Impact of endocrine disrupting chemicals on reproduction in Japanese quail

Many environmental contaminants can interact with the endocrine system, thereby potentially disrupting the reproductive fitness of individuals. In avian species, the egg-yolk is a major route for excretion of lipophilic compounds by the adult female bird and embryos are exposed to contaminants that have been deposited in the eggs. The reproductive and neuroendocrine system of Japanese quail undergoes sexual differentiation during embryo development. The phenotypic sex, including sex-specific adult behavior, is hormonally imprinted already before hatching. The sexual differentiation of the brain in quail is sensitive to estrogens and the presence of estrogen results in a female phenotype. The relatively low concentration of estrogens in male embryos, on the other hand, results in a male behavioral phenotype. The behavior of male quail can be demasculinized by estrogen exposure during the period of sexual differentiation, and estrogen-exposed males are not able to display a male-typical behavior as adults. Also, differentiation of the reproductive organs is sensitive to hormones during embryogenesis, and an excess of estrogens can for instance induce persistent morphological changes in the reproductive organs of females. Our research has focused on effects in adult birds after embryonic estrogen exposure. We have studied sexual behavior and other reproductive variables in adult quail after in ovo injection of known and suspected estrogenic compounds. Synthetic estrogens and insecticides, such as o,p'-DDT altered the development of the neural system and resulted in demasculinization of male quail. In females, o,p'-DDT caused morphological changes of the oviduct and egg laying was reduced. Our studies suggest that the neural system and the female reproductive system of avian embryos are very sensitive to the effects of chemicals with estrogenic activity.     

The biomarker and endocrine disruptors in mammals

The compounds that bind steroid hormone receptors including estrogen receptors (ERs), progesterone receptor (PR) or androgen receptor (AR), and induce or modulate a steroid hormone receptor-mediated response could be defined as endocrine disruptors (EDs). Currently, there are no standard methods to determine whether a chemical is an endocrine disruptor or not. Most results of in vitro and in vivo data are derived from assays that measure estrogenic activity, thus fewer data are available from assays that measure androgenic and progestogenic activities. In this review, we introduce a novel in vivo model to detect EDs using immature rats in the induction of Calbindin-D(9k) (CaBP-9k) mRNA and protein by estrogenic compounds. In addition, we summarize other biomarkers and screening methods for EDs in mammals to describe the usefulness of indicated biomarkers, although mammalian models are very few based on experimental findings.     

Combined action of plant estrogens, F-2 toxin and natural estrogens

The investigation concerns the interaction of plant estrogens, F-2 toxin, and natural estrogen (estradiol-benzoate) as causative factors of eostrogenic changes. Plant estrogens and F-2 toxin were applicated at three and estradiol-benzoate at two dosage levels. The estrogenic effect was studied by bioassay on immature rats taking uterine weight, uterine liquid and vaginal opening as criteria. When uterine weight is taken as a criterion, plant estrogens and F-2 toxin, no matter if they are administered alone or together, regularly increase the effect of estradiol-benzoate, but when uterine fluid is taken as a criterion they always decrease the effect of estradiol-benzoate. According to the present results it seems obvious, that the mode of action of estrogenic substances occurring in plants considerably differ from that of natural estrogens. Thus the mechanisms which cause disturbances may be different from those of natural estrogens.     

The effect of storage on the estrogenic effect of red clover silage.

Pure red clover was selected for plant estrogen analyses from the fresh fodder crops of 1972 and 1973. The investigations were made on fresh red clover and on the same red clover after periods of storage of varying length. The red clover was stored in manilla bags in a silo among the ordinary silge fodder, use being of the "green solution method" (Farmos Oy). In the studies the estrogenic effect of the fodder was ascertained by means of bioassay, the criterion being the increase in murine uterine weight. The known plant estrogens were determined by thin layer chromatography and by liquid chromatography. The estrogenic effect of the red clover silage fodder of 1972 was greater in all the silage fodder samples than it was in the fresh red clover. The quantity of individual isoflavones and "transformed" estrogen too, was greater in many of the silage fodder samples than it was in the respective fresh red clover. The estrogenic effect of the silage fodder made from the red clover of 1973 varied considerably; in some samples it was greater but in most it was smaller than the estrogen effect of the fresh red clover. However, apart from a few exceptions, the quantity of individual isoflavones and of transformed estrogens was smaller than it was the fresh red clover.     

A sensitive bioassay for detection of dietary estrogens in animal feeds

Estrogen-responsive proliferation in the MCF-7 cell line was used as a bioassay for detection of dietary estrogens. The bioassay procedure was adapted to screen for estrogenic activity in feedstuffs that have been associated with hyperestrogenism in livestock. Methanolic feed extracts were added to the cell culture medium at microliter/ml concentrations for 4 days, after which the cell proliferation response was measured as DNA content. The half-maximal response for estradiol occurred at 2 pM, or 0.54 pg/ml. For zearalenone, a weaker estrogen, the half-maximal response occurred at approximately 200 pM, or 64 pg/ml. The bioassay was calibrated against a number of known estrogens (estradiol, diethylstilbestrol, zearalenone, zearalanol [cattle implant], beta-zearalenol, zearalane), including the naturally occurring phytoestrogens (formononetin, genistein, daidzein, biochanin A, and coumestrol). The estrogenic activity of feed samples was expressed as equivalents of zearalenone (ppm zearalenone) that would have to be present to equally stimulate proliferation of the MCF-7 cells. The sensitivity of the bioassay was 0.05-0.1 ppm equivalents of zearalenone in feed, well below the threshold level associated with reproductive problems. The feed additive melengestrol acetate (MGA) showed no estrogenic activity in this assay. Estrogenic activity of feed extracts was confirmed by competitive inhibition with the antiestrogens tamoxifen or LY156758 (keoxifene) to show that stimulation of growth by feed extracts was through an estrogenic mechanism. Confirmation of known estrogens was by tandem mass spectroscopy. The assay is a sensitive and reliable screening procedure for detecting estrogenic activity in feedstuffs.     

Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k

Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17beta-estradiol (E2; 9-fold) and 17alpha-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10(-9) M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10(-6) M up to 10(-4) M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10(-4) M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.     

雌激素对哺乳动物卵泡发育的影响研究进展

雌激素在卵泡发育过程中所起的作用,已经得到人们的普遍认可。雌二醇(E2)及其类似物在卵泡的体细胞的增殖和分化中起重要作用,但其作用机理目前还不完全清楚。一些最新研究结果雌激素受体β(ERβ)的发现、雌激素受体基因敲除以及雌激素缺失动物模型的建立等,使我们能够进一步探讨雌激素在卵泡发育过程中的作用机理。结合这些最新研究动态,探讨雌激素在卵泡发育过程中的作用及雌激素对卵巢內的体细胞表型分化的影响。     

Effect of Estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.     

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.     

The effect of phytoestrogens on the reproductive tract

Environmental estrogens may be derived from plants (phytoestrogens), pharmaceuticals or synthetic compounds. They exert estrogenic and/or potentially antiestrogenic effects on farm animals, wildlife and humans. Exposure to these compounds results in some abnormalities in the reproductive tract, changes in the estrous cycle, and possibly protection against the development of hormone-dependent cancer. The data obtained from animal studies suggest that the timing of exposure to phytoestrogens is important, and neonatal exposure causes the most pronounced effects.     

Assessing estrogenic activity of pyrethroid insecticides using in vitro combination assays

Pyrethroid insecticides are among the most commonly used classes of insecticides worldwide, but their endocrine disrupting activities remain unclear. Therefore, in the present study, we examined the estrogenic activities of pyrethroid insecticides in E-screen and competition binding assays. In addition, we measured estrogen receptor (ER) protein and pS2 mRNA levels in human breast cancer cells (MCF-7 BUS) to clarify the mechanism of their estrogenicity. Seven pyrethroid insecticides (bioallethrine, cypermethrin, deltamethrin, fenvalerate, permethrin, sumithrin, and tetramethrin) were tested because of their worldwide usage. In addition, 17beta-estradiol was tested as a positive control. As expected, 17beta-estradiol significantly increased MCF-7 BUS cell proliferation at concentrations of 10(-11) M and above. Of the pyrethroid insecticides tested, only sumithrin increased MCF-7 BUS cell proliferation in a dose-dependent manner; the maximum induction of cell proliferation was observed at a dose of 10(-5) M. In the anti-estrogenic activity test, bioallethrin, fenvalerate, and permethrin significantly inhibited 17beta-estradiol-induced MCF-7 BUS cell proliferation at 10(-6) M, a concentration comparable to the effective dose (10(-9) M) of ICI 182,780, a pure ER antagonist. However, none of the pyrethroid insecticides competitively inhibited the binding of [(3)H]estradiol to rat uterus ERs in competition binding assays. Both 17beta-estradiol (10(-10) M) and sumithrin (10(-5) M) decreased the levels of cytosolic ERalpha and ERbeta protein expression significantly as compared with the vehicle control. In addition, 17beta-estradiol (10(-10) M) increased pS2 mRNA expression markedly, and sumithrin significantly increased pS2 mRNA levels in a dose-dependent manner. The other six compounds tested in the present study did not affect ER protein levels or pS2 mRNA levels. These results suggest that certain pyrethroid insecticides may be considered to be estrogen-like chemicals that act through pathways other than direct ER binding, and may function as endocrine modulators in both wildlife and humans.     

Board-invited review: Estrogen and progesterone signaling: genomic and nongenomic actions in domestic ruminants

Progesterone and estrogens play key roles in regulating various physiological phenomena related to normal growth, development, and reproduction of domestic animals. This review focuses on the mechanisms by which progesterone and estrogens regulate the reproductive processes in these animals. The majority of research on the actions of progesterone and estrogens on the reproductive systems of cattle, sheep, and pigs has been genomic in nature and represents attempts to better understand how these steroids regulate gene expression. Results of recent research suggest that progesterone and estrogens can alter target cell responses nongenomically via membrane receptors. The characteristics of membrane receptors for progesterone and estrogen in various cell types are described and the intracellular signal pathways defined. Estrogens acting via membrane receptors can suppress LH secretion by gonadotropes and stimulate rapid increases in uterine blood flow. Progesterone acting via a membrane receptor has been shown to inhibit binding of oxytocin to oxytocin receptors in isolated endometrial plasma membranes and stimulate capacitation of spermatozoa. Results of research suggest that progesterone and estrogens can act nongenomically to alter target cell responses in domestic animals. The biological implications of this mode of action in these animals are discussed.     

Androgen and estrogen receptors in bovine skeletal muscle: relation to steroid-induced allometric muscle growth

The presence of free androgen (AR) and estrogen receptors (ER) was demonstrated in bovine skeletal muscle. Androgen receptor concentrations in neck muscle from cattle of different sexes and stages of development were related to hormonal status. In mature bulls (mean weight 600 kg), no free AR was detectable. Highest AR concentrations were measured in mature bulls (517 kg) castrated 24 h prior to slaughter (.85 +/- .21 fmol/mg protein). In female calves (155 kg), AR concentrations (.56 +/- .14 fmol/mg) were greater (P less than .01) than in male calves (.20 +/- .08 fmol/mg) of the same weight. Androgen receptors and ER in skeletal muscle of neck, shoulder, abdomen and hind leg of female and male calves were compared. There was no significant difference between AR concentrations in the neck, shoulder and hind leg, but concentrations were lower (P less than .05) in abdominal muscle. Estrogen receptor concentrations in neck, shoulder, abdomen and hind leg were not different between sexes (P less than .05). In male calves, ER content was lower (P less than .05) in abdominal than in other muscles. Estrogen receptor concentrations in muscles of female calves did not differ (P less than .05). The pronounced sensitivity to estrogens and androgens in the neck, shoulder, and hind leg of calves, being free of the respective hormone, may partly explain the characteristic conformation in calves treated with estrogenic and androgenic steroids and the sexual dimorphism of muscle growth.     

猕猴脾脏中雌激素受体的表达及其性别差异

选取健康成年雌雄猕猴各3只,采用免疫组织化学SP法研究了雌激素受体(ER)在脾脏中的分布,观察猕猴脾脏中ER的表达及性别差异.结果显示,ER免疫阳性反应物主要分布于脾脏红髓区,脾小结相对较少,而动脉周围淋巴鞘、血管内皮、脾小粱等组织结构内仅有少量分布.ER阳性产物主要定位于细胞核中.部分存在于胞浆和胞膜上.雌性猕猴脾脏中的阳性细胞数量显著高于雄性,表达强度也较雄性强.这表明,ER参与雌激素对脾脏的免疫功能调控.ER阳性产物分布特点表明雌激素发挥作用主要是通过经典基因组机制,同时也通过非基因组机制途径.而ER表达的明显性别差异提示体内雌激素水平可能对脾脏中B淋巴细胞的功能有正调节作用.     

Calbindin-D9k mRNA expression in the rat uterus following exposure to methoxychlor: a comparison of oral and subcutaneous exposure

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein that is induced by estrogenic compounds possibly through estrogen receptors. We compared CaBP-9k mRNA expression in the uterus with uterotrophic response in immature rats exposed to methoxychlor (MC), an environmental chemical with estrogenic activity. MC was orally or subcutaneously administered to 3-week-old female Sprague-Dawley rats for 3 days. The weights of the uterus and vagina significantly increased in the oral treatment group at a dose of 50, 100 and 200 mg/kg, but those of the subcutaneous (SC) treatment group only increased at 200 mg/kg. Northern blot analysis showed that CaBP-9k mRNA expression was significantly induced in a dose-dependent manner at doses of 50, 100 and 200 mg/kg/day in the oral treatment group. SC administration of MC induced significant expression at only a dose of 200 mg/kg/day; this was similar to the uterotrophic response. MC has an estrogenic effect on the uterus as shown by the increase in weight and induction of CaBP-9k mRNA expression, which were much greater following exposure via oral gavage than via the SC route. The strong correlation between the results of in vivo uterotrophic assay and CaBP-9k mRNA expression suggests that CaBP-9k mRNA expression in the rat uterus may be used as an early gene marker for detection of the estrogenic effects of putative environmental chemicals.     

Predictive modes of action of pesticides in uterine adenocarcinoma development in rats

Endometrial adenocarcinoma in the uterine corpus is a malignant cancer that occurs in menopausal women and aged rodents. Because of the similarities in pathogenesis and morphology of endometrial adenocarcinoma in rodents and humans, prediction of the modes of action (MOA) in uterine carcinogenesis is important for extrapolation of rodent data to humans. Three MOAs have been accepted as major pathways for uterine carcinogenesis in rodents: 1) estrogenic activity, 2) increased serum 17beta-estradiiol (E2) to progesterone (P4) ratio and 3) modulation of estrogen metabolism to produce 4-hydroxyestradiol via P450 induction. Inhibition of estrogen excretion and increased aromatase in situ in the tumor are also a potential pathway. Here, chemicals showing uterine carcinogenicity were chosen from approximately 300 pesticides evaluated in Japan within the past decade, and their mechanisms were predicted using parameters from mechanistic and toxicity studies. Seven pesticides increased uterine tumor formation in rats, and the pathways of 4 pesticides could be predicted based on various mechanistic studies. The MOAs of cyenopyrafen and benthiavalicarb-isopropyl were predicted to be modulation of estrogen metabolism, while those of pyriminobac-methyl and spirodiclofen were predicted to be increased E2 to P4 ratio. The driven pathways of metazosulfuron and isopyrazam could not be predicted using several mechanistic studies. No mechanistic studies have been reported for sedaxane, which has a chemical structure and toxicological profile similar to isopyrazam. Our results indicated that appropriate mechanistic studies are useful for mechanism prediction in risk assessment. From this analysis, a flowchart showing a decision tree for predictive MOAs in uterine carcinogenesis was proposed.     

Analysis of the influence of temperature on the stability of sex steroids in cow feces

Up to the present natural and synthetic steroids have only rarely been considered a cause of disturbances of sexual behavior and anomalies of sex organs. The increasing environmental contamination with chemicals showing estrogenic effects underlines the importance of further investigations in that matter. Therefore, the concentrations of estrogens and progesterone in the faeces of cattle were examined over a 13 weeks period. The values obtained are presented as equivalents of the standard substances estrone and 4-pregnene-20 beta-ol-3-one. The samples were stored in a refrigerator or incubator at temperatures of 5 degrees C and 30 degrees C, respectively, and the investigations were carried out by using an enzyme-immunoassay. Estrogen and progesterone concentrations decreased more rapidly when incubation was performed at 30 degrees C as compared to storage at 5 degrees C, and estrogen values declined more slowly than those of progesterone. When keeping the samples at 5 degrees C the estrogen concentrations decreased to below the basal values only beyond the eleventh week of storage. In contrast, comparable concentrations were already obtained during the third week of the experiment if the samples were stored at 30 degrees C. Progesterone levels decreased to below the basal values during the fourth week of storage at 5 degrees C and during the second week at 30 degrees C.     

唇形科植物提取物对单胃动物的抗氧化作用及其机制

动物在患病、应激或特殊生理条件下,机体内会大量产生自由基,自由基与机体内的脂质、ＤＮＡ等产生反应,可对动物造成氧化损伤。唇形科植物提取物中的抗氧化物主要活性成分是酚类化合物,它和抗坏血酸及类胡萝卜素能够阻止自由基引发的氧化伤害。本文阐述了唇形科植物提取物对猪、鸡体外和体内抗氧化作用的实例与抗氧化功能,分析了其抗氧化作用机制。