotype. In rats, play behavior is sexually dimorphic. Administration of exogenous androgens during the perinatal period results in masculine-like play behavior of juveniles. Recently, there has been increasing concern about the potential for environmental endocrine-disrupting chemicals (EDCs) to alter sexual differentiation in mammals. One such EDC is the fungicide and androgen receptor (AR) antagonist, vinclozolin. We tested whether developmental exposure to an EDC could alter androgen-dependent behaviors such as play. To examine this possibility, neonatal male rats were injected from Postnatal Days (PND) 2 to 3 with corn oil, pharmacological antiandrogen flutamide (50 mg/kg/day) or vinclozolin (200 mg/kg/day); whereas neonatal females were treated either with corn oil or testosterone propionate (TP, 250 microg/kg/day). At PNDs 36-37, animals were observed for social play. Behaviors associated with general social activity, such as sniffing and dorsal contact, were unaffected by treatment or sex. However, play behavior in males treated with flutamide or vinclozolin was significantly reduced to near-female levels when compared to control males. Play behavior in females exposed to TP during the neonatal period was significantly increased when compared with control females. Hence, this study suggests that perinatal exposure to vinclozolin, an environmental antiandrogen, can alter androgen-dependent behavior, such as play, in the male rat.

Cumulative and Antagonistic Effects of a Mixture of the Antiandrogens Vinclozolin and Iprodione in the Pubertal Male Rat

Vinclozolin and iprodione are dicarboximide fungicides that display antiandrogenic effects in the male rat, which suggests that a mixture would lead to cumulative effects on androgen-sensitive end points. Iprodione is a steroid synthesis inhibitor, but androgen receptor antagonist activity, which is displayed by vinclozolin, has not been fully evaluated. Here, we demonstrate that iprodione binds to the human androgen receptor (IC(50) = 86.0 microM), reduces androgen-dependent gene expression, and reduces androgen-sensitive tissue weights in castrated male rats (Hershberger assay). Since vinclozolin and iprodione affect common targets in the pubertal male rat, we tested the hypothesis that a mixture would have cumulative antiandrogenic effects. An iprodione dose, that does not significantly affect androgen-dependent morphological end points, was combined with vinclozolin doses (2 x 5 factorial design). Sprague-Dawley rats were dosed by gavage with vinclozolin at 0, 10, 30, 60, and 100 mg/kg/day with and without 50 mg iprodione/kg/day from postnatal day (PND) 23 to 55-57 (n = 8 per group). The age at puberty (preputial separation [PPS]), organ weights, serum hormones, and ex vivo testis steroid hormone production were measured. Vinclozolin delayed PPS, reduced androgen-sensitive organ weights, and increased serum testosterone. The addition of iprodione enhanced the vinclozolin inhibition of PPS (PND 47.5 vs.49.1; two-way ANOVA: iprodione main effect p = 0.0002). The dose response for several reproductive and nonreproductive organ weights was affected in a cumulative manner. In contrast, iprodione antagonized the vinclozolin-induced increase in serum testosterone. These results demonstrate that these fungicides interact on common targets in a tissue-specific manner when coadministered to the pubertal male rat.

Repeated exposure to the polychlorinated biphenyl (Aroclor 1254) elevates the basal serum levels of corticosterone but does not affect the stress-induced rise.

Previous studies indicate that repeated exposure of weanling male Fischer 344 rats to Aroclor can cause immune system alterations but the pattern of effects suggested the release of corticosteroids may have played a role. Rats were exposed daily by gastric intubation to the polychlorinated biphenyl (PCB) Aroclor 1254 at 0.1, 1.0, 10, or 25 mg/kg for exposure durations of 5, 10 or 15 weeks. By the 15th week of dosing all groups displayed an elevation in the basal level of serum corticosterone but no change in adrenal weight. Further, rats exposed to Aroclor 1254 for 15 weeks and subjected to stress prior to serum collection displayed elevations in corticosterone levels equivalent to stressed control rats. The failure to observe altered adrenal structure indicative of hyperactivity in the presence of increased serum levels of corticosterone suggest these basal increases may be indirect rather than direct effects of Aroclor 1254.

The postnatal effects of prenatal exposure to low doses of nitrogen (2,4-dichlorophenyl-p-nitrophenyl ether) in Sprague-Dawley rats☆

Nitrofen was administered to pregnant Sprague-Dawley rats by gavage on days 8-16 of gestation at 5 different dose levels--0, 0.46, 1.39, 4.17 and 12.5 mg/kg/day. Diaphragmatic hernias were found in pups that died immediately after birth at the 3 highest dose levels. At the 1.39-mg/kg dose level 3 of the 4 pups examined had diaphragmatic hernias, at the 4.17-mg/kg dose level 2 out of 3 pups had diaphragmatic hernias, and at the 12.5-mg/kg dose level all 5 pups found dead had diaphragmatic hernias. Locomotor activity of the offspring was measured on postnatal days 17 and 24, and hyperactivity was evident at the 3 highest dose levels. However, when the rats were later tested at 45, 49 and 90 days of age they had apparently recovered from this earlier hyperactivity. In the female rat, nitrofen did not delay the onset of puberty as measured by the age of vaginal opening or the age at first estrus. At necropsy of the offspring which began on postnatal day 133, Harderian gland weight reduction and hydronephrosis were seen at the 4.17- and 12.5-mg/kg dose levels, while no effects were found in body, liver, testes, seminal vesicle, kidney, or lung weights. Results of the present study and earlier studies demonstrate that rats are more sensitive than mice to the teratogenic effect of nitrofen (Gray et al., Science, 215 (1982) 293 and Gray et al., Toxicol. Appl. Pharmacol., 67 (1983) 1). In general, nitrofen affects the same organ systems in rats as it does in mice, but the rank order of sensitivity of these effects differs from those described earlier in the mouse by Gray et al. (Toxicol. Appl. Pharmacol., 67 (1983) 1).

In utero exposure to an AR antagonist plus an inhibitor of fetal testosterone synthesis induces cumulative effects on F1 male rats

Risk assessments are typically conducted on a chemical-by-chemical basis; however, many regulatory bodies are developing frameworks for assessing the cumulative risk of chemical mixtures of chemicals. The current investigation examined how chemicals that disrupt rat sex differentiation via two diverse mechanisms disrupt F1 male rat reproductive development, when administered together orally on days 14-18 of gestation. Experiment 1 used a mixture of 50 mg/kg-d procymidone and 500 mg/kg-d dibutyl phthalate (DBP), whereas experiment 2 used 150 mg/kg-d procymidone and 1125 mg/kg-d DBP (top dose), or 0, 4.17, 8.33, 16.7, 33.3, 50, 66.7, and 83.3% of the top dose. When we compared the dose and response addition predictions to the observed effects we found that dose addition models were more accurate than response addition models, indicating that compounds that act by different mechanisms of toxicity produce cumulative dose-additive effects.

Dibutyl phthalate: Maternal effects versus fetotoxicity

Dibutyl phthalate (DBP), a plasticizer, is a teratogen in mice and rabbits but produces fetal loss in the rat. Long-term dosing studies indicating reduced fertility in the rat suggested a maternal effect of the compound. The decidual cell response (DCR) and pregnant rats were used to examine whether DBP affects maternal physiological parameters independent of the compounds fetotoxic effect. DBP has no effect on the DCR, pregnant uterine weight, number of implantation sites, ovarian weight, or serum progesterone concentration during early pregnancy or pseudopregnancy. These data show that short-term dosing with DBP has no direct maternal effect in the rat and suggest that the viability of preimplantation embryos is not compromised.

High throughput adjustable 96-well plate assay for androgen receptor binding: a practical approach for EDC screening using the chimpanzee AR.

The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor.