Abstract: Available toxicological evidence indicates that environmental contaminants with strong affinity to the aryl hydrocarbon receptor (AhR) have anti-estrogenic properties in both mammalian and non-mammalian in vivo and in vitro studies. The primary objective of the present study was to investigate the interactions between the AhR and estrogen receptor (ER) in salmon in vitro system. Two separate experiments were performed and gene expression patterns were analyzed using real-time PCR, while protein analysis was done by immunoblotting. Firstly, salmon primary hepatocytes were exposed to the dioxin-like PCB126 at 1, 10 and 50 pM and ER agonist nonylphenol (NP) at 5 and 10 muM, singly or in combination. Our data showed increased levels of ER-mediated gene expression (vitellogenin: Vtg, zona radiata protein: Zr-protein, ERalpha, ERbeta and vigilin) as well as increased cellular ERalpha protein levels after treatment with NP and PCB126, singly or in combination. PCB126 treatment alone produced, as expected, increased transcription of AhR nuclear translocator (Arnt), CYP1A1 and AhR repressor (AhRR) mRNA, and these responses were reduced in the presence of NP concentrations. PCB126 exposure alone did not produce significant effect on AhR2alpha mRNA but increased (at 1 and 50 pM) and decreased (at 10 pM) AhR2beta mRNA below control level. For AhR2delta and AhR2gamma isotypes, PCB126 (at 1 pM) produced significant decreases (total inhibition for AhR2gamma) of mRNA levels but was indifferent at 10 and 50 pM, compared to control. NP exposure alone produced concentration-dependent significant decrease of AhR2beta mRNA. In contrast, while 5 muM NP produced an indifferent effect on AhR2delta and AhR2gamma, 10 muM NP produced significant decrease (total inhibition for AhR2gamma) and the presence of NP produced apparent PCB126 concentration-specific modulation of all AhR isotypes. A second experiment was performed to evaluate the involvement of ER isoforms in PCB126 mediated estrogenicity. Here, cells were treated with the different concentrations of PCB126, alone or in combination with ICI182,780 (ICI) and sampled at 12, 24 and 48 h post-exposure. Our data showed that PCB126 produced a time- and concentration-specific increase of ERalpha and Vtg expressions and these responses were decreased in the presence of ICI. In general, these responses show a direct PCB126 induced transcriptional activation of ERalpha and estrogenic responses in the absence of ER agonists. Although not conclusive, our findings represent the first study showing the activation of estrogenic responses by a dioxin-like PCB in fish in vitro system and resemble the "ER-hijacking" hypothesis that was recently proposed. Thus, the direct estrogenic actions of PCB126 observed in the present study add new insight on the mechanisms of ER-AhR cross-talk, prompting a new wave of discussion on whether AhR-mediated anti-estrogenicity is an exception rather than rule of action.

Abstract: In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 mug/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERalpha and ERbeta), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERalpha, Vtg, and Zr-protein mRNA were detectable in the skin. ERbeta was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment.

Abstract: Hydroxylated metabolites of PCBs [OH-PCBs] represent new health and environmental concern because they have been shown to have agonist or antagonist interactions with hormone receptors (HRs) or hormone-receptor mediated responses. The present study was designed to investigate the estrogenic potency based on anti-AhR signalling effect of three 4-OH substituted PCB congeners (#107, #146 and #187), one 3-OH substituted congener (#138), and the pharmaceutical synthetic estrogen, ethynylestradiol (EE2) in fish in vitro system using primary culture of Atlantic salmon hepatocytes. The effects were studied by quantifying changes in transcripts with gene-sequence primer pairs for a suite of gene responses (AhRalpha, ARNT, CYP1A1, CYP3A, UGT and GST) belonging to the xenobiotic biotransformation system. Our data show that OH-PCB congeners and EE2, decreased AhRalpha and ARNT transcript levels, and CYP1A1, UGT and GST gene expressions, together with CYP3A gene expression. The decreased expression of transcripts for xenobiotic biotransformation system is related to the concentration of individual OH-PCB congener and these responses are typical of reported estrogenic and estrogen-like effects on the CYP system. Modulation of biotransformation pathways by OH-PCBs may alter xenobiotic metabolism leading to the production of toxic reactive molecules, altering pharmacokinetics and diminishing the clearance rate of individual chemicals from the organism.

Abstract: The synthetic antioxidant ethoxyquin (EQ) is a widely used additive in animal feeds, including farmed fish feed. The use of EQ as food additive is prohibited and it is also undesirable in farmed meat and fish products. The possible negative aspects of EQ in fish feeds, such as modulation of hepatic detoxifying enzymes and possible effects through "carry-over" to edible parts of fish are not known. In addition, the subsequent consequences for human consumers have not been previously studied. In the present work, the alteration in gene and protein expression patterns, and catalytic activities of phase I and II hepatic biotransformation enzymes due to prolonged exposure to graded levels of dietary EQ in the range of 11-1800 mg EQ/kg feed were studied. The kinetics of parent EQ and its major metabolite, ethoxyquin dimer (EQDM) was also studied. In general two weeks seem to be the critical point in the entire toxicological response of salmon to dietary consumed EQ. Biotransformation of EQ to EQDM is shown to be a rapid process. However, the decrease in biotransformation rate results in the accumulation of EQ metabolites, high concentration of which was postulated to alter translation and post-translational modification of CYP3A, GST and UDPGT at feeding day 14 and 42, with subsequent decreases in the biotransformation of consumed EQ. Decrease in the biotransformation of consumed EQ produced the retention of un-metabolized EQ rather than metabolites in salmon liver. This may be considered as undesirable effect, since it could lead to the transport and accumulation in other organs and edible tissues. It may also cause a new wave of biotransformation with formation of metabolites inhibiting detoxifying enzymes. In general, these processes may prolong the excretion of dietary EQ from the fish body and produce EQ-derived residues in the ready-to-consume salmon or fish products. These EQ residues may have higher toxicological effects for human consumers than the parent compound and therefore need to be studied in more detail.

Abstract: The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B's role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 microM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17beta (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450scc and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 microM, and decreased at 50 and 100 microM. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity.

Abstract: Multiple biological effects of tributyltin (TBT) on juvenile salmon have been investigated. Fish were exposed for 7 days to waterborne TBT at nominal concentrations of 50 and 250 microg/L dissolved in dimethyl sulfoxide (DMSO). Hepatic samples were analyzed for gene expression patterns in the hormonal and xenobiotic biotransformation pathways using validated real-time PCR method. Immunochemical and several cytochrome P450 (CYP)-mediated enzyme activity (ethoxyresorufin: EROD, benzyloxyresorufin: BROD, methoxyresorufin: MROD and pentoxyresorufin: PROD) assays were analyzed. Our data show that TBT produced concentration-specific decrease of estrogen receptor-alpha (ERalpha), vitellogenin (Vtg), zona radiata protein (Zr-protein) and increase of estrogen receptor-beta (ERbeta) and androgen receptor-beta (ARbeta) in the hormonal pathway. In the xenobiotic biotransformation pathway, TBT produced apparent increase and decrease at respective low and high concentration, on aryl hydrocarbon receptor-alpha (AhRalpha), AhR nuclear translocator (ARNT) and AhR repressor (AhRR) mRNA. The expression of CYP1A1 and GST showed a TBT concentration-dependent decrease. The AhRbeta, CYP3A and uridine diphosphoglucuronosyl transferase (UGT) mRNA expressions were significantly induced after exposure to TBT. Immunochemical analysis of CYP3A and CYP1A1 protein levels confirmed the TBT effects observed at the transcriptional levels. The effect of TBT on the biotransformation enzyme gene expressions partially co-related but did not directly parallel enzyme activity levels for EROD, BROD, MROD and PROD. In general, these findings confirm previous reports on the endocrine effects of TBT, in addition to effects on hepatic CYP1A isoenzyme at the transcriptional level that transcends to protein and enzymatic levels. The induced expression patterns of CYP3A and UGT mRNA after TBT exposure, suggest the involvement of CYP3A and UGT in TBT metabolism in fish. The effect of TBT on CYP3A is proposed to represent another hormonal effect of TBT not previously reported in any fish or lower vertebrate. The proposed androgenic effect is supported by the observation that TBT also induced ARbeta mRNA expression in a concentration-specific manner. To our knowledge, this is the first study that has simultaneously studied multiple responses after exposure to TBT in fish.

Abstract: In toxicogenomics, gene arrays are valuable tools in the identification of differentially expressed genes and potentially identify new gene biomarkers altered by exposure of organisms to xenobiotic compounds, either singly or as complex mixtures. In this study, we investigated the mechanisms of interaction between estrogen receptor (ER) and aryl hydrocarbon receptor (Ah receptor or AhR) signalling pathways using toxicogenomic approaches. First, we generated cDNA libraries using suppressive subtractive hybridization (SSH) of clones containing differentially expressed genes from Atlantic salmon (Salmo salar) separately exposed to ER and AhR agonists. Second, a targeted gene array (SalArray) was developed based on true-positive differentially expressed genes. In the experimental setup, primary cultures of salmon hepatocytes isolated by a two-step perfusion method were exposed for 48 h to nonylphenol (NP; 5 microM) and 3,3',4,4'-tetrachlorobiphenyl (TCB; 1 microM), singly and combined, in the absence or presence of antagonists. Using a targeted SalArray, we demonstrate that exposure of salmon to NP singly or in combination with TCB produced differential gene expression patterns in salmon liver. Array analysis showed that exposure of hepatocytes to NP mainly altered genes involved in the estrogenic pathway, including genes for steroid hormone synthesis and metabolism. The anti-estrogenic properties of TCB were demonstrated in the array analysis as genes induced by NP were decreased by TCB. To study the effects of TCB on ER-mediated transcription, hepatocytes were treated for 48 h with tamoxifen (Tam; 1 microM) and ICI182,780 (ICI; 1 microM). The effect of AhR on ER-mediated transcription was investigated by blocking AhR activity with alpha-naphthoflavone (ANF; 0.1 and 1 microM). Quantitative real-time polymerase chain reactions confirmed the changes in expression of ERalpha, ERbeta, vitellogenin (Vtg), zona radiata protein (Zr-protein), and vigilin for the ER pathway and AhRalpha, AhRbeta, AhRR, ARNT, CYP1A1, UDPGT, and a 20S proteasome beta-subunit for the AhR pathway. We found that exposure to NP and TCB both singly and in combination produced gene expression patterns that were negatively influenced by individual receptor antagonists. TCB caused decreased ER-mediated gene expression, and NP caused decreased AhR-mediated responses. Inhibition of AhR with ANF did not reverse the effect of TCB on ER-mediated transcription suggesting that AhRs do not have a direct role on TCB-mediated decreases of ER-mediated responses. In contrast, the inhibition of ER with Tam and ICI reversed the transcription of AhR-mediated responses (except AhRR). Taken together, the findings in the present study demonstrate a complex mode of ER-AhR interaction, possibly involving competition for common cofactors. This complex mode of interaction is further supported by the observation that the presence of ER antagonists potentiated the transcription of AhR isoforms and their mediated responses when TCB was given alone (more so for AhRbeta). Thus, the inhibitory ER-AhR interactions can be used to further investigate specific genes found to be affected in our targeted SalArray chip that are important for the reproductive effects of endocrine disruptors.

Abstract: Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Abstract: Gonadal steroids are known to modulate both the synthesis and the release of gonadotropins by the pituitary and influence several brain functions that are apparently responsible for gender-specific differences in the regulation of the hypothalamus-pituitary-gonadal (HPG) axis. It is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and subsequent conversion to pregnenolone by P450-mediated cholesterol side chain cleavage (P450 scc). In the present study, we have evaluated the effects of 17alpha-ethynylestradiol (EE2) on salmon previtellogenic oocytes using an in vitro culture system and molecular, histological, and physiological methods. The in vitro culture technique was based on an agarose floating method recently validated for xenoestrogens in our laboratory. Tissue was cultured in a humidified incubator at 10 degrees C for 3, 7, and 14 days with different concentrations of EE2 [0 (control), 0.01, 0.1, and 1 microM] dissolved in ethanol (0.1%). The StAR, P450 scc, P450 arom isoforms, and insulin-like growth factor 2 (IGF-2) mRNA expressions were performed using validated real-time polymerase chain reaction (PCR) with specific primers, and immunohistochemistry of the StAR and P450 scc proteins was performed using antisera prepared against synthetic peptide for both proteins and estradiol-17beta (E2); testosterone (T) and 11-ketotestosterone (11-KT) tissue levels were performed using enzyme immunoassay (EIA). Our data show that EE2 produced time- and concentration-specific effects on the StAR protein, P450 scc, P450 arom isoforms, and IGF-2 gene expressions in salmon gonadal tissues. Cellular expression of the StAR and P450 scc proteins was mainly demonstrated in follicular cells of the oocyte membrane, showing time- and EE2 concentration-dependent differences in staining intensities. Tissue levels of E2, T, and 11-KT in salmon were differentially modulated by EE2 in a time- and concentration-specific manner. Although an apparent negative relationship between E2 and T that reflected aromatization of T to E2 was observed at day 3 postexposure, T and 11-KT showed an apparent concentration-dependent effect after EE2 exposure at day 14. The consistencies between our data at day 14 postexposure suggest that the EE2 modulates steroidogenesis by targeting the initial and rate-limiting step that involves the StAR protein. In general, these findings show that the synthetic pharmaceutical endocrine disruptor and ubiquitous environmental pollutant also produce variations in key gonadal steroidogenic and growth-regulating pathways. These effects and the hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Abstract: BACKGROUND: The estrogenic and xenobiotic biotransformation gene expressions are receptor-mediated processes that are ligand structure-dependent interactions with estrogen-receptor (ER) and aryl hydrocarbon receptor (AhR), probably involving all subtypes and other co-factors. The anti-estrogenic activities of AhR agonists have been reported. In teleost fish, exposure to AhR agonists has been associated with reduced Vtg synthesis or impaired gonadal development in both in vivo- and in vitro studies. Inhibitory AhR and ER cross-talk have also been demonstrated in breast cancer cells, rodent uterus and mammary tumors. Previous studies have shown that AhR-agonists potentiate xenoestrogen-induced responses in fish in vivo system. Recently, several studies have shown that AhR-agonists directly activate ER alpha and induce estrogenic responses in mammalian in vitro systems. In this study, two separate experiments were performed to study the molecular interactions between ER and AhR signalling pathways using different concentration of PCB-77 (an AhR-agonist) and time factor, respectively. Firstly, primary Atlantic salmon hepatocytes were exposed to nonylphenol (NP: 5 microM--an ER agonist) singly or in combination with 0.001, 0.01 and 1 microM PCB-77 and sampled at 48 h post-exposure. Secondly, hepatocytes were exposed to NP (5 microM) or PCB-77 (1 microM) singly or in combination for 12, 24, 48 and 72 h. Samples were analyzed using a validated real-time PCR for genes in the ER pathway or known to be NP-responsive and AhR pathway or known to be PCB-77 responsive. RESULTS: Our data showed a reciprocal inhibitory interaction between NP and PCB-77. PCB-77 produced anti-NP-mediated effect by decreasing the mRNA expression of ER-responsive genes. NP produced anti-AhR mediated effect or as inhibitor of AhR alpha, AhRR, ARNT, CYP1A1 and UDPGT expression. A novel aspect of the present study is that low (0.001 microM) and medium (0.01 microM) PCB-77 concentrations increased ER alpha mRNA expression above control and NP exposed levels, and at 12 h post-exposure, PCB-77 exposure alone produced significant elevation of ER alpha, ER beta and Zr-protein expressions above control levels. CONCLUSION: The findings in the present study demonstrate a complex mode of ER-AhR interactions that were dependent on time of exposure and concentration of individual chemicals (NP and PCB-77). This complex mode of interaction is further supported by the effect of PCB-77 on ER alpha and ER beta (shown as increase in transcription) with no concurrent activation of Vtg (but Zr-protein) response. These complex interactions between two different classes of ligand-activated receptors provide novel mechanistic insights on signalling pathways. Therefore, the degree of simultaneous interactions between the ER and AhR gene transcripts demonstrated in this study supports the concept of cross-talk between these signalling pathways.

Abstract: Pharmaceuticals are ubiquitous pollutants in the aquatic environment where their potential effects on non-target species like fish has only recently become subject of systematic investigations. In the present study, experiments were undertaken to examine the effects of a synthetic pharmaceutical endocrine disruptor, ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at days 0 (control), 3 and 7 after exposure, on hepatic phase I and II biotransformation and hormonal pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR), Vtg ELISA and 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. Our data show that EE2 produced time- and concentration-specific modulation of estrogen receptor isoforms (ERalpha, ERbeta) and androgen receptor-beta (ARbeta). EE2 produced a concentration-specific induction of vitellogenin (Vtg) and zona radiata protein (Zr-protein) at day 3 after exposure. At day 7, Vtg and Zr-protein mRNA (and plasma Vtg protein) expression were significantly decreased in the group given 5 ng EE2/L, compared to dimethyl sulfoxide (DMSO) control group. In the xenobiotic biotransformation pathway, EE2 produced a significant increase of aryl hydrocarbon receptor-alpha (AhRalpha) at day 3 in the group given 5 ng EE2/L and AhRbeta was decreased at the same concentration at day 7. While CYP3A was not significantly affected by EE2 exposure, the CYP1A1, AhR nuclear translocator (Arnt) and AhR repressor (AhRR) mRNA showed an apparent EE2 concentration and time-dependent decrease. The expression of uridine diphosphoglucuronosyl transferase (UGT) and glutathione S-transferase class pi-like (GSTpi-like) mRNA were decreased after exposure to 50ng EE2/L at both day 3 and 7 after exposure. The effect of EE2 on the CYP1A1 gene expressions paralleled effect on EROD and AhRR mRNA, suggesting a direct role of EE2 in controlling cellular detoxification machinery. Interestingly, the carrier vehicle, DMSO produced significant time-dependent induction of estrogenic (ERalpha, Vtg and Zr-protein) responses, compared with blank (i.e. without DMSO) controls at day 7 post-exposure. The effect of DMSO totally underscored the observed EE2 effect at day 7 after exposure. In general, these findings support previous reports on the endocrine effects of EE2, in addition to effects on hepatic biotransformation system. In view of the data presented here and our recent studies, the use of DMSO as carrier vehicle in endocrine toxicological experimental studies should be re-evaluated.

Abstract: Pharmaceuticals are ubiquitous pollutants in the aquatic environment, where their potential effects on nontarget species like fish has only recently become subject of systematic investigations. Recently, it was shown that the documented xenoestrogen nonylphenol produced variations in brain steroidogenic acute regulatory (StAR) protein, cytochrome P-450-mediated cholesterol side-chain cleavage (P450scc), and cytochrome P-45011beta hydroxylase (CYP11beta) gene transcripts of exposed juvenile salmon (Arukwe, 2005). In the present study, experiments were undertaken to examine the effect of the synthetic pharmaceutical endocrine disruptor ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at d 0 (control), 3, and 7 after exposure, on these key and rate-limiting brain and interrenal steroidogenic pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR). Our data, which are based on nominal exposure concentrations, show that brain and head kidney StAR and P450scc expression were modulated by EE2 in a time- and concentration-specific manner. While the StAR protein and P450scc showed EE2 concentration-dependent transcriptional increases in the brain and head kidney at d 3 after exposure, no significant effect was observed at d 7. The EE2 induced effects at d 7 were underscored because the carrier solvent (dimethyl sulfoxide, DMSO) produced significant induction of the StAR protein and P450scc in both the brain and head kidney at d 7 compared to d 3 postexposure. CYP11beta transcript was detected in the brain and head kidney, where the expression patterns were modulated by EE2 in a concentration-and time-specific manner. In the brain, DMSO produced significant changes in the CYP11beta gene expression at d 7 compared to d 3 after exposure. These changes in the levels of StAR, P450scc, and CYP11beta mRNA levels in important steroidogenic organs suggest that the experimental animals are experiencing a time-dependent impaired steroidogenesis. Thus, the StAR protein, P450scc, and CYP11beta might represent sensitive diagnostic tools for short-term and acute exposure to endocrine disrupting chemicals. In view of the present study and high concentrations of EE2 reported in effluents and surface waters from Europe and the United States, pharmaceuticals in the environment represent potentially more serious health concern both to humans and wildlife than earlier anticipated.

Abstract: The modulations of reproductive biomarker responses, such as vitellogenin (Vtg) and zona radiata protein (Zr-protein) by xenoestrogens are mediated through the estrogen receptors (ERs). The ERalpha and ERbeta belong to the nuclear receptor (NR) superfamily of ligand activated transcription factors that modulate specific gene expression. By interacting with estrogen and estrogen like molecules (including estrogen mimics), the ERs exert a diverse array of biological effects. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis through the aromatase enzyme (CYP19 or P450arom). The aim of this study was to investigate the effects of nonylphenol on the expression patterns of ER and aromatase (P450arom) isoform genes in the brain as sensitive molecular biomarker of response to endocrine disrupting chemicals. Immature Atlantic salmon were exposed to continuous nominal waterborne nonylphenol at 15 microg/L. Brain samples were collected after 7 days exposure. A quantitative RT-PCR method was used to analyze mRNA expression patterns. Nonylphenol caused a significant induction of P450arom and ER isoform patterns in the brain and represent quantitative gene expression biomarkers of exposure to endocrine disrupting chemicals in the environment.

Abstract: The organ-specific gene expression patterns of P450arom isoforms have been studied in European common frog, Rana temporaria after exposure to DDE, using real-time PCR. Four groups of frogs were subcutaneously injected with DDE at 0.01, 0.1, 1 and 10 mg/kg body weight and one group, serving as the control was injected with pure corn oil. Brain, kidney, testis and liver P450aromA and P450aromB gene expressions were evaluated at day 14 after exposure. P450aromB data show that 0.1 and 10 mg DDE/kg doses caused 76% and 63% (testis) and 79% and 80% reductions, respectively, of mRNA levels. Brain P450aromB mRNA decreased 10% and 34%, respectively, after exposure to 0.01 and 0.1 mg DDE/kg. Thereafter, a 185% and 177% induction response of brain P450aromB was observed in the groups treated with 1 and 10 mg DDE/kg, respectively. In the kidney, 0.01, 0.1, 1 and 10 mg DDE/kg induced a 516%, 178%, 466% and 247% induction of P450aronB mRNA, respectively. P450aromA expression was not quantified in any of the organs. The relative abundance of P450aromB gene expression in different organs is in the order: kidney > brain > liver > testis. The present data suggest potential detrimental effect of organochlorine pesticides (OCs) and their metabolites on the European frog steroidogenic pathways. Given the high persistency in the environment and continued use in developing countries coupled with the tendency for global atmospheric transport, OCs and their metabolites such as DDE will remain a focus of concern both for scientific and societal reasons.

Abstract: Nonylphenol is a degradation product of alkylphenol polyethoxylates (APEs). APEs represent an important class of non-ionic surfactants widely used in many detergent formulations and plastic products for industrial and domestic use. Nonylphenol interacts with xenobiotic- and drug-metabolizing CYP forms, including members of the CYP3A and CYP1A1 subfamilies in fish. The pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) are known regulators of the induction of CYP3A and CYP1A1, respectively. Previously in our laboratory, we have shown that nonylphenol, in addition to inducing plasma egg-yolk and eggshell proteins, also modulates hepatic CYP3A- and CYP1A1-mediated enzyme activities in juvenile Atlantic salmon. Given the potential for indirect actions of nonylphenol on xenobiotic and steroid metabolism, studies were carried out to determine the effects of nonylphenol on the expression of two critical enzyme systems using juvenile salmon and waterborne nonylphenol at environmentally relevant concentrations (5, 15 and 50 microg/L) and ethanol solvent control and sampled at different time intervals (days 0 (control), 3 and 7) post-exposure. Our data show that nonylphenol-induced CYP3A and CYP1A1 mRNA levels correlate with PXR and AhR at day 7 post-exposure to 5 and 15 microg NP/L. For CYP1A1 and AhR, nonylphenol caused a temporal decrease at day 3 post-exposure, thereafter CYP1A1 and AhR mRNA levels were significantly induced at day 7. 50 microg NP/L caused a more pronounced effect on CYP1A1 by decreasing the mRNA levels at days 3 and 7, compared to control. Despite the decreased CYP1A1 mRNA levels at day 7, 50 microg NP/L caused a significant induction of AhR mRNA at the same time period. This study suggests a possible regulation by xenoestrogens of fish hepatic CYP3A and CYP1A1 enzymes via PXR and AhR, respectively, and may have impact on the metabolism of endogenous and exogenous substrates. This is the first study that simultaneously examined CYP3A, CYP1A1, PXR and AhR transcripts following nonylphenol treatment in any aquatic species or earlier diverging vertebrates.

Abstract: The molecular basis for vitellogenin (Vtg) and Zr-protein gene (and protein) expression shows that the Vtg and Zr-protein gene activations are receptor-mediated responses that are ligand structure-dependent interactions with ER, probably involving all isoforms, in addition to other co-activators. In addition to their effect as direct agonist to the ERs, the endocrine-disrupting effects of environmental chemicals are sometimes interpreted as interference with steroidogenesis and with the steroidal regulation of the normal development and function of the male and female reproductive tracts. In vertebrates, the cytochrome P450 aromatase (P450arom) is a crucial steroidogenic enzyme catalyzing the final step in the conversion of androgens to estrogens. The present study was undertaken to investigate the effects of nonylphenol on brain and liver ER and P450arom isotypes gene expressions as sensitive biomarker of effect. Fish were exposed to static and continuous waterborne nonylphenol at 5, 15 and 50 microg/L. Blood, brain and liver samples were collected after 0 (control), 3 and 7 days post-exposure. Plasma levels of Vtg and Zr-proteins were analyzed using immunoblotting and enzyme linked immunosorbent assay (ELISA) methods. A quantitative RT-PCR method was used to analyse gene expression patterns using cloned plasmid containing amplicons of interest as standards. Our data demonstrate that both ER and P450arom gene isotypes showed differential organ-specific, nonylphenol concentration- and time-dependent expression patterns after exposure to environmental relevant concentrations of nonylphenol. Liver and plasma Vtg and Zr-protein gene and protein expression showed a concentration- and time-dependent induction at day 3 and 7 post-exposure. This is the first study evaluating, in parallel, the ERs, P450aroms and protein xenoestrogen biomarkers of effect in any fish species or lower vertebrate. Therefore, the present study shows that the brain and hepatic ER and P450arom isotypes evaluated using state-of-the-art molecular approaches are sensitive xenoestrogen biomarkers of effect. These responses should be evaluated in parallel with plasma protein biomarkers such as Vtg and Zr-proteins.

Abstract: It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3',4,4'-tetrachlorobiphenyl, PCB-77) and estrogen receptor agonist (ER; nonylphenol, NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 microM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 microM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERalpha, ERbeta, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRalpha mRNA (except 1 microM PCB-77, which caused the induction of AhRalpha mRNA levels). AhRbeta mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 microM NP restored the PCB-77-inhibited AhRbeta mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.

Abstract: Although polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE) flame retardants are important organic contaminants in the tissues of marine mammals, including those species from the Arctic, there is exceedingly little direct evidence on congener-specific biotransformation. We determined and compared the in vitro metabolism of environmentally relevant PCB (4,4'-di-CB15, 2,3',5-tri-CB26, 2,4,5-tri-CB31, 2,2',5,5'-tetra-CB52, 3,3',4,4'-tetra-CB77, 2,2',4,5,5'-penta-CB101, 2,3,3',4,4'-penta-CB105 and 2,3',4,4',5-penta-CB118), and PBDE (4,4'-di-BDE15, 2,4,4'-tri-BDE28, 2,2',4,4'-tetra-BDE47, 2,2',4,5'-tetra-BDE49, 2,2',4,4',5-penta-BDE99, 2,2',4,4',6-penta-BDE100, 2,2',4,4',5,5'-hexa-BDE153, 2,2',4,4',5,6'-hexa-BDE154 and 2,2',3,4,4',5',6-hepta-BDE183) congeners using hepatic microsomes of a beluga whale (Delphinapterus leucas) from the Arviat (western Hudson Bay) area of the Canadian Arctic. Ortho-meta bromine-unsubstituted BDE15, BDE28 and BDE47 were significantly metabolized (100%, 11% and 5% depleted, respectively) by beluga, whereas control rat microsomes (from pooled male Wistar Han rats) metabolized BDE28, BDE49, BDE99 and BDE154 (13%, 44%, 11% and 17% depleted, respectively). CB15 and CB77 (putative CYP1A substrates) were more rapidly metabolized (100% and 93% depleted, respectively) by male beluga than CB26 and CB31 (CYP1A/CYP2B-like) (25% and 29% depleted, respectively), which were more rapidly metabolized than CB52 (CYP2B-like) (13% depleted). Higher chlorinated CB101 and CB105 showed no depletion. Rat control microsomes metabolized CB15 to a lesser extent (32% depleted) than beluga, but much more rapidly transformed CB52 (51% depleted, respectively). Within the 90 min in vitro assay time frame, the preference was towards metabolism of ortho-meta unsubstituted congeners (for both PCBs and PBDEs) in beluga whale, whereas for rat controls, meta-para unsubstituted congeners also substantially metabolized. For both beluga whale and rat, metabolic rates were inversely associated with the degree of halogenation. For the rapidly biotransformed CB15 and BDE15, water-soluble OH-metabolites were detected after incubation. These results indicate that CYP-mediated oxidative hepatic biotransformation is a metabolic pathway in the toxicokinetics of both PCB and PBDE congeners in beluga whales and in the rat model. This may suggest that the formation of potentially toxic oxidative PCB and PBDE products (metabolites), in addition to the parent pollutants, may be contributing to contaminant-related stress effects on the health of beluga whale.

Abstract: In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestradiol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses, such as brain estrogen receptor (ERalpha) and plasma vitellogenin (Vtg) levels. Fish were exposed to nominal waterborne EE2 (5 and 50 ng/l) and TBT (50 and 250 ng/l) concentrations dissolved in dimethyl sulfoxide (DMSO), singly and in combination. Gene expressions were quantified using real-time PCR with gene-specific primers, aromatase activity was analyzed using the tritiated water-release assay, and plasma Vtg was analyzed using competitive ELISA. Our data show that EE2 induced a concentration-specific modulation of P450aromA, P450aromB, and aromatase activity in addition to ERalpha and plasma Vtg levels in juvenile salmon at day 3 postexposure. TBT exposure caused both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity levels, depending on concentration, at day 7 postexposure. TBT elevated and inhibited ERalpha and plasma Vtg and also antagonized EE2-induced expression of the studied variables at day 7 postexposure. Interestingly, the carrier vehicle DMSO modulated the receptor-mediated and non-receptor-mediated estrogenic responses at day 7 postexposure, compared to day 3. In general, these findings suggest that the exposed animals are experiencing impaired steroidogenesis and modulations of receptor-mediated endocrine responses. Given the integral role of neurosteroids in homeostatic process, growth, metabolism, reproduction, and development of central nervous system and function, these effects may have serious impact on this endocrine pathway and potentially affect organismal reproductive performance and health. In conclusion, the regulation of steroidogenesis is a fundamental mechanism involved in the biosynthesis of important biological compounds, irrespective of organ; therefore, the search for the molecular targets of xenoestrogens, given singly and also in combination, in these pathways will increase our understanding of organismal endocrine disruption and potential consequences.

Abstract: It is known that estrogen-like environmental pollutants can feminise gonadal differentiation in frogs resulting in female-biased sex-ratios at metamorphosis. The long-term effects on reproductive function in frogs following larval exposure to pollutants are less known. Amphibian test systems which allow life-cycle studies are therefore needed. The aim of the present study was to characterise long-term estrogenic effects on the reproductive system of the emerging model species Xenopus (Silurana) tropicalis following larval exposure to ethynylestradiol (EE(2)). EE(2) is a synthetic estrogen that has been detected in sewage effluents and in surface waters. Newly hatched tadpoles (Niewkoop Faber (NF) stage 48) were exposed to the nominal EE(2) concentrations 0 (control), 1, 10, and 100 nM (with analytical chemistry support) until complete metamorphosis (NF stage 66). Effects on the reproductive organs were determined in juveniles (1 month after metamorphosis) and in 9-month-old frogs. Larval exposure to EE(2) caused female-biased phenotypic sex-ratios in both juvenile and adult frogs, which is in agreement with previous work on other frog species. Nearly all (97%) of the 63 EE(2)-exposed 9-month-old frogs had ovaries. Histological evaluation of the gonads of the 9-month-old frogs showed that they were sexually mature. Among the adult frogs with ovaries there was a dose-dependent increase in the frequency of individuals lacking oviducts. Adult frogs exposed to 100 nM EE(2) that had ovaries but no oviducts had lower levels of estrogen receptor alpha (ERalpha) mRNA in the brain than control animals and those exposed to 100 nM EE(2) that had ovaries as well as oviducts. EE(2) exposure did not cause any significant changes in ERalpha mRNA levels in the ovaries of the adult frogs. The reduced level of ERalpha mRNA in the brain of individuals with ovaries lacking oviducts suggests an organizing effect of EE(2) on the central nervous system. The results show that transient early life-stage exposure to an environmental pollutant can induce effects on the reproductive organs and the central nervous system that persist into adulthood. Overall, our data suggest that X. tropicalis, which has a shorter generation time than the well-established model species Xenopus laevis, is a suitable model organism for research on developmental reproductive toxicity in anuran species.

Abstract: It is assumed that the expression of housekeeping genes is constant regardless of experimental conditions. In toxicology, this assumption has indeed become a misconception of reasonable concern, as these so-called housekeeping genes vary considerably across different experimental conditions and thereby lead to an erroneous interpretation of the expression profile of a target gene. Given that the choice of reference gene will ultimately influence statistical interpretation of toxicological data, it is essential to validate potential reference genes prior to their use, to establish their suitability for a specific experimental purpose. Therefore, the aim of this study is to quantitatively evaluate the most commonly used housekeeping genes in toxicology research for their suitability as reference endpoints, and thus provide toxicology researchers who have little experience in molecular biology but find themselves interested or involved with gene expression analysis with a summary of information necessary for re-evaluating their procedures. We show that the expression pattern of beta-actin, beta-tubulin, 18S ribosomal RNA (18S rRNA), and elongation factor-lalpha (EF-lalpha), representing commonly used housekeeping genes in toxicology, was modulated on the basis of random exposure condition and time, in both in vivo and in vitro test systems of Atlantic salmon (Salmo salar). Based on the data presented herein and several other reports by other researchers, there are very few biological justifications to refer to anything as a housekeeping gene in real-time PCR assays for toxicological research. However, given the absolute need for normalization genes to correct for sample-to-sample variations, the choice of internal control gene should be determined empirically on the basis of the individual exposure condition and by the individual researcher.

Abstract: The effects of environmental contamination on amphibians are of particular concern because there are reports of declining numbers of species and individuals in most parts of the world during the last 50 years. During the last decade there has been increased focus on the role of persistent organic pollutants as retinoid (vitamin A) disrupters, and their effects on development, growth and sexual differentiation. To study the effects of p,p'-DDE, one of the most persistent metabolites of the pesticide DDT, on retinol homeostasis, we subcutaneously exposed adult male European common frogs (Rana temporaria) to different doses of p,p'-DDE (0, 0.01, 0.1, 1 and 10 mg/kg body mass) and studied the effect of a short term exposure (14 days) on hepatic retinoid levels and CYP26 gene and protein expression. Hepatic retinol concentrations, CYP26 gene and protein levels were analysed using HPLC, quantitative RT-PCR and indirect ELISA, respectively. Our results showed a significant p,p'-DDE dose-specific increase in the hepatic retinol concentration. CYP26 gene and protein expression were reduced in an apparent p,p'-DDE dose-specific manner. The results suggest that p,p'-DDE may interfere with the hepatic metabolism of retinol in adult frogs by decreasing CYP26 expression patterns.

Abstract: Dimethyl sulfoxide (DMSO) has been frequently used as carrier solvent in toxicological experiments where the most compelling DMSO attributes are its exceptionally low toxicity and environmental impact. We were inspired by recent and consistent observations that ethanol and DMSO modulate endocrine-disruptor biomarker responses in both in vitro and in vivo studies in our laboratory, to take a critical evaluation of these effects. Quantitative (real-time) polymerase chain reaction (PCR) method with specific primer pairs was used in this study to measure DMSO-induced time-dependent modulation of estrogen receptor (ER) isoforms, vitellogenin (Vtg) and zona radiata-protein (Zr-protein) gene expression patterns in primary culture of salmon hepatocytes. In addition, immunochemical analysis, using indirect enzyme linked immunosorbent assay (ELISA) with monoclonal (Vtg) and polyclonal (Zr-proteins) antibodies was used to detect and measure Vtg and Zr-proteins secreted in culture media. Salmon hepatocytes were isolated by a two-step collagenase perfusion method and exposed to 0.1% or 10 microL/L of DMSO after 48 h pre-culture. Cells were harvested at 12, 24, 48 and 72 h after exposure and analysed for ERalpha, ERbeta, Vtg and Zr-protein gene expression using real-time PCR method. Media samples were collected at similar time-intervals for protein analysis. Our data show that DMSO-induced significant increase in ERalpha, ERbeta, Vtg and Zr-protein genes in a time-dependent manner. Indirect ELISA analysis showed a time-specific effect of DMSO. The use of DMSO as carrier solvent in fish endocrine disruption studies should be re-evaluated. We recommend more investigation, using other endocrine-disruptor biomarkers in order to validate the suitability of common carrier solvents used in toxicology with the aim of setting new maximum allowable concentrations. In particular, given the high sensitivity of genomic approaches in toxicology, these results may have serious consequences for the interpretation of biomarker responses.

Abstract: The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHbeta) and thyroid hormone receptor (TRalpha and TRbeta) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHbeta in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRalpha mRNA steady-state, the expression of TRbeta was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRbeta mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRbeta expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.

Abstract: The synthetic antioxidant ethoxyquin (EQ) is increasingly used in animal feeds and has been candidate for carcinogenicity testing. EQ has the potential for toxicological and adverse health effects for both fish and fish consumers through "carryover" processes. The toxicological aspects of EQ have not been systematically investigated. The present study was performed to investigate the hepatic metabolism, metabolite characterization, and toxicological aspects of EQ in salmon during a 2-week depuration after a 12-week feeding period with 18 mg (low), 107 mg (medium), and 1800 mg/kg feed (high). The alteration in gene expressions and catalytic activities of hepatic biotransformation enzymes were studied using real-time polymerase chain reaction with specific primer pairs and by kinetics of two identified hepatic metabolites. Analysis of EQ metabolism was performed using high performance liquid chromatography (HPLC) method and showed the detection of four compounds of which two were quantified, parent EQ and EQ dimer (EQDM). Two metabolites were identified as de-ethylated EQ (DEQ) and quinone imine, but these were not quantified. The concentration of the quantified EQ-related compounds in the liver at day 0 showed a positive linear relationship with measured dietary EQ (R2= 0.86 and 0.92 for parent EQ and EQDM, respectively). While the low-EQ-feeding group showed a time-specific increase of aryl hydrocarbon receptor (AhR) mRNA expression, the medium-dose group showed decreased AhR mRNA at depuration day 7. Expression of CYP1A1 was decreased during the depuration period. Consumption of dietary EQ produced the expression of CYP3A, glutathione S-transferase (GST), and uridine diphosphate glucuronosyl-transferase (UDPGT) mRNA during the depuration period. A similar pattern of effect was observed for both CYP3A and phase II genes and supports our previous postulation of common regulation of these enzymes by the same inducer, namely EQ metabolites. The increase of CYP3A, UDPGT, and GST gene expressions at day 7 was in accordance with the low concentration of DEQ. The low concentration of putative DEQ may induce the CYP3A with subsequent increase in the biotransformation of EQ into DEQ. The increase in UDPGT may seem to be a synchronizing mechanism required for the excretion of DEQ. The biotransformation of dietary EQ is proven by simultaneous induction of both phase I and II detoxification system in the liver of Atlantic salmon. Therefore, the apparent low concentration of putative DEQ may account for the induced phase I and II detoxifying enzymes at least during depuration. This speculated hypothesis is currently a subject for systematic investigation in our laboratory using in vitro and genomic approaches.

Abstract: The present study investigated the effects of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) on the thyroid and steroid-metabolizing system in Atlantic salmon (Salmo salar) parr. Fish were exposed to waterborne DDE and thyroxine (T4), both singly and in combination, for 5 d. Thyroid-stimulating hormone (TSHbeta), T4 deiodinase (T4ORD), thyroid receptors (TRalpha and TRbeta), and insulin-like growth factor type 1 receptor (IGF-1R) were analyzed using quantitative (real-time) polymerase chain reaction in liver, brain, and kidney, whereas cytochrome P4503A (CYP3A) and pregnane X receptor (PXR) mRNA levels were analyzed only in the liver. Exposure to DDE and T4, both singly and in combination, inhibited TSHbeta expression in the brain. The DDE induced TSHbeta in the liver, and T4 inhibited TSHbeta in the liver and kidney, both singly and in combination with DDE. The DDT-metabolite DDE induced T4ORD expression in the kidney and liver, and combined exposure with T4 inhibited T4ORD expression in the brain, kidney, and liver. The IGF-1R and TRalpha expressions were induced by DDE and T4 singly in the brain, whereas combined exposure with both compounds did not affect IGF-1R and TRd transcript levels. Whereas T4 inhibited TRbeta expression in the liver, exposure to DDE, both singly and in combination with T4, induced TRbeta transcript levels in the liver. Exposure to T4 and DDE, both singly and in combination, resulted in a parallel pattern of CYP3A and PXR mRNA induction in the liver. These results indicate that DDE alters thyroid hormone-dependent genes and hepatic CYP3A and PXR levels. The hepatic modulation of CYP3A and PXR transcript levels by DDE represents a novel aspect of DDE toxicity that, to our knowledge, has not been demonstrated previously in fish. Therefore, the present study demonstrates some possible physiological and endocrine consequences from exposure to endocrine-disrupting chemicals for salmon parr during smoltification.

Abstract: Gene expression patterns for key brain steroidogenic (StAR, P450scc, CYP11beta) and xenobiotic- and steroid-metabolizing enzymes (CYP1A1 and CYP3A) have been investigated in waterborne nonylphenol (5, 15, and 50 microg/ L) treated juvenile Atlantic salmon (Salmo salar), in addition to carrier vehicle (ethanol) exposed fish, sampled at different time intervals (0, 3, and 7 days) after exposure. Gene expression patterns were studied using the quantitative polymerase chain reaction (real-time PCR). Treatment of juvenile salmon with nonylphenol caused significant induction of steroidogenic acute regulatory (StAR) protein mRNA at day 7 postexposure in the group receiving 15 microg of nonylphenol/L. P450scc was first induced in the group treated with 5 microg of nonylphenol/L at day 7; thereafter, an apparent nonylphenol-concentration-dependent decrease in P450scc mRNA was observed. CYP11beta mRNA was significantly induced at day 3 after exposure to 5 betag of nonylphenol/L; thereafter, CYP11beta mRNA levels were inhibited below control levels in the 15 and 50 microg of nonylphenol/L groups at day 3. At day 7, significant induction of CYP11beta mRNA was observed only in the group exposed to 15 microg of nonylphenol/L. For CYP1A1 mRNA, apparent nonylphenol-concentration-dependent decreases were observed at day 7 postexposure. CYP3A mRNA was significantly induced by all nonylphenol exposure concentrations at day 7. When exposed groups were compared, CYP3A transcript was significantly induced between 5 and 15 microg of nonylphenol/ L, and decreased between 15 and 50 microg of nonylphenol/ L. The ethanol control showed a significant reduction of CYP3A mRNA at day 3 postexposure. The present study has demonstrated variations in three key steroidogenic proteins and xenobiotic- and steroid-metabolizing CYP isoenzyme gene transcripts in the brain of nonylphenol-exposed juvenile salmon. Therefore, the present study represents a novel aspect of neuroendocrine effects of nonylphenol in fish not previously demonstrated and should be studied in more detail.

Abstract: Induction of blood plasma vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) in male and juvenile of oviparous vertebrates was proposed and shown to be sensitive biomarkers for exposure to estrogen mimic. The time- and dose-dependent expression of Vtg and Zr-proteins in nonylphenol (NP) exposed juvenile Atlantic salmon (Salmo salar) is reported. Fish were exposed continuously to waterborne nonylphenol at 5, 15 and 50 microg/L. Blood and surface mucus samples were collected after 3 and 7 days post-exposure. Nonylphenol-induced plasma and surface mucus levels of Vtg and Zr-protein were analysed using immunochemical methods (Western blot and enzyme-linked immunosorbent assay; ELISA). Both Vtg and Zr-protein levels in plasma and surface mucus showed similar and parallel nonylphenol-induced expression patterns after waterborne nonylphenol exposure and in a concentration- and time-dependent manner. Zr-proteins were significantly induced at the lowest concentration of nonylphenol after 3 and 7 days of exposure both in plasma and in surface mucus. We conclude that the detection of Vtg and Zr-proteins directly in the surface mucus of fish, and the correlation of these values with plasma protein biomarker values in xenoestrogen-treated fish represents a sensitive non-invasive system for the detection of these known endocrine disruptor biomarkers. The demonstration of detectable Vtg and Zr-protein levels from surface mucus is a potential biomarker for estrogenic compounds, and their presence should be considered as an improvement in the methods for detection of endocrine disrupting chemicals (EDCs) and related pollutants in the environment.

Abstract: Cytochromes P450 (CYP, phase I) and conjugating (phase II) enzymes can be induced by and influence the toxicokinetics (metabolism) and toxicity of xenobiotic contaminants in exposed organisms. Beluga whale (Delphinapterus leucas) from the endangered St. Lawrence (SL) River Estuary population exhibit deleterious health effects and various severe pathologies that have been associated with contaminant exposure. In contrast, such effects (e.g. reproductive and immunological impairment) are generally less frequent in less exposed populations in the Canadian Arctic (CA). In the present study, opportunistic sampling resulted in the collection immediately after death of liver tissue from a single female neonate SL beluga (SL6) and male and female CA beluga (n=10) from the Arviat region of western Hudson Bay, in addition to sampling of stranded carcasses of male and female SL beluga (n=5) at least 12 h postmortem. We immunologically characterized cross-reactive proteins of hepatic microsomal CYP1A, CYP2B, CYP3A, CYP2E, epoxide hydrolase (EH) and uridine diphosphoglucuronosyl transferase (UDPGT) isozymes. Cross-reactive proteins were found in all SL and CA beluga using anti-rat CYP1A1, anti-rainbow trout CYP3A, anti-human CYP2E1, anti-rabbit EH and anti-human UDPGT1A1 polyclonal antibodies (Abs), whereas faintly cross-reactive CYP2B proteins were only found in SL6 and the CA samples using an anti-rabbit CYP2B1 Ab. In corresponding catalytic activity assessments, only SL6 and all CA beluga microsomal samples exhibited CYP1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activity (51-260 pmol/mg/min), CYP3A-mediated activity (113-899 pmol/mg/min) based on the formation of 6beta-hydroxytestosterone using a testosterone hydroxylase assay, and UDPGT activity (830-4956 pmol/mg/min) based on 1-naphthylglucuronide formation. The marginal cross-reactivity with the anti-CYP2B1 Ab and lack of catalytically measurable hydroxytestosterone isomers associated with CYP2B-type activity in all the SL and CA animals is suggestive of low CYP2B-type enzyme expression in beluga. The absence of measurable total P450 enzyme levels and catalytic activities in samples from the stranded SL belugas suggested catalytically inactive enzymes as a consequence of tissue degradation related due to the time delay of sample collection after death. However, all SL and CA animals demonstrated similar, immunologically cross-reactive phase I and II hepatic enzyme profiles, which is suggestive of the importance of metabolism in the toxicokinetics and fate of xenobiotics in animals from both populations

Abstract: The oocyte is the starting point for a new generation. Most of the machinery for DNA and protein synthesis needed for the developing embryo is made autonomously by the fertilized oocyte. However, in fish and in many other oviparous vertebrates, the major constituents of the egg, i.e. yolk and eggshell proteins, are synthesized in the liver and transported to the oocyte for uptake. Vitellogenesis, the process of yolk protein (vitellogenin) synthesis, transport, and uptake into the oocyte, and zonagenesis, the synthesis of eggshell zona radiata proteins, their transport and deposition by the maturing oocyte, are important aspects of oogenesis. The many molecular events involved in these processes require tight, coordinated regulation that is under strict endocrine control, with the female sex steroid hormone estradiol-17beta in a central role. The ability of many synthetic chemical compounds to mimic this estrogen can lead to unscheduled hepatic synthesis of vitellogenin and zona radiata proteins, with potentially detrimental effects to the adult, the egg, the developing embryo and, hence, to the recruitment to the fish population. This has led to the development of specific and sensitive assays for these proteins in fish, and the application of vitellogenin and zona radiata proteins as informative biomarkers for endocrine disrupting effects of chemicals and effluents using fish as test organisms. The genes encoding these important reproductive proteins are conserved in the animal kingdom and are products of several hundred million years of evolution.

Abstract: A complementary DNA (cDNA) encoding the eggshell zona radiata protein (RBTZR: AF407574) has been cloned from the liver of estradiol-17beta (E(2))-treated rainbow trout (Oncorhynchus mykiss) by reverse-transcriptase polymerase chain reaction (RT-PCR). A set of degenerate primers homologous to the highly conserved cysteine-rich region of the zona radiata protein gene from salmon, winter flounder, medaka and carp were used for the initial RT-PCR. The resulting PCR product was cloned, sequenced and identified as the Zrp gene fragment based on amino acid sequence similarities. Based on the Zrp sequence from the initial PCR, a pair of gene-sequence primers was designed for 3'- and 5'- random amplification of cDNA ends (RACE). Cloning and sequencing of RACE products showed a 1349-bp Zrp gene encoding a 403-amino acid protein with a theoretical molecular mass of approximately 45 kDa. Alignment of the deduced amino acid sequence reveals that RbtZR is similar to piscine and mammalian zona pellucida proteins. The RbtZR gene, together with the estrogen receptor (ER) and vitellogenin (Vtg) genes, was further characterized and comparatively studied for transcriptional and translational expression in xenoestrogen- (nonylphenol, NP) and E(2)-treated juvenile rainbow trout in a time-course experiment. Northern and slot blot analysis showed that the RbtZR mRNA was expressed, in parallel with the ER and Vtg mRNA, in both NP- and E(2)-treated juvenile rainbow trout. Indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody raised against Atlantic salmon Zrp indicated the translational expression of RbtZR protein in blood plasma samples from NP- and E(2)-treated juvenile trout. The differential time-dependent transcriptional and translational expression and use of Zrp, ER and Vtg as sensitive biomarkers in environmental monitoring of endocrine disrupters in fish is discussed.

Abstract: Previously, it has been demonstrated that nonylphenol (NP) has a dual function in regulating reproductive hormones by: (1) increasing the activity of steroid metabolizing enzymes at low concentration, that does not induce vitellogenin (Vtg) and zona radiata proteins and (2) decreasing the activity of these enzymes at higher concentration [Environ. Health Perspect. 105 (1997) 109; Environ. Toxicol Chem. 16 (1997) 2576]. To more precisely understand the estrogenic effects of NP in fish and a possible interference with steroid hormone metabolism, we investigated in the Atlantic salmon the identity of the cytochrome P450 enzymes involved in NP hydroxylation. Up to 9 metabolites were separated by radio-HPLC when [R-(14)C]-4n-NP was incubated with hepatic microsomes from juvenile salmon. In control fish the major metabolites were identified as monohydroxylated products at omega-, (omega-1)- and (omega-2)-positions of the alkyl chain of 4n-NP. Salmon hepatic microsomes formed omega-, (omega-1)- and (omega-2)-lauric acid (LA) hydroxylation products. The potency of alpha-naphthoflavone, ketoconazole and ethynylestradiol (non-specific, but strong inhibitors of CYP1A, 2K and 3A, respectively) on LA and NP hydroxylations were assessed in the present study. Ketoconazole inhibited omega-, (omega-1)- and (omega-2)-hydroxylations of LA and 4n-NP and was the only inhibitor of omega-hydroxylation of both substrates. Ethynylestradiol specifically inhibited (omega-1)- and (omega-2)-hydroxylations of LA as well as 4n-NP. Interestingly, the lowest NP dose (1 mg/kg) was the most potent inducer of NP-metabolites formation. These results suggest the involvement of CYP2M- and 2K-like enzymes in terminal and subterminal hydroxylations of 4n-NP respectively, and was confirmed by the competitive inhibition between LA and 4n-NP. The production of one unidentified 4n-NP metabolite was not affected by any of the chemicals used, suggesting a possible ring hydroxylation with involvement of another cytochrome P450 isoform. Our data reveal a novel aspect of CYP isozymes involvement in NP metabolism that may complicate the assessment of its endocrine effects. Hence, the regio-selective hydroxylation of endocrine disruptors, such as NP, by CYP isozymes is revealed as a possible new marker of estrogenicity.

Abstract: The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and cta-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days I and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n = 5 for each group) and 14 (n = 6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYPIA were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17beta for Vtg and Zrp expression, and beta-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.

Abstract: Cytochrome p4501A induction and subsequent enzyme expression is used as a biomarker for exposure to aryl hydrocarbon receptor active contaminants in fish and other species. In the present study, CYP1A cDNA (1912 bp, GenBank accession number AF364076) was cloned, sequenced and characterized from the liver of a beta-naphthoflavone (betaNF)-treated teleost, Atlantic salmon (Salmo salar), by reverse-transcriptase polymerase chain reaction (RT-PCR). The salmon CYP1A sequence contained a 5'-flanking region of 99 bp, an open reading frame of 1566 bp that encodes a 521 amino acid protein, a stop codon, and a 3'-untranslated region of 346 bp, and a single polyadenylated signal. The theoretical molecular mass and isoelectric point was 58.6 kDa and 6.17, respectively. Comparison of the deduced amino acid sequence of salmon cDNA with reported CYP1A genes showed identities of 73-88% with fish CYP1A, 51-54% with mammalian CYP1A1, 47-51% with mammalian CYP1A2 and 54% with frog p450. Phylogenetic analysis showed that salmon CYP1A clustered in the tree with rainbow trout CYP1A1 and eel CYP1A sequences. CYP1A mRNA induction in beta-naphthoflavone-treated salmon showed differential organ expression with a distinct single transcript pattern. A new specific molecular tool has been developed for the monitoring of environmental pollution using CYP1A mRNA from salmon as a biomarker.

Abstract: Zonagenesis and vitellogenesis (eggshell zona radiata protein (Zrp) and vitellogenin (Vtg) production, respectively), are two estrogen-regulated processes in oviparous vertebrates that are crucial for oocyte maturation. Treatment of juvenile Atlantic salmon (Salmo salar) with nonylphenol (NP; 25 mg kg(-1)) alone or in combination with 3,3',4,4'-tetrachlorobiphenyl (TCB; 0.1 mg kg(-1)) resulted in pronounced elevations of plasma eggshell Zrp and Vtg and their respective liver mRNA levels in two separate experiments. TCB treatment alone caused the elevation of CYP1A mRNA, protein and enzyme levels (7-ethoxyresorufin O-deethylase (EROD)). In experiment 3, which also included the time factor, exposure of juvenile salmon to 10 and 25 mg NP per kg in combination with TCB generally resulted in reduced plasma Zrp and Vtg levels, compared with NP treatments alone. In a fourth experiment, juvenile salmon were exposed to different doses of TCB either 2 days before or 2 days after a single dose (25 mg kg(-1)) of NP. Samples were always collected 5 days after the NP exposure and analyzed for mRNA and protein levels. Generally, TCB doses given 2 days after NP exposure resulted in the elevation of Vtg and Zrp protein and mRNA levels. Vtg and Zrp mRNA levels were also elevated in the groups treated with 0.1 mg TCB 2 days before NP exposure. In all experiments, TCB injection resulted in the induction of liver CYP1A mRNA, CYP1A protein and EROD activity, but no Zrp or Vtg protein/mRNA inductions were observed when given alone. The present study documents for the first time the apparent stimulation of xenoestrogen-induced responses by an antiestrogenic CYP1A-inducer, in fish or any other lower vertebrate. However, the stimulatory or inhibitory effect of TCB on NP-induced responses appear to be dependent on the ratio of NP and TCB doses, and temporal sequence of exposure. Fish hepatic zonagenesis and vitellogenesis continue to provide interesting models for further studies on the mechanisms and possible interactions between endocrine disruptors and CYP1A-inducers, their antiestrogenic and/or estrogen potentiating effects.

Abstract: Anthropogenic chemicals in the aquatic environment are known to cause reproductive disturbances in vertebrate and invertebrate organisms, by interfering with the endocrine systems. Laboratory-based in vivo and in vitro studies have indicated that several of the anthropogenic and other naturally occurring chemicals in the environment can cause adverse reproductive effects. Various definite or possible reproductive abnormalities caused by endocrine disruption have been identified, but in majority of the reported cases, it is not known whether adverse effects have occurred in the population level of biological organization. Disruption of the hormonal functions in fish may have effects on a number of events, including sexual maturation, gamete production and transport, sexual behaviour, fertility, gestation, lactation or modifications in other functions that are dependent on the integrity of the reproductive system. Although several reproductive effects have been reported, but the degree of causality established between the abnormalities observed and exposure to particular chemicals is variable, and understanding of the mechanism(s) is limited. Fishes are a vital source of proteins and lipids for humans and domestic animals, forming the basis for economically important fisheries and aquaculture. Large efforts have recently been denoted to dissect the mechanisms of action of xenobiotics in aquatic species, with the ultimate aim of detecting, controlling and possibly intervening in chemical exposure and its effects on the aquatic ecosystem and humans. In this context, we ought to be concerned with the health and safety of aquatic species per se, as well as a resource for human needs.

Abstract: The time- and dose-dependent transcriptional and translational expression of biomarker genes in nonylphenol (NP) and estradiol-17beta (E(2)) treated juvenile rainbow trout is reported. Fish were exposed to NP (1, 5 and 25 mg/kg) and E(2) (5 mg/kg) and killed at 2, 6, 12, 24, 48 and 72 h after exposure. The estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) gene expressions were analyzed in total liver RNA using Northern and slot hybridization with specific cDNA probes. Plasma Vtg and Zr-protein levels were evaluated using indirect ELISA. While Zr-protein gene showed an induction only at 24 h post-exposure, the plasma protein levels showed a time-dependent increase in the 25-mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25-mg NP treated fish. Time-dependent increases in Vtg and Zr-protein gene expressions without the corresponding increases in ER gene transcription was observed in E(2)-treated fish at 2, 6 and 12 h post-exposure. Induction of ER gene transcripts was observed from 24 h and did not change significantly at 48 and 72 h. In the E(2)-treated fish, induction of plasma Vtg levels was observed at 48 and 72 h, while plasma Zr-protein was induced at 24, 48 and 72 h, after exposure. We conclude that the E(2)- and NP-induced Vtg and Zr-protein gene expressions at the early time intervals after exposure are not dependent on increase in the transcriptional activity of the ER gene and that Vtg and Zr-protein gene transcriptions require only basal or minimal ER concentration, in addition to other mechanisms.

Abstract: In the environment, nonylphenol (NP) occurs predominantly as a degradation product of nonylphenol ethoxylate (NPE). They can be found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges. Because of its enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms NP has been regarded as the most critical metabolite of APEs. We have studied the in vivo and in vitro metabolism and organ distribution of NP in juvenile salmon. Fish were exposed in vivo to waterborne [3H]-4-n-NP for a period up to 72 h or were administered a single oral dose of [3H]-4-n-NP. In vitro biotransformation of NP was studied by exposure of cultured salmon hepatocytes to [3H]-4-n-NP in the presence or absence of a CYP1A-inducer, beta-naphthoflavone (betaNF). Our results show that 4-n-NP was mainly metabolized in vivo, to its corresponding glucuronide conjugates and hydroxylates. The major route of excretion was the bile. The half-life of residues in carcass and muscle was between 24 and 48 h in both waterborne and dietary exposure. In whole body autoradiography, intragastric administered [3H]-4-n-NP was mainly present in the gastrointestinal tract and bile. NP-derived radioactivity in fish exposed via water was more evenly distributed in the organs compared to intragastric exposure and were observed in the intestinal contents, liver, kidney, gills, skin, abdominal fat and brain. In vitro pretreatment of hepatocytes with betaNF had no effect on rates or patterns of NP biotransformation. The in vitro metabolic rate of NP were 118 pmol NP metabolized/h/0.5x10(6) cells without betaNF, and 98 pmol NP metabolized/h/0.5x10(6) cells when betaNF was added to the culture medium.

Abstract: 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.

Abstract: 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 micrograms) and sampled at 24, 48 and 72 h after exposure. Metabolites were separated by radio-high-performance liquid chromatography and tentatively identified by cochromatography 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.

Abstract: : A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17beta (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.

Abstract: Alkylphenol ethoxylate degradation products such as nonylphenol and octylphenol are shown to have estrogenic effects. Nonylphenol induces synthesis of vitellogenin (a precursor of egg yolk proteins) and zona radiata proteins (eggshell proteins) in juvenile and/or male fish. Little is known about the molecular mechanisms of estrogenicity of environmental chemicals such as nonylphenol. To study the mechanisms of estrogenic effects of 4-nonylphenol (NP), we examined its in vivo effects on the expression of the estrogen receptor (ER), vitellogenin (Vtg) and zona radiata protein (Zrp) genes in juvenile Atlantic salmon liver. We show that the ER mRNA synthesis is induced by NP in a dose-dependent manner in juvenile Atlantic salmon liver. The induction of the ER mRNA synthesis is followed by the induction of Zrp and Vtg mRNA synthesis. The ER transcripts reach peak levels earlier than the Zrp and Vtg mRNA and proteins, which is in agreement with the physiological effects of estradiol during zonagenesis and vitellogenesis. Various studies have also shown that NP competitively inhibits the binding of 17 beta-estradiol (E2) to ER. Our results further suggest that NP directly mimics E2 in inducing the ER, Zrp and Vtg genes in salmon liver.

Abstract: The in vivo estrogenic potency of zearalenone (ZEA), a mycotoxin produced by different strains of Fusarium fungi, and its metabolites (alpha- and beta-zearalenol), have been studied in fish. Estrogenicity was evaluated using an in vitro competitive receptor binding assay and in vivo induction of vitellogenesis and zonagenesis, two estrogen receptor (ER)-mediated responses that are integral aspects of fish oogenesis. The ER binding affinities of alpha-zearalenol and ZEA in rainbow trout (Oncorhynchus mykiss) were approximately 1/150 and 1/300 to that of estradiol, respectively. Juvenile salmon (Salmo salar) were exposed to a single intraperitoneal injection of ZEA, alpha-zearalenol and beta-zearalenol (each at 1 and 10 mg/kg) and compared to fish injected with estradiol-17 beta (E2; 5 mg/kg) and controls. Using indirect enzyme-linked immunosorbent assay (ELISA) with homologous antibodies, a dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr-proteins) were observed 7 days after exposure to ZEA and alpha-zearalenol. beta-Zearalenol did not elevate plasma Vtg levels, but a non-significant elevation of plasma Zr-proteins levels was observed at the highest dose (10 mg/kg). Generally, alpha-zearalenol and ZEA possess estrogenic potencies that are approximately 50% compared to that of E2, and their order of estrogenic potency (in both in vitro receptor competitive binding and in vivo induction of Vtg and Zr-proteins levels) is: alpha-zearalenol > ZEA > beta-zearalenol. Our results show that blood plasma analysis of Vtg and Zr-proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of ZEA and its metabolites.

Abstract: Environmental estrogens have recently caused great concern because of their ability to mimic natural hormones and influence vital endocrine functions in humans and wildlife. The induction of vitellogenin (Vtg) synthesis by environmental estrogens in viviparous vertebrates has been proposed as an effective and sensitive biomarker of estrogenicity. Immunochemical analysis of plasma from Atlantic salmon (Salmo salar) exposed to 4-nonylphenol (NP) or to effluent from oil refinery treatment plant (ORTP), shows that NP and ORTP effluent induces Vtg and zona radiata proteins (Zrp) in a dose-dependent manner. However, Zrp-beta cross-reactive proteins are more responsive than Zrp-alpha, Zrp-gamma, and Vtg. The sensitivity of Zrp induction points to the zona radiata proteins as alternate biomarkers of estrogenicity.