A two-year toxicology study of bisphenol A (BPA) in Sprague-Dawley rats: CLARITY-BPA core study results.

We report the data from the guideline-compliant two-year toxicology study conducted as part of the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA). BPA (0, 2.5, 25, 250, 2,500, and 25,000 µg/kg body weight (bw)/day) was administered daily by gavage in 0.3% carboxymethylcellulose vehicle to NCTR Sprague-Dawley rats from gestation day 6 through the start of parturition and then directly to pups from the day after birth until postnatal day 21 (stop-dose arm) or continuously until termination at one or two years. The stop-dose arm was included to assess the potential for any BPA effects that were due to developmental exposure. No BPA-related effects were evident in the in-life and non-histopathology data. Neoplastic and nonneoplastic lesions diagnosed in both females and males were common age-associated lesions that were variable across control and BPA-treated groups. The lack of consistent responses within the continuous- and stop-dose arms within and across tissues brought into question the plausible relationship of most of these lesions to BPA treatment. There was a possible relationship between the increased incidences of lesions in the female reproductive tract and the male pituitary and exposure to the 25,000 µg BPA/kg bw/day dose level.

NTP toxicity report of reproductive dose range-finding study of Genistein (CAS No. 446-72-0) administered in feed to Sprague-Dawley rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A series of short-term studies with genistein was conducted with two goals: 1) to obtain data necessary to establish dose levels for subsequent multigeneration reproductive and chronic toxicity studies and 2) to evaluate the effects of genistein on endpoints outside the reproductive tract. The data generated from these studies have been reported previously in the peer-reviewed literature or in technical reports (Appendix C). In addition, selected data from these studies were analyzed and discussed in the National Toxicology Program's Report of the Endocrine Disruptors Low-Dose Peer Review (NTP, 2001). The present report focuses on the reproductive and general toxicology endpoints evaluated. Data obtained in separate evaluations of behavioral, neuroanatomical, neurochemical, and immunological endpoints, as well as the assessment of serum genistein levels, are also discussed to put in better perspective the selection of doses for the multigenerational and chronic studies. Genistein was administered in an irradiated soy- and alfalfa-free diet (Purina 5K96) at exposure concentrations of 0, 5, 25, 100, 250, 625, or 1,250 ppm to 10 vaginal plug-positive, female Sprague-Dawley rats starting on gestation day 7 and continuing throughout pregnancy. These dietary exposure concentrations resulted in ingested doses of approximately 0.3, 1.7, 6.4, 16, 38, and 72 mg genistein/kg body weight to dams in the 5, 25, 100, 250, 625, and 1,250 ppm groups, respectively. Dietary exposure of the dams continued through lactation, during which time ingested doses were approximately 0.6, 3.5, 14, 37, 84, and 167 mg/kg per day. Pups from five litters, culled to eight per litter with an equal sex distribution on postnatal day (PND) 2, were maintained on the same dosed feed as their mothers after weaning until sacrifice at PND 50. Ingested doses were approximately 0.6, 3, 11, 29, 69, and 166 mg/kg per day for male pups and 0.6, 3, 12, 31, 73, and 166 mg/kg per day for female pups. Body weight and feed consumption of the treated dams prior to parturition showed decreasing trends with increasing dose, and both parameters were significantly less than those of the controls in the 1,250 ppm group. A significant exposure concentration-related effect on litter birth weight was observed, but no exposed group differed significantly from the control group in pairwise comparisons. Pups in the 1,250 ppm group had significantly decreased body weights relative to controls at the time of sacrifice (males, 9% decrease; females, 12% decrease). The most pronounced organ weight effects in the pups were decreased ventral prostate weight (absolute weight, 28% decrease; relative weight, 20% decrease) in males at 1,250 ppm and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at exposure concentrations greater than 250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at exposure concentrations of 25 ppm or greater for hypertrophy and 250 ppm or greater for hyperplasia. Abnormal cellular maturation (mucocyte metaplasia) in the vagina was observed at 625 and 1,250 ppm, and abnormal ovarian antral follicles were observed at 1,250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1,250 ppm. Histologic evaluation indicated a deficit of sperm in the epididymis at 625 and 1,250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these exposure concentrations. Control females showed a high incidence of renal tubule mineralization, and the severity of this lesion was significantly increased at exposure concentrations of 250 ppm or greater. Males showed no renal tubule mineralization below 250 ppm, but incidence and severity increased with increasing exposure concentration at 250 ppm and greater. The primary goal of the current study was to provide information for the selection of exposure concentrations to be used in subsequent multigenerational and chronic studies. These long-term studies were designed to address multiple aspects of the endocrine disruptor hypothesis, that is, the hypothesis that exposures of human and wildlife populations to endocrine-active compounds contribute to adverse reproductive tract effects and cancers of hormone-sensitive organs. In particular, the long-term consequences of low dose exposures that may produce subtle initial effects, the magnification of those effects across generations, and the reversibility of those effects were to be investigated. The goal was to select a high exposure concentration that would not induce overt toxicity in the dams or pups but would induce observable effects in the reproductive organs of the pups without severely impairing fertility in the F1 generation. The 1,250 ppm exposure concentration was clearly ruled out for further testing based on the effects on body weights, histopathologic observations in males and females, and a reduction in the proportion of mated dams producing litters. While the effects observed at 625 ppm would not be predicted to significantly impair reproduction, the observation of significant effects at 250 ppm (hyperplasia in the mammary gland of both sexes), together with the suggestion of subtle effects at this exposure concentration and less in the parallel immunotoxicity and neuroanatomical surveys, a high exposure concentration between 250 and 625 ppm was deemed appropriate for the purposes of the multigenerational reproductive toxicology study and the chronic study of genistein. A high exposure concentration for the multigenerational and chronic studies was thus set at 500 ppm. A low exposure concentration of 5 ppm, where no significant effects were observed in the reproductive dose range-finding, and an intermediate exposure concentration of 100 ppm were also selected. Synonyms: 4',5,7-Trihydroxyisoflavone.

Long term dietary methoxychlor exposure in rats increases sodium solution consumption but has few effects on other sexually dimorphic behaviors.

Methoxychlor is an insecticide with estrogen-like activity, thus exposure during development might cause sexually dimorphic behavioral alterations. To evaluate this, pregnant rats consumed diets containing 0, 10, 100 or 1000 ppm methoxychlor from gestational day 7, and offspring continued on these diets until postnatal day (PND) 77. Assessments of sexually dimorphic behaviors in offspring indicated that intake of a 3.0% sodium chloride solution was significantly increased (41%) in males and females of the 1000 ppm group. No treatment group differed from controls in open field nor running wheel activity, play behavior, nor 0.3% saccharin solution intake. Offspring of the 1000 ppm group showed significantly decreased body weight, reaching 17% less than controls at PND 77, but not clearly related to their salt solution intake. During pregnancy, 1000 ppm dams consumed 23% less food and weighed 10% less than controls, but this did not affect litter outcomes. These results indicate that in rodents, developmental and chronic exposure to dietary methoxychlor alters the sexually dimorphic behavior of salt-solution intake in young adults of both sexes. Similar behavioral alterations with other xenoestrogens, and the potential for interactions among xenoestrogens, suggest that this report may minimize the true effects of dietary methoxychlor exposure.

Myelotoxicity in genistein-, nonylphenol-, methoxychlor-, vinclozolin- or ethinyl estradiol-exposed F1 generations of Sprague-Dawley rats following developmental and adult exposures.

The myelotoxicity of five endocrine active chemicals was evaluated in F1 generation of Sprague-Dawley rats following developmental and adult exposures at three concentration levels. Rats were exposed to genistein (GEN: 25, 250 and 1250 ppm), nonylphenol (NPH: 25, 500 and 2000 ppm), methoxychlor (MXC: 10, 100 and 1000 ppm), vinclozolin (VCZ: 10, 150 and 750 ppm) and ethinyl estradiol (EE2: 5, 25 and 200 ppb) gestationally and lactationally through dams from day 7 of gestation and through feed after weaning on postnatal day (PND) 22 to PND 64. The parameters examined included the number of recovered bone marrow cells, DNA synthesis, and colony forming units (CFU) in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin. Except for the EE2, the concentrations of other individual chemicals in the diet were in an approximate range that allowed for a comparison to be made in terms of myelotoxic potency. Decreases in the DNA synthesis, CFU-GM and CFU-M seemed to be the common findings among the alterations induced by these compounds. Using the numbers of alterations induced by each chemical in the parameters examined as criteria for comparison, the order of myelotoxic potency in F(1) males was: GEN>MXC>NPH>VCZ; the order in females: GEN>NPH>VCZ. Additionally, some of the functional changes induced by these compounds were gender-specific or dimorphic. Overall, the results demonstrated that developmental and adult exposures of F1 rats to these endocrine active chemicals at the concentrations tested had varied degrees of myelotoxicity with GEN being the most potent. Furthermore, the sex-specific effects of these chemicals in F1 male and female rats suggest that there may be interactions between these compounds and sex hormone in modulating these responses.

Dietary methoxychlor exposure modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in F0 and F1 generations of Sprague Dawley rats.

Methoxychlor, a chlorinated hydrocarbon pesticide, is a persistent environmental contaminant that has been identified in human reproductive tissues. Methoxychlor has been shown to be estrogenic in both in vivo and in vitro studies. As an endocrine disrupter, it may have the potential to adversely affect endocrine, reproductive, and immune systems in animals. The present study evaluated methoxychlor's immunotoxic potential in F0 (dams) and F1 generations of Sprague Dawley rats exposed to an isoflavone-free diet containing methoxychlor at concentrations of 10, 100, and 1000 ppm. In dams, exposure to methoxychlor from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the NK activity (1000 ppm) and the percentages of T cells (1000 ppm), helper T cells (1000 ppm) and macrophages (100 and 1000 ppm). In contrast, a decrease in the numbers of splenocytes and B cells was observed at the 100 and 1000 ppm concentrations. In F1 males, exposure to methoxychlor gestationally, lactationally and through feed from postnatal day 22-64 (78 days total exposure) produced an increase in the spleen IgM antibody-forming cell response to sheep red blood cells (100 and 1000 ppm) and the activity of NK cells (1000 ppm). However, there was a decrease in the terminal body weight (1000 ppm), spleen weight (1000 ppm), thymus weight (100 and 1000 ppm), and the numbers of splenocytes (1000 ppm), B cells (100 and 1000 ppm), cytotoxic T cells (1000 ppm) and NK cells (100 and 1000 ppm). In F1 females, exposure to methoxychlor produced a decrease in the terminal body weight (1000 ppm) and the percentages of cytotoxic T cells (10, 100 and 1000 ppm). These results demonstrate that developmental and adult dietary exposure to methoxychlor modulates immune responses in Sprague Dawley rats. Immunological changes were more pronounced in the F1 generation male rats that were exposed during gestation and postpartum, when compared to the F0 and F1 generation females. Increases in antibody-forming cell response and NK cell activity, and altered spleen cell subpopulation numbers were observed in the F1 generation male rats, without similar changes to the F1 generation females.

Increased volume of the calbindin D28k-labeled sexually dimorphic hypothalamus in genistein and nonylphenol-treated male rats.

The adult rat brain develops through an interplay of neuronal proliferation and programmed cell death. Steroid hormones and growth factors may alter the balance between these competing processes. "Endocrine disrupters" (EDs) may also alter brain development, by mimicry or modulation of endogenous hormone systems. Under control conditions, the sexually dimorphic nucleus (SDN) of the medial preoptic hypothalamus becomes larger in adult males than females, but its final volume may also reflect the hormonal conditions prevailing during development. Two EDs that have recently been studied in protocols involving lifespan exposures are the phytoestrogen genistein and the weakly estrogenic compound para-nonylphenol, which is used in the production of many surfactants and plastics. Experimental dietary exposure of adult female rats to genistein or p-nonylphenol began 28 days prior to their mating at concentrations of 5 ppm, 100 ppm, and 500 ppm for genistein or 25 ppm, 200 ppm, and 750 ppm for p-nonylphenol. Exposure of the offspring continued throughout gestation and lactation, as well as in their chow after weaning, until they were sacrificed at 140 days of age for immunohistochemical labeling of the calbindin D28k-labeled subdivision of the SDN: the CALB-SDN. Both genistein and nonylphenol were found to increase the volume of the CALB-SDN in male rats (p's < 0.01), but not in female rats.

Nonylphenol alters the activity of splenic NK cells and the numbers of leukocyte subpopulations in Sprague-Dawley rats: a two-generation feeding study.

Nonylphenol (NP) has been identified at low levels in surface waters throughout North America. This industrial chemical is primarily used for the production of certain non-ionic surfactants, and has been reported to have weak estrogen-like activity. As estrogen has immunoregulatory properties and is crucial for normal fetal development, it was hypothesized that adult and developmental exposures to NP had the potential to adversely affect the immune system. Furthermore, developmental exposure to NP might also produce differential immunomodulation in F(1) male and female rats. Thus, a two-generation feeding study was conducted to evaluate the potential for NP to modulate certain immune parameters. Pregnant female Sprague-Dawley rats were exposed to NP (0, 25, 500, and 2000 ppm) in their feed for 65 days, beginning 7 days into gestation. The F(1) generation male and female offspring were exposed in utero at the respective treatment levels, commencing the 7th day of gestation, and continuing through to 64 days of age. Changes in splenic antibody-forming cell response, natural killer cell activity, and leukocyte numbers were used to evaluate NP immunotoxicity. The results from the present study indicate that dietary exposure to NP can increase splenic natural killer (NK) cell activity and splenocyte subpopulation numbers in the F(1) generation rats, without similar changes to the F(0) generation. The immunological changes that were observed in the F(1) generation also appeared to be gender-specific.

Vinclozolin modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in sprague dawley rats: a two-generation feeding study.

The potential effects of the fungicide vinclozolin (VCZ) on the immune system were evaluated in F(0) (dams) and F(1) generations of Sprague Dawley rats exposed to a soy-free diet containing VCZ at 10, 150 and 750 ppm. In dams, exposure to VCZ at the highest concentration from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the numbers of splenocytes, B cells, T cells, helper T cells and cytotoxic T cells and a decrease in the percentage of NK cells. In F(1) males, exposure to VCZ gestationally, lactationally and through feed from postnatal day 22 to 64 (78 days total exposure) produced no effect on spleen or thymus weights or splenocyte subsets. However, increases in the spleen IgM antibody-forming cell response to sheep red blood cells (150 and 750 ppm) and the activity of NK cells (150 ppm) were observed. In F(1) females, exposure to VCZ produced a decrease in the activity of NK cells in all the treatment groups. Although decreases in the number of cytotoxic T cells (150 ppm) and the percentages of NK cells (10 ppm) and cytotoxic T cells (150 ppm) were also observed, the lack of a dose-related response suggested that these findings might not be biologically meaningful. In conclusion, these results demonstrate that exposure to VCZ at the concentrations tested modulates the immune responses in Sprague Dawley rats. Furthermore, the differential effect of VCZ in F(1) male and female rats is consistent with the reported anti-androgenic properties of VCZ.

Genistein modulates splenic natural killer cell activity, antibody-forming cell response, and phenotypic marker expression in F(0) and F(1) generations of Sprague-Dawley rats.

The potential effects of the phytoestrogen genistein (GEN) on the immune system were evaluated in both F(0) (dams) and F(1) generations of Sprague-Dawley rats exposed to a soy-free diet containing low (L: 25 ppm), middle (M: 250 ppm), and high (H: 1250 ppm) levels of GEN. In dams, exposure to GEN from Gestation Day 7 to Postpartum Day 51 (totally 65 days) produced a significant increase in NK cell activity (M and H), while a decrease in the percentage of helper T cells (H). In F(1) males, exposure to GEN gestationally, lactationally, and through feed from Postnatal Days 22 to 64 (total 78 days) produced an increase in the relative weights (% body) of spleen (L and H) and thymus (L). Furthermore, exposure to GEN increased the number of splenic B cells (H), T cells (L, M, and H), and T-cell subsets (L, M, and H). Although GEN decreased the percentages of splenic NK cells (L, M, and H), no effect on the activity of NK cells was observed. In F(1) females, exposure to GEN produced a decrease in terminal body weight (H), with an increase in the relative weight of spleen (L, M, and H). Exposure to GEN also increased the number of splenic B cells (L), macrophages (L and M), T cells (H), helper T cells (L and H), and cytotoxic T cells (M and H). Additionally, exposure to GEN increased the percentages of T cells (M and H), helper T cells (H), and cytotoxic T cells (M and H). Moreover, the spleen IgM antibody-forming cell response to sheep red blood cells was enhanced (H), although the percentages of B cells were decreased (M and H). No effect on the activity of NK cells was observed; however, the percentages of splenic NK cells were decreased by GEN (L and H). In conclusion, these results demonstrate that exposure to GEN can modulate the immune responses in Sprague-Dawley rats. Furthermore, the sexual dimorphic effects of GEN in F(1) male and female rats suggest that there may be interactions between GEN and the responses modulated by sex hormones.

Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression.

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

Polycystic kidney disease induced in F(1) Sprague-Dawley rats fed para-nonylphenol in a soy-free, casein-containing diet.

para-Nonylphenol (NP; CAS #84852-15-3), an alkylphenol with a 9-carbon olefin side chain, is widely used in the manufacture of nonionic surfactants, lubricant additives, polymer stabilizers, and antioxidants. Due to its wide commercial use and putative endocrine activity in humans and wildlife, the NTP elected to assess its effects on reproduction in multigenerational studies. To avoid known estrogenic activity of phytoestrogens in soy and alfalfa, a soy- and alfalfa-free, casein-containing diet was used in a range-finding study to determine the doses of NP to be tested further. NP was administered to Sprague-Dawley rats in the diet at 0, 5, 25, 200, 500, 1000, or 2000 ppm to F(0) dams beginning on gestation-day 7. The F(1) pups were weaned at postnatal day (PND) 21, and their exposure via diet was continued at the same dose level as their respective dams. Pup weights from birth through weaning were not significantly different from controls in any dose group, but the average weight of both sexes was significantly less compared to controls, beginning with the PND 28 weighing. The F(1) rats were sacrificed on PND 50 (n = 15, 3 pups of each sex from 5 litters for all dose groups). Terminal body weights of males and females in the 2000-ppm dose group were 74% and 85% of controls, respectively. Severe polycystic kidney disease (PKD) was present in 100% of the 2000 ppm-exposed male and female rats. At 1000 ppm, 67% of males and 53% of females had mild to moderate PKD versus none of either sex in the control and lower-dose groups. The no-adverse-effect level (NOAEL) for PKD was determined to be 500 ppm. Previous studies with comparable duration and route of exposure, but using soy-containing diets, reported either no or only mild PKD at 2000 ppm NP. We conclude that the renal toxicity of NP is highly dependent on the diet on which the animals are maintained. The potential interaction of diet and test compounds on nonreproductive as well as reproductive endpoints should be considered when contemplating the use of special diets formulated to minimize exogenous "hormone" content for the study of the effects of putative endocrine disruptive chemicals.

Behavioral responses of rats exposed to long-term dietary vinclozolin.

Vinclozolin is a fungicide used on food crops with human exposure estimated at approximately 2 microg/kg/day from ingestion; occupational exposure, however, may be greater. The metabolites of vinclozolin have been reported to act as antiandrogens and have adverse effects on reproductive physiology and behavior in animals. Here, pregnant rats were fed soy-free diets containing 0, 10, 150, or 750 ppm of vinclozolin (approximately 0, 0.8, 12, and 60 mg/kg/day for an adult) beginning on gestational day 7, and offspring were continued on these diets through sacrifice at postnatal day 77. Male and female offspring were assessed for changes in several nonreproductive sexually dimorphic behaviors: open field and running wheel locomotor activity, play behavior, and consumption of saccharin- and sodium chloride-flavored solutions. There was a significant interaction of sex with vinclozolin exposure on running wheel activity, which indicated that females in the high-dose exposure group were hypoactive compared to same-sex controls. There was a significant overall effect of vinclozolin exposure on fluid consumption, and high-dose animals showed increased intake of the saccharin solution and decreased intake of plain water while saccharin was available. Effects were more pronounced in females, which drank 40.8% more saccharin than control females, whereas males drank 6.2% more than control males. There were no effects of vinclozolin treatment on play behavior or sodium solution intake. Gestational duration, total and live pups per litter, litter sex ratios, and birth weight were also not significantly affected, nor were body weight and food intake for dams and offspring. These results indicate that long-term dietary exposure to vinclozolin does not have severe toxicological consequences on the nonreproductive behaviors measured here. However, exposure may cause subtle alterations in locomotor activity and consumption of saccharin-flavored solution.

Placental transfer of the soy isoflavone genistein following dietary and gavage administration to Sprague Dawley rats.

Genistein, the principal soy isoflavone, has estrogenic activity and is widely consumed by humans for putative beneficial health effects. The goal of the present study was to measure placental transfer of genistein in rats as a possible route of developmental exposure. Pregnant Sprague-Dawley rats were administered genistein orally, either by diet or by gavage. Concentrations of genistein aglycone and conjugates were measured in maternal and offspring serum and brain using HPLC with isotope dilution electrospray tandem mass spectrometry. Although fetal or neonatal serum concentrations of total genistein were approximately 20-fold lower than maternal serum concentrations, the biologically active genistein aglycone concentration was only 5-fold lower. Fetal brain contained predominately genistein aglycone at levels similar to those in the maternal brain. These studies show that genistein aglycone crosses the rat placenta and can reach fetal brain from maternal serum genistein levels that are relevant to those observed in humans.

Maternal and offspring toxicity but few sexually dimorphic behavioral alterations result from nonylphenol exposure.

Nonylphenol ethoxylates are used in the production of surfactants and are found in numerous manufactured substances. para-Nonylphenol (NP) is a suspected endocrine disruptor, exhibiting estrogen-like activity and might cause alterations with developmental exposure. To evaluate such effects, pregnant Sprague-Dawley rats consumed diets containing 0 (n = 11), 25 (n = 10), 500 (n = 10), or 2,000 (n = 9) ppm NP beginning on gestational day (GD) 7. At postnatal day (PND) 21, offspring continued on the same maternal diets until PND 77 and were evaluated for behavioral alterations (open-field activity at PNDs 22-24, 43-45, 64-66, play behavior at PND 35, running wheel activity at PND 63-77, flavored solution intake at PND 69-75). During pregnancy and lactation, dams in the 25-, 500-, and 2,000-ppm groups consumed 9 to 25% less food, which was associated with a 17% less weight gain during GDs 1 to 21 in dams of the 2,000-ppm group, although this effect was not statistically significant. Gestation duration, birth weight, sex ratio of live pups, and number of live or dead pups per litter did not differ between treatment groups. Offspring body weight and food consumption were decreased in the 2, 000-ppm group beginning at PND 28; however, an effect of feed aversion could not be eliminated. Behavioral assessments of offspring indicated no consistent NP-related effects in open-field activity at PNDs 22-24, 43-45, and 65-67 nor in running wheel activity at PNDs 63-75. Play behavior at PND 35 and intake of a 0.3% saccharin-flavored solution at PNDs 69-71 did not differ with respect to treatment groups. However, intake of a 3% sodium-flavored solution at PNDs 73-75 was significantly increased in offspring of the 2,000-ppm group and intake of regular water during this same time was also significantly increased. These results indicate that developmental NP treatment results in maternal and offspring toxicity as evidenced by decreased food intake and weight gain. However, behavioral alterations were evident only in increased intake of a sodium solution.

Mass spectrometric determination of Genistein tissue distribution in diet-exposed Sprague-Dawley rats.

Genistein, the principal soy isoflavone, was administered in the diet to male and female Sprague-Dawley rats as part of a multigeneration study of potential endocrine modulation. The rats were exposed to genistein in utero, through maternal milk, and as adults through postnatal d 140 via essentially isoflavone-free feed (approximately 0.5 microg/g) fortified at 5, 100 and 500 microg/g with genistein aglycone. Analytical methods based on liquid chromatography, mass spectrometry and the use of deuterated genistein were developed and validated for use in measuring genistein in serum and tissues. Pharmacokinetic analysis of serum genistein showed a significant difference (P < 0.001) in the elimination half-life and area under the concentration-time curve between male [2.97 +/- 0.14 h and 22.3 +/- 1.2 micromol/(L. h), respectively] and female rats [4.26 +/- 0.29 h and 45.6 +/- 3.1 micromol/(L. h), respectively, +/- SEM]. Endocrine-responsive tissues including brain, liver, mammary, ovary, prostate, testis, thyroid and uterus showed significant dose-dependent increases in total genistein concentration. Female liver contained the highest amount of genistein (7.3 pmol/mg tissue) and male whole brain contained the least (0.04 pmol/mg). The physiologically active aglycone form was present in tissues at fractions up to 100%, and the concentration was always greater than that observed in serum in which conjugated forms predominated (95-99%). These results for measured amounts of genistein, present as aglycone and conjugates, in putative target tissues provide a link with other studies in which blood concentrations and physiologic effects of genistein are measured.

A comparison of the inflammatory response to a polydimethylsiloxane implant in male and female Balb/c mice.

The implantation of biomaterials elicits a host response that influences the long-term behavior of implanted medical devices. This foreign body response is governed by cells of the immune system. Because sexual dimorphism in the immune system is well-established, a comparative study of the foreign body response in male and female mice was initiated. Eight-week-old male and female Balb/c mice received two subcutaneous implants in the interscapular region of a smooth peroxide-catalyzed polydimethylsiloxane (PDMS) and were sacrificed at 2, 14, 42, 70, and 105 days after implantation (n = 6 per sex per time point). Controls for each time point underwent the surgical procedure but received no implant. Tissue from the implant or surgical site was fixed, processed, and paraffin-embedded for histopathological evaluation and immunohistochemical (IHC) staining for tumor necrosis factor-alpha TNF-alpha) and interleukin-1 beta (IL-1beta). In control animals, an inflammatory response was observed at 2 days that was decreased by 14 days and absent after 42 days. At 2 and 14 days after PDMS implantation, a mild to moderate inflammatory reaction was observed around implants. The peak response was seen at 14 days, and granulation tissue, composed primarily of fibroblasts, macrophages, and neutrophils, was first observed at this time. After 105 days, the implantation site was surrounded by mature connective tissue, which had minimal numbers of macrophages and neutrophils, with severity scores that did not differ significantly in males and females. The immunostaining for TNF-alpha and IL-1beta followed similar temporal patterns, with both reaching a peak at the two week time point and remaining elevated, compared to level of expression in the controls, throughout the 105 day observation period. Staining for both cytokines in the implanted animals was generally higher in females than in males, although this difference was significant only for IL-1beta. These results suggest subtle differences between males and females in the activity of peri-implant inflammatory cells.

Effects of genistein exposure on sexually dimorphic behaviors in rats.

The phytoestrogen genistein, the principal isoflavone in soybeans, has adverse effects on animal reproduction. As adult physiology and behavior are sensitive to perturbation by developmental estrogens, exposure to genistein during development may produce behavioral alterations as well. Pregnant rats were fed soy-free diets containing 0, 25, 250, or 1250 ppm genistein (approximately 0, 2, 20, or 100 mg/kg/day) beginning on gestational day 7, and offspring continued on these diets through postnatal day (PND) 77. Male and female offspring were assessed for levels of sexually dimorphic behaviors: open field activity, play behavior, running wheel activity, and consumption of saccharin- and sodium chloride-flavored solutions. Consumption of the salt solution was affected by genistein, with animals in the 1250-ppm group drinking significantly more than controls; consumption of plain water was unaffected. Genistein treatment also significantly affected play behavior; although no treated group was significantly different from controls, and the effect was not sexually dimorphic. Running wheel activity and saccharin solution consumption showed significant sex differences, but no effects of genistein treatment. Gestational duration, total and live pups per litter, and total and live litter sex ratios were not significantly affected by genistein. However, average weight per live pup at birth and offspring body weights from PND 42-77 were significantly decreased in the 1250-ppm group. Body weight and food intake for the dams were also significantly decreased in the 1250-ppm group. These results indicate that developmental genistein treatment, at levels that decrease maternal and offspring body weight, causes subtle alterations in some sexually dimorphic behaviors.

Differences in the response to oxidative stress and mutant frequency in CD (Sprague-Dawley) and Fisher 344 rats due to an induced inflammatory response.

In this study, the rodent air pouch model was used to examine the production and processing of oxidative DNA damage in two strains of rats commonly used in toxicity testing. An inflammatory response was induced by injecting zymosan A (50 mg) into an air pouch on male CD (Sprague-Dawley [S-D]) and Fisher 344 (F-344) rats, and the animals were then sacrificed at 1, 3, 7, 14, and 28 days (n = 6 per time point per strain). Tissues from the lining of the air pouch were collected for 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis and for paraffin embedding. Significant (P < 0.01) increases in 8-OH-dG were observed after 1 day in the DNA from cells lining the air pouch of zymosan A-treated versus control S-D (101.5 +/- 27.1 vs. 23.1 +/- 2. 7 8-OH-dG/dG x 10(5)) and F-344 (51.4 +/- 5.3 vs. 14.4 +/- 0.6 8-OH-dG/dG x 10(5)) rats. By 28 days, 8-OH-dG levels had returned to background in S-D rats, but remained elevated in F-344 rats. The frequency of apoptosis was evaluated using the in situ end-labeling (TUNEL) assay, which revealed that zymosan A-treated S-D rats had a significantly (P < 0.05) higher frequency of apoptosis compared to zymosan A-treated F-344 rats. To examine the potential consequences of these differences in endogenously produced DNA damage and apoptosis, we measured mutations at the hprt locus in fibroblasts of the pouch lining and observed a significant (P < 0.05) increase in the mutant frequency at day 28 in F-344 rats (54.2 +/- 13.6 mutants per 10(6) cells) compared to controls (4.5 +/- 2.0 mutants per 10(6) cells). The mutant frequency was not increased in S-D rats. These data demonstrate that strain differences in the production and processing of oxidative DNA damage due to an inflammatory response may impact the long-term pathologic consequences of chronic inflammation. Environ. Mol. Mutagen. 35:336-342, 2000 Published 2000 Wiley-Liss, Inc.