Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray

Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.     

Effects of ginsenosides on organogenesis and expression of glutathione peroxidase genes in cultured rat embryos

Ginseng has been extensively used around the world for several thousand years as a food or drug. However, recently, several reports have indicated that the organogenesis of cultured embryos is inhibited by treatment with ginsenoside, the principal component of ginseng. In this study, we evaluated the morphological changes of embryos and the gene expression patterns of antioxidant enzymes, 3 types of glutathione peroxidases [GPx; cytosolic (cGPx), plasma (pGPx) and phospholipid hydroperoxide (phGPx) forms], in cultured rat embryos (embryonic days 9.5-11.5) exposed to ginsenosides Rb1, Rg1, Re and Rc at levels of 5, 50 and 100 microg/ml. With regard to total morphological scores, no significant differences were noted in the embryos exposed to all doses of ginsenosides, with the exception of 50 microg/ml of Rc. In the cultured embryos exposed to Rg1, a majority of the developmental parameters were normal, but growth of the hind- and mid- brains and the caudal neural tube was significantly increased compared with that observed in the control group (P<0.05). Furthermore, Rc significantly enhanced the growth of a variety of developmental parameters in the cultured embryos, with the exception of the hindlimbs. According to the results of our semiquantitative RT-PCR analysis, the levels of cGPx and phGPx mRNA in the cultured embryos were unaffected by treatment with the ginsenosides. However, the levels of pGPx mRNA increased significantly in the embryos treated with ginsenosides Re, Rc and Rb1 compared with the control group (P<0.05). These findings indicate that ginsenosides may exert a stimulatory effect on the growth of embryos via differential expression of GPx genes.     

Immunohistochemical Study of Steroidogenic Enzymes in the Ovary and Placenta During Pregnancy in the Dog

Using the immunohistochemical technique, we attempted to identify the source of secretion of steroid hormones between the mid- and late-terms of gestation in dogs by investigating steroid converting enzymes such as cholesterol side-chain cleavage enzyme (SCC), 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17 alpha-hydroxylase/C17, 20lyase (c17), and aromatase in the ovaries and placenta. Aromatase positive cells were slightly confirmed in luteal cells in the mid-term of gestation (day 40), whereas, in the late-stage (day 50 and 60), the number of aromatase positive cells had increased. However, the oestrogen precursor (c-17 positive cells), could barely be identified in the marginal regions of the corpora lutea (CL) and completely disappeared in the late-stage of gestation. The androgen precursors, convertase SCC and 3 beta-HSD, were confirmed in all regions of the CL during the mid-stage of gestation (day 40), showing particularly strong cell reactions in the marginal region of the CL. Yet, these positive reactions of SCC and 3 beta-HSD in the marginal region of the CL disappeared in the late-stage of gestation. Moreover, it was discovered that the number of SCC and 3 beta-HSD positive cells had decreased in all regions of the CL. None of the enzymes were detected in the placenta. The above results indicated that the source of oestrogen secretion in pregnant dogs is considered to be the CL, and that, compared with the mid-stage of gestation, there was an increased number of oestrogen synthesizing cells within the CL in the late-stage. However, the biosynthetic site of oestrogen precursors from the luteal cells during the late-stage of gestation is still unknown.     

Maternal exposure to bisphenol a during late pregnancy resulted in an increase of Calbindin-D9k mRNA and protein in maternal and postnatal rat uteri

It has been reported that Calbindin-D9k (CaBP-9k) is rapidly and strongly induced by environmental estrogenic compounds, possibly through estrogen receptors (ERalpha) in the uterus of mammals. CaBP-9k can be evaluated as an early gene marker for assaying estrogenic effects of putative environmental chemicals in the rat uterus. This study was undertaken to investigate CaBP-9k mRNA and protein expression in the postnatal rat uterus following maternal exposure to 17beta-estradiol (E2) and bisphenol A (BPA) during the neonatal period. Treatment with a high dose of BPA (600 mg/kg body weight (BW) per day) resulted in a 3-fold increase in CaBP-9k mRNA expression for 3 days, while a single dose of E2 (40 microg/kg BW per day) induced 2-fold increase of this gene in the maternal uterus. In an agreement with maternal CaBP-9k mRNA, postnatal CaBP-9k mRNA in the uterus increased 4-fold when treated with BPA (600 mg/kg BW per day). In addition, treatment with increasing concentrations of BPA resulted in significant increases in CaBP-9k protein in the maternal rat uterus. It is of interest that increasing doses of BPA induced a significant ERalpha mRNA increase in the postnatal uterus. Furthermore, immunohistochemistry revealed that treatment with BPA induced CaBP-9k protein in the maternal uterus. We demonstrated that maternal exposure to BPA during late pregnancy induced CaBP-9k mRNA and protein in maternal and postnatal rat uteri. These results suggest that rapid absorption and distribution of environmental estrogenic compounds occurs in maternal and neonatal rat uteri and these chemicals can easily pass though the placenta during pregnancy to affect postnatal reproductive functions.     

Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa

Virkel, G., Carletti, M., Cantiello, M., Della Donna, L., Gardini, G., Girolami, F., Nebbia, C. Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa. J. vet. Pharmacol. Therap. 33 , 295–303. The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C‐ and 3A‐mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin‐containing monooxygenases (methimazole S‐oxidation), and uridindiphosphoglucuronyltransferases (1‐naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S‐transferase (GST)‐dependent conjugation of 1‐chloro‐2,4‐dinitrobenzene; besides, the GST‐mediated conjugation of ethacrinic acid (GSTπ) or cumene hydroperoxide (GSTα) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4‐dichloronitrobenzene, a protein cross reacting with anti‐rat GSTμ antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes.     

孕鼠暴露双酚A对仔鼠存活率、生殖激素及相关基因的影响

为了研究孕鼠在孕期暴露双酚A（bisphenol A,BPA）对仔鼠生殖激素及相关基因的影响,试验将40只昆明孕鼠随机分为A、B、C、D共4组,每组10只。其中A组为对照组,饲喂普通鼠粮;B、C、D组孕鼠整个妊娠期（妊娠1 d至分娩）分别按每只鼠每天50、500、2 500 mg/kg体重给予BPA,待孕鼠自然分娩,观察记录仔鼠死亡情况。至仔鼠性成熟（56日龄）剖杀仔鼠,摘取睾丸或卵巢称重并计算脏器指数,HE染色观察卵巢或睾丸组织结构的变化,ELISA试剂盒分析仔鼠血清睾酮（T）、促卵泡素（FSH）及雌二醇（E₂）水平,免疫组织化学方法检测仔鼠睾丸或卵巢Bax、Bcl-2蛋白的表达,实时荧光定量PCR检测仔鼠睾丸StAR、CYP11a或卵巢AMH、Kitlg mRNA表达。结果显示,孕鼠暴露BPA极显著增加了仔鼠死亡率（P<0.01）,显著降低了仔鼠睾丸重（P<0.05）。ELISA检测结果表明,孕鼠暴露BPA极显著降低了仔鼠T（♂）及FSH（♀）含量（P<0.01）,极显著升高了仔鼠（♀） E₂水平（P<0.01）。HE染色结果显示,随BPA剂量增加,仔鼠睾丸组织损伤严重,间质细胞减少;卵巢组织结构随BPA剂量增大,空泡逐渐增多,黄体颗粒数量逐渐减少。免疫组化结果显示,孕鼠暴露BPA增加了仔鼠睾丸或卵巢组织中Bax阳性蛋白表达,减少了Bcl-2阳性蛋白表达,显著降低了雄性仔鼠StAR mRNA表达量（P<0.05）;B、D组雄性仔鼠CYP11a mRNA表达量极显著降低（P<0.01）,而C组CYP11a mRNA表达量极显著升高（P<0.01）;C、D组雌鼠Kitlg mRNA表达极显著降低（P<0.01）,AMH mRNA表达量显著升高（P<0.05）。本试验结果表明,孕鼠妊娠期暴露不同剂量BPA增加了仔鼠死亡率,扰乱了生殖激素平衡和睾丸/卵巢中相关凋亡蛋白及生殖基因表达。     

Effects of Maternal Exposure to Bisphenol A on Survival Rate,Reproductive Hormones and Relative Genes of Offspring Mice

This study was aimed to investigate the reproductive toxicity of bisphenol A (BPA) exposed to the mother on the offsprings mice. Forty pregnant Kunming mice were randomly divided into 4 groups, i.e. groups A, B, C and D with 10 mice in each group. Group A was the control group and the mice received conventional feeds, mice in groups B, C and D were given 50,500 and 2 500 mg/kg BW BPA in feedstuffs during the whole gestation period (from 1 d to parturition), respectively. The death rates of the offsprings were calculated every week. The offspring mice were sacrificed at 56 days of age (at puberty). The morphology of ovary and testicular tissues were observed with hematoxylin-eosin (HE) staining. The levels of estradiol (E₂), follicle stimulating hormone (FSH), testosterone (T) in mice serum were detected with ELISA Kit. The protein levels of Bax and Bcl-2 in ovary or testicular tissues were detected with immunohistochemistry, and the StAR,CYP11a mRNA levels in testicular tissues, the AMH, Kitlg mRNA levels in ovary were measured using Real-time PCR. The results showed that exposure of BPA to the mother extremely significantly increased the mortality (P<0.01),and significantly reduced the testicular weight of offspring mice (P<0.05). Maternal exposure to BPA extremely significantly reduced the levels of T (♂) and FSH(♀) (P<0.01),and extremely significantly elevated E₂ (♀) level in offspring mice (P<0.01). BPA exposure damaged the testicular with less leydig cells and ovarian tissues with more vacuoles and less corpus granules in offspring mice. Immunohistochemistry results revealed that maternal exposure to BPA increased the Bax protein level and decreased the Bcl-2 protein level of testicular and ovary tissues in offspring mice. BPA significantly reduced the StAR mRNA expression in male offsprings (P<0.05). However, the mRNA level of CYP11a in groups B and D extremely significantly decreased while group C showed an significant elevation in male offsprings (P<0.01). The expression levels of Kitlg mRNA in groups C and D were decreased extremely significantly in female offsprings (P<0.01), the AMH mRNA expression in groups C and D increased significantly (P<0.05). The conclusion indicated that pregnant mice exposed to different doses of BPA had harmful effects on survival rate in offspring mice, and impact the reproductive hormones, proteins and genes expression.     

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.     

Gene expression profile by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the liver of wild-type (AhR+/+) and aryl hydrocarbon receptor-deficient (AhR-/-) mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic environmental pollutants that cause various biological effects on mammals. The purpose of our study was to identify the genes involved in hepatotoxicity and hepatocarcinogenesis caused by TCDD. C57BL/6 (AhR+/+, wild type) and B6.129-AhR/J (AhR-/-, knock out) mice were injected i.p. with a single treatment of TCDD at the dose of 100 microg/kg body weight. Relative liver weight was significantly increased at 72 hr after TCDD treatment without an apparent histopathological change in AhR+/+ mice (p<0.05). TCDD treatment also significantly increased activity of serum alanine aminotransferase in AhR-/- mice (p<0.05). The liver was analyzed for gene expression profiles 72 hr later. As compared with AhR-/- mice, the expression of 51 genes (>3-fold) was changed in AhR+/+ mice; 28 genes were induced, while 23 genes were repressed. Most of the genes were associated with chemotaxis, inflammation, carcinogenesis, acute-phase response, immune responses, cell metabolism, cell proliferation, signal transduction, and tumor suppression. This study suggests that the microarray analysis of genes in the liver of AhR+/+ and AhR-/- mice may help to clarify the mechanism of AhR-mediated hepatotoxicity and hepatocarcinogenesis by TCDD.     

Effects of bisphenol A administration to pregnant mice on serum Ca and intestinal Ca absorption

Bisphenol A (BPA) is a xenoestrogen commonly used in food storage plastics. The present study was conducted to clarify the effects of BPA administration to pregnant mice on serum calcium (Ca) and Ca metabolism of the gut and kidney. From 6.5 to 16.5 days post coitus (dpc), pregnant mice were administered at 2 mg or 20 mg/kg body weight/day of BPA. Serum Ca was decreased in mice treated with 20 mg BPA at 17.5 dpc, but no remarkable differences were detected in the alkaline phosphatase activity and vitamin D receptor protein expression in the duodenum and jejunum. The messenger RNA (mRNA) expressions of calcium binding protein (CaBP‐9k) and active vitamin D synthesis enzyme (CYP27B1) in the kidney were increased in mice treated with 20 mg BPA. The mRNA expressions of occludin and junction adherence molecular A (JAM‐A) in the duodenum and ileum, which regulate paracellular transport, were increased in mice treated with 20 mg BPA. However, the administration of 2 mg BPA had no effect on serum Ca and mRNA expressions of relative genes in Ca metabolism. These results imply that BPA administration at 20 mg/kg body weight/day during pregnancy decreases serum Ca in pre‐delivery mice, which may be partly due to decreased paracellular Ca absorption.     

Comparison of aryl hydrocarbon receptor gene expression in laser dissected granulosa cell layers of immature rat ovaries

In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more β-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.     

Expression of tumor necrosis factor-alpha and cyclooxygenase-2 mRNA in porcine split-thickness wounds treated with epidermal growth factor by quantitative real-time PCR

Epidermal growth factor (EGF) accelerates the re-epithelialization of damaged epidermal cell layers in a wound, so it especially shortens the duration of wound healing. The effect of EGF on pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2), levels during wound healing has not been reported. We investigated the relationship between exogenous EGF treatment and the expression of TNF-alpha and COX-2 mRNA in porcine split-thickness wounds by real-time PCR. Twenty split-thickness wounds were created on the back of five pigs. Fifteen wounds were treated twice daily with EGF ointments (1 microg/g, 10 microg/g, and 50 microg/g) for 10 days and five wounds were untreated. Healing time until full-epithelialization was evaluated. We performed a quantitative analysis of TNF-alpha and COX-2 mRNA expression in wound biopsies using real-time PCR. Topical application of 1 microg/g EGF accelerated re-epithelialization more than treatments of EGF at 10 microg/g and 50 microg/g, and no treatment. The levels of TNF-alpha and COX-2 mRNA were significantly greater in wounds treated with 1 microg/g than those with 10 microg/g and 50 microg/g EGF, and no treatment. Topical treatment of EGF influences the level of TNF-alpha and COX-2 mRNA within porcine split-thickness wounds. EGF-dependent slightly up-regulation of TNF-alpha and COX-2 mRNA expression during the inflammatory phase of healing may create an optimal molecular environment for wound healing.