Lactational coumestrol exposure increases ovarian apoptosis in adult rats.

This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81-84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135-140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health.

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.

Neurotoxic effects of alcohol and acetaldehyde during embryonic development.

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.

Multigeneration reproductive and developmental toxicity study of bar gene inserted into genetically modified potato on rats.

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive-developmental ability.

Assessment of estrogenic and androgenic activities of tetramethrin in vitro and in vivo assays.

Tetramethrin, a synthetic pyrethroid insecticide, is used globally for agriculture, and thus potential environmental exposure to tetramethrin is a concern. Environmental chemicals that are hormonally active (particularly estrogen or androgen) may adversely affect the reproductive and endocrine systems. However, little is known about the estrogenic and androgenic activities of tetramethrin. In this study, uterine CaBP-9k gene expression assay and a uterotrophic assay were conducted for estrogenic activity assessment of tetramethrin, and a Hershberger assay was conducted for androgenic activity. Estrogen receptor (ERalpha and ERbeta) protein levels were also measured in tetramethrin-treated rat uteri. Northern blot analysis showed reduction in uterine CaBP-9k mRNA levels in response to tetramethrin, as well as when rats were given both tetramethrin and 17beta-estradiol (E2). In the uterotrophic assay using 18-d-old female Sprague-Dawley rats, subcutaneous treatment with tetramethrin (5 to 800 mg/kg/day) for 3 d led to a statistically significant decrease in absolute and relative uterine wet weights at all doses tested. Moreover, tetramethrin blocked the effect of E2 on uterine weights. In addition, tetramethrin reduced absolute and relative vaginal wet weights, and also inhibited the increases of vaginal weights produced by E2. Tetramethrin showed no androgenic on antiandrogenic activities in the Hershberger assay. These results suggest that tetramethrin might exert endocrine-disrupting effects on female rats through antiestrogenic action.

Potential estrogenic and antiandrogenic effects of permethrin in rats.

Many environmental chemicals including pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. Permethrin, a synthetic pyrethroid insecticide, is used worldwide, which provides potential environmental exposure. However, relatively few studies have reported on hormonal activities, particularly estrogenic and androgenic activities of permethrin, and the results of these studies are in some respects contradictory. Therefore, this study investigated the potential estrogenic and androgenic activities of permethrin in vitro and in vivo. We conducted an uterine Calbindin-D9k (CaBP-9k) gene expression assay and an uterotrophic assay for estrogenic activity, and a Hershberger assay for androgenic activity. The CaBP-9k gene, one of the intracellular calcium binding proteins, is estrogen-responsive in the uterus. The rat uterotrophic and Hershberger assays are generally used as in vivo short-term screening assays for detecting the estrogenic and androgenic activities of chemicals, although these assays are still being validated by the Organization for Economic Cooperation and Development (OECD). Northern blot analysis showed the induction of uterine CaBP-9k mRNA level in response to permethrin as well as co-administration of permethrin with E2. In the uterotrophic assay using 18-day-old female rats, subcutaneous treatments with permethrin (10 to 800 mg/kg) for three days increased relative uterine wet weights, and E2-induced uterine weights. These effects were statistically significant at 800 and 200 mg/kg, respectively. Moreover, permethrin-induced uterine weights were inhibited by the co-administration of ICI 182,780, an antiestrogen. In the Hershberger assay, the administration of permethrin orally to testosterone propionate-treated castrated male rats led to statistically significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights at all doses tested (10, 50 and 100 mg/kg). These results suggest that permethrin might have estrogen-like effects on female rats, but antiandrogen-like effects on males.

Neuronal differentiation of embryonic midbrain cells by upregulation of peroxisome proliferator-activated receptor-gamma via the JNK-dependent pathway.

Our previous study showed that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist 15-deoxy-PGJ(2) has the promoting ability to differentiate neuronal PC12 cells. To expand our study, the effect of 15-deoxy-PGJ(2) on the differentiation of embryonic midbrain cells into dopaminergic neuronal cells was investigated in this study. The relationship between cell differentiation with activation of PPAR-gamma and the possible signal pathway were also investigated. 15-Deoxy-PGJ(2) increased neurite extension, a typical characteristic of the differentiation of embryonic midbrain cells isolated from 12-day rat embryos in a dose-dependent manner. The expression of differentiation markers, neurofilament, tyrosine hydroxylase, and nestin, was also increased by the treatment of 15-deoxy-PGJ(2). Consistent with the increasing effect on cell differentiation, 15-deoxy-PGJ(2) increased the expression and transcriptional activity of PPAR-gamma in cultured embryonic midbrain cells. In addition, the expression of PPAR-gamma and NeuN in the differentiated neuron of fetus (17 days) and adult rat brain was co-localized. Furthermore, treatment of PPAR-gamma antagonist bisphenol A diglycidyl ether blocked 15-deoxy-PGJ(2)-induced neuronal differentiation of embryonic midbrain cells and expression of PPAR-gamma. To elucidate the possible signal pathway, the activation of mitogenic-activated protein (MAP) kinase family was determined. 15-Deoxy-PGJ(2) (0.5 microM) increased activation of Jun N-terminal kinase (JNK) and p38 kinase but not extra-signal response kinase (ERK). In addition, NGF (50 ng/ml) further increased the 15-deoxy-PGJ(2)-induced JNK activation. Moreover, pretreatment of specific inhibitor of JNK SP600125 blocked the 15-deoxy-PGJ(2)-induced JNK activation. This inhibition correlated well with the inhibition of neurite extension and expression of PPAR-gamma induced by 15-deoxy-PGJ(2). The present results therefore indicate that 15-deoxy-PGJ(2) stimulates differentiation of embryonic midbrain cells into dopaminergic neuronal cells, and its effect may be PPAR-gamma and JNK signal pathway dependent.

No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats.

Several studies have demonstrated that bisphenol A (BPA) exhibited weak estrogenic activity in the 3-day uterotrophic assay using ovariectomized (OVX) and immature rats (Toxicol. Lett. 115 (2000) 231; Regul. Toxicol. Pharmacol. 32 (2000) 118; J. Toxicol. Sci. 26 (2001) 111) and BPA also possessed anti-androgenic activity in in vitro yeast based assays (J. Endocrinol. 158 (1998) 327). To investigate anti-androgenic effects of BPA. a rodent Hershberger assay was carried out using immature Sprague-Dawley male rats. An androgen agonist, testosterone (0.4 mg/kg per day), was administered for 7 consecutive days by subcutaneous (s.c.) injection as a positive control. Additionally, a pure androgen antagonist, flutamide (1, 5. 10 mg/kg per day. oral) was co-administered with testosterone (0.4 mg/kg per day s.c.). BPA was also administered orally with or without testosterone (0.4 mg/kg per day, s.c.) for 7 consecutive days. In the testosterone treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani plus bulbocavernosus muscles (LABC) weights were significantly increased compared with control. However. flulamide dose-dependently inhibited the testosterone-induced re-growth of seminal vesicles, ventral prostate, and LABC, with a significant decrease at flutamide 1.0 mg/kg and above (P<0.05). Serum LH levels were also significantly increased (5 mg/kg and above, P<0.05), but no changes in serum testosterone levels. In contrast, BPA had no effects on the re-growth of seminal vesicles, ventral prostate and LABC induced by testosterone, and no significant differences were observed in serum LH and testosterone levels. In summary, the Hershberger assay could be a sensitive method for detecting androgenic or anti-androgenic chemicals, but BPA did not exhibit any androgenic or anti-androgenic activities in Hershberger assay.

Inhibitory effect of peroxisome proliferator-activated receptor gamma agonist on ochratoxin A-induced cytotoxicity and activation of transcription factors in cultured rat embryonic midbrain cells.

The effects of 15-deoxy-delta12,14-prostaglandin J2 (15-deoxy PGJ2) on ochratoxin A (OTA)-induced neurotoxicity and on the activation of transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) were investigated in cultured rat embryonic midbrain cells. Twelve-day rat embryo midbrain cells were cultured for 48 h. OTA (0.5 or 1 microg/ml) and/or 1.5-deoxy PGJ2 (0.5 microM) were then added for 48 h. Cell number and neurite outgrowth were determined to assess the neurotoxicity of OTA. AP-1 and NF-kappaB activation was determined by gel mobility shift assay after 3 h of exposure to OTA and/or 15-deoxy PGI2. OTA caused concentration-dependent reductions in neurite outgrowth and cell number, and induced AP-1 and NF-kappaB activation. Cotreatment with 15-deoxy PGJ2 (0..5 microM) blocked OTA-induced decrease in neurite outgrowth and cell number and inhibited AP-1 and NF-kappaB activation. 15-Deoxy PGJ2 (0.5 microM) caused the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the cells. Results show that 1.5-deoxy PGJ2 blocked OTA-induced neurotoxicity by inhibiting AP-1 and NF-kappaB activation in cultured rat embryonic midbrain cells.

Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo.

This study was undertaken to compare the sensitivity of screening test methods and to investigate the structure-activity relationships of the estrogenic activity of alkylphenolic compounds (APs) using in vitro and in vivo assays. Two in vitro systems, MCF-7 cell proliferation (E-screen assay) and competitive binding assay to estrogen receptor (ER), were selected to evaluate the estrogenic effects. Uterotrophic assay and Calbindin-D9K (CaBP9K) mRNA expression were also examined in ovariectomized Sprague-Dawley female rats. A series of APs with various alkyl groups were examined, namely, 4-propylphenol, 4-butylphenol, 4-t-butylphenol, 4-pentylphenol, 4-nonylphenol, 4-octylphenol, 4-t-octylphenol, and 4-phenylphenol, and 17beta-estradiol (E2) was used as a positive control. In the E-screen assay, E2 was found to induce maximum proliferation of MCF-7 cells at 1 nM. Among the APs, 4-t-octylphenol and 4-nonylphenol were found to be considerably more potent than any other compound and estrogenic effects were detectable at 1 and 10 microM, respectively. 4-t-Octylphenol and 4-nonylphenol inhibited the binding of E2 to the ER of MCF-7 cells in a competitive ER binding assay. The uterotrophic effects to APs (10, 50, 200, and 400 mg/kg/d) were compared to E2 (1 microg/kg) in ovariectomized rats after treatment for 3 d. 4-Nonylphenol, 4-t-octylphenol, and 4-phenylphenol produced dose-dependent increases in the uterine weights of ovariectomized rats. In the CaBP-9K mRNA expression test, CaBP-9K mRNA levels were detected in the uteri of ovariectomized rats treated with 4-pentylphenol (400 mg/kg), 4-nonylphenol, 4-phenylphenol (200 and 400 mg/kg), and 4-t-octylphenol (50 mg/kg and above), respectively. In the dot blot assay, CaBP-9K mRNA levels were significantly increased in rats exposed to 4-t-octylphenol (200 and 400 mg/kg), 4-pentylphenol, 4-nonylphenol, and 4-phenylphenol (400 mg/kg), respectively. Among the APs, compounds with bulky alkyl groups or higher carbon numbers possessed higher estrogenic capacity. In addition, the pattern of CaBP-9K expression correlated with that of the 3-d uterotrophic assay. Therefore, our results suggest that the CaBP-9K gene might be used as a potential biomarker for the screening of endocrine disruptors.