Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10(-8) M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17beta (E(2)) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E(2) in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.     

Reciprocal inhibiting interactive mechanism between the estrogen receptor and aryl hydrocarbon receptor signaling pathways in goldfish (Carassius auratus) exposed to 17β-estradiol and benzo[a]pyrene

In the aquatic environment, both the estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) responses are established biomarkers for assessing exposure to pollutants. These receptor responses can also be affected by the presence of other classes of pollutants and may result in misinterpretation of existing pollution. In this study, we investigated the interaction between ER-vitellogenin (VTG) and AhR-cytochrome P450 1A (CYP1A) signaling pathways in goldfish (Carassius auratus) after 10 days exposure to pollutants. 17β-Estradiol (E(2)) and benzo[a]pyrene (BaP) were selected as the ER and AhR agonists, respectively. The messenger RNA (mRNA) expression of ER-VTG and AhR-CYP1A in liver was determined using quantitative real-time polymerase chain reaction (QRT-PCR). VTG, endogenous E(2) and 7-ethoxyresorufin-O-deethylase (EROD) were also studied. Exposure to E(2) and BaP alone significantly induced the gene expression of ERα-VTG and AhR2-CYP1A, respectively. Moreover, the obvious expression of related proteins was also observed. However, these inductions were significantly reduced after combined exposure to E(2) and lower concentrations of BaP (20 and 50 μg/L), indicative of a reciprocal inhibiting ER-AhR interaction. However, high concentrations (100 μg/L) of BaP did not affect the E(2)-induced gene expression. Changes in VTG protein were in accordance with the expression of VTG mRNA, and more VTG protein was observed in liver than in serum. The induced endogenous E(2) levels were suppressed by the presence of BaP. While the gene expression of CYP1A showed a concentration-dependent increase, EROD induction exhibited a bell-shaped concentration-response curve. Taken together, these results demonstrate a reciprocal inhibiting mode of ER-AhR interactions and may lead to a possible underestimation of actual exposure.     

Delayed gametogenesis and progesterone levels in soft-shell clams (Mya arenaria) in relation to in situ contamination to organotins and heavy metals in the St. Lawrence River (Canada)

Wild carp, Cyprinus carpio, were sampled in January and March 2000 in a section of the Anoia River (NE Spain) known to be polluted by estrogenic compounds. At each sampling time, three groups were distinguished: (1) apparently normal males; (2) apparently normal females; and (3) affected fish. The latter were characterized by the simultaneous development of male and female tissue in their gonads at a macroscopical level (six out of 31 fish sampled at this particular point), or testicular atrophy (three out of 31). Plasmatic and hepatic vitellogenin (VTG) levels and plasma testosterone (T) and estradiol (E2) were measured to observe the particular estrogenic response of the affected fish. Moreover, the response in the xenobiotic metabolizing capacity in liver was tested. This involved the analysis of mixed function oxygenase (MFO) system such as: total cytochrome P450 content, NAD(P)H cytochrome c reductases and the associated CYP1A1, EROD activity. Also, glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) as detoxifying enzymes were measured. Our results showed: (1) a highly variable VTG content in all fish groups; (2) an increase in sex hormones content in March for the female group; and (3) an enhanced xenobiotics metabolism in the affected fish group, measured as total cytochrome P450, EROD activity in the January survey and cytosolic GST in March. The observed increase in VTG, sex hormones and in most of the enzymatic activities from January to March that could also be attributed to higher water temperature.     

Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor.

Hepatic CYP1A expression in fish can be modulated by the female sex hormone, 17beta-estradiol (E2), however neither the mechanism of E2 suppression of CYP1A nor the capacity for hormonal regulation to overcome CYP1A induction by xenobiotics are known. The present study investigates for the first time in fish if the estrogen receptor (ER) is involved in the suppressive action of E2 on CYP1A gene expression. The study further examines, if the E2 effect is able to overcome xenobiotic induction of CYP1A. As experimental model, in vitro cultures of rainbow trout, Oncorhynchus mykiss, hepatocytes were used. The effect of E2 on CYP1A was assessed by measuring the CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity, and CYP1A mRNA contents. E2 at non-cytotoxic concentrations caused a significant time- and concentration-dependent decline of basal but not of induced hepatic EROD activities. The inhibitory action of E2 on basal CYP1A was also evident at the mRNA level. The presence of the ER antagonist tamoxifen abolished the inhibitory action of E2 on CYP1A expression. The results from these in vitro experiments provide evidence (a) that the ER is involved in the suppressive action of E2 on CYP1A, and (b) that E2 inhibitory action does not overcome xenobiotic induction of CYP1A.     

Induction of 7-ethoxyresorufin-O-deethylase activity by planar chlorinated hydrocarbons and polycyclic aromatic hydrocarbons in cell lines from the rainbow trout pituitary

The induction of 7-ethoxyresorufin-o-deethylase (EROD) activity was examined in three rainbow trout pituitary cell lines: RTP-91E, RTP-91F and RTP-2. RTP-91E and RTP-91F were developed from the pituitary of a male and have epithelial-like and fibroblast-like morphologies, respectively. RTP-2, which was described previously, was developed from the pituitary of a female and has an epithelial-like shape. In all cell lines EROD activity was induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Immunoblotting with the polyclonal antibody, anti-trout CYP1A1(277-294)/KLH, confirmed induction of a 58-kDa polypeptide. Potential inhibitors of the aryl hydrocarbon receptor, geldanamycin and alpha-naphthoflavone, inhibited EROD induction by TCDD. Other compounds inducing EROD activity were 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3-methylcholanthrene (3MC). When judged by the concentration eliciting 50% of the maximal response (EC50), induction was similar in RTP-2 and RTP-91E, and less effective in RTP-91F. Regardless of the cell line, the rank order from most to least potent inducer on the basis of EC50 value was TCDD> or =PCDD>TCDF>PCB 126>3MC. When induction potencies were expressed relative to TCDD, the values obtained with the pituitary cell lines were similar to previously published values derived with a rainbow trout liver cell line.     

Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro-in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E(2) treatment where we observed a trigger in ERβ2 expression upon E(2) exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.     

Potency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures

A number of highly toxic environmental pollutants including certain polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and 'dioxin-like' polychlorinated biphenyls (PCB) are among the most potent agonists of the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A1 (CYP1A1) in mammalian cell culture is widely used as a functional parameter for AHR activation providing an estimate for 'dioxin-like' inducing equivalents in extracts from environmental samples. Since a number of polycyclic aromatic hydrocarbons (PAHs) also act as AHR-agonists, the CYP1A1-inducing potencies, measured as induction of 7-ethoxyresorufin O-deethylase (EROD) activity in rat hepatocyte cultures were analyzed for 16 PAHs frequently present in environmental samples. Among these, seven PAHs including benzo[a]pyrene were relatively potent inducers allowing the determination of Induction Equivalency Factors (IEF). For three PAHs including benzo[k]fluoranthene which acted as weak inducers, IEFs were estimated, while six PAHs including acenaphthylene were classified as inactive. Based on different efficacies the concentration-response characteristics of CYP1A1 induction were analyzed in more detail for benzo[a]pyrene, benzo[k]fluoranthene, and acenaphthylene. Benzo[k]fluoranthene was markedly less effective than benzo[a]pyrene as inducer of EROD activity but even more effective than benzo[a]pyrene as inducer of CYP1A1 protein and mRNA. Acenaphthylene was highly more effective on the level of mRNA than on the levels of protein or EROD activity. Further analysis revealed that the low efficacy of acenaphthylene as inducer of CYP1A1 protein and EROD activity is due to its marked cytotoxicity while no clear-cut explanation was found for the differences in efficacy between benzo[k]fluoranthene and benzo[a]pyrene. The EROD-inducing potency of a mixture of 16 PAH was about 2-fold higher than that calculated on the basis of IEFs of the individual constituents of the mixture.     

Cytochrome P450 1A induction in gudgeon Gobio gobio : Laboratory and Field Studies

The induction of cytochrome P450 1A was studied in gudgeon (Gobio gobio), a common European cyprinid, using both farm-raised and field-caught fish. The effects of sex, reproductive status and past exposure to xenobiotics were assessed. When exposed to beta-naphthoflavone (bNF), reared gudgeon showed a dose-dependent increase of EROD activity with a plateau observed at doses from 20 mg kg-1 (females) and 5 mg kg-1 (males). The sexual difference in EROD activity was related to the gonadosomatic index (GSI) of the female whatever the level of induction. Dose and sex effects were confirmed by the immunodetection of CYP1A protein. More than 1 month was necessary for EROD activity to decrease to baseline levels. A second bNF injection after 32 days gave similar levels of induction, suggesting that EROD induction by bNF was not impaired by a pretreatment. Wild fish were brought from two sites in the Rhone river basin: a low contaminated site (Ain) and a highly contaminated site (Rhone). Wild gudgeon were highly induced by bNF in laboratory conditions, except males from the Rhone site which exhibited EROD levels as high as the EROD plateau found in laboratory conditions. A 2- month depuration period in clean water was necessary for EROD activity in wild gudgeon to decrease to baseline levels. These results provide better knowledge of the main factors of modulation of the induction in gudgeon as well as on the influence of the history of exposure to inducers.     

Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta . During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml⁻¹) and female (plasma VTG levels >800 μg ml⁻¹) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.     

Effects of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on plasma sex steroids and vitellogenin in rockfish (Sebastes schlegeli)

We examined the estrogenic effect of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on the rockfish, Sebastes schlegeli. We measured levels of plasma estradiol-17beta (E(2)) and testosterone (T) by radioimmunoassay (RIA). Plasma concentrations of T and 17alpha-hydroxyprogesterone in female and male fish injected with PCB 153 using two dosages (0.16 mg/kg body weight. and 0.57 mg/kg) did not differ significantly between sexes or from sham-injected controls of the same sex. Plasma concentrations of E(2) in females injected with PCB 153 (both levels) increased at 12 and 24 h. Concentrations of plasma vitellogenin (VTG) in females increased 72 h after injection with PCB 153 and reached 0.38 and 1.25 mg/mL, respectively. No VTG was detected in males injected with the same dosage. These results suggest that PCB 153 may lead to the production VTG in female rockfish through a synergistic effect with E(2), resulting in indirect disruption of the aromatization process.     

Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol

Vitellogenin (VTG) produced in male fish has been used for a biomarker to study endocrine disrupters. However, the characteristics of VTG produced in male fish have not been studied well. In this study, we investigated the localization of VTG in the liver and the testis of male medaka (Oryzias latipes) treated with 17beta-estradiol (E2) and p-nonylphenol (NP). The male fish were exposed to 1 microg/L E2 and 500 microg/L NP for 1-12 days. Control groups were kept in water including only vehicle. The frozen sections of the liver and the testis were stained with immunohistochemical methods using an antiserum against medaka VTG as the first antibody. In the E2 and NP treated liver, the hepatocytes showed immunoreactivity. In particular, the cytoplasm close to the cell membrane surrounding the sinusoids was strongly immunopositive. In the testis of both treatments, the interstitial tissues and the cells (spermatocytes) in the seminiferous tubules were immunopositive. The concentration of VTG became gradually higher in both tissues with longer treatments. These results suggest that germ cells in the testis treated with E2 and NP are able to incorporate and accumulate VTG.     

Retinoic acid‐metabolizing enzyme cytochrome P450 26a1 (cyp26a1) is essential for implantation: Functional study of its role in early pregnancy

Vitamin A (VA) is required for normal fetal development and successful pregnancy. Excessive VA intake during pregnancy may lead to adverse maternal and fetal effects. Cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)‐metabolizing enzyme, is involved in VA metabolism. It has been shown that cyp26a1 is expressed in female reproductive tract, especially in uterus. In order to investigate the role of cyp26a1 during pregnancy, we constructed a recombinant plasmid DNA vaccine encoding cyp26a1 protein and immunized mice with the plasmid. Compared to control groups, the pregnancy rate of the cyp26a1 plasmid‐immunized mice were significantly decreased (P < 0.01). Further results showed that both cyp26a1 mRNA and protein were specifically induced in the uterus during implantation period and localized in the uterine luminal epithelium. Importantly, the number of implantation sites was also significantly reduced (P < 0.05) after the uterine injection of cyp26a1‐specific antisense oligos or anti‐cyp26a1 antibody on day 3 of pregnancy. Accordingly, the expression of RA‐related cellular retinoic acid binding protein 1 and tissue transglutaminase was markedly increased (P < 0.05) in the uterine luminal epithelium after intrauterine injection treatments. These data demonstrate that uterine cyp26a1 activity is important for the maintenance of pregnancy, especially during the process of blastocyst implantation. J. Cell. Physiol. 223: 471–479, 2010. © 2010 Wiley‐Liss, Inc.     

Cytochrome P450 1A isoenzymes in brain cells: Expression and inducibility in cultured rat brain neuronal and glial cells

Studies initiated to determine the expression of CYP1A1/1A2 isoenzymes in the primary cultures of rat brain neuronal and glial cells revealed significant activity of CYP1A-dependent 7-ethoxyresorufin-o-dealkylase (EROD) in microsomes prepared from both rat brain neuronal and glial cells. RT-PCR and immunocytochemical studies demonstrated constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes in cultured neuronal and glial cells. Cultured neurons exhibited relatively higher constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes, associated with higher activity of EROD than the glial cells. Induction studies with 3-methylchlorantherene (MC), a known CYP1A-inducer, resulted in significant concentration dependent increase in the activity of EROD in cultured rat brain cells with glial cells exhibiting a greater magnitude of induction than the neuronal cells. This difference in the increase in enzyme activity was also observed with RT-PCR and immunocytochemical studies, indicating relatively higher increase in CYP1A1 and 1A2 mRNA as well as protein expression in the cultured glial cells when compared to the neuronal cells. The greater magnitude of induction of CYP1A1 in glial cells is of significance, as these cells are components of the blood-brain barrier and it is suggested that they have a potential role in the toxication-detoxication mechanism. Our data indicating differences in the expression and sensitivity of CYP1A1 isoenzymes in cultured rat brain cells will not only help in identifying and distinguishing xenobiotic metabolizing capability of these cells but also in understanding the vulnerability of these specific cell types towards neurotoxicants.