Anne S. Mortensen

Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to water-soluble fraction of crude oil

The induction of CYP enzyme activities, particularly CYP1A1, through the aryl hydrocarbon receptor (AhR) in most vertebrate species is among the most studied biochemical response to planar and aromatic organic contaminant exposure. Since P450 families play central roles in the oxidative metabolism of a wide range of exogenous and endogenous compounds, interactions between the biotransformation processes and reproductive physiological responses are inevitable. Steroidogenesis is the process by which specialized cells in specific tissues, such as the gonad, brain (neurosteroids) and kidney, synthesize steroid hormones. In the present study, we evaluated the effects of water-soluble fraction (WSF) of crude oil on the xenobiotic biotransformation and steroidogenic processes in the head (brain) and whole-body tissue of a model species by transcript analysis using quantitative (real-time) polymerase chain reaction (qPCR), enzyme activities and steroid hormone (testosterone: T and 17beta-estradiol: E2) levels using enzyme immune assay (EIA). Our data showed that exposure of fish to WSF produced an apparent concentration-specific increase of AhR1, CYP1A1 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA levels, and decrease of AhR2. On the activity level, WSF produced concentration-specific increase of ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin (BROD) methoxyresorufin (MROD) and pentoxyresorufin (PROD) activities in whole-body tissue. In the steroidogenic pathway, WSF exposure produced apparent concentration-specific decrease of ER* and ERbeta, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc), P450aromA and P450aromB mRNA expression. For steroid hormones, while T levels decreased, E2 levels increased in an apparent WSF concentration-specific manner. In general, the xenobiotic biotransformation and estrogenic responses showed negative relationship after exposure of zebrafish to WSF, suggesting an interaction between these physiological pathways. The relationship between WSF mediated changes in brain StAR, P450scc, 3beta-HSD, ER*alpha, ERbeta, P450aromA, P450aromB and whole-body steroid hormone levels suggests that the experimental animals might be experiencing altered neurosteroidogenesis probably through increased activity level of the biotransformation system. Thus, these responses might represent sensitive diagnostic tools for short-term and acute exposure of fish or other aquatic organisms to WSF.

Interactions Between Estrogen- and Ah-Receptor Signalling Pathways in Primary Culture of Salmon Hepatocytes Exposed to Nonylphenol and 3,3',4,4'-Tetrachlorobiphenyl (Congener 77)

BackgroundThe 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 ERα 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 μM – an ER agonist) singly or in combination with 0.001, 0.01 and 1 μM PCB-77 and sampled at 48 h post-exposure. Secondly, hepatocytes were exposed to NP (5 μM) or PCB-77 (1 μM) 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.ResultsOur 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 AhRα, AhRR, ARNT, CYP1A1 and UDPGT expression. A novel aspect of the present study is that low (0.001 μM) and medium (0.01 μM) PCB-77 concentrations increased ERα mRNA expression above control and NP exposed levels, and at 12 h post-exposure, PCB-77 exposure alone produced significant elevation of ERα, ERβ and Zr-protein expressions above control levels.ConclusionThe 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 ERα and ERβ (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.

Gene Expression Patterns in Estrogen (Nonylphenol) and Aryl Hydrocarbon Receptor Agonists (PCB-77) Interaction Using Rainbow Trout (Oncorhynchus Mykiss) Primary Hepatocyte Culture

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 µM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 µM. 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 ERα, ERβ, 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 AhRα mRNA (except 1 µM PCB-77, which caused the induction of AhRα mRNA levels). AhRβ mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 µM NP restored the PCB-77-inhibited AhRβ 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.

Lipid peroxidation and oxidative stress responses of salmon fed a diet containing perfluorooctane sulfonic- or perfluorooctane carboxylic acids.

The present study was conducted to evaluate the effects of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on lipid β-oxidation and oxidative stress responses in Atlantic salmon liver and kidney tissues. We quantified changes in the expression levels of peroxisome proliferator-activated receptors (PPARs) and acyl-CoA oxidase (ACOX1) enzyme whose transcription is induced by PPARs. In addition, we analyzed gene expression patterns for enzymatic antioxidants (superoxide dismutase: SOD, catalase: CAT and glutathione peroxidase: GPx). Thiobarbituric acid reactive substances (TBARS) were analyzed as a measure for lipid peroxidation. Juvenile Atlantic salmon were repeatedly force-fed food spiked with PFOA or PFOS at 0.2mg/kg, and samples were collected after 0, 2, 5 and 8 days and after a 7 days recovery period. Our data showed that exposure of salmon to PFOS or PFOA produced changes (either increased or decreased) in mRNA expression for PPARs, ACOX1, oxidative stress responses and lipid peroxidation (TBARS) and these responses showed marked organ differences, associated with tissue bioaccumulation patterns and dependent on exposure time. Given that a classical reaction during reactive oxygen species (ROS)-induced damage involves the peroxidation of lipids, our study demonstrates that salmon continuously exposed to dietary PFOS or PFOA dose showed alteration in peroxisomal responses and oxidative stress responses, with higher severity in the kidney, compared to liver. Overall, our data suggest that ROS-mediated oxidative damage maybe a significant and putative toxic effect of PFOA and PFOS in fish as has been reported in mammals.

Tissue bioaccumulation patterns, xenobiotic biotransformation and steroid hormone levels in Atlantic salmon (Salmo salar) fed a diet containing perfluoroactane sulfonic or perfluorooctane carboxylic acids

In the present study, groups of juvenile Atlantic salmon (Salmo salar) were fed gelatine capsules containing fish-food spiked with PFOA or PFOS (0.2 mg kg(-1) fish) and solvent (methanol). The capsules were given at days 0, 3 and 6. Blood, liver and whole kidney samples were collected prior to exposure (no solvent control), and at days 2, 5, 8 and 14 after exposure (Note: that day 14 after exposure is equal to 7d recovery period). We report on the differences in the tissue bioaccumulation patterns of PFOS and PFOA, in addition to tissue and compound differences in modulation pattern of biotransformation enzyme genes. We observed that the level of PFOS and PFOA increased in the blood, liver and kidney during the exposure period. Different PFOS and PFOA bioaccumulation patterns were observed in the kidney and liver during exposure- and after the recovery periods. Particularly, after the recovery period, PFOA levels in the kidney and liver tissues were almost at the control level. On the contrary, PFOS maintained an increase with tissue-specific differences, showing a higher bioaccumulation potential (also in the blood), compared with PFOA. While PFOS and PFOA produced an apparent time-dependent increase in kidney CYP3A, CYP1A1 and GST expression, similar effects were only temporary in the liver, significantly increasing at sampling day 2. PFOA and PFOS exposure resulted in significant decreases in plasma estrone, testosterone and cortisol levels at sampling day 2, and their effects differed with 17α-methyltestostrerone showing significant decrease by PFOA (also for cholesterol) and increase by PFOS. PFOA significantly increased estrone and testosterone, and no effects were observed for cortisol, 17α-methyltestosterone and cholesterol at sampling day 5. Overall, the changes in plasma steroid hormone levels parallel changes in CYP3A mRNA levels. Given that there are no known studies that have demonstrated such tissue differences in bioaccumulation patterns with associated differences in toxicological responses in any fish species or lower vertebrate, the present findings provide some potential insights and basis for a better understanding of the possible mechanisms of PFCs toxicity that need to be studied in more detail.

Neural aromatase transcript and protein levels in Atlantic salmon (Salmo salar) are modulated by the ubiquitous water pollutant, 4-nonylphenol

At present, there are no known direct occurrences of nonylphenol (NP) in nature. Therefore, its presence in nature is solely a consequence of human activities. NP is generated through degradation of alkylphenol ethoxylates released mainly from textile, metal working, institutional cleansing and laundry cleaning, but few data on the amount of the release is available. These compounds have been shown to affect several biological processes, including the endocrine systems, in a wide number of species. The cytochrome P450 aromatase (Cyp19) is the rate-limiting step in estrogen production, and is known to be a potential target for endocrine-disrupting chemicals (EDCs) such as NP. Teleost fish generally have a high brain aromatase activity, and the effects of EDCs in fish brain is not thoroughly investigated. In this study, juvenile Atlantic salmon (Salmo salar) were exposed to waterborne concentrations of the synthetic pharmaceutical and xenoestrogen 17alpha-ethynylestradiol (EE2; 5ng/L) and the xenoestrogen 4-nonylphenol (NP; 5 and 50microg/L) for 72h. Brain tissue and blood were sampled from individual fish. Gene expression patterns of Cyp19 isoforms were determined by quantitative PCR, aromatase protein immunoreactivity in the brain was evaluated by immunohistochemistry and immunoblotting, and aromatase activity was analyzed using the tritiated water-release assay. Plasma estradiol (E2) and testosterone (T) levels were measured by EIA. In the brain, EE2 increased the mRNA expression of Cyp19b almost threefold compared to the solvent control, whereas Cyp19a levels were unaffected by EE2 treatment. In contrast, both NP concentrations produced significant reduction of Cyp19a expression. Immunohistochemical aromatase protein reactivity was localized in several brain regions, but no apparent quantitative effects of the exposures were observed. Immunoblotting analysis showed that EE2 and NP produced a slight increase in brain immunoreactive aromatase protein band, compared with controls. Plasma levels of E2 increased twofold when treated with EE2 and 5microg NP/L, and threefold when exposed to 50microg NP/L. In general, the present study shows that the parallel biochemical, transcriptional and cellular detection of neural aromatase for endocrine-disrupting effects from EE2 and NP may be observed at specific levels of the biological organization.

The persistent DDT metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, alters thyroid hormone-dependent genes, hepatic cytochrome P4503A, and pregnane x receptor gene expressions in atlantic salmon (Salmo salar) parr

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.

Estrogenic effects of selected hydroxy polychlorinated biphenyl congeners in primary culture of Atlantic Salmon (Salmo salar) hepatocytes.

Many persistent organic pollutants are known to have endocrine-disrupting effects in several aquatic and terrestrial species. In this regard, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) represent serious health and environmental concern because they are shown to act agonistic or antagonistic at hormone receptors (HRs) or to cause hormone-receptor-mediated responses. In the present study, salmon primary hepatocytes were used to study alterations in an estrogen signaling pathway resulting from exposure to four hydroxylated (4OH-CB 107, 4OH-CB146, 4OH-CB187, and 3OH-CB138) metabolites of PCB at different concentrations using quantitative real-time polymerase chain reaction. The effects of the PCB metabolites were compared to the mRNA expression in 17α-ethynylestradiol (EE2)-treated cells. Concentration-specific increase of vitellogenin (Vtg) mRNA transcription after exposure to OH-PCBs was observed. Decreased mRNA transcription was observed for zona radiata protein (Zr-protein) and cytochrome P450 side-chain cleavage (P450scc) enzyme. For estrogen receptor β (ERβ), the mRNA expression pattern was OH-PCB-metabolite congener-specific. A novel aspect of this study is that OH-PCBs produced effects on hepatic steroidogenic pathways by targeting the StAR protein and P450scc genes. Given that endocrine toxicology research mainly has focused on estrogenicity involving direct ER-mediated effects and that steroidogenic enzyme and proteins are highly tissue- and cell-type-specific and controlled by different promoters and second-messenger pathways, the present findings provide potential new targets for interaction with xenobiotics such as hydroxylated congeners of certain chemicals. The quantitative expression patterns of hepatic and extrahepatic steroidogenic genes and proteins after exposure to environmental contaminants are the subject of systematic investigations in our laboratory.

Biotransformation of PCBs in Arctic seabirds: Characterization of phase I and II pathways at transcriptional, translational and activity levels

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO(2)-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher SigmaOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

Effects of tributyltin (TBT) on in vitro hormonal and biotransformation responses in Atlantic salmon (Salmo salar).

The mechanisms by which the biocide tributyltin (TBT) and its metabolites affect the hormonal and xenobiotic biotransformation pathways in aquatic species are not well understood. In this study hepatocytes isolated from salmon were used to evaluate the mechanistical effects of TBT on fish hormonal and xenobiotic biotransformation pathways. Cells were exposed to 0.01, 0.1, 1, or 5 μM TBT and samples were collected at 0, 12, 24, or 48 h following exposure. Gene expression patterns were evaluated using quantitative polymerase chain reaction (PCR), and cytochrome P-450 (CYP)-mediated enzyme activities were evaluated by ethoxyresorufin, benzyloxyresorufin, and pentoxyresorufin O-deethylase (EROD, BROD, and PROD, respectively) activity assays. Generally, exposure of hepatocytes to 1 μM (at 48 h) and 5 μM TBT (at 12, 24, and 48 h) consistently produced reductions in all mRNA species investigated. TBT produced significant decreases of vitellogen (Vtg) expression at 48 h and modified the expression patterns of estrogen receptors (ERα and ERβ) and androgen receptor-β (ARβ) that were dependent on time and TBT concentration. In the xenobiotic biotransformation pathway, TBT produced differential expression patterns that were dependent on exposure time and concentration for all salmonid AhR2 isoforms (AhR2α, AhR2β, AhR2δ, and AhR2γ). For CYP1A1, CYP3A, AhRR, and Arnt mRNA, TBT produced exposure- and time-specific modulations. Catalytic CYP activities showed that BROD activity increased in an apparent concentration-specific manner in cells exposed to TBT for 12 h. Interestingly, EROD activity showed a TBT concentration-dependent increase at 24 h and PROD at 12 and 48 h of exposure. In general our data show that TBT differentially modulated hormonal and biotransformation responses in the salmon in vitro system. The apparent and consistent decrease of the studied responses with time in 1 and 5 μM exposed hepatocytes suggest a possible transcription inhibitory effect of TBT.