Transgenerational effects of Di (2-ethylhexyl) phthalate in the male CRL:CD(SD) rat: added value of assessing multiple offspring per litter.

In the rat, some phthalates alter sexual differentiation at relatively low dosage levels by altering fetal Leydig cell development and hormone synthesis, thereby inducing abnormalities of the testis, gubernacular ligaments, epididymis, and other androgen-dependent tissues. In order to define the dose-response relationship between di(2-ethylhexyl) phthalate (DEHP) and the Phthalate Syndrome of reproductive alterations in F1 male rats, Sprague-Dawley (SD) rat dams were dosed by gavage from gestational day 8 to day 17 of lactation with 0, 11, 33, 100, or 300 mg/kg/day DEHP (71-93 males per dose from 12 to 14 litters per dose). Some of the male offspring continued to be exposed to DEHP via gavage from 18 days of age to necropsy at 63-65 days of age (PUB cohort; 16-20/dose). Remaining males were not exposed after postnatal day 17 (in utero-lactational [IUL] cohort) and were necropsied after reaching full maturity. Anogenital distance, sperm counts and reproductive organ weights were reduced in F1 males in the 300 mg/kg/day group and they displayed retained nipples. In the IUL cohort, seminal vesicle weight also was reduced at 100 mg/kg/day. In contrast, serum testosterone and estradiol levels were unaffected in either the PUB or IUL cohorts at necropsy. A significant percentage of F1 males displayed one or more Phthalate Syndrome lesions at 11 mg/kg/day DEHP and above. We were able to detect effects in the lower dose groups only because we examined all the males in each litter rather than only one male per litter. Power calculations demonstrate how using multiple males versus one male/litter enhances the detection of the effects of DEHP. The results at 11 mg/kg/day confirm those reported from a National Toxicology Program multigenerational study which reported no observed adverse effect levels-lowest observed adverse effect levels of 5 and 10 mg/kg/day DEHP, respectively, via the diet.

Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats.

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.

Chronic di-n-butyl phthalate exposure in rats reduces fertility and alters ovarian function during pregnancy in female Long Evans hooded rats.

Testis function in fetal and peripubertal male rats is disrupted by subchronic exposure to phthalate esters (PEs). In contrast to the male rat, it is generally held that reproduction in female rats is much less sensitive to phthalate-induced disruption. However, the current study demonstrates that oral administration of dibutyl phthalate (DBP) to female Long Evans (LE) hooded rats from weaning, through puberty, mating, and gestation disrupts pregnancy maintenance at dose levels similar to those that affect testis function in male rats. Administration of 500 and 1000 mg DBP/kg/day, but not 250 mg DBP/kg/day, to female LE rats induced midpregnancy abortions. The percentage of females delivering live pups was reduced by more than 50% at 500 mg/kg/day and by 90% at 1000 mg/kg/day in the absence of overt toxicity, whereas the ages at vaginal opening and first estrus, estrous cyclicity, and mating indices (N mated/N paired or N pregnant/N mated) were not significantly affected. On gestational day 13, prior to the stage when litters were being aborted, ex vivo ovarian hormone production was significantly decreased by in vivo DBP treatment at 500 and 1000 mg/kg/day. These results should be considered when evaluating mechanisms of reproductive toxicity for the PE because it is likely that these reproductive alterations in the female rat arise via a mode of action similar to that operative in male rats.

Late gestational exposure to the fungicide prochloraz delays the onset of parturition and causes reproductive malformations in male but not female rat offspring.

Prochloraz (PZ) is an imidazole fungicide that displays multiple endocrine activities. It inhibits steroid synthesis via P450 modulation and acts as an androgen receptor (AR) antagonist, but its effects on male sexual differentiation have not been described. The purpose of the current study was to expand in vitro observations and to determine whether PZ affected sexual differentiation. PZ effects on AR-mediated gene expression were tested using a cell line (MDA-kb2) containing endogenous AR and stably transfected with an MMTV-luc reporter. PZ concentrations greater than 1 microM caused a dose-dependent inhibition of dihydrotestosterone-induced gene expression. PZ also inhibited R1881 binding to the rat AR (IC50 approximately 60 microM). In vivo, pregnant rats received PZ by gavage from Gestational Day 14 to 18 at doses of 31.25, 62.5, 125, and 250 mg/kg of body weight per day. PZ delayed delivery in a dose-dependent manner and resulted in pup mortalities at the two highest doses. In male offspring, anogenital distance and body weight were slightly reduced at 3 days of age. Additionally, female-like areolas were observed at 13 days of age at frequencies of 31%, 43%, 41%, and 71% in the lowest-dose to highest-dose groups, respectively. Weights of androgen-dependent tissues showed dose-dependent reductions. Hypospadias and vaginal pouches were noted in all males treated with 250 mg/kg, whereas those defects were observed in 12.5% and 6.25%, respectively, of males treated with 125 mg/kg. Treatment did not affect age of preputial separation in animals without penile malformations. Despite severe malformations in males, no malformations were noted in females. Together, these results indicate that PZ alters sexual differentiation in an antiandrogenic manner.

Hershberger assay to investigate the effects of endocrine-disrupting compounds with androgenic or antiandrogenic activity in castrate-immature male rats.

The protocol described in this unit is designed to evaluate the effects of androgenic and antiandrogenic endocrine-disrupting compounds (EDCs) in the castrate-immature male rat. Continuous 10-day exposure of the prepubertal male to chemicals is used to identify androgenic or antiandrogenic activities based on the weights of several androgen-dependent tissues on the day after treatment is ended. Androgen-dependent organ weights and growth, along with kidney, liver, and adrenal weights are measured at necropsy. The androgen-dependent tissues include the ventral prostate, seminal vesicles (with fluid plus coagulating glands), levator ani plus bulbocavernosus muscles, Cowper's glands, and glans penis. Optional measures, which facilitate interpretation of the data when positive effects are detected, include serum testosterone and luteinizing hormone levels. Androgenic chemicals cause increases in one or all of the androgen-dependent tissue weights after being administered by oral gavage or subcutaneously (s.c.) to castrate-immature male rats. Antiandrogenic chemicals cause reductions in androgen-dependent tissue weights after being coadministered with testosterone propionate (s.c.).

Phthalate ester-induced gubernacular lesions are associated with reduced insl3 gene expression in the fetal rat testis.

Targeted inactivation of the insulin-like hormone 3 (insl3) gene in male mice results in altered gubernacular development, disrupted testis decent, and cryptorchidism. Cryptorchidism is a fairly common human malformation, being displayed in about three males per 100 at birth, but only a small percentage can be linked directly to genetic defects. The phthalate esters (PEs) are high production volume, ubiquitous environmental chemicals, some of which when administered during sexual differentiation, induce male rat reproductive tract malformations including gubernacular agenesis. We hypothesized that phthalate-induced gubernacular lesions likely result from an inhibition of insl3 gene expression. Three phthalates, di-n-ethylhexyl phthalate (DEHP), dibutyl phthalate (DBP) and benzyl butyl phthalate (BBP) were administered orally to the dam on gestation day 14 through 18 (GD14-18) and the fetal testes examined on GD18 for effects on steroid hormone production and insl3 gene expression. Compared to chemicals like vinclozolin, linuron, and prochloraz that act as AR antagonists and/or inhibit fetal Leydig cell testosterone production, only the three phthalates significantly reduced both ex vivo testosterone production and insl3 gene expression when quantified by real-time rtPCR. These results provide the first demonstration of PE-induced alteration of insl3 mRNA in the fetal male rat testis.

Transgenerational (in utero/lactational) exposure to investigate the effects of endocrine disrupting compounds (EDCS) in rats.

This protocol is designed to evaluate the effects of endocrine disrupting compounds (EDCs) through fetal (transplacental) and/or neonatal (via the dam's milk) exposure during the critical periods of reproductive organogenesis in the rat. Continued direct exposure to the F1 pups after weaning is an option that can also be included. Reproductive indices, growth, and viability are monitored throughout the life of the F1 generation and an extensive necropsy is conducted after sexual maturity is attained. The endpoints evaluated are designed to detect compounds such as androgen receptor antagonists or steroid hormone synthesis inhibitors which inhibit normal development of the male reproductive system and growth stimulating hormones which masculinize females and facilitate development of male reproductive tissues. Data collected from these studies can be used to determine the no and lowest observed effects levels and predict potential adverse consequences of EDCs in human and wildlife populations. The information also provides a foundation for future studies designed to identify mechanism(s) of action, specific genes and or proteins involved, and susceptible populations these compounds might target.

Effects of prenatal testosterone propionate on the sexual development of male and female rats: a dose-response study.

Testosterone plays a major role in male sexual development. Exposure of females to testosterone in utero can induce masculine characteristics such as anovulation, increased anogenital distance (AGD), absence of nipples, retention of male-like tissues, and agenesis of the lower vagina. In addition, high levels of androgens during fetal development can lead to toxic effects such as reduced litter size and viability. The study of the effects of testosterone administration during sexual differentiation provides a foundation for understanding the effects of environmental androgens on fetuses, a sensitive subpopulation. In the current study, we investigated the ability of a range of concentrations of testosterone propionate (TP) administered prenatally to masculinize female and alter male offspring, and measured maternal and fetal T levels. Pregnant Sprague-Dawley rats were dosed by sc injection on gestational day (GD) 14-19 (GD 1= day of plug) with either corn oil (vehicle; 0.1 ml/rat) or with 0.1 ml of TP solution at 0.1, 0.5, 1, 2, 5, or 10 mg/0.1 ml. Parturition was delayed at 2, 5, and 10 mg TP, litter size was reduced at 5 and 10 mg TP, and pup weight was significantly reduced in both sexes at 0.5 mg TP and higher doses. Viability of offspring was unaffected at any dosage level. Androgenic effects seen at 0.5 mg TP in females included increased AGD at weaning and adulthood, reduced number of areolas and nipples, cleft phallus, small vaginal orifice, and presence of prostate tissue. This dose of TP elevated maternal T levels 10x but had no effect on fetal T levels. At 1 mg TP and above, female AGD on postnatal day (PND) 2 (or postcoital day 24 [gestation length = 22(1/2)]) was increased; areolas and nipples were virtually eliminated; levator ani muscle, bulbourethral glands, and seminal vesicles (2 mg TP and above) were present; none of the females developed a vaginal orifice and many females in the 1 and 2 mg TP dose groups developed a greatly distended, fluid-filled uterus after puberty. Maternal T levels at 1 mg TP were elevated 30x, and female fetal T levels showed an 80% increase. Male offspring displayed a reduced AGD and body weight on PND 2 at 0.5 mg TP and higher doses. These effects were not evident by weaning and male offspring displayed no malformations. We conclude that gestational administration of 0.5 and 1 mg TP masculinizes female offspring without greatly affecting pup viability or pregnancy of the dam. This study provides a useful model for in utero testing of environmental androgens for their potential to induce developmental abnormalities.