Aryl hydrocarbon receptor (AhR) ligands and heavy metals are environmental co-contaminants and their molecular interaction may disrupt the coordinated regulation of AhR-dependent phase I and II drug metabolizing enzymes. To determine the effect of heavy metals on the AhR-regulated genes: cytochrome P4501A1 (Cyp1a1), NAD(P)H: quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were treated with increasing concentrations of As3+ (1–10 μM), Cd2+ (1–25 μM) and Cr6+ (1–25 μM) with or without the AhR ligands: 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 μM), β-naphthoflavone (10 uM), or benzo[a]pyrene (1 μM). Our results show that AhR ligands alone and As3+ or Cd2+ alone increased the catalytic activities and mRNA levels of all AhR-regulated genes. When metals were co-administered with an AhR ligand, all three metals inhibited the induction of Cyp1a1 activity by the AhR ligands but potentiated its mRNA and protein expression. In addition, all metals enhanced QOR and GST Ya at the activity and mRNA levels but modulated their induction by AhR ligands in a concentration, metal, and AhR ligand-dependent manner. Generally, Cr6+ inhibited while As3+ and Cd2+ potentiated the induction of QOR and GST Ya activities and mRNA levels. The three metals enhanced the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results show that the ability of metals to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II enzymes will influence the carcinogenicity and mutagenicity of the AhR ligands.

Perfluorooctane sulfonic acid (PFOS) is widely distributed in the environment including in the tissues of wildlife and humans, however, its mechanism of action remains unclear. Here, the Affymetrix rat genome U34A genechip was used to identify alterations in gene expression due to PFOS exposure. Rat hepatoma cells were treated with PFOS at 2–50 mg/L (4–100 μM) for 96 h. Sprague-Dawley rats were orally dosed with PFOS at 5 mg/kg/day for 3 days or 3 weeks. Genes that were significantly (P <0.0025) induced were primarily genes for fatty acid metabolizing enzymes, cytochrome P450s, or genes involved in hormone regulation. Consistent expression profiles were obtained for replicate exposures, for short-term and long-term in vivo exposures, and for acute and chronic exposures. One major pathway affected by PFOS was peroxisomal fatty acid β-oxidation, which could be explained by the structural similarity between PFOS and endogenous fatty acids.

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.

The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human alpha (Gal4-hERalphadef), mouse alpha (Gal4-mERalphadef), mouse beta (Gal4-mERbetadef), chicken (Gal4-cERalphadef), green anole (Gal4-aERalphadef), Xenopus (Gal4-xERdef) or rainbow trout alpha ERs (Gal4-rtERalphadef). The efficacy of 17beta-estradiol (E2) in inducing reporter gene expression was similar among the different constructs overall, with EC(50) values ranging from 0.05 to 0.7nM. However, Gal4-rtERalphadef had an EC(50) value at 37 degrees C of 28nM, though at 20 degrees C an EC(50) value of 1nM was observed. Despite a similar response to E2 treatment among the ERs, many differences were observed in the magnitude of the response to other structurally diverse chemicals. For example, coumestrol induced Gal4-mERbetadef- and Gal4-aERdef-mediated reporter gene expression 164- and 8-fold greater, respectively, than mediated with the other Gal4-ERs. As well, in contrast to results with other Gal4-ERs, alpha-zearalenol consistently induced Gal4-rtERalphadef-mediated reporter gene activity at lower concentrations than did E2. Overall, the results demonstrate that selected estrogenic compounds exhibit a differential ability to induce reporter gene activity mediated by ERs from different vertebrate species. These data also highlight the importance of incubation temperature when examining rtERalpha-mediated activity.

Zonagenesis (zona radiata protein synthesis) and vitellogenesis (yolk protein synthesis) are two reproductive responses that are integral aspects of fish oogenesis. This study examines the responses of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) to five environmental pollutants; 4-nonylphenol (NP) and o,p'-DDT [both at 25 mg/kg], lindane (gamma-HCH) [10 mg/kg], a technical PCB mixture (Aroclor 1254; A1254) and bisphenol A (BPA) [both at 5 mg/kg] in juvenile Atlantic salmon (Salmo salar). Fish were given intraperitoneal injections of o,p'-DDT, gamma-HCH, A1254 or BPA; singly, in combination with NP, and as a cocktail of all five chemicals, and later compared to NP-treated and untreated fish. In a separate experiment, fish were exposed to BPA in a dose-response manner (1, 5, 25 and 125 mg/kg fish). Based on previous studies, blood and liver samples were collected 2 weeks after injection. Zrp and Vtg levels were analyzed in plasma using immunoblotting and enzyme-linked immunosorbent assay. Liver cytochrome P4501A was analyzed by 7-ethoxyresorufin O-deethylase (EROD) activity. NP caused pronounced elevations in plasma Zrp and Vtg levels. In comparison, when NP was given in combination with gamma-HCH, Vtg levels was significantly reduced, compared to NP treatment alone. Using o,p'-DDT, A1254 and BPA, significant elevations of plasma Zrp and Vtg were seen when chemicals were given in combination with NP, but not when administered by themselves. An apparent dose-response induction of Vtg and Zrp levels were observed in BPA treated juvenile salmon. In immunoblots, one component of molecular weight approximating the Zrp-beta was detected when either o,p'-DDT, gamma-HCH, A1254 or BPA were given singly. A1254 significantly induced hepatic EROD activity when administered alone. However, when given as a mixture with all the other xenobiotics, reduction of EROD activity was observed. The data suggest a pattern of xenobiotics action which may complicate assessment of their reproductive effects. Zrp (the beta monomer) was more responsive to the xenoestrogens than Vtg, and provides a sensitive means of detecting exposure to environmental estrogens.

Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and β-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp β-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17β (E2) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or α-zearalenol (α-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E2 or α-ZEA. ZR was more responsive to low levels of E2 and α-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.

Nonylphenol (NP) is a breakdown product of alkylphenol polyethoxylates (APEs), an important class of non-ionic surfactants that are widely used in many detergent formulations and plastic products for industrial and domestic use. A complex microbial degradation pattern, characterized by the formation of several metabolic products that are more toxic than the parent compound, has been established for APEs. We have studied the in vivo metabolism and organ distribution of NP in juvenile salmon. Fish were exposed to a single oral dose of [3H]-4-n-NP (1295 KBq, 25 μg) and sampled at 24, 48 and 72 h after exposure. Metabolites were separated by radio-high-performance liquid chromatography and tentatively identified by co-chromatography with standards characterized by mass spectrometry. Our results show that 4-n-NP was mainly metabolized in vivo to its corresponding glucuronide conjugate and to a lesser extent to various hydroxylated and oxidated compounds. Biliary excretion at 72 h after dosing amounted to 2.83±0.75% of the administered radioactivity. Kinetic analysis shows that NP-glucuronide accounted for 83, 95 and 81% of total radioactivity in the HPLC-injected bile sample at 24, 48 and 72 h, respectively, after exposure. The half-life of residues in carcass and muscle was between 24 and 48 h after exposure.

Mounting evidence confirms that hepatic biosynthetic processes are essential for female sexual maturation in fish, which is directly controlled by estrogens. These oogenetic events (zonagenesis and vitellogenesis) are induced in both sexes by estrogens. In this paper, we report the induction of zona radiata (zr) proteins and vitellogenin in primary hepatocytes from Atlantic salmon (Salmo salar L.) exposed to xenoestrogens and mycotoxins. Cells were treated with doses of 1, 5, and 10 microM 4-nonylphenol (4-NP), o, p'-DDT, lindane ([gamma]-HCH), and bisphenol A (BPA), which all induced zr proteins and vitellogenin in an approximate dose-dependent manner. Hepatocytes were also treated with combinations of xenoestrogens at 1 or 2 microM, resulting in elevated levels of both zr proteins and vitellogenin, compared to single treatment. The estrogenic activity of the mycotoxin zearalenone (ZEA) and its metabolites [alpha]-ZEA) and ss-zearalenol (ss-ZEA)], with regard to zonagenesis and vitellogenesis, was assessed in this assay system. Mycotoxins were used at concentrations of 10, 100, or 1,000 nM. All induced zr proteins and vitellogenin, with [alpha]-ZEA being the strongest inducer. When cells were treated with xenoestrogens or mycotoxins in combination with an estrogen receptor inhibitor (ICI 182,780), the induction of both zr proteins and vitellogenin was inhibited in all cases. Thus, the reported estrogen effects are bonafide estrogen responses. Zona radiata proteins were more responsive than vitellogenin to both xenoestrogens and mycotoxins. The versatility and sensitivity of the hepatocyte assay demonstrates that biosynthesis of zr proteins provides a new supplementary method for estimating xenoestrogenicity and mycotoxin action.

In Atlantic salmon (Salmo salar L) female sexual maturation entails both zonagenesis and vitellogenesis, both of which are controlled by increasing levels of estradiol-17 beta (E-2). Antibodies against salmon zona radiata proteins (eggshell zr-proteins) and vitellogenin were used to monitor induction of oogenesis in juvenile salmon. Molecular weights of zr-monomers were estimated to about 66, 61, and 55 kDa, and to about 180 kDa for vitellogenin. Xenobiotics such as the pesticide DDT impair biological reproduction. The o,p'-DDT (1,1,1-trichloro-2-[2-chlorophenyl]-2-[4-chlorophenyl]ethane) isomer seems to be a xenoestrogen. Serum levels of zr-proteins and vitellogenin, and hepatocytic biosynthesis of these components, were determined after in vivo treatment of salmon with DDT (technical, p,p'-(1,1,1-trichloro-2,2-bis[4-chlorophenyl]ethane) or o,p'-DDT) or E-2. Exposing fish to frequent doses of o,p'-DDT (25 mg/kg b.w. (body weight) twice a week, six times totally) resulted in induction of all three zr-protein monomers, but not of vitellogenin. In contrast, three weekly injection of 10 mg/kg b.w of either of the three DDT preparations did not induce typical zr-proteins or vitellogenin in serum. In vivo studies with combined DDT + E-2 injections showed that none of the DDT preparations influenced E-2-induced biosynthesis of zr-proteins or vitellogenin. E-2 induction of these oogenetic processes was not blocked even by a high concentration (125 mg/kg b.w.) of o,p'-DDT. Furthermore, pretreatment of salmon with o,p'-DDT for 2 weeks, followed by one injection of E-2, did not antagonize biosynthesis of zr-proteins, but serum concentration of vitellogenin was decreased. The data indicate that in juvenile salmon o,p'-DDT may be xenoestrogenic with regard to zonagenesis, but weakly anti-(xeno)estrogenic with regard to vitellogenesis. These findings suggest new complexities in fish reproductive toxicology of xenoestrogens. Compared to vitellogenesis, zonagenesis is a more sensitive parameter for monitoring reproductive effects of xenoestrogens. J. Exp. Zool. 281:346-353, 1998. (C) 1998 Wiley-Liss, Inc.

Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of fish oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268, 59–70), and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418–422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p′-DDT (both at 25 mg kg−1, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond significantly stronger to NP treatment alone and in combination with o,p′-DDT compared to control and o,p′-DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p′-DDT.

Fish oogenesis represents pleiotropic cytodifferentiative programs including hepatic synthesis of the molecular components for both the eggshell and the oocytic energy deposits. Both hepatic processes are directly controlled by plasma levels of estradiol (E2), and injected E2 induces both biogenetic processes in prepubertal fish of both sexes. This work compares the temporal pattern of E2-induced biosynthesis of zona radiata proteins (zr-proteins) and vitellogenin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. We monitored the presence of plasma zr-proteins and vitellogenin, using homologous polyclonal antiserum to zr-proteins and a monoclonal antibody to vitellogenin. Zr-proteins were induced by all E2 concentrations (0.001-1.1 mg/kg body weight (bw)) within one week of exposure while vitellogenin was not induced until two weeks post-injection and then only in plasma from fish injected with high E2 concentrations (0.4 mg or 1.1 mg/kg bw). After E2 treatment, hepatocytes isolated from male fish synthesized zr-proteins and vitellogenin in vitro. However, zr-proteins were secreted into the medium two days before vitellogenin, as measured by ELISA. The data indicate a preferential induction of zr-proteins compared with vitellogenin, both with regard to E2 sensitivity and response time to E2 treatment. These findings suggest an obligate sequence in salmon oogenesis. During sexual maturation low E2 levels at first induce only zonagenesis, while increasing levels of E2 subsequently induce both zonagenesis and vitellogenesis. In nature, the interval between zonagenesis and vitellogenesis may, therefore, be considerable. The data suggest new control mechanisms in fish oogenesis.