Establishment of killer whale (Orcinus orca) primary fibroblast cell cultures and their transcriptomic responses to pollutant exposure.

Populations of killer whale (Orcinus orca) contain some of the most polluted animals on Earth. Yet, the knowledge on effects of chemical pollutants is limited in this species. Cell cultures and in vitro exposure experiments are pertinent tools to study effects of pollutants in free-ranging marine mammals. To investigate transcriptional responses to pollutants in killer whale cells, we collected skin biopsies of killer whales from the Northern Norwegian fjords and successfully established primary fibroblast cell cultures from the dermis of 4 out of 5 of them. Cells from the individual with the highest cell yield were exposed to three different concentrations of a mixture of persistent organic pollutants (POPs) that reflects the composition of the 10 most abundant POPs found in Norwegian killer whales (p,p'-DDE, trans-nonachlor, PCB52, 99, 101, 118, 138, 153, 180, 187). Transcriptional responses of 13 selected target genes were studied using digital droplet PCR, and whole transcriptome responses were investigated utilizing RNA sequencing. Among the target genes analysed, CYP1A1 was significantly downregulated in the cells exposed to medium (11.6 µM) and high (116 µM) concentrations of the pollutant mixture, while seven genes involved in endocrine functions showed a non-significant tendency to be upregulated at the highest exposure concentration. Bioinformatic analyses of RNA-seq data indicated that 13 and 43 genes were differentially expressed in the cells exposed to low and high concentrations of the mixture, respectively, in comparison to solvent control. Subsequent pathway and functional analyses of the differentially expressed genes indicated that the enriched pathways were mainly related to lipid metabolism, myogenesis and glucocorticoid receptor regulation. The current study results support previous correlative studies and provide cause-effect relationships, which is highly relevant for chemical and environmental management.

Alterations in the Atlantic cod (Gadus morhua) hepatic thiol-proteome after methylmercury exposure.

Proteomic studies in general have demonstrated that the most effective and thorough analysis of biological samples requires subfractionation and/or enrichment prior to downstream processing. In the present study, Atlantic cod (Gadus morhua) liver samples were fractionated using activated thiol sepharose to isolate hepatic proteins containing free/reactive cysteines. This subset of proteins is of special interest when studying the physiological effects attributed to methylmercury (MeHg) exposure. Methylmercury is a persistent environmental contaminant that has a potent affinity toward thiol groups, and can directly bind proteins via available cysteine residues. Further, alterations in the cod thiol-proteome following MeHg exposure (2 mg/kg body weight) were explored with two-dimensional gel electrophoresis combined with downstream mass spectrometry analyses for protein identifications. Thirty-five protein spots were found to respond to MeHg exposure, and 13 of these were identified when searching cod-specific databases with acquired mass spectrometry data. Among the identified thiol-containing proteins, some are known to respond to MeHg treatment, including constituents of the cytoskeleton, and proteins involved in oxidative stress responses, protein synthesis, protein folding, and energy metabolism. Methylmercury also appeared to affect cod heme metabolism/turnover, producing significantly altered levels of hemoglobin and hemopexin in liver following metal exposure. The latter finding suggests that MeHg may also affect the hematological system in Atlantic cod.

Precision-cut liver slices of Atlantic cod (Gadus morhua): an in vitro system for studying the effects of environmental contaminants.

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 µm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, ß-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner.

Reducing persistent organic pollutants while maintaining long chain omega-3 fatty acid in farmed Atlantic salmon using decontaminated fish oils for an entire production cycle.

Oily fish are an important source of health promoting nutrients such as the very long chain marine omega-3 (VLC-n3) fatty acids and simultaneously a source of potentially hazardous contaminants. Fish oils that are used in fish feed are the main source for both contaminants and VLC-n3. Decontamination techniques have recently been developed to effectively remove persistent organic contaminants from fish oils. The aim of the present study was to assess the level of potentially hazardous contaminants and the health beneficial fatty acids in Atlantic salmon reared on novel decontaminated feeds. Atlantic salmon were fed for 18 months (an entire seawater production cycle) on diets based on decontaminated or non-treated (control) fish oils until market size (approximately 5 kg). The level of known notorious persistent organic pollutants (POPs, i.e. dioxins, dioxin-like polychlorinated biphenyls (DL-PCBs), non dioxin-like PCBs, poly brominated diphenyl ethers (PBDE), and organochlorine pesticides), as well as fatty acid composition were analysed in fish oils, the two diets, and Atlantic salmon fillet. The oil decontamination process was a two-step procedure using active carbon and short path distillation. The fillet levels of POPs in market size fish were reduced by 68-85% while the concentration of very long chain omega-3 fatty acids was reduced by 4-7%. No differences in biomarkers of dioxin-like component exposures, such as hepatic gene expression of CYP1A or AhR2B, CYP1A protein expression and 7-ethoxyresorufin O-deethylase (EROD) activity, were observed between salmon raised on normal or decontaminated feeds, thus indicating that the difference in POPs levels were of no biological significance to the fish. Atlantic salmon reared on decontaminated feeds had sum polychlorinated dibenzodioxins/furans (PCDD/Fs) and DL-PCB concentrations that were comparable with terrestrial food products such as beef, while the level of marine omega-3 fatty acids remained as high as for commercially farmed Atlantic salmon.

A new ELISA for the three-spined stickleback (Gasterosteus aculeatus L.) spiggin, using antibodies against synthetic peptide.

The aim of this study was to develop an enzyme-linked immunosorbent (ELISA) assay to quantify spiggin in the three-spined stickleback. Spiggin is a glue protein produced in the kidney of male three-spined stickleback under the control of androgens during the breeding period. Disturbances of spiggin production in male fish and abnormal induction of spiggin in female fish are considered as valuable biomarkers of exposure to (anti-)androgenic chemicals. Polyclonal antibodies against a peptide sequence of spiggin (HRD-16) were used and the specificity of the antibodies was verified by Western blotting and direct ELISA experiments. By using HRD-16 antibodies and spiggin standard preparation, a competitive ELISA was set-up and validated. This assay appears sensitive, with a detection limit of 0.5 U/mL, and specific, as shown by the competition curves, obtained by serial dilution of male and female kidney homogenates, that were parallel to the spiggin standard curves. The ability of the spiggin ELISA to quantify spiggin induction was achieved by exposing male and female three-spined sticklebacks to 0.1 and 1 microg/L of methyltestosterone. The results show a significant dose-dependent induction of spiggin in methyltestosterone-exposed female fish compared to controls.

Environmental agent susceptibility assessment using existing and novel biomarkers as rapid noninvasive testing methods.

This study is part of a project aimed at developing and validating novel noninvasive methods for the detection of biomarkers of endocrine disrupters (EDs) directly in the mucus of aquatic species, to identify novel functional biomarker(s) for EDs, and to verify their applicability for field studies. The multidisciplinary approach chosen aims at the development of an integrated testing strategy utilizing in vitro protocols to identify water and sediment fractions with potential endocrine-disrupting activity; the identification, characterization, and measurement of new biomarker(s) for EDs; the development and validation of a dipstick-based test method; and the development of (computer-assisted) predictive models. Some results of the first year of the project are presented here.

Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis.

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of approximately 49 kDa size, and weak signals for proteins of approximately 41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of approximately 56 and 59 kDa size, and also a weak immunoreaction with a protein of approximately 43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14-40 ng/g DW in controls to 373-638 ng/g DW in exposed mussels.

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses.

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.

Cytochrome P4501A responses in two tropical fish species, riverine catfish (Rita rita) and marine mudfish (Apocryptes bato).

The present study focuses on the establishment of methods for biomarker studies in freshwater and marine fish species as a basis for monitoring the extent of contamination of fisheries resources in tropical waters. Riverine catfish (Rita rita) and marine mudfish (Apocryptes bato) were given a single intraperitoneal injection with two selected inducing compounds; beta-naphthoflavone (BNF, 50 mg/kg) and a polychlorinated biphenyl mixture (Clophen A50, 20 mg/kg), and the heavy metal compound cadmium chloride (CdCl2, 1 mg/kg). Effects on cytochrome P4501A (CYP1A) were determined in post-mitochondrial supernatants (PMS) of liver at days 3 and 10 after treatment. EROD (7-ethoxyresorufin O-deethylase) activity and CYP1A protein level by indirect non-competitive enzyme-linked immunosorbent assay (ELISA) and Western blotting using a monoclonal antibody against fish CYP1A, were measured. BNF and Clophen A50 resulted in induction upto 9.5- and 5-fold, respectively, of CYP1A protein compared to control, while CdCl2 showed significant inhibition in these species. The present study examined the phase-I cytochrome P450 monooxygenase activity and response in these two tropical fish species for the first time.

Regio-specific hydroxylation of nonylphenol and the involvement of CYP2K- and CYP2M-like iso-enzymes in Atlantic salmon (Salmo salar).

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.

The effect of stress on toxicant-dependent cytochrome p450 enzyme responses in the Arctic charr (Salvelinus alpinus).

In the present study we investigated the effect of stress and cortisol on cytochrome P450 (CYP) expression in Arctic charr exposed to benzo[a]pyrene (BaP). Expression of hepatic CYP1A and CYP3A was monitored 8 d after a single oral dose of BaP (10 mg/kg fish) and compared to that in unexposed fish. During this period the fish were subjected to one of the following stress regimes: no stress, no stress and cortisol implantation, 10 min of daily handling and confinement stress, and confinement stress during the last 6 h before sampling. In BaP-exposed fish daily stress resulted in significantly lower (53%) CYP1A protein levels as compared to those in unstressed fish. For CYP1A catalytic activity (measured as 7-ethoxyresorufin-O-deethylase [EROD] activity), the suppressive response to stress was less pronounced. These results contrast to previous findings of a potentiation by corticosteroids on xenobiotic-dependent CYP1A induction in vitro in cultured fish hepatic cells. No effects of high cortisol levels or BaP were found on the steroid-metabolizing CYP3A enzyme levels. The lack of any alterations in the CYP3A protein level indicates that CYP3A expression is not inducible by cortisol in the Arctic charr under the conditions used here. The conclusion was made that short-term stress associated with sampling (i.e., 6 h of confinement stress before sampling) of wild charr does not compromise the EROD activity as a reliable biomarker.

In vivo modulation of nonylphenol-induced zonagenesis and vitellogenesis by the antiestrogen, 3,3'4,4'-tetrachlorobiphenyl (PCB-77) in juvenile fish.

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.

Immunochemical and catalytic characterization of hepatic microsomal cytochrome P450 in the sperm whale (Physeter macrocephalus).

Liver samples from three live-stranded adult male sperm whales, that could be sampled and frozen in liquid nitrogen within 18 h post mortem, provided an opportunity to learn more about the basic properties of their cytochrome P450 (CYP) system. All samples were catalytically active and showed sharp bands of the different CYP enzymes after Western blotting, indicating that degradation of the proteins was negligible. All three sperm whales showed a similar immunochemical CYP pattern: bands of CYP1A1/2, CYP3A and CYP4A were present, but CYP2B1/2 was not detected. Significant biotransformation of the polychlorinated aromatic hydrocarbons 4, 4'-dichlorobiphenyl (CB-15), 2,7-dichlorodibenzodioxin and 1,2,3,4,8-pentadibenzofuran was measured in an in vitro biotransformation assay. In contrast, 3,3',4,4'-tetrachlorobiphenyl (CB-77) and two chlorobornanes (CHB-32 and CHB-62) occurring in the insectide toxaphene(R), were not metabolised.

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes.

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.

Identification and distribution of nitric oxide synthase in the brain of adult zebrafish.

Nitric oxide (NO) is proposed to be involved in developmental and plastic processes. We investigated the presence and distribution of nitric oxide synthase (NOS) in the zebrafish (Danio rerio) using molecular and histochemical techniques. A partial gene sequence corresponding to the neuronal NOS isoform (nNOS) was identified, and in situ hybridization revealed cellular nNOS mRNA expression throughout the brain of adult zebrafish, distributed in distinct central nuclei and in proliferation zones. NOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase activity partly coincided with the nNOS mRNA expression, however was present also in additional neuronal and non-neuronal cell types. The results indicate the occurrence of different NOS isoforms in the adult brain, of which nNOS may participate in neurotransmission, and in mechanisms related to the continuous growth and neuronal plasticity of the teleost brain.

Partial cloning of constitutive and inducible nitric oxide synthases and detailed neuronal expression of NOS mRNA in the cerebellum and optic tectum of adult Atlantic salmon (Salmo salar).

Studies of different species have implicated nitric oxide (NO) synthase (NOS) in various physiological and pathological events. Three major NOS isoforms are present in the brain of mammals; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). Little is known about the significance of the presence of these proteins in the brain. We report the first investigation into the presence of nNOS and iNOS isoforms in a teleost, adult Atlantic salmon (Salmo salar). Complementary DNA was synthesized from cerebellum and thymus mRNA using RT-PCR techniques with primers against conserved regions of NOS. Cloning and sequencing revealed a partial gene sequence of 560 bp corresponding to mammalian nNOS from cerebellum cDNA. The predicted protein sequence of identified salmon nNOS possessed 85% identity to that of mammalian nNOS. Northern blot analysis of different tissues revealed expression in brain and heart, and indicated expression of three different nNOS mRNAs in the brain. In addition, a 389 bp sequence corresponding to iNOS was identified in thymus cDNA. Salmon iNOS is almost identical to rainbow trout iNOS (95%), but shows much less amino acid identity to goldfish (65%) and mammalian (52%) iNOS. Phylogenetically, all vertebrate nNOS and iNOS homologues are clustered separately. Expression studies by means of in situ hybridization revealed abundant nNOS mRNA transcripts in distinct neuronal populations throughout the Purkinje cell layer of the corpus cerebellum and the periventricular layer of the optic tectum. Our data show that adult Atlantic salmon possess a gene encoding an nNOS isoform and putative alternatively spliced forms, which are expressed in distinct neuronal populations in the cerebellum and optic tectum, and in yet unidentified cell types in the heart. The data suggest that the arising of different vertebrate NOS isoforms is an evolutionary old event. The well conserved sequences present in salmon and mammalian nNOS may reflect their importance in protein function, whereas interspecies distributional differences in cellular expression of nNOS and sequence differences of iNOS may reflect variations and specializations in routes of NO action in the vertebrate phylogeny.

Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo salar).

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.

In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar).

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.

Organochlorines in top predators at Svalbard--occurrence, levels and effects.

Alarmingly high polychlorinated biphenyl (PCB) levels have been found in the top predators such as glaucous gull (Larus hyperboreus) and polar bear (Ursus maritimus) at Svalbard [Gabrielsen, G.W., Skaare, J.U., Polder, A., Bakken, V., 1995. Chlorinated hydrocarbons in glaucous gull (Larus hyperboreus). Sci. Total Environ. 160/161, 337-346; Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U., 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press). ]. Studies of the possible toxic effects, particularly on the immune system and reproduction, of the very high PCB levels in these species are currently being investigated. Data obtained in the field (f.i. reproductive success in polar bears and intestinal nematodes in glaucous gulls), as well as levels of various biochemical and physiological parameters (f.i. thyroid hormones, retinol, EROD activity, CYP1A, IgG), have been coupled with the PCB levels [Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23 (in Norwegian); Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Bernhoft, A., Skaare, J.U., Wiig, O., Derocher, A.E., Larsen, H.J., 2000. Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard (in press); Henriksen, E.O., Gabrielsen, G.W., Skaare, J.U., Skjegstad, N., Jensen, B.M., 1998a. Relationship between PCB levels, hepatic EROD activity and plasma retinol in glaucous gull, Larus hyperboreus. Marine Environ. Res. 46, 45-49; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U. , 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press); Sagerup, K., Gabrielsen, G.W., Skorping, A., Skaare, J.U., 1998. Association between PCB concentrations and intestinal nematodes in glaucou gulls, Larus hyperboreus, from Bear Island. Organohalogen compounds 39, 449-451; Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23. (in Norwegian)].

Metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar).

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.