Injection Of Bisphenol A Research Articles

Adolescent bisphenol-A exposure decreases dendritic spine density: role of sex and age.

Bisphenol-A (BPA), a common environmental endocrine disruptor, modulates estrogenic, androgenic, and antiandrogenic effects throughout the lifespan. We recently showed that low dose BPA exposure during adolescence increases anxiety and impairs spatial memory independent of sex. In this study, six week old Sprague Dawley rats (n=24 males, n=24 females) received daily subcutaneous injections (40 µg/kg bodyweight) of BPA or vehicle for one week. Serum corticosterone levels in response to a 1 h restraint stress and spine density were examined at age 7 (cohort 1) and 11 (cohort 2) weeks. Adolescent BPA exposure did not alter stress dependent corticosterone responses but decreased spine density on apical and basal dendrites of pyramidal cells in the medial prefrontal cortex (mPFC) and hippocampal CA1 region (CA1). Sex differences in spine density were observed on basal dendrites of the mPFC and CA1 with females having greater spine density than males. This sex difference was further augmented by both age and treatment, with results indicating that BPA-dependent decreases in spine density were more pronounced in males than females on mPFC basal dendrites. Importantly, the robust neuronal alterations were observed in animals exposed to BPA levels below the current U.S.E.P.A. recommended safe daily limit. These results are the first demonstrating that BPA given during adolescence leads to enduring effects on neural morphology at adulthood. Given that humans are routinely exposed to low levels of BPA through a variety of sources, the decreased spine density reported in both male and female rats after BPA exposure warrants further investigation.

Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice

Bisphenol-A (BPA), an environmental estrogen, adversely affects female reproductive health. However, the underlying mechanisms remain largely unknown. We found that oral administration (p.o.) of BPA (20 μg/kg) to adult female mice at proestrus, but not at estrus or diestrus, significantly increased the levels of plasma E₂, LH and FSH, and Gnrh mRNA within 6 h. The administration of BPA at proestrus, but not at diestrus, could elevate the levels of Kiss1 mRNA and kisspeptin protein in anteroventral periventricular nucleus (AVPV) within 6 h. In contrast, the level of Kiss1 mRNA in arcuate nucleus (ARC) was hardly altered by BPA administration. In addition, at proestrus, a single injection (i.c.v.) of BPA dose-dependently enhanced the AVPV-kisspeptin expression within 6 h, this was sensitive to E₂ depletion by ovariectomy and an estrogen receptor α (ERα) antagonist. Similarly, the injection of BPA (i.c.v.) at proestrus could elevate the levels of plasma E₂, LH, and Gnrh mRNA within 6 h in a dose-dependent manner, which was blocked by antagonists of GPR54 or ERα. Injection of BPA (i.c.v.) at proestrus failed to alter the timing and peak concentration of LH-surge generation. In ovariectomized mice, the application of E₂ induced a dose-dependent increase in the AVPV-Kiss1 mRNA level, indicating 'E₂-induced positive feedback', which was enhanced by BPA injection (i.c.v.). The levels of Erα (Esr1) and Erβ (Esr2) mRNAs in AVPV and ARC did not differ significantly between vehicle-and BPA-treated groups. This study provides in vivo evidence that exposure of adult female mice to a low dose of BPA disrupts the hypothalamic-pituitary-gonadal reproductive endocrine system through enhancing AVPV-kisspeptin expression and release.

Bisphenol A does not affect memory performance in adult male rats.

Bisphenol A (BPA) is an estrogenic endocrine disruptor used for producing polycarbonate plastics and epoxy resins. This study investigated the effects of oral BPA administration on memory performance, general activity, and emotionality in adult male Sprague Dawley rats using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial memory performance. In addition, in order to confirm the effects of BPA on spatial memory performance, we examined whether intrahippocampal injection of BPA affects spatial memory consolidation. In the MAZE test, although oral BPA administration at 10 mg/kg significantly altered the number of entries into the incorrect area compared to those of vehicle-treated rats, male rats given BPA through either oral administration or intrahippocampal injection failed to show significant differences in latencies to reach the reward. Also, oral BPA administration did not affect fear-motivated memory performance in the step-through passive avoidance test. Oral BPA administration at 0.05 mg/kg, the lowest dose used in this study, was correlated with a decrease in locomotor activity in the open-field test, whereas oral administration at 10 mg/kg, the highest dose used in this study, was correlated with a light anxiolytic effect in the elevated plus-maze test. The present study suggests that BPA in adulthood has little effect on spatial memory performance in male rats.

Adolescent exposure to Bisphenol-A increases anxiety and sucrose preference but impairs spatial memory in rats independent of sex

The endocrine disruptor Bisphenol-A (BPA) has been shown to modulate estrogenic, androgenic, and anti-androgenic effects. The effects of BPA exposure during early organizational periods of development have been well documented. The current study focuses on the effects of short term, low-dose BPA exposure on anxiety, spatial memory and sucrose preference in adolescent rats. Seven week old Sprague Dawley rats (n=18 male, n=18 female) received daily subcutaneous injections (40µg/kg body weight) of BPA or vehicle for 12 days. Starting on day 6 of injections, subjects were tested on the elevated plus maze which provides a measure of anxiety, the open field test which provides a measure of anxiety and locomotor activity, and object placement, a measure of spatial memory. On the twelfth day of BPA administration, sucrose preference was tested using a standard two-bottle choice (tap versus sucrose solution). All rats gained weight during the study; there was a main effect of sex, but not BPA treatment on body weight. The results indicate that BPA exposure, regardless of sex, increased anxiety on both the elevated plus maze and open field. Spatial memory was impaired on the object recognition task with BPA animals spending significant less time with the object in the novel location than controls. Finally, a significant increase in sucrose consumption for both male and female subjects exposed to BPA was observed. The current data shows that short term BPA exposure, below the current reference safe daily limit of 50µg/kg day set by the United States Environmental Protection Agency, during adolescent development increases anxiety, impairs spatial memory, and increases sucrose consumption independent of sex.

High Specificity of Molecularly Imprinted Polymer Particles toward Target Compound in Competitive Environmental Binding

Molecularly imprinted polymer (MIP) particles tailored for bisphenol A (BPA) were prepared using a 1:8:7 molar ratio of BPA the template, methacrylic acid (MAA) the functional monomer, and ethylene glycol dimethacrylate (EGDMA) the cross-linker. These colloidal particles were tested for rapid competitive binding by capillary electrophoresis with ultraviolet detection (CE-UV). Good interface binding efficiencies were obtained for BPA even in the presence of 2-hydroxy-4-methoxy benzophenone (HMB) as a structurally related compound at high concentrations. The specifically bound BPA could be desorbed rapidly out of the interface cavities by 5% triethylamine in methanol. However reusability was demonstrated by repeated injections of BPA that bound to MIP particles without regeneration of the cavities.

Bisphenol-A (BPA) Affects Reproductive Formation across Generations in Mice

To understand effects of Bisphenol-A (BPA) exposure on the reproductive organ across generations, we analyzed morphology of the uterus and ovary, and the methylation pattern of HOXA10 gene of the 2(nd) generation. Pregnant mice (F0) were treated with sc injection of BPA in sesame oil at various doses of 0-1,000 mg/kg Bwt on days 12-16 of gestation. Their offspring (F1) were bred by foster mice, and the offspring (F2) from F1 mice were prepared. That is, F1 mice experienced in utero BPA exposure during the developmental period of reproductive organs, while F2 mice did not at all. Using these F2 mice, the present study was carried out. Comparing to the control, the body weights in BPA exposure groups were significantly increased. Correlating with the increase of body weight, the relative weights of the ovary and uterus in each group were decreased. The histological analysis revealed expansion or emphraxis of the uterine lumen and partial loss of the uterine epithelium. Unmethylation of HOXA10 gene in the uterus was observed in the intron region. The present study suggested that BPA exposure to F0 mice could affect reproductive organ of F2 mice who were not exposed to BPA.

Developmental programming: Impact of fetal exposure to endocrine-disrupting chemicals on gonadotropin-releasing hormone and estrogen receptor mRNA in sheep hypothalamus

Bisphenol-A (BPA) and methoxychlor (MXC), two endocrine-disrupting chemicals (EDCs) with estrogenic and antiandrogenic effects, disrupt the reproductive system. BPA has profound effects on luteinizing hormone (LH) surge amplitude, and MXC has profound effects on on LH surge timing in sheep. The neural mechanisms involved in the differential disruption of the LH surge by these two EDCs remain to be elucidated. We tested the hypothesis that the differential effects of BPA and MXC on LH surge system involved changes in hypothalamic gonadotropin-releasing hormone (GnRH) and estrogen receptors (ESR), ESR1 and ESR2, mRNA expression. Pregnant sheep were given daily injections of cottonseed oil (controls), MXC, or BPA (5 mg/kg/day) from day 30 to 90 of gestation (term 147 d). Offspring from these animals were euthanized as adults, during the late follicular phase following synchronization of estrus with prostaglandin F 2α, just before the expected onset of preovulatory LH surge and changes in mRNA expression of hypothalamic GnRH, ESR1, and ESR2 quantified following in situ hybridization. GnRH mRNA expression was significantly lower in both groups of EDC-treated females compared to controls. ESR1 expression was increased in prenatal BPA- but not MXC-treated females in medial preoptic area relative to controls. In contrast, ESR2 expression was reduced in the medial preoptic area of both EDC-treated groups. Differences in expression of ESR1/ESR2 receptors may contribute to the differential effects of BPA and MXC on the LH surge system. These findings provide support that prenatal exposure to EDCs alters the neural developmental trajectory leading to long-term reproductive consequences in the adult female.

Effects of Endocrine Disruptors onBombina orientalisP450 Aromatase Activity

To assess the effects of endocrine-disrupting chemicals on the expression and activity of aromatase in the gonads of Bombina orientalis, a common amphibian, we intraperitoneally injected nonylphenol or bisphenol-A and then examined aromatase mRNA levels by RT-PCR as well as aromatase enzymatic activity by tritiated water release assays. To design primers for the RT-PCR, we cloned the B. orientalis aromatase gene using RT-PCR and degenerate primers. The full-length cDNA was obtained by 5'- and 3'-RACE PCR. The complete sequence of the B. orientalis aromatase gene revealed an open reading frame of 1500 bp encoding a deduced protein of 500 amino acids. Semi-quantitative RT-PCR indicated that nonylphenol or bisphenol-A injection did not significantly affect the expression of B. orientalis aromatase mRNA. However, a 48-hr treatment with nonyphenol or bisphenol A reduced aromatase activity to 47% and 32% of the control, respectively. These results suggest that endocrine disrupters can effectively modulate the activity of B. orientalis aromatase without affecting its mRNA levels.

Effects of Neonatal Exposure to Bisphenol A on Steroid Regulation of Vascular Endothelial Growth Factor Expression and Endothelial Cell Proliferation in the Adult Rat Uterus1

Hormonally controlled vascular changes play a key role in endometrial development and in the differentiation process necessary for implantation. Vascular endothelial growth factor (VEGF) has emerged as one of the central regulators of the uterine vasculature. Hormonal perturbations during neonatal development may alter sex steroid-dependent regulation of VEGF and may ultimately affect fertility later in life. The aim of this study was to determine whether neonatal exposure to the environmental estrogenic chemical bisphenol A (BPA) affects the adult rat uterine response to hormonal stimuli. Newborn female rats were given s.c. injections of vehicle, BPA (0.05 mg/kg per day or 20 mg/kg per day) or diethylstilbestrol (0.2 microg/kg per day) on Postnatal Days 1, 3, 5, and 7. To evaluate the long-term effects, rats were ovariectomized at Postnatal Day 80 and submitted to hormonal replacement. Rats neonatally exposed to xenoestrogens showed a decreased induction of uterine endothelial proliferation and a decreased Vegf mRNA expression in response to ovarian steroid treatment. Also, although the estrogen receptor alpha (ESR1) expression was lower in subepithelial cells than in controls, a higher expression of silencing mediator of retinoic acid and thyroid hormone receptor (NCOR1, also known as SMRT) corepressor was evidenced in the same compartment. The results indicate that disturbed Vegf expression in BPA rats could be the result of changes in endocrine pathways, such as an altered induction of ESR1 and/or NCOR1 expression. Because of the importance of VEGF in the implantation process, our data suggest that neonatal BPA exposure might have negative consequences on female fertility.

275. The endocrine disruptor, bisphenol A, alters gene expression in the hypothalamus of the ram lamb but does not alter LH secretion

Bisphenol A (BPA) is an endocrine disruptor, which has both oestrogenic and anti-oestrogenic actions. It has profound effects on development, especially of sex organs but also on the brain. The actions on the brain are poorly understood and little is known about males. Most work has been conducted in fish & rodents, but the lifespan of domestic ruminants means that BPA may accumulate in tissues over many years. This study tested the hypothesis that treatment of ram lambs with BPA would alter LH secretion as well as gene activity in various hypothalamic regions. Ram lambs were treated from 1 month old with thrice weekly injections (im) of BPA (3.5 mg/kg) or vehicle (n = 10/group) for 80days. Weekly bodyweights and jugular blood samples were collected and on day 79, blood samples were taken every 15 min for 8 h for LH RIA. The sheep were killed, body & testicular weights collected and the hypothalamus dissected into anterior, dorsal and ventral blocks, then frozen in liquid nitrogen. Bodyweight (Cont. 26.0 ± 0.5 kg v. BPA 25.2 ± 0.7 kg) and paired testicular weight (Cont. 31.6 ± 1.2 g v. BPA 29.3 ± 1.4 g) did not significantly differ (P > 0.05). LH secretion also did not differ, in either mean LH concentrations (Cont 0.31 ± 0.14 v. BPA 0.29 ± 0.12ng/mL) or pulses/8 h (Cont 1.8 ± 0.4 v. BPA 2.0 ± 0.4). Total RNA was extracted from the basal hypothalamus (ventral block) and RNA from 2 treated and 2 control animals was hybridised on 44K Agilent bovine microarrays. Preliminary analysis suggests 118 probes were differentially expressed. Gene ontology analysis of these differentially expressed probes revealed involvement in a broad range of biological processes and cellular components. This study suggests that gene array analysis is a suitable tool with which to study BPA actions in the sheep hypothalamus. Future work will expand this pilot microarray study to include all animals and all hypothalamic regions.

Bisphenol‐A disruption of the endocrine pancreas and blood glucose homeostasis

The link between endocrine disruptors and altered blood glucose homeostasis has been recently suggested. Epidemiological studies have correlated levels of phthalates, dioxins and persistent organic pollutants with alterations of blood glucose homeostasis in humans. Environmentally relevant doses of the ubiquitous endocrine disruptor bisphenol-A (BPA) have profound effects on mice endocrine pancreas--an essential tissue involved in glucose metabolism. BPA exerts rapid non-genomic effects on insulin releasing beta-cells and glucagon releasing alpha-cells within freshly isolated islets of Langerhans. In vivo, a single BPA injection of 10 microg/kg rapidly increases plasma insulin and concomitantly decreases glycaemia. When mice were treated with BPA 100 microg/kg/day for 4 days, the environmental oestrogen produced an increase in beta-cell insulin content along with a post-prandial hyperinsulinaemia and insulin resistance. The results reviewed here demonstrate that doses well below the current lowest observed adverse effect level considered by the US-EPA, disrupt pancreatic beta-cell function producing insulin resistance in male mice. Therefore, this altered blood glucose homeostasis by BPA exposure may enhance the risk of developing type II diabetes.

Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract

The developing fetus is uniquely sensitive to perturbation by chemicals with hormone-like activity. The adverse effects of prenatal diethylstilbestrol (DES) exposure are a classic example. Since concern has been mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical with estrogenic activity used in the synthesis of plastics, we investigated its long-term effects in an experimental animal model that was previously shown useful in studying the adverse effects of developmental exposure to DES. Outbred female CD-1 mice were treated on days 1–5 with subcutaneous injections of BPA (10, 100 or 1000 μg/kg/day) dissolved in corn oil or corn oil alone (Control). At 18 months, ovaries and reproductive tract tissues were examined. There was a statistically significant increase in cystic ovaries and cystic endometrial hyperplasia (CEH) in the BPA-100 group as compared to Controls. Progressive proliferative lesion (PPL) of the oviduct and cystic mesonephric (Wolffian) duct remnants were also seen in all of the BPA groups. More severe pathologies of the uterus following neonatal BPA treatment included adenomyosis, leiomyomas, atypical hyperplasia, and stromal polyps. These data suggest that BPA causes long-term adverse effects if exposure occurs during critical periods of differentiation.

Induction of Uterine Calbindin-D9k Through an Estrogen Receptor-Dependent Pathway Following Single Injection with Xenobiotic Agents in Immature Rats

Various environmental chemicals, both natural and synthetic, are believed to act as endocrine disruptors (EDs) in mammals. In this study, a new in vivo model of immature rats was used to explore the induction of calbindin-D9k (CaBP-9k) following a single injection of EDs. In a time-dependent experiment, immature rats at postnatal day 16 were treated with high doses (600 mg/kg body weight [BW]) of 4-tert-octyphenol (OP), p-nonylphenol (NP), or bisphenol A (BPA), and euthanized at different time points (3, 6, 12, 24, or 48 h). For a dose-dependent study, immature rats were given different doses (200, 400, or 600 mg/kg BW) and euthanized at 24 h after injection. After treatment with these EDs, the effects on CaBP-9k mRNA and protein were examined by Northern and Western blot analyses, respectively. An anti-estrogen, ICI 182,780, was employed to examine the potential involvement of estrogen receptor (ER) in the induction of estrogen receptor-mediated physiologic responses in vivo. A single treatment with each of the chemicals, at 600 mg/kg BW, resulted in a significant increase in the expression of CaBP-9k mRNA and protein 24 h after injection. In addition, treatment with OP, NP, or BPA resulted in a positive uterotrophic response. Cotreatment with the ER antagonist ICI 182,780 completely prevented the ED-induced uterine weight gain. Taken together, these results demonstrate that a single injection of OP, NP, or BPA results in an increase of CaBP-9k mRNA and protein via an ER-dependent pathway in the uterus of immature rats. This new model may be important to elucidate the mechanism of action of xenoestrogens on estrogen-sensitive tissue.

Effects of Bisphenol A (BPA) on Placentation and Survival of the Neonates in Mice

The effects of bisphenol A (BPA) on placentation have not been fully determined. The aim of this study was to clarify the structural changes of the placenta, abortion rate, and survival of neonates after BPA administration in mice. BPA (10 mg/kg/day) was administered to pregnant mice (BPA mice) subcutaneously from the first day of pregnancy (Day 0) to Day 7 (8 days total). The number of embryos and weights of whole uteri were measured on Days 10 and 12. Morphological changes in the placentae were examined by light microscopy on the corresponding days of pregnancy. The number of neonates was also counted. Survival rates were periodically calculated for neonates from the first day after parturition (P-Day 0) to P-Day 56. The number of embryos and weight of the uterus on Days 10 and 12 were significantly decreased by BPA injection. No notable differences were recognized between the left and right uteri. The proportion of the labyrinthine zone per whole placenta in the BPA mice became lower than that in the controls, and that of the metrial gland was higher in the BPA mice. The intervillous spaces of the placenta were narrower in the BPA mice. Degenerative changes were found in the trophoblastic giant cells and spongiotrophoblast layers of the BPA mice. The number of BPA mouse neonates was drastically decreased within 3 days after birth, and no mice survived after P-Day 56. The results suggest that BPA not only disrupts placental functions and leads to abortion through chronic stimulation of gene expression by binding to DNA but that it also affects the mortality of neonates through indirect exposure of embryos.

Differential effects of the endocrine-disrupting compounds Bisphenol-A and Octylphenol on gonadotropin secretion, in prepubertal ewe lambs

This study examined the effects of long-term exposure to the endocrine-disrupting compounds (EDCs) Bisphenol-A (BPA) and Octylphenol (OP) on gonadotrophin secretion in pre-pubertal female sheep. Four-week-old, female lambs were randomly allocated to four groups ( n=6), and twice each week treated with i.m. injections of either corn oil (vehicle controls), diethylstilbestrol (DES; 0.175 mg/kg), BPA (3.5 mg/kg) or OP (3.5 mg/kg). After 5 weeks of treatment, animals were ovariectomized (ovx) and ovary weights recorded. Two weeks later, blood samples were collected from lambs every 15 min for 6 h, for LH pulse analysis. Animals were then euthanased and adrenal and kidney weight recorded. An age-related increase in tonic LH secretion was noted in Control, BPA- and OP-treated lambs, but was absent in DES-treated lambs. Following ovx, LH secretion increased in all except DES-treated lambs; FSH concentrations increased in all groups. BPA and DES significantly suppressed LH pulse frequency (C: 6.7±0.3 pulses/6 h, DES: 1.5±0.8 pulses/6 h, BPA: 2.3±0.8 pulses/6 h) and amplitude (C: 7.1±1.0 ng/ml, DES: 1.9±0.6 ng/ml, BPA: 1.6±0.4 ng/ml). OP had no effect on LH secretion (Frequency: 5.8±0.5 pulses/6 h, amplitude: 8.0±2.0 ng/ml). Ovary weight was similar among all groups. Results show that chronic in vivo exposure of prepubertal female lambs to BPA, at levels lower than those reported previously, can have significant effects on LH secretion that are comparable to those seen following exposure to the known xenoestrogen, DES. Exposure to an equal dose of the EDC OP, over the equivalent period of time was without effect on gonadotropin secretion in the prepubertal ewe lamb. These results indicate that exposure of prepubertal female lambs to the EDC BPA can induce significant effects on gonadotropin secretion, the potential long-term effects of exposure and the effects of these changes on reproductive performance and efficacy, therefore, merit further study.

Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues

We investigated the modifications in endogenous antioxidant capacity, including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, oxidative stress index, reduced glutathione (GSH), glutathione disulfide (GSSG), and thiobarbituric acid-reactive substance (TBARS) in the brain, liver, kidney, and testes of mice under bisphenol A (BPA), an endocrine disrupter, treated for 5 days. BPA was administrated intraperitoneally at doses of 25 and 50 mg/kg/day. The TBARS levels were not affected by BPA administrations. The SOD activities increased and the catalase activities decreased in the liver after BPA administration. The GPx activity decreased in the kidney. The levels of GSH+GSSG increased in the brain, kidney, liver, and testes, while, the levels of GSH decreased in the testes. SOD converts superoxide into hydrogen peroxide, and catalase and GPx convert hydrogen peroxide into hydrogen oxide. Our results suggest that the injection of BPA induces overproduction of hydrogen peroxide in the mouse organs. Hydrogen peroxide is easily converted to hydroxy radical. The decrease of GSH and the increase of GSSG may be caused by the hydroxy radical. BPA may show its toxicity by increasing hydrogen peroxide.

Bisphenol A Increases Progesterone Receptor Immunoreactivity in the Hypothalamus in a Dose‐Dependent Manner and Affects Sexual Behaviour in Adult Ovariectomized Rats

Recently, we reported that bisphenol A (BPA), an endocrine disrupter, increased progesterone receptor (PR) mRNA in the preoptic area (POA) in adult ovariectomized rats. In the present study, we examined whether BPA also induced expression of PR proteins in both the POA and the ventromedial hypothalamic nucleus (VMH), and whether those proteins were involved in the induction of sexual behaviour. Two weeks after ovariectomy, rats received a subcutaneous (s.c.) injection of BPA, 17 beta-oestradiol or vehicle. Twenty-four hours after the injection, the rats were killed and their tissues were examined by immunocytochemistry. Some rats that received a s.c. injection of BPA, E2 or vehicle alone on the day before were injected with progesterone at 15.00 h and examined for sexual behaviour 5-7 h later. As expected, injection of 10 microg E2 significantly increased the number of PR immunoreactive cells in both the POA and the VMH compared to the number after injection of vehicle alone. In both the POA and the VMH, injection of BPA at a dose of 10 mg also significantly increased the number of PR immunoreactive cells compared to the number after injection of sesame oil alone. Furthermore, BPA induced a dose-dependent increase in the number of PR immunoreactive cells in both the POA and the VMH, demonstrating that the number of PR cells was significantly increased by as little as 100 microg of BPA. Ovariectomized (OVX) rats that were primed with 10 mg of BPA, followed by 1 mg of progesterone, displayed mainly rejection behaviour, but not lordosis as typically observed in OVX rats primed with E2 followed by progesterone. The present study suggests that BPA influences reproductive functions, including sexual behaviour even in adulthood, by altering the PR system in the hypothalamus.

Endocrine disrupter bisphenol a induces orphan nuclear receptor Nur77 gene expression and steroidogenesis in mouse testicular Leydig cells.

The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear receptor superfamily, which plays an important role in the regulation of LH-mediated steroidogenesis in testicular Leydig cells. The aim of the current study was to investigate the potential role of bisphenol A (BPA) on orphan nuclear receptor Nur77 gene expression and steroidogenesis. Northern blot analysis demonstrated that BPA transiently induced Nur77 mRNA expression, and protein kinase inhibitor H-89 and PD98059 strongly inhibited the induction of BPA-mediated Nur77 gene expression in mouse Leydig tumor cell line, K28. Moreover, BPA increased the activation of mitogen-activated protein kinase. Transient transfection assay demonstrated that BPA increased Nur77 gene promoter activity and Nur77 transactivation, whereas BPA did not significantly affect the interaction of Nur77 with its corepressor. Furthermore, BPA increased progesterone biosynthesis in K28 cells, which was suppressed by overexpression of dominant negative Nur77. Finally, BPA injection to prepubertal mice revealed that the expression of Nur77 mRNA was elevated, and this induction was correlated with increased concentration of testicular T in vivo. Taken together, these results demonstrated that BPA induces Nur77 gene expression and subsequently alters the steroidogenesis in testicular Leydig cells. These observations provide a novel mechanism by which BPA acts as an endocrine disrupting chemical.

Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice

Reproductive tract development is influenced by estrogen. The aim of this study was to determine the effects of an environmental estrogenic chemical bisphenol-A (BPA) on prenatal and postnatal development of female mouse reproductive organs. In the prenatal treatment group, BPA or the synthetic estrogen diethylstilbestrol (DES) were given by subcutaneous (s.c.) injections to pregnant mice during gestational days 10–18. Some offspring treated prenatally with 10 and 100 mg/kg bw BPA or 0.67 and 67 μg/kg bw DES were ovariectomized at 30 days and sacrificed at 40 days of age. Vaginal smears were examined in the remaining offspring, then these offspring were mated with normal males. Prenatal exposure to 10 mg/kg BPA reduced the number of mice with corpora lutea compared to sesame oil controls at 30 days, but more than 80% of mice from either prenatally exposed BPA group were fertile at 90 days. Mice exposed prenatally to maternal doses of 67 μg/kg DES were sterile and showed ovary-independent vaginal and uterine epithelial stratification; however, mice exposed prenatally to BPA did not show ovary-independent vaginal and uterine changes. The number of offspring and litter sex ratio from mice exposed prenatally to BPA (10 or 100 mg/kg) or 0.67 μg/kg DES were not different compared to controls. In postnatal treatment group, female mice were given s.c. injections of BPA (15 or 150 μg/pup) or DES (0.3 or 3 μg/pup) for 5 days from the day of birth, then some mice were ovariectomized at 30 days and examined at 40 and 90 days. In the remaining mice, vaginal smears were examined from 61 to 90 days and ovarian histology was evaluated at 90 days. Mice exposed postnatally to 150 μg BPA exhibited ovary-independent vaginal epithelial stratification. Postnatal DES (0.3 and 3 μg) treatment also induced ovary-independent vaginal stratification. Polyovular follicles having more than one oocyte in a follicle were induced by postnatal injections of BPA (150 μg) or DES (0.3 or 3 μg) at 30 days. These findings indicate for the first time that a large dose of BPA can induce ovary-independent vaginal epithelial changes when given postnatally but not prenatally.

The Endocrine Disrupters Butyl Benzyl Phthalate and Bisphenol A Increase the Expression of Progesterone Receptor Messenger Ribonucleic Acid in the Preoptic Area of Adult Ovariectomized Rats

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), so-called endocrine disrupters, are known to mimic the action of estrogens: they are thus liable to influence reproductive functions. Since little is known about their action on gene expression in the adult hypothalamus, we examined the effects of these chemicals on the expression of estrogen-regulated mRNAs, i.e., progesterone receptor (PR) mRNA, preproenkephalin (PPE) mRNA and neurotensin (NT) mRNA, in the hypothalamus and pituitary of adult female rats. Two weeks after ovariectomy, rats were subcutaneously injected with 10 mg BBP, 10 mg BPA, or 10 µg 17β-estradiol (E₂) in sesame oil, or with sesame oil alone as a control. Twenty-four hours after the injection, tissues including the preoptic area (POA), mediobasal hypothalamus (MBH) and anterior pituitary were collected. Northern blot revealed that injection of E₂ resulted in expected changes, i.e., significant increases in PR mRNA in the POA, MBH and anterior pituitary, and in PPE mRNA in the MBH. We also found that injection of BPA significantly increased PR mRNA in the POA and anterior pituitary, while injection of BBP increased PR mRNA in the POA and anterior pituitary, although the increase in the anterior pituitary was not significant. No significant effect of E₂, BPA, or BBP on NT mRNA in the POA was detected. The present study demonstrates that the two endocrine disrupters BPA and BBP can increase the expression of PR mRNA in the POA of adult ovariectomized rats.