Preputial Separation Research Articles

Effects of in utero through lactational exposure to dicyclohexyl phthalate and p,p′-DDE in Sprague–Dawley rats

Anti-androgenic chemicals alter sexual differentiation by a variety of mechanisms, and the mechanisms between phthalate esters and p,p'-DDE are considered to be different. We performed an in utero through lactational exposure assay using dicyclohexyl phthalate and p,p'-DDE to investigate the sexual differentiation of these chemicals. Pregnant CD (SD) IGS rats were given dicyclohexyl phthalate or p,p'-DDE orally from gestational day (GD) 6 to postnatal day (PND) 20, and the endocrine-mediated effects in dams and their offspring were examined. The reproductive performance of offspring was also examined. The doses of dicyclohexyl phthalate were 0, 20, 100, and 500 mg/kg/day, and those of p,p'-DDE were 5, 15, and 50mg/kg/day. Using the dicyclohexyl phthalate, a dam in the 500 mg/kg group showed dystocia and died. The viability index of offspring on PND 4 decreased in the 500 mg/kg group. Prolonged preputial separation, reduced ano-genital distance, increased areolas/nipple retention, hypospadia, decreased ventral prostate and levator ani/bulbocavernosus muscle weights and decreased testicular germ cells were observed in male offspring in the 500 mg/kg group. In the assay using p,p'-DDE, decreased viability index of offspring on PND 21, prolonged preputial separation in male offspring and early vaginal opening in female offspring were observed in the 50mg/kg group. The copulation and fertility indices decreased in the reproductive performance of offspring in the 50mg/kg group. The endocrine-mediated effects were detected in offspring of dams given 100mg/kg dicyclohexyl phthalate, and in offspring of dams given 20mg/kg p,p'-DDE. Our results suggest that the in utero through lactational exposure assay is a useful method to detect endocrine-mediated effects and that further comparative study between this assay and two-generation reproductive test are necessary when this assay becomes one of the definitive tests.

P23: Histopathological assessment of juvenile rat tissues treated with the food contaminant semicarbazide: Preliminary findings

Semicarbazide (SEM) is a food contaminant, by-product of azodicarbonamide sealing for gaskets of jar lids for baby foods. Toxicological data on SEM are insufficient; studies in rodents showed osteolathyrogenic effects in foetuses and potential carcinogenicity, but not genotoxicity. To characterize hazards for children, juvenile Sprague-Dawley rats of both sexes are exposed po to SEM from PND 23 to PND 60 at dose levels of 0 (vehicle only – distilled water), 40, 75 and 140 mg/kg bw day. During the treatment, body weight gain and food consumption are recorded; vaginal opening and preputial separation are observed to evaluate the development of reproductive system. At sexual maturity rats are sacrificed and tissues/organs are collected for histological analysis, with attention to endocrine, immune and skeletal systems: thyroid, adrenals, testes, spleen, uteri and ovaries, thymus, coxal-femoral joint. Body weight gain is reduced in males at 75–140 mg/kg. Food consumption is reduced in both sexes at 140 mg/kg and increased in females at 75 mg/kg. Vaginal opening is delayed in all treated groups; preputial separation is anticipated at 40 and 75 mg/kg and delayed in the high dose group. Histology of thyroid showed in all treated males increased desquamation in follicular lumen; in females this is present only at 140 mg/kg. Adrenal alterations are present in females only: cortex vacuolization (all dose groups) and blurred zonation architecture (high dose only), in medulla increased cell degeneration (high dose). In the spleen, increased haematopoiesis is present in males (high dose), with a dose-related trend at mid-dose. Such preliminary observations show alterations in endocrine and immune tissues. Altered timing in sexual maturation, adrenal cortex and thyroid effects are present also at 40 mg/kg, without evident general toxicity, supporting the need for toxicological studies on food and environmental contaminants targeted to juvenile lifestages.

Reproductive Effects of Di(2-ethylhexyl)phthalate in Immature Male Rats and Its Relation to Cholesterol, Testosterone, and Thyroxin Levels

Phthalates are chemicals employed in several industrial products and there is a growing body of evidence demonstrating that they induce numerous adverse effects on the reproductive system. This study was carried out to assess possible alterations induced by the plasticizer di(2-ethylhexyl phthalate (DEHP) on cholesterol, testosterone, and thyroxine (total T4) levels, as well as to discuss the significance of these data in global changes observed in the reproductive tract of pubertal animals. Wistar rats aged 21 days received DEHP orally at 0, 250, 500, and 750 mg/kg/day for 30 days and were examined for different reproductive endpoints. At the end of the treatment, significant decreases in relative weight of testosterone-dependent organs, delayed preputial separation, and low serum testosterone were observed at the highest DEHP dose. The plot of the relationship between DEHP dose and serum cholesterol revealed a biphasic effect. The concentration of cholesterol in serum was significantly reduced at 250 mg/kg/day DEHP but returned to control values at 750 mg/kg/day. Cholesterol levels measured in testicular tissue increased with DEHP treatment. Serum T4 levels were not affected by DEHP at any dose, indicating the absence of a link between total thyroxin concentration and phthalate effects on cholesterol levels. Taken together these results indicate that effects observed in serum and testicular cholesterol levels may reflect distinct effects of DEHP on cholesterol synthesis and usage. These results confirm and extend previously reported findings showing that alterations in cholesterol balance may play a role in the suppression of steroidogenesis induced by DEHP in rats.

Sex differences in effects on sexual development in rat offspring after pre- and postnatal exposure to triphenyltin chloride

Consumers are exposed to organotin compounds (OTCs) via contaminated fish and seafood due to the accumulation of these compounds in marine organisms. Certain OTCs are immunotoxic and may also have endocrine disrupting properties resulting in adverse effects on the reproductive tract in mollusks and mammals. Since effects of in utero exposure to endocrine disrupting chemicals on the reproductive system are dependent on the critical window of exposure during its development, we conducted a comprehensive study with the aim to identify the most sensitive window of exposure to TPTCl and to investigate the effects of pre- and postnatal treatment on sexual development in rats. Male and female offspring rats were exposed to 2 or 6mg TPTCl/kg b.w. and day either in utero and during lactation (gestation day 6 until weaning on PND 21) or from gestation day 6 until termination. As previously reported, offspring in the 6mg TPTCl dose group exhibited high perinatal mortality and therefore no further evaluation was carried out at this dose level (Grote, K., Hobler, C, Andrade, A.J.M., Wichert Grande, S., Gericke, C., Talsness, C.E., Appel, K.E., Chahoud, I., 2007. Effects of in utero and lactational exposure to triphenyltin chloride on pregnancy outcome and postnatal development in rat offspring. Toxicology 238, 177–185). In the present paper, results on postnatal development obtained from surviving offspring of dams exposed to 2mg TPTCl/kg b.w. are reported. Male offspring were sacrificed on PND 64 or 65 and female offspring at first estrus after PND 58. A clear sex difference in response to treatment was observed. Male postnatal development was severely affected with decreases in body weight gain, reproductive organ weights and testosterone concentration as well as a significant delay in the age at preputial separation. In contrast, females exhibited a precocious completion of vaginal opening while all other endpoints were unaffected. Most of these effects were already present in animals that were only exposed until weaning indicating that these effects may be irreversible and continued treatment until termination had contributed less than expected to the severity of the observed effects. The results of the present study suggest that the sensitive window for the evaluated endpoints seems to be the period of prenatal development and that male offspring rats were more susceptible to treatment.

A two-generation oral gavage reproduction study with potassium perfluorobutanesulfonate (K +PFBS) in Sprague Dawley rats

Perfluorobutanesulfonate (PFBS) is a surfactant and degradation product of substances based on perfluorobutanesulfonyl fluoride. A two-generation reproductive rat study has been conducted with potassium PFBS (K +PFBS). Parental-generation (P) rats were dosed orally by gavage with 0, 30, 100, 300 and 1000 mg K +PFBS/kg/day for 10 weeks prior to and through mating (males and females), as well as during gestation and lactation (females only). First generation (F1) pups were dosed similarly, beginning at weaning. Second generation (F2) pups were not directly dosed but potentially exposed to PFBS through placental transfer and nursing, and the study was terminated 3 weeks after their birth. Endpoints evaluated included body weight, food consumption, clinical signs, estrus cycling, sperm quality, pregnancy, natural delivery, litter outcomes, and developmental landmarks. The no-observable-adverse effect dose level (NOAEL) in the parental generations (P and F1) was 100 mg/kg/day. In the 300 and 1000 mg/kg/day dose group rats, there were (1) increased liver weight (absolute or relative) and corresponding increased incidence of adaptive hepatocellular hypertrophy (male only) and (2) increased incidence of minimal to mild microscopic findings in the medulla and papilla of the kidneys (male and female). There were no K +PFBS treatment-related effects on fertility or reproduction among the P or the F1 rats. There were no microscopic changes in male or female reproductive organs, and no biologically relevant effects on sperm parameters, mating, estrous cycles, pregnancy, and natural delivery in the P- or F1-generations. There were no K +PFBS treatment-related effects on survival of pups in the two-generation study. Litter size and average pup birth weight per litter were not statistically significantly different from controls in any dose group. In the F1-generation, terminal body weight was reduced in males at 1000 mg/kg/day. Preputial separation was slightly delayed (approximately 2 days) at this dose, a finding consistent with the body weight reduction. Essentially no effects were observed in the F1 females. F2 pups had normal body weights. The reproductive NOAEL was >1000 mg/kg/day in both generations.

A exposição ao glifosato-Roundup causa atraso no início da puberdade em ratos machos

The effects of herbicides and it's adjuvants can be responsible for several alterations in reproductive organs of male and female. Since glyphosate-Roundup penetrates in the cell, it reduces the activity of StAR protein and aromatase enzime. This study evaluated the development of pubertal characteristics in animals receiving oral dosages of glyphosate-Roundup by gavage, by daily body weight measurement and verification of preputial separation (PPS). 32 male rats were allocated in 4 groups of treatments (0, 5, 50 e 250 mg/kg) from 23 to 53 days of age. The body weight from 23 to 53 days was compared using analysis of MANCOVA, Kruskall-Wallis was used to compare the day at PPS and ANOVA was used to compare the body weight at PPS. Age at PPS was different between groups 50 mg/kg and 0 mg/kg (36,6±0,5; 36 days; p<0,05), 250 mg/kg and 0 mg/kg (37,2±0,4; 36 days; p<0,001) and 5 mg/kg and 250 mg/kg (36,5±0,5; 36 days; p<0,05). The body weight at PPS was higher in group of 250 mg/kg n comparison to groups of 0 mg/kg and 5 mg/kg (142,7 ± 9,3; 128,9 ± 5,4; 126,1 ± 8,8 g, respectively; p<0,001). The body weight of group 50 mg/kg (134,1 ± 9,2g) wasn't differ from another groups. Probably, the daily exposure to glyphosate-Roundup causes endocrine disruption in hypothalamic-pituitary-gonad axis during sexual maturation, because it delayed the onset of puberty.

The Effects of Triclosan on Puberty and Thyroid Hormones in Male Wistar Rats

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a potent antibacterial and antifungal compound that is widely used in personal care products, plastics, and fabrics. Recently triclosan has been shown to alter endocrine function in a variety of species. The purpose of this study was to determine effects of triclosan on pubertal development and thyroid hormone concentrations in the male rat. Weanling rats were exposed to 0, 3, 30, 100, 200, or 300 mg/kg of triclosan by oral gavage from postnatal day (PND) 23 to 53. Preputial separation (PPS) was examined beginning on PND 33. Rats were killed on PND 53, organ weights were recorded and serum was collected for subsequent analysis. Triclosan did not affect growth or the onset of PPS. Serum testosterone was significantly decreased at 200 mg/kg, however no effects were observed on androgen-dependent reproductive tissue weights. Triclosan significantly decreased total serum thyroxine (T4) in a dose-dependent manner at 30 mg/kg and higher (no observed effect level of 3 mg/kg). Triiodothyronine (T3) was significantly decreased only at 200 mg/kg, but thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly increased at 100 mg/kg triclosan and above suggesting that the induction of hepatic enzymes may have contributed to the altered T4 and T3 concentrations, but it does not appear to correlate with the T4 dose-response. This study demonstrates that triclosan exposure does not alter androgen-dependent tissue weights or onset of PPS; however, triclosan exposure significantly impacts thyroid hormone concentrations in the male juvenile rat.

Diisobutyl phthalate impairs the androgen-dependent reproductive development of the male rat

Diisobutyl phthalate (DIBP) is the branched isomer of DBP; DBP side chains have a four-carbon backbone (C4), whereas DIBP has its four-carbon alkyl side chains rearranged into a three-carbon backbone (C3) with a methyl branch. Di- n-butyl phthalate (DBP), and several other ortho-phthalate esters with side-chain lengths of C4–C6, are known to disrupt the androgen-dependent sexual differentiation in the male rat. This study was performed to determine whether in utero exposure to DIBP would induce permanent and dose-responsive alterations of male reproductive development. Pregnant Sprague–Dawley rats were administered olive oil (vehicle control), DIBP or DBP, by gavage on gestation Days 12–21, at doses of 125, 250, 500, 625 mg DIBP/(kg day) and 500 mg DBP/(kg day). DIBP caused no overt maternal toxicity, nor reduced litter size. Male offspring displayed reduced neonatal anogenital distance (Postnatal day 1, PND) at 250 mg DIBP/(kg day) and higher doses, and dose-related retention of areolas/nipples (PND 12–14). Preputial separation (onset of puberty) was delayed in male offspring at 500 and 625 mg DIBP/(kg day). Hypospadias, cleft prepuce, and undescended testis were observed in males (11–12 or 16–17 weeks old) exposed in utero to 500 and 625 mg DIBP/(kg day). Histopathological lesions were also present in adult testes, mainly consisting in seminiferous tubule degeneration. Our results show that DIBP can cause severe and specific adverse effects on the male rat reproductive development, with a pattern similar to that of DBP. However, DIBP appeared slightly less potent than DBP in inducing malformations.

Gestational Exposure to Atrazine: Effects on the Postnatal Development of Male Offspring

Atrazine is an herbicide used worldwide to control grasses and weeds. Previous studies have shown that, depending on atrazine's administered dose, exposure of male rats during the early postnatal or peripubertal periods can result in alterations in endocrine function. The gestational period is particularly vulnerable to environmental agents; however, the possible effects of atrazine exposure during this period have received only limited attention. Herein we examine the dose effects of atrazine exposure during Sprague-Dawley rat gestation on the postnatal development of male offspring. Pregnant dams were treated by oral gavage with atrazine at 0 to 100 mg/kg/d from gestational day 14 to parturition. Thereafter, neither the pups nor the dams received atrazine. Atrazine had no effect on the number of live births per dam. Neonatal pup survival was affected, however, with increased pup death seen at doses of 10 mg/kg/d and higher. There was no effect of atrazine on the testosterone concentration within the testes of newborn pups. Anogenital distance, an androgen-dependent process, decreased from the control level at the 75 and 100 mg/kg/d doses, with the decrease reaching significance at 100 mg/kg/d. Preputial separation, also an androgen-dependent process, was delayed significantly compared with that in controls in response to the 50 and 100 mg/kg/d doses. At postnatal day 60, serum testosterone concentrations were reduced significantly from controls in the 50 to 100 mg/kg/d groups. However, these decreases had little effect on seminal vesicle or ventral prostate weights. These results, taken together, are suggestive of antiandrogenic effects of gestational atrazine exposure on male offspring, although for most parameters, the doses used in this study are unlikely to be experienced under any but experimental conditions.

Two-Generation Reproductive Toxicity Study of Dietary Bisphenol A in CD-1 (Swiss) Mice

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

Two-Generation Reproductive Toxicity Evaluation of Dietary 17β-Estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) Mice

No information exists on reproductive/developmental effects in mice exposed to dietary 17beta-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm ( approximately 0, 0.2, 1, 10, 30, or 100 mug E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, approximately 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 ( approximately 1 mug/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 mug/kg/day.

One-generation reproductive toxicity study of dietary 17β-estradiol (E2; CAS No. 50-28-2) in CD-1 ® (Swiss) mice

There is no information on reproductive/developmental effects in mice from dietary estrogen. Therefore, 10 adult CD-1 ® mice/sex/group were administered dietary 17β-estradiol (E2) at 0, 0.005, 0.05, 0.5, 2.5, 5, 10, and 50 ppm for 2-week prebreed, mating, gestation, lactation. F1 weanlings (3/sex/litter) were necropsied and 2/sex/litter were retained, with exposure, until vaginal patency (VP) or preputial separation (PPS) and then necropsied. Results included complete infertility at 2.5–50 ppm with normal mating indices. At 0.5 ppm (and above), F0 adult female uterus plus cervix plus vagina weights (UCVW) were increased. At 0.5 ppm: prolonged gestational length; increased F1 stillbirth index; reduced live birth index and litter size; decreased testes and epididymides weights at weaning; unaffected AGD on pnd 0 and 21; delayed PPS; increased undescended testes; unaffected prostate weight; accelerated VP; enlarged vaginas; fluid-filled uteri. At 0.05 ppm: no F0 reproductive effects, increased F1 weanling UCVW; delayed PPS. The NOEL was 0.005 ppm (∼1 μg/kg/day).

Two-generation reproductive toxicity study of the rubber accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide in rats

Male and female Crl:CD(SD) rats were fed a diet containing rubber accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation, and lactation periods for two generations. At 4500 ppm, decreases in the body weight, body weight gain, and food consumption were found in F0 males and females. No changes in the estrous cyclicity, copulation index, fertility index, gestation index, delivery index, number of implantations, precoital interval, or gestation length were observed in any generation at any dose of DCBS. Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation. A transient change in performance in a water-filled multiple T-maze was found at 600 and 4500 ppm in F1 females. There were no compound-related changes in number of pups delivered, sex ratio of pups, viability of pups, anogenital distance, surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna unfolding, incisor eruption, or eye opening in the F1 and F2 generations. The body weight of F1 and F2 male and female pups was lowered at 4500 ppm. Reduced uterine weight of the weanlings was noted in the F1 generation at 4500 ppm and in the F2 generation at 600 and 4500 ppm. The data indicate that the NOAEL of DCBS for two-generation reproductive toxicity is 80 ppm (5.2 mg/kg bw per day) in rats.

Iprodione delays male rat pubertal development, reduces serum testosterone levels, and decreases ex vivo testicular testosterone production

Iprodione (IPRO) is a dichlorophenyl dicarboximide fungicide similar to procymidone and vinclozolin. All three of these fungicides induce Leydig cell tumors in the rat testis in long-term studies and an endocrine mode of action has been hypothesized to mediate this effect. Although both procymidone and vinclozolin antagonize the androgen receptor (AR) in vitro and in vivo, IPRO does not appear to be an AR antagonist. We proposed that pubertal exposure to IPRO would delay male rat pubertal development and reduce testosterone production within the testis. Sprague–Dawley weanling rats were dosed by gavage with 0, 50, 100, or 200 mg/kg /day of IPRO from post-natal day (PND) 23 to 51/52. The onset of puberty (progression of preputial separation (PPS)) was measured starting on PND 37. Organ weights, serum hormones, and ex vivo testis steroid hormone production under stimulated (+human chorionic gonadotropin (hCG)) and unstimulated (−hCG) conditions were measured at necropsy. IPRO delayed PPS at 100 and 200 mg/kg/day and decreased androgen sensitive seminal vesicle and epididymides weights at 200 mg/kg/day. Furthermore, IPRO increased adrenal and liver weights at 200 mg/kg/day, presumably by different mechanism(s) of action. Serum testosterone levels were decreased along with serum 17α-hydroxyprogesterone and androstenedione whereas serum LH was unaffected. IPRO reduced ex vivo testis production of testosterone and progesterone. Taken together, these results suggest that IPRO affects steroidogenesis within the testis, not through disruption of LH signaling, but possibly through enzyme inhibition of the steroidogenic pathway before CYP17. These data, along with the reported failure of IPRO to elicit an AR antagonism in vitro, provide evidence that IPRO differs from the dicarboximides procymidone and vinclozolin in that the effects on male rat pubertal development result from an inhibition of steroidogenesis and not AR antagonism.

Biphasic Effects of Postnatal Exposure to Diethylhexylphthalate on the Timing of Puberty in Male Rats

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP), which are commonly found in cosmetics and in flexible plastics distributed by the food, construction, and medical products industries, have been classified as anti-androgens. High-dose DEHP exposure in utero is associated with decreased androgen levels. However, when administered after birth, low doses of DEHP (eg, 10 mg/kg body weight) may stimulate androgen production. In the present study, the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 +/- 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 +/- 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 +/- 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 +/- 0.37 ng/mL) and seminal vesicle weights (0.33 +/- 0.02 g) compared with control serum T (1.98 +/- 0.20 ng/mL) and seminal vesicle weight (0.26 +/- 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 +/- 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 microM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels.

EFFECT OF 2,4-D EXPOSURE ON PUBERTAL DEVELOPMENT AND THYROID FUNCTION IN THE MALE WISTAR RAT

2,4-Dichlorophenoxyacetic acid (2,4-D) is used extensively in agriculture as a herbicide and has been suspected to have endocrine disrupting activity. Because 2,4-D has been reported to alter CNS monoaminergic function, we hypothesized that it may also modify hypothalamic regulation of pituitarygonadal function. Therefore, we evaluated the effects of 2,4-D on pubertal development and thyroid function in the juvenile male Wistar rat. The rats were exposed to 0, 100 or 200 mg/kg of 2,4-D by oral gavage from postnatal day (PND) 23 to 53. Preputial separation (PPS) was evaluated beginning on PND 33. The rats were necropsied on PND 53 and tissue weights recorded. Serum was collected at necropsy and analyzed for thyroid, testicular and pituitary hormone concentrations. PPS was significantly delayed for 2.7 days in the 200 mg/kg group as compared to controls. However, no effect on PPS was observed at 100 mg/kg. The mean weight of the ventral prostate and levator ani plus bulbocavernosus muscles (LABC) was significantly decreased in the 200 mg/kg group. Testosterone and androstenedione were significantly decreased following exposure to the highest dose of 2,4-D, but luteinizing hormone and prolactin were not altered at either dose. Triiodothyronine (T3) and thyroxine (T4) were significantly decreased at both 100 and 200 mg/kg of 2,4-D. The altered pubertal onset and reduced growth of the androgen-dependent tissues appear to be the result of a suppression of the androgenic hormones. Further studies are needed to determine if the pubertal alterations are due to changes in the CNS regulation of pituitarygonadal function or to direct effects on testicular function. This abstract does not necessarily reflect EPA policy. (platform)

IPRODIONE DELAYS MALE RAT PUBERTAL DEVELOPMENT, REDUCES SERUM TESTOSTERONE, AND DECREASES EX VIVO TESTICULAR TESTOSTERONE PRODUCTION

Iprodione (IPRO) is a dichlorophenyl dicarboximide fungicide similar to procymidone and vinclozolin, which are both known to antagonize the androgen receptor (AR). Based upon a pilot study, we hypothesized that pubertal exposure to IPRO would delay male rat pubertal development and reduce testosterone production within the testis. Sprague-Dawley weanling rats were dosed by gavage with 0, 50, 100, or 200 mg/kg/day of IPRO from postnatal day (PND) 23 to 51/52. The progression of preputial separation (PPS) was measured starting at PND 37. Organ weights, serum hormones, and ex vivo testis production under stimulated (+human chorionic gonadotropin (hCG)) and unstimulated (−hCG) conditions were measured at necropsy. IPRO delayed PPS at 100 and 200 mg/kg/day and decreased androgen sensitive seminal vesicle and epididymides weights. Furthermore, IPRO increased adrenal and liver weights. Serum testosterone levels were decreased along with serum progesterone and 17alphahydroxyprogesterone whereas serum LH was unaffected. IPRO reduced ex vivo testis production of testosterone, progesterone, and 17alphahydroxyprogesterone. Taken together, these results suggest that IPRO affects steroidogenesis within the testis, not through disruption of LH signaling, but possibly through inhibition of CYP11A (P450scc), Steroidogenic Acute Regulatory protein (StAR), or at some other point in the steroidogenic pathway before CYP17. These data, along with the reported failure of IPRO to elicit an AR antagonism response, provide evidence that IPRO differs from the dicarboximides procymidone and vinclozolin in that the effects on male rat pubertal development results from an inhibition of steroidogenesis and not AR antagonism. CRB supported through USEPA/NCSU Cooperative Training Agreement. This abstract does not necessarily reflect US EPA policy. (poster)

Kiss1 −/− Mice Exhibit More Variable Hypogonadism than Gpr54−/− Mice

The G protein-coupled receptor Gpr54 and its ligand metastin (derived from the Kiss1 gene product kisspeptin) are key gatekeepers of sexual maturation. Gpr54 knockout mice demonstrate hypogonadotropic hypogonadism, but until recently, the phenotype of Kiss1 knockout mice was unknown. This report describes the reproductive phenotypes of mice carrying targeted deletions of Kiss1 or Gpr54 on the same genetic background. Both Kiss1 and Gpr54 knockout mice are viable but infertile and have abnormal sexual maturation; the majority of males lack preputial separation, and females have delayed vaginal opening and absence of estrous cycling. Kiss1 and Gpr54 knockout males have significantly smaller testes compared with controls. Gpr54 knockout females have smaller ovaries and uteri than wild-type females. However, Kiss1 knockout females demonstrate two distinct phenotypes: half have markedly reduced gonadal weights similar to those of Gpr54 knockout mice, whereas half exhibit persistent vaginal cornification and have gonadal weights comparable with those of wild-type females. FSH levels in both Kiss1 and Gpr54 knockout males and females are significantly lower than in controls. When injected with mouse metastin 43-52, a Gpr54 agonist, Gpr54 knockout mice fail to increase gonadotropins, whereas Kiss1 knockout mice respond with increased gonadotropin levels. In summary, both Kiss1 and Gpr54 knockout mice have abnormal sexual maturation consistent with hypogonadotropic hypogonadism, although Kiss1 knockout mice appear to be less severely affected than their receptor counterparts. Kiss1 knockout females demonstrate a bimodal phenotypic variability, with some animals having higher gonadal weight, larger vaginal opening, and persistent vaginal cornification.

Effects of maternal nuclear genome on the timing of puberty in mice offspring

The timing of puberty is a complex trait which is regulated by environmental and genetic factors, but the detailed regulatory mechanism remains elusive. Maternal nutrition administration during late gestation in rats revealed that the time of onset of puberty in daughter rats was influenced by the mother's nutritional and physiological status during the embryonic development period. In this study, the potential effects of the maternal nuclear genome on the timing of puberty of offspring were investigated. Two inbred strains of mice (C3H/HeJ (C3H) and C57BL/6J (B6)) were used to set up two pedigrees (direct and reciprocal crosses), and the timing of puberty in all these mice (parent, F1 and F2) was recorded (the females were assessed by vaginal opening (VO) and the males by balano preputial separation (BPS)). The results from data of 822 mice showed that: 1) in female mice, the heritability of the timing of puberty in direct and reciprocal crosses is 68.51% and 63.97% respectively; 2) in female mice, a significant difference in the timing of puberty is observed between B6 and C3H (P = 3.7 x 10(-13)) mice as well as between direct and reciprocal F1 hybrids (P = 5.4 x 10(-3)), but not between direct and reciprocal F2 hybrids (P = 0.0941); 3) in male mice, direct and reciprocal F1 hybrids differ significantly from each other in the timing of BPS (P = 2.7 x 10(-7)), while such differences vanish in their male progenitor and progeny. The significant discrepancy between direct and reciprocal crosses in F1 but not in either cross of F2 hybrids reveals that the maternal nuclear genome has effects on the timing of puberty in mice progeny, probably through imprinting genes or the genes associated with intra-uterine physiological status.

Prochloraz Inhibits Testosterone Production at Dosages below Those that Affect Androgen-Dependent Organ Weights or the Onset of Puberty in the Male Sprague Dawley Rat

Prochloraz (PCZ) is an imidazole fungicide that inhibits gonadal steroidogenesis and antagonizes the androgen receptor (AR). We hypothesized that pubertal exposure to PCZ would reduce testosterone production and delay male rat reproductive development. Sprague Dawley rats were dosed by gavage with 0, 31.3, 62.5, or 125 mg/kg/day of PCZ from postnatal day (PND) 23 to 42 or 51. There was a significant delay in preputial separation (PPS) at 125 mg/kg/day PCZ and several of the androgen-dependent organ weights were decreased significantly, but the significant organ weight effects were not consistent between the 2 necropsies (PND 42 vs. 51). At both ages, serum testosterone levels and ex vivo testosterone release from the testis were significantly decreased whereas serum progesterone and 17alpha-hydroxyprogesterone levels were significantly increased at dose levels below those that affected PPS or reproductive organ weights. The hormone results suggested that PCZ was inhibiting CYP17 activity. In a second pubertal study (0, 3.9, 7.8, 15.6, 31.3, or 62.5 mg/kg/day PCZ), serum testosterone levels and ex vivo testosterone production were significantly reduced at 15.6 mg/kg/day PCZ. In order to examine the AR antagonism effects of PCZ, independent of its effects on testosterone synthesis, castrated immature male rats were dosed with androgen and 0, 15.6, 31.3, 62.5, or 125 mg/kg/day PCZ for 10-11 days (Hershberger assay). In this assay, androgen-sensitive organ weights were only significantly decreased at 125 mg/kg/day PCZ. These data from the pubertal assays demonstrate that PCZ decreases testosterone levels and delays rat pubertal development, as hypothesized. However, the fact that hormone levels were affected at dosage eightfold below that which delayed the onset of puberty suggests that rather large reductions in serum testosterone may be required to delay puberty and consistently reduce androgen-dependent tissue weights.