Several members of the phthalate ester family have antiandrogenic properties, yet little is known about how exposure to these ubiquitous environmental contaminants early in development may affect sexual development. We conducted experiments to determine effects of in utero and lactational exposure to the most prevalent phthalate ester, di(2-ethylhexyl) phthalate (DEHP), on male reproductive system development and sexual behavior. Sprague-Dawley rats were dosed with corn oil or DEHP (0, 375, 750, or 1,500 mg/kg/day, per os) from gestation day 3 through postnatal day (PND) 21. Dose-related effects on male offspring included reduced anogenital distance, areola and nipple retention, undescended testes, and permanently incomplete preputial separation. Testis, epididymis, glans penis, ventral prostate, dorsolateral prostate, anterior prostate, and seminal vesicle weights were reduced at PND 21, 63, and/or 105-112. Additional dose-related effects included a high incidence of anterior prostate agenesis, a lower incidence of partial or complete ventral prostate agenesis, occasional dorsolateral prostate and seminal vesicle agenesis, reduced sperm counts, and testicular, epididymal, and penile malformations. Many DEHP-exposed males were sexually inactive in the presence of receptive control females, but sexual inactivity did not correlate with abnormal male reproductive organs. These results suggest that in utero and lactational DEHP exposure also inhibited sexually dimorphic central nervous system development. No major abnormalities were found in any of eight control litters, but DEHP caused severe male reproductive system toxicity in five of eight litters at 375 mg/kg/day, seven of eight litters at 750 mg/kg/day, and five of five litters at 1,500 mg/kg/day. These results demonstrate that the male reproductive system is far more sensitive to DEHP early in development than when animals are exposed as juveniles or adults. The effects of DEHP on male reproductive organs and sexual behaviors and the lack of significant effects on time to vaginal opening and first estrus in their littermates demonstrate that DEHP (and/or its metabolites) affects development of the male reproductive system primarily by acting as an antiandrogen. The pattern of effects of in utero and lactational DEHP exposure differed from patterns caused by other phthalate esters, and the preponderance of anterior prostate agenesis appears to be unique among all chemicals. These results suggest that DEHP acts partly by mechanisms distinct from those of other antiandrogens.

In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in wild-type but not aryl hydrocarbon receptor (AhR) null mutant C57BL/6 mice. Experiments have now been conducted to test the hypothesis that TCDD causes an AhR-dependent inhibition of the earliest visible stage of prostate development, the formation of prostatic buds by urogenital sinus (UGS) epithelium. A novel method for viewing budding was developed that uses scanning electron microscopy of isolated UGS epithelium instead of three-dimensional reconstruction of serial histological sections of intact UGS. In the initial experiment, the time course for prostatic epithelial bud formation in vehicle- and TCDD-exposed wild-type C57BL/6J mice was determined. A single maternal dose of TCDD (5 mug/kg) on gestation day 13 delayed the appearance of dorsal, lateral, and anterior buds by about one day, reduced dorsolateral bud number, and prevented ventral buds from forming. No such effects were seen in TCDD-exposed AhR null mutant fetuses, while AhR null mutation, alone, had no detectable effect on budding. Treatment of wild-type dams with sufficient 5alpha-dihydrotestosterone (DHT) to masculinize female fetuses failed to protect against the inhibition of budding caused by TCDD. These results demonstrate that in utero TCDD exposure causes an AhR-dependent inhibition of prostatic epithelial bud formation commensurate with its inhibitory effects on ventral and dorsolateral prostate development, and that the inhibition of budding is not due to insufficient DHT. Inhibited bud formation appears to be the primary cause of abnormal prostate development in TCDD-exposed mice.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1) was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to re-entrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD, 1 μg/kg BW) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

The aryl hydrocarbon receptor (AhR) is a transcription factor that mediates the inhibitory effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on prostate growth and also modulates normal prostate development. This is evidenced by AhR null mice (Ahr-/-) having smaller dorsolateral and anterior prostates, even though all prostate lobes remain histologically normal. To test the hypothesis that loss of the AhR increases the rate of prostate carcinogenesis, the incidence of macroscopic prostate tumors was determined in Ahr+/+, Ahr+/- and Ahr-/- C57BL/6J transgenic adenocarcinoma of the mouse prostate (TRAMP) mice at 35, 70, 105, 140, 175 and 210 days of age. From 140 days, prostate tumor incidence was greater in Ahr-/- (60%) and Ahr+/- (43%) mice than in Ahr+/+ mice (16%). Allele quantification did not indicate a loss of the wild-type Ahr allele in heterozygous TRAMP tumors, suggesting that tumor formation in these mice was not due to a loss of Ahr heterozygosity. Prostatic SV40 large T antigen mRNA expression and protein localization were comparable in TRAMP mice of each Ahr genotype. Prostates from all mice of each Ahr genotype were histologically indistinguishable, exhibiting diffuse epithelial hyperplasia by 105 days of age. mRNA expression and protein localization for molecular markers of neuroendocrine differentiation, including chromogranin A and neuropilin-1, were elevated in prostate tumors compared to tumor-free ventral prostates, regardless of Ahr genotype or age. Taken together, these results demonstrate that the Ahr inhibits prostate carcinogenesis in C57BL/6J TRAMP mice by interfering with neuroendocrine differentiation.

Experiments were conducted to determine the effects of aryl hydrocarbon receptor (AhR) null mutation and in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, alone and in combination, on prostate and seminal vesicle development in C57BL/6 mice. AhR heterozygous (Ahr+/-) mice were mated, and pregnant females were dosed orally on gestation day 13 with TCDD (5 microg/kg) or vehicle. Pups underwent necropsy on postnatal days (PNDs) 35 and 90. Comparison of vehicle-exposed AhR knockout (AhRKO;Ahr-/-) with wild-type (Ahr+/+) pups revealed that the AhR is necessary for normal dorsolateral prostate, anterior prostate, and seminal vesicle development but apparently not for ventral prostate development. In wild-type mice,in utero and lactational TCDD exposure reduced ventral prostate weight by 79-87% and mRNA expression for its major androgen-dependent secretory protein (MP25) by 99%. Yet high levels of mRNA for a secretory protein normally produced primarily by the lateral prostate (PSP94) were expressed. These effects were predominantly AhR dependent because TCDD had little if any effect in AhRKO mice. TCDD reduced dorsolateral prostate weight in wild-type but not AhRKO mice and had no significant effect on expression of mRNA for PSP94 or for probasin, a major androgen-dependent secretory protein. The PSP94 results suggest that TCDD may have caused a respecification of prostatic gene expression. TCDD reduced anterior prostate weight by more than half, and expression of mRNA for its major androgen-dependent secretory protein (renin-1) was greatly reduced. These effects were AhR dependent. Seminal vesicle weight was reduced by TCDD in wild-type mice but was increased in AhRKO mice on PND 35 and decreased on PND 90 (relative weight only). Androgen receptor mRNA levels were not significantly altered in any prostate lobe, and all organs appeared histologically normal in all groups. Serum testosterone concentrations were unchanged, and modest reductions in serum 5alpha-androstane-3alpha,17beta-diol concentrations could not account for the effects on sex organs. Collectively, these results indicate that the AhR signaling pathway plays a role in normal accessory sex organ development and thatin utero and lactational TCDD exposure disrupts development of these organs via spatially and perhaps temporally specific mechanisms.