Abstract: The aim of this study was to examine the transcriptional levels of selected genes in liver and head kidney of Atlantic cod Gadus morhua sampled in Store Lungegårdsvann, a seawater recipient situated in the middle of the city of Bergen, Norway, for effects of contaminants released from municipal sewage effluents and former dump sites. Five males and six females were caught with fish traps in Store Lungegårdsvann in 2006. Cod from a location near Jondal in the Hardanger Fjord were used as controls (five males and four females). The following 12 genes were picked as potential markers of contaminant exposure: cytochrome P-450 1A (CYP1A), cytochrome P-450 2C33-like (CYP2C33-like), cytochrome P-450 3C (CYP3C), glutathione S-transcriptase pi (GST) (detoxification and biotransformation), Mn superoxide dismutase (Mn SOD), glutathione reductase (GR), heat-shock protein 70 (HSP70) (oxidative stress), vitellogenin A (VtgA), vitellogenin B (VtgB), zona pellucida 2 (ZP2) (effects of estrogen disruptors), B-cell lymphoma 2 (Bcl-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A) (radiation). The results showed that two males caught in Store Lungegårdsvann possessed high transcriptional levels of VtgA, VtgB, and ZP2 mRNA in the liver. In addition, CYP1A was 4.9-fold higher expressed in males from Store Lungegårdsvann compared to males from the reference population. CYP2C33-like mRNA expression was significantly higher (1.8-fold) in females from Store Lungegårdsvann than in females from the reference population. CYP1A was significantly lower (4.7-fold) expressed in head kidney of females from Store Lungegårdsvann than in females from Hardanger Fjord. In a follow-up examination with sexually mature cod sampled in Store Lungegårdsvann in 2007, the livers were shown to contain high levels of polychlorinated biphenyls (PCB) and dioxin-like PCB. In conclusion, fish inhabiting Store Lungegårdsvann are exposed not only to endocrine disruptors but also to other contaminants that affect the transcription of phase I biotransformation genes.
Abstract: Cytochrome P4501A (CYP1A), benzo(a)pyrene (B(a)P) activation and biliary elimination, phase II activities, and peroxisomal and antioxidant activities of turbot (Scophthalmus maximus) were studied in a long-term controlled experiment. Fish were serially exposed in water on day 1 and on completion of months 3, 6 and 9 to 0.1, 0.2, 0.1 and 0.1mg B(a)P/l, respectively, while another group was identically treated with additional PCB77 (3,3',4,4'-tetrachlorobiphenyl) at 1% of concomitant B(a)P (w/w). Temporally persistent responses were obtained by sampling on week 3 and 3 months from each latest exposure. Serial exposure to B(a)P+PCB77 progressively induced liver 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A protein levels (ELISA, western blotting) towards months 9, 12 and gill EROD activity on month 12. It associated with an apparent increase in liver benzo(a)pyrene diol epoxide (BPDE)-DNA adduct levels (ultrasensitive enzyme radioimmunoassay), and elevated bile B(a)P metabolite levels on month 9 females as compared to males. In contrast, B(a)P alone did not cause (p>0.05) comparable effects on liver EROD, CYP1A, adducts nor on bile metabolites. Both exposed groups demonstrated evidence for lasting oxidative stress as hepatic superoxide dismutase, catalase and glutathione peroxidase activities were significantly altered (p<0.05) with symptomatic pro-oxidant associations among them. Both treatments affected liver somatic index similarly (increase on month 3, decrease on month 9 in males). Continued exposure on month 18 (0.2mg B(a)P/l, 1% PCB77) followed by sampling 6 months later showed sustained induction (p<0.001) of hepatic EROD in B(a)P+PCB77 group, which was not seen in B(a)P alone treatment. Thus, PCB77 co-exposure prolonged CYP1A induction and contributed to a persistent oxidative challenge in B(a)P-exposed turbot. The results indicate synergistic effects of polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) exposure in the aquatic environment.
Abstract: An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.
Abstract: 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.
Abstract: A collaborative study was conducted on the Biosense amnesic shellfish poisoning (ASP) enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins in shellfish in order to obtain interlaboratory validation data for the method. In addition, a method comparison study was performed to evaluate the ASP ELISA as an alternative to the current liquid chromatography (LC) reference method for DA determination. The study material comprised 16 shellfish samples, including blue mussels, Pacific oysters, and king scallops, spiked with contaminated mussel homogenates to contain 0.1-20 mg DA/kg shellfish flesh. The shellfish samples were extracted with 50% aqueous methanol, and the supernatants were directly analyzed. Sixteen participating laboratories in 10 countries reported data from the ASP ELISA, and 4 of these laboratories also reported data from instrumental LC analysis. The participating laboratories achieved interlaboratory precision estimates for the 8 Youden paired shellfish samples in the range of 10-20% for RSD(r) (mean 14.8 +/- 4%), and 13-29% for RSDR (mean 22.7 +/- 6%). The precision estimates for the ELISA data did not show a strong dependence on the DA concentration in the study samples, and the overall precision achieved was within the acceptable range of the Horwitz guideline with HorRat values ranging from 1.1 to 2.4 (mean HorRat 1.7 +/- 0.5). The analysis of shellfish samples spiked with certified reference material (CRM)-ASP-MUS-b gave recoveries in the range of 88-122%, with an average recovery of 104 +/- 10%. The estimate on method accuracy was supported by a correlation slope of 1.015 (R2 = 0.992) for the determined versus the expected DA values. Furthermore, the correlation of the ASP ELISA results with those for the instrumental LC analyses of the same sample extracts gave a correlation slope of 1.29 (R2 = 0.984). This indicates some overestimation of DA levels in shellfish by the ELISA, but it is also a result of apparent low recoveries for the LC methods. This interlaboratory study demonstrates that the ASP ELISA is suitable for the routine determination and monitoring of DA toxins in shellfish, and that it offers a rapid and cost-effective methodology with high sample throughput.
Abstract: To identify possible CYP1A-immunopositive proteins in bivalves, we used anti-fish CYP1A antibodies combined with one- and two-dimensional gel electrophoresis and mass spectrometry, and found that two of the main CYP1A-immunopositive proteins in digestive gland of Mytilus edulis, were cytoskeletal actin (42 kDa) and major vault protein (102 kDa), while the main CYP1A-immunopositive protein in the clam Chamaelea gallina was the cytoskeletal protein tropomyosin (33 kDa). Anti-CYP1A cross-reactive bands of 48-54 and 75 kDa in M. edulis were observed but not identified in this study. Sequence alignments with one of the most conserved CYP1A regions (NIRDITDSLIDHCED) from fish revealed similarities with tropomyosin and actin sequences from mussels, which could explain the immunological cross-reactivity. Changes in isoforms of tropomyosin after exposure to Aroclor1254 and Cu(II), could indicate modifications due to oxidative stress. Effects of pollutant related oxidative stress on the cytoskeleton require further studies. (c) 2006 Elsevier B.V. All rights reserved.
Abstract: Recent research demonstrated how endocrine-disrupting chemicals (EDCs) may disturb wildlife populations and possibly also represent a human health risk. Much of the focus has been on (anti-)estrogenic and (anti-)androgenic effects, and these effects are thought to be mediated through the estrogen (ER) and androgen (AR) receptors, respectively. The seriousness of the problem has led international bodies such as the Organization for Economic Cooperation and Development (OECD) and the European Union (EU) to initiate large research programs and developments toward new guidelines and regulations. EDCs have both synthetic and natural sources. The mechanisms of action of EDCs can be divided into: (1) agonistic/antagonistic effect ("hormone mimics"), (2) disruption of production, transport, metabolism, or secretion of natural hormones, and (3) disruption of production and/or function of hormone receptors. However, the number of nuclear hormone receptors being potential targets for EDCs has increased dramatically the last decade, opening up new avenues for possible endocrine disruptor effects. In studies with Atlantic salmon, data showed that 4-nonylphenol, a model xenoestrogen previously used in large volumes, for example, in paints and detergents, acts as an estrogen mimic, as a steroid metabolism disruptor, and by modulating estrogen receptor (ER) levels, indicating that one single compound exerts all of these three mechanisms, depending on the dose given to the organism. A hypothesis explaining this observation is that the nature of the effect of an EDC is determined by dose-dependent routing and cross-talk between different classes of nuclear receptors.
Abstract: The possible use of cytoskeletal components as biomarkers of organic pollution in mussels has been investigated. Responses of non-muscular actin and tropomyosin (TM), two bivalve proteins that were recently demonstrated to cross-react with anti-fish-CYP1A, were analysed in digestive tissue of blue mussels (Mytilus sp.) exposed to a wide range of organic contaminants. The results were evaluated with ELISA and Western blot assays, utilising commercial monoclonal antibodies, and compared with expression of Hsp70, a marker of chemical stress. Furthermore, mussels were sampled from the Baltic Sea at sites with different degrees of pollution to assess the expression of these proteins, and to monitor seasonal changes in relation to energy reserves and water temperature. The results demonstrated that expression of microsomal actin was significantly higher (p<0.02) in mussels exposed to a brominated flame retardant (BDE-47), and lower, however not significantly, in specimens exposed to crude oil, alone and spiked with alkylphenols and PAHs. Hsp70 was strongly induced in all exposure groups, which also included bisphenol A and diallylphthalate. Furthermore, microsomal actin exhibited seasonal variations, and expression was negatively correlated with water temperature. No correlation was seen between actin and the microfilament-binding protein TM, indicating that regulation of these two cytoskeletal components are not coupled. Furthermore, parallel and significant (p<0.05) up-regulations of TM and Hsp70 were seen in individuals sampled from a strongly polluted field site, whereas the seasonal analysis showed that TM expression was positively correlated with energy reserves (total glycogen content) in mussels, suggesting the use of TM as a marker of growth. In conclusion, this study has demonstrated the cytoskeleton to be a target of contaminants in mussels, calling for further attention. Exposure-induced increase of microsomal actin can be interpreted either as stimulated actin synthesis, or re-arrangements of the dynamic microfilaments.
Abstract: Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of exposure to (anti-)oestrogens and their mimics in fish [Sumpter, J.P., 1995. Feminized responses in fish to environmental estrogens. Toxicol. Lett. 82, 737-742; Arukwe, A., Goksøyr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. ]. In some instances, links have been drawn between high level induction of Vtg and adverse health effects in fish [Herman, R.L., Kincaide, H.L., 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture 72, 165-172; Schwaiger, J., Spieser, O.H., Bauer, C., Ferling, H., Mallow, U., Kalbfus, W., Negele, R.D., 2000. Chronic toxicity of nonylphenol and ethinyloestraiol: haematological and histopathological effects in juvenile common carp (Cyprinus carpio). Aquat. Toxicol. 51, 69-78]. The widespread use of Vtg as a biomarker has led to the development of a variety of assays to quantitatively measure Vtg concentrations in tissue samples from fish, and hence a need for a standardization of the performance criteria and validation of such assays [Goksøyr, A., Eidem, J.K., Kristiansen, S.I., Nilsen, B.M., 2003. On the need for a standardized set-up for validation studies of fish vitellogenin assays as an endpoint in endocrine disruptor testing and screening-a proposal. ]. One of the most popular test fish species for assessing chemical effects is the fathead minnow (Pimephales promelas), which is now used widely for studies into endocrine disruption [Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti)estrogenic substances. Environ. Toxicol. Chem. 21, 319-326; Hutchinson, T.H., Yokota, H., Hagino, S., Ozato, K., 2003. Development of fish tests for endocrine disruptors. Pure Appl. Chem. 75, 2343-2353]. This paper describes the development and validation of a new, homologous enzyme-linked immunosorbent assay (ELISA) for quantification of Vtg in this fish species.
Abstract: To identify possible CYP1A-immunopositive proteins in bivalves, we used anti-fish CYP1A antibodies combined with one- and two-dimensional gel electrophoresis and mass spectrometry, and found that two of the main CYP1A-immunopositive proteins in digestive gland of Mytilus edulis, were cytoskeletal actin (42 kDa) and major vault protein (102 kDa), while the main CYP1A-immunopositive protein in the clam Chamaelea gallina was the cytoskeletal protein tropomyosin (33 kDa). Anti-CYP1A cross-reactive bands of 48-54 and 75 kDa in M. edulis were observed but not identified in this study. Sequence alignments with one of the most conserved CYP1A regions (NIRDITDSLIDHCED) from fish revealed similarities with tropomyosin and actin sequences from mussels, which could explain the immunological cross-reactivity. Changes in isoforms of tropomyosin after exposure to Aroclor1254 and Cu(II), could indicate modifications due to oxidative stress. Effects of pollutant related oxidative stress on the cytoskeleton require further studies.
Abstract: Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.
Abstract: 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.
Abstract: BACKGROUND: Xenoestrogens and antifungal azoles probably share a common route of metabolism, through hepatic cytochrome P450 (CYP) enzymes. Chemical interactions with metabolic pathways may affect clearance of both xenobiotics and endobiotics. This study was carried out to identify possible chemical interactions by those substances on CYP1A and CYP3A, in Atlantic cod liver. We investigated effects of two xenoestrogens (nonylphenol and ethynylestradiol) and of the model imidazole ketoconazole, alone and in combination. RESULTS: Treatment with ketoconazole resulted in 60% increase in CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity. Treatment with nonylphenol resulted in 40% reduction of CYP1A activity. Combined exposure to ketoconazole and nonylphenol resulted in 70% induction of CYP1A activities and 93% increase in CYP1A protein levels. Ketoconazole and nonylphenol alone or in combination had no effect on CYP3A expression, as analyzed by western blots. However, 2-dimensional (2D) gel electrophoresis revealed the presence of two CYP3A-immunoreactive proteins, with a more basic isoform induced by ketoconazole. Treatment with ketoconazole and nonylphenol alone resulted in 54% and 35% reduction of the CYP3A-mediated benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activity. Combined exposure of ketoconazole and nonylphenol resulted in 98% decrease in CYP3A activity. This decrease was greater than the additive effect of each compound alone. In vitro studies revealed that ketoconazole was a potent non-competitive inhibitor of both CYP1A and CYP3A activities and that nonylphenol selectively non-competitively inhibited CYP1A activity. Treatment with ethynylestradiol resulted in 46% decrease in CYP3A activity and 22% decrease in protein expression in vivo. In vitro inhibition studies in liver microsomes showed that ethynylestradiol acted as a non-competitive inhibitor of CYP1A activity and as an uncompetitive inhibitor of CYP3A activity. CONCLUSIONS: Ketoconazole, nonylphenol and ethynylestradiol all interacted with CYP1A and CYP3A activities and protein expression in Atlantic cod. However, mechanisms of interactions on CYP1A and CYP3A differ between theses substances and combined exposure had different effects than exposure to single compounds. Thus, CYP1A and CYP3A mediated clearance may be impaired in situations of mixed exposure to those types of compounds.
Abstract: The possible ecotoxicological effects of a paper and pulp mill effluent were investigated by measuring selected contaminants and biomarkers in caged Nile tilapia (Oreochromis niloticus) and sharptooth catfish (Clarias gariepinus) from the Karnaphuly River, Bangladesh. Fish were caged for 28 d at two upstream reference sites (2 and 4 km) and four downstream sites (3, 5, 8, and 12 km) from the effluent outlets. Organochlorine contaminants and bile biomarkers were bioaccumulated to higher levels at downstream polluted sites than at the reference sites, including hexachlorobenzene (HCB), hexachlorocyclohexane (HCH), polychlorinated biphenyls (PCBs) in liver, and chlorophenolic compounds like 2,4-dichlorophenol and 2,4,6-trichlorophenol in bile. Levels of glucose, protein, and aspartate aminotransferase were analyzed in plasma, whereas cytochrome P4501A (CYP1A) levels were determined in S-12 supernatants of liver. The results, including CYP1A induction and bile biomarkers, clearly indicated that, in addition to the paper and pulp mill effluents, the downstream sites appear to receive other inputs of contaminants. This field assessment in a Bangladeshi river demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of industrial effluents in fish of developing countries.
Abstract: 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.
Abstract: The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1-63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5-63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25-16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.
Abstract: BACKGROUND: In the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERalpha). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERalpha genes, in primary cultured salmon hepatocytes, have been investigated. RESULTS: The expression of the genes encoding VTG and ERalpha were strongly induced by 17beta-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor alpha-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERalpha mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERalpha genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERalpha genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERalpha mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERalpha. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERalpha or that activated AHR blocked the ability of ERalpha to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA. CONCLUSION: In fish hepatocytes E2 induces ERalpha and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERalpha. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERalpha- and AHR-signalling pathways is unidirectional.
Abstract: 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.
Abstract: Proteomics has been used in the clam Chamaelea gallina as a preliminary screening of changes in protein expression caused by pollutants, potentially useful as new biomarkers. Clams were exposed in water for seven days to four model contaminants, Aroclor 1254, copper(II), tributyltin (TBT), and arsenic(III), and cytosolic fractions were initially analyzed by two-dimensional (2-D) electrophoresis in 7 cm IPG strips (pH 4-7). On average, about 1000 spots were resolved and altered expression was qualitatively detected in 9-26 spots per treatment. Aroclor 1254, Cu(II) and As(III) had a mainly upregulating effect, in contrast to TBT. Altered protein expression was confirmed in 18 cm gels (at narrow pH ranges). The 15 spots more drastically altered were excised and analyzed by mass spectrometry (MS), and four proteins were identified. Aroclor 1254 and Cu(II) upregulated putative isoforms of tropomyosin and light chain of myosin. Actin was downregulated by Aroclor and Cu(II) but upregulated by TBT and As(III), while the opposite behavior was shown by a truncated actin form, homologous to the Drosophila act87E gene product. The exclusive identification of cytoskeletal proteins could reflect their relative abundance, their prevalence in databases in molluscs, or their role as major targets of pollutant-related oxidative stress.
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: 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: 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.
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: Vitellogenin (VTG) induction has proved to be a valuable biomarker for assessing exposure to environmental estrogens in fish. The widespread use of VTG in this regard has lead to the need for standardized assays to quantify VTG, and monoclonal antibodies have the potential to help accomplish this. A VTG enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody prepared against Atlantic salmon (Salmo salar) VTG (MAb BN-5) and its ability to quantify VTG in the rainbow trout (Oncorhynchus mykiss) compared with a rainbow trout vitellogenin (rt-VTG) ELISA that employed homologous polyclonal antibodies (PAb). In routine protocols, the working range of the homologous rt-PAb VTG ELISA was between 9 ng/ml and 70 ng/ml (80- 20% relative maximum binding [B/Bo]) with a 50% B/Bo of 25+/-0.9 ng/ml and inter- and intraassay variations at 50% B/Bo of 7% (n = 7) and 8% (n = 15), respectively. The working range of the MAb BN-5 VTG ELISA was between 60 ng/ml and 850 ng/ml (80-20% B/Bo) with a 50% B/Bo of 227+/-22 ng/ml and inter- and intraassay variations at 50% B/Bo of 5% (n = 10) and 9% (n = 12), respectively. In the routine protocols, detection limits for measurement of plasma VTG in rainbow trout (at 80% B/Bo; and given the requirement to dilute plasma to a minimum of 1:10 for the assays) were 90 ng/ml for the polyclonal rt-VTG assay and approximately 600 ng/ml in the monoclonal antibody assay. In juvenile female rainbow trout exposed to a series of doses of estradiol-17beta (E2) and 4-tert nonylphenol (4-NP), there were no differences in the vitellogenic responses measured in the PAb and MAb BN-5 VTG ELISAs. The monoclonal MAb BN-5 VTG ELISA is likely to be of considerable value for studies on environmental estrogens in juvenile female rainbow trout in standardized tests.
Abstract: In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.
Abstract: 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.
Abstract: 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.
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: 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.
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: 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.
Abstract: 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)].
Abstract: Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination:in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.
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: 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.
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: Concentrations of total cytochrome P450 and cytochrome P450 IA (CYP 1A) and activities of ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) were measured in the liver of prespawning, spawning and postspawning dab (Limanda limanda) from the German Eight. Between all P450-dependent parameters measured significant correlations were found. Generally, during prespawning and spawning season higher values were measured in the liver of males compared to females, but the ratio between sexes changed during spawning time, when concentrations and activities in the liver of males decreased and increased in the liver of females. The activity and the signal-to-noise ratio decrease in the order EROD, ECOD and PROD. This decrease is accompanied by an increase in K-m. The findings indicate that the different activities can be attributed to the strongly overlapping substrate specificity and the different enzyme affinities of one enzyme, CYP 1A, towards the three substrates. A biphasic kinetic of ECOD indicates that in addition to CYP 1A a second isozyme catalyses the O-deethylation of ethoxycoumarin in the liver of dab. Interestingly, the ratio between EROD activity and CYP 1A concentration varied seasonally but did not differ significantly between sexes. (C) 1999 Elsevier Science Inc. All rights reserved.
Abstract: Induction of vitellogenin (Vtg) in oviparous vertebrates has been used as a biomarker of response for environmental oestrogens. This study reports the cellular localization of oestrogen- and xenoestrogen-induced Vtg synthesis in the liver of juvenile Atlantic salmon (Salmo salar). Paraffin-embedded liver sections were incubated with homologous monoclonal antibody against Atlantic salmon Vtg. Following intraperitoneal (ip) exposure of fish to estradiol-17 beta [E-2; 5 mg kg(-1)] or 4-nonylphenol [NP; 125 mg kg(-1)], Vtg induction was primarily demonstrated immunohistochemically in the cytoplasm of hepatocytes, endothelial cells and within hepatic sinusoids. Vtg staining of hepatocytes was not evenly distributed, as there was a high degree of polarization toward the sinusoid. The intensity of positive Vtg staining was stronger in the liver sections of E-2-treated fish, compared with NP-treated fish. Hepatocytes of E-2-, NP- and vehicle (control)-treated fish showed normal cellular structures, thus showing no evidence of histopathological changes. In parallel, indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis of plasma Vtg levels show significant induction of Vtg in E-2- and NP-treated fish, as compared with untreated (control) fish. The present study demonstrates the applicability of immunohistochemistry in studies of cellular structures, processes and responses of fish exposure to oestrogen and oestrogen-mimicking compounds.
Abstract: The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 microg l(-1) propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.
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: Exposure to a variety, of xenobiotics, including polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), results in the induction of CYP1A and related biological activity. Historically, antibodies against purified CYP1A have been raised for ELISA and western immunoblotting techniques to quantitate this protein. However, recent advances in molecular cloning and sequencing of CYP1A genes, followed by Hopp-Woods hydrophilicity plotting, have allowed investigators to raise polyclonal antibodies to specific peptide sequences of interest [Myers et al. (1993) Environmental Toxicology and Chemistry 12, 1619-1626]. We sensitized RBF/dnj mice with KLH-conjugated peptide 277-294 of rainbow trout CYP1A1. Splenic lymphocytes were then fused with FOX myeloma cells, resulting in hybridoma mixtures secreting antibodies that reacted strongly, with BSA-conjugated peptide and PCB-126 induced channel catfish hepatic microsomes. Using ELISA, dot immunoblots, and SDS-PAGE/immunoblotting as screening techniques, hybridomas were cloned that secrete monoclonal antibodies exhibiting cross-reactivity with CYP1A from several species of fish. To date, these include rainbow trout (Onchorhynchus mykiss), Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), pinfish (Lagodon rhomboides), killifish (Fundulus sp.), carp (Cyprinus carpio), channel catfish (Ictalurus punctatus) and others. Studies characterizing inhibitory effects of these antibodies on CYP1A-related enzyme activity (EROD) are underway. (C) 1998 Elsevier Science Ltd.
Abstract: We investigated the ability of the Edible crab (Cancer pagurus) to biotransform polycyclic aromatic hydrocarbon (PAH) compounds in vivo and in vitro. In vivo the metabolism of benzo(a)pyrene (BaP) was analysed by direct fluorimetry of hepatopancreas cytosol, demonstrating the formation of water-soluble metabolites of this PAH compound. A pyrene hydroxylase assay was developed for in vitro studies. The activity was dependent upon artificially supported NADPH-cytochrome P450 reductase, but no induction effect was observed in crabs treated with BaP (5 mg/kg) up to 30 d after treatment. (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: Heme oxygenase (HO) was measured in fish liver and spleen by a coupled catalytic assay and by immunochemical analysis using an anti-mouse HO-1 antibody. In Atlantic salmon (Salmo salar) treated i.p. with cadmium, arsenite and phenylhydrazine, increased levels of HO activity and HO-cross-reacting protein were observed in liver microsomes. In the spleen, although no HO activity could be detected, increased levels of HO-cross-reacting protein Mere detected bit western blotting. The cross-reacting protein in liver and spleen had a relative molecular weight of approx. 30 kDa, slightly lower than the mammalian counterpart. Pn mackerel (Scomber scombrus) fed a high lipid diet, increased levels of HO activity were observed in a continuously fed group compared with fish starved for 2 months and wild mackerel. The results indicate that HO may be developed into a diagnostic biomarker for certain heavy metals and oxidative stress in fish. This application relies on the production of fish-specific antibodies for this enzyme. (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: Fixed wavelength fluorescence (FF) for detection of polyaromatic hydrocarbon (PAH) metabolites in fish bile has been studied and is presented through a laboratory and a field experiment. The method, which can be regarded as a biomarker of exposure to PAH, has proved to be a simple, rapid and cost effective method for monitoring PAH contamination. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.
Abstract: Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently,, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of fish oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268, 59-70), and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418-422). This study, compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p'-DDT (both at 25 mg kg(-1), singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond significantly stronger to NP treatment alone and in combination with o,p'-DDT compared to control and o,p'-DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination, with o,p'-DDT (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16d. The highest BaP concentration ill the edible portion of the fish, 16.5 +/- 4.3 mu g BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement. (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: Fish maturation and reproduction are complex biological processes that are regulated by endogenous substances (hormones), and synchronized by exogenous factors (photoperiod and temperature), thus ensuring that reproduction occurs at a time of the year optimal for survival of the offspring. The survival of any fish species is ultimately determined by the ability of its members to reproduce successfully in a fluctuating environment and thereby maintain a viable population. Several reports have documented that many compounds introduced into the environment by human activity either deliberately or unintentionally are capable of affecting reproductive processes in fish. Zonagenesis and vitellogenesis (eggshell protein and egg yolk precursor production, respectively) are two estrogen-regulated processes that are integral aspects of fish oogenesis. Several in vivo and in vitro studies have reported that some xenobiotics (xenoestrogens) possess the ability to mimic natural estrogens and therefore initiate precocious or unscheduled zonagenesis and vitellogenesis. Aspects of these effects acid other xenobiotically-induced responses will be discussed here, with special reference to their possible consequences for fish populations.
Abstract: Two monoclonal antibodies (KB-1 and BN-5) and one polyclonal antibody (AA-1) were prepared against purified vitellogenin (Vtg) from Atlantic salmon (Salmo salar), and tested for their ability to bind Vtg from plasma of various other fish species. The MAb KB-1 was found to bind specifically to Vtg from the Salmo species, Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). The MAb BN-5 showed a much wider cross-reactivity, binding to Vtg from both Salmoniformes species (Atlantic salmon, brown trout, rainbow trout and Arctic charr) and Pleuronectiformes species (turbot and halibut). The widest cross-reactivity Iras observed with the polyclonal antibody AA-1 which, in addition to the species recognized by the MAb BN-5, also bound to Vtg from Atlantic cod (Gadiformes). (C) 1998 Elsevier Science Ltd. All rights reserved.
Abstract: There is an increasing understanding that polynuclear aromatic hydrocarbons (PAHs) and organochlorine compounds (like polychlorinated biphenyls (PCBs), certain pesticides and dioxins) in the aquatic environment may lead to physiological and pathological effects such as immunological disturbances, effects on reproduction and development, and even neoplasms. Exposure to pollutants may have consequences at all levels in the biological organization, from the cellular level over effects on the individual organism, population, to the entire ecosystem. The cytochrome P450 system (CYP or P450) has an essential function in the biotransformation of endogenous and exogenous compounds. The fact that many different environmental pollutants induce de novo synthesis of cytochrome P450 1A (CYP1A) proteins in fish, gives these enzymes an interesting position in aquatic toxicology. Many investigations concerning the CYP1A system in fish have been performed over the last two decades, demonstrating its usefulness as a biomarker for aquatic pollution. A general overview of the biochemical and toxicological aspects concerning the cytochrome P450 system will be given here, followed by a more detailed description of CYP1A induction responses in fish. Ecotoxicological consequences of CYP1A induction and the use of immunochemical techniques for CYP1A detection as a biomarker in environmental monitoring will be discussed.
Abstract: Hepatic microsomal biotransformation reactions with xenobiotic and steroid substrates have been investigated in 4-non-ylphenol (NP; 1, 5, 25, and 125 mg/kg body weight)-treated juvenile Atlantic salmon (Salmo salar), in addition to control and estradiol-17 beta (5 mg/kg, positive control)-treated fish. Treatment of juvenile salmon with NP caused an initial increase and an apparent dose-dependent decrease in progesterone 6 beta-, 16 alpha, and 17 alpha-hydroxylase activities in liver microsomes. 7-Ethoxyresorufin O-deethylase and UDP-glucuronosyltransferase activities were also reduced. Plasma levels of estradiol-17 beta (E-2) were lowered 24-43% as a result of NP treatment. Immunochemical analysis of CYP1A, CYP2K-like, and CYP3A like proteins showed 18%, 47%, and 30% reductions in enzyme-linked immunosorbent assay absorbance levels, respectively, in the groups treated with 125 NP/kg fish. The group treated with E, also showed similar reductions. In summary, the present study has demonstrated variations in steroid hydroxylases, cytochrome P450 isozymes, and conjugating enzyme levels in NP-treated juvenile salmon. These results represent a novel aspect of NP effects not previously demonstrated with an environmental estrogen in any fish species or lower vertebrate.
Abstract: Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I), In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had;U-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity, Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP LA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP LA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera, Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH. (C) 1997 Federation of European Biochemical Societies.
Abstract: Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I). In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had AI-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity. Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP IA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP IA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera. Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH.
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.
Abstract: Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg(-1)), 2,3,3',4,4'5-hexachlorobiphenyl (PCB-156, 2.5 mg kg(-1)), and cadmium (cadmium, 1 mg kg(-1)), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB-treated flounder after 2 and 8 days, respectively. The strongest induction (44-fold) was caused by BaP No further induction was observed after additional treatment with PCB-156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre-treated with cadmium, whereas induction by PCB-156 appeared to be unaffected by pre-treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre-treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S-transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB-156 prior to cadmium. GST selenium-independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.
Abstract: Sexual differentiation of the hepatic cytochrome P450 system was characterized in 2-year-old farmed turbot (Scophthalmus maximum L.) during their first spawning period (January-September). The fish were kept in tanks supplied with continuously flowing seawater (34.5 parts per thousand, ppt) at a constant temperature of 16 degrees C and natural photoperiod (60 degrees N). Sampling of liver samples (n = 4-6) was performed once every month for 9 months. Pronounced sex differences were recorded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), and NADPH-cytochrome P450 reductase during spawning (May-July). EROD activity in female fish decreased gradually towards the onset of ovulation in May-July to rise again in the postspawning period. The decrease correlated with increasing gonadosomatic index and estradiol-17 beta (E(2)) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52 kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higher protein levels in male compared to female fish from April/May-June, and significant differences were observed in June (CYP2K-like also in April and May). Analysis of monthly variations within sexes during the reproductive cycle shows significant monthly changes in all parameters in both female and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated in male fish during spawning in June. To study the induction response during spawning, beta-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes in June. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levels but had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-like proteins. This study documents, for the first time in any fish species or lower vertebrate, the sexual differentiation in the liver of three different CYP subfamilies during sexual maturation and spawning. (C) 1997 Wiley-Liss, Inc.
Abstract: Responses in flounder (Platichthys flesus) towards benzo [a]pyrene (BaP), 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156), and cadmium (Cd) were investigated in time-course and dose-response studies of selected biomarkers. Measurements of biliary fluorescent BaP metabolites and hepatic concentrations of PCB-156 and cadmium showed that the injected toxicants were rapidly mobilized from the muscle to the liver, but a depot effect was indicated in the highest dose groups of BaP and PCB-156 (12 mg kg(-1) bodyweight). Clearest biomarker responses were found in the induction of hepatic cytochrome P450 1A (CYP1A) enzymes as a response towards BaP and PCB-156 exposure. Maximum induction of CYP1A dependent 7-ethoxyresorufin-O-deethylase (EROD) activity was observed after 2 and 8 days in BaP and PCB-156-treated flounder, respectively. Positive dose-effect relationships were observed towards both compounds, but the CYP1A induction was move persistent with PCB exposure than with BaP exposure. In Cd-exposed fish, the hepatic lever of metallothionein responded more slowly with highest levels observed after 16 days in the time-study, in the combined BaP + Cd treatment, the CYP1A induction was only slightly suppressed. Aspartate aminotransferase in serum appeared to be responsive towards BaP, but also towards the acetone vehicle in controls in the first part of the exposure period. Hematocrit as well as hepatic activities of aldrin epoxidase, glutathione S-transferase, and UDP-glucuronyl transferase were not responsive to any treatment in the present study. in general, the results demonstrate that selected biomarkers in flounder are responsive to PAH, PCB, and heavy metal pollutant exposure, indicating the applicability of this species in future environmental pollution monitoring programmes.
Abstract: Mummichog (Fundulus heteroclitus), an estuarine, teleost fish, were exposed for 456 hr to environmentally relevant concentrations of aqueous (10 micrograms/liter) and dietary (10 micrograms/g) benzo[a]pyrene (BP) in static renewal aquaria. Cellular expression of BP-inducible cytochrome P4501A (CYP1A) was evaluated several times during exposure by immunohistochemistry in longitudinal histologic sections of whole fish. CYP1A-associated staining intensities in tissues were scored by a subjective rating system similar to that used previously for qualitative information. Exposure to aqueous BP resulted in high levels of CYP1A-associated immunohistochemical staining in gill pillar cells, heart endothelium, and vascular endothelium. Exposure to dietary BP resulted in only mild to moderate staining in these tissues but high-intensity staining in gut mucosal epithelium. CYP1A induction in hepatocytes appeared most sensitive to aqueous exposure. Route-specific patterns of CYP1A expression were also observed in other cells including gill epithelia, pseudobranch, and skin. Expression of CYP1A in renal tubules and interrenal tissues was not affected by either treatment. Coexposure to both aqueous and dietary BP resulted in a pattern of induction reflecting both routes of exposure. In addition to the subjective rating system for scoring CYP1A expression, we developed a photometric approach that was used to obtain quantitative data on CYP1A-associated staining intensity. Photometric values of CYP1A staining intensity revealed patterns essentially the same as those observed during subjective ranking but were amenable to statistical analysis. The results of this study support the use of tissue-specific patterns of CYP1A expression in identification of target sites and exposure routes for polycyclic aromatic hydrocarbons and other compounds.
Abstract: The effects of piperonyl butoxide (PBO) and beta-naphthoflavone (BNF) on cytochrome P4501A (CYP1A) expression and activity in juvenile Atlantic salmon (Sabo salar L.) with regard to time and temperature was investigated. The time exposure study was performed at 8 degrees C, and the results show that PBO, although acutely functioning as a CYP1A inhibitor, is able to induce CYP1A expression in salmon liver. Both PBO and BNF give highest induction of CYP1A mRNA 48 h after intraperitoneal injection (five- and 14-fold, respectively). The mRNA levels induced by PBO and BNF were sustained during the 8 d of the experiment (four- and 11-fold, respectively). The CYP1A protein content measured by enzyme-linked immunosorbent assay demonstrated the highest induction by PBO 8 d after exposure (eightfold) and by BNF 4 d after exposure (11-fold). Activity of CYP1A measured by ethoxyresorufin-O-deethylase (EROD) demonstrated inhibition after PBO treatment the first 24 h after exposure, followed by threefold induction from 48 h and to the end of the experiment (8 d). beta-Naphthoflavone strongly induced EROD activity, with the highest levels occurring 4 d after treatment (56-fold) and 8 d after treatment (22-fold). In the temperature study, the results demonstrated temperature compensation, as salmon acclimated to 7 degrees C for 3 weeks had a significantly higher EROD activity than those acclimated to 11 and 15 degrees C. This was not reflected in significantly higher levels of reduced nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity or CYP1A protein. The inductive properties of PBO and BNF on CYP1A expression was also demonstrated in primary cultures of salmon hepatocytes.
Abstract: The primary aim of this study was to select a set of relevant biomarkers in feral eel for the biological assessment of inland water pollution. A suite of biochemical parameters in eel (hepatic biotransformation enzymes and cofactors, antioxidant enzymes, PAH metabolites, DNA adducts, serum transaminases) was measured in order to determine their response to xenobiotic compounds in the environment. The results of the analyses of organic trace pollutants in sediments and eel from six Amsterdam freshwater sites with different pollution levels have been discussed in the first part of this paper (Van der Oost, R., Opperhuizen, A., Satumalay, K., Heida, H. and Vermeulen, N.P.E., 1996a. Biomonitoring aquatic pollution with feral eel (Anguilla anguilla): I. Bioaccumulation: biota-sediment ratios of PCBs, OCPs, PCDDs and PCDFs. Aquat. Toxicol., 35: 21-46). The main conclusions drawn from the trends found for the levels and activities of biochemical parameters in eel were the following: the phase I biotransformation enzymes in eel liver appeared to be the most sensitive to environmental xenobiotics. Cytochrome b(5) (Cyt b(5)), cytochrome P450 1A (CYP1A), ethoxyresurofin-O-deethylase (EROD) and EROD turnover (EROD/P450) in eel liver showed significant responses to contamination, and can therefore be used as biomarkers. Levels of a CYP3A-like protein were significantly elevated in eel from three moderately polluted sires, but since this protein was not induced in eel from the most polluted site its relevance as a biomarker remains unclear. Phase II enzymes and cofactors in eel liver were less susceptible to pollutants than phase I enzymes. The activity of UDP glucuronyl transferase (UDPCT) in eel may, however, be a useful biomarker, while glutathione S-transferase (CST) activity and levels of oxidized glutathione (GSSG) were less sensitive. The reduced glutathione (GSH) cofactor levels in eel liver are most probably not reliable as biomarkers. Hepatic activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and GSH-peroxidase (GPOX)) in eel did not show any response to pollution and are therefore not feasible as biomarkers. The level of 1-hydroxypyrene (1-OH pyrene) in eel bile might be a useful biomarker to determine short-term PAH exposure. The hepatic level of DNA adducts in eel liver seems to be a sensitive biomarker for exposure to (and possible effects of) mutagenic and carcinogenic xenobiotics. In feral eel, serum transaminases are not usable as biomarkers of chronic intoxication. The proposed set of the most relevant biomarkers for the assessment of inland water pollution with feral eel thus consists of the following six parameters: cyt b(5), CYP1A, EROD, EROD/P450, UDPGT and DNA adducts.
Abstract: The ecotoxicological effects of a textile mill effluent were investigated by caging tilapia (Oreochromis niloticus) in the Volta River, Ghana, and by exposing mudfish (Clarias anguillaris) to sediment collected from the same river. Tilapia were caged for 3 weeks at three locations (0.6, 4, and 8 km) downstream from the effluent outlet. Mudfish were exposed in the laboratory for 2 weeks to sediment collected from the vicinity of the effluent outlet and 8 km downstream. Upstream reference locations 2 km (tilapia) and 10.2 km (mudfish) were included. Liver cytochrome P4501A (CYPIA) monooxygenase activity (measured as activity of 7-ethoxyresorufin O-deethylase, EROD, and CYP1A protein level) and two conjugation enzymes, UDP-glucuronosyl transferase (UDP-GT) and glutathione S-transferase (GST), were analysed. A distance-related decrease in EROD activity and CYPIA protein level was observed. EROD activity was 21-fold higher in tilapia caged at the site nearest the effluent outlet and 25-fold higher in mudfish exposed to sediment collected from the vicinity of the outlet, compared with the respective reference values. UDP-GT and GST levels increased significantly by 70 and 27%, respectively, in tilapia while the respective levels in mudfish were 73 and 28%, compared with reference values. The results clearly indicate that the textile mill effluent contains some highly potent inducers of biotransformation enzymes. This first assessment of the biological effects of organic pollutants in the Volta River demonstrates the utility of the CYP1A system as a valuable early warning biomarker of industrial effluents and also as a biomarker to detect exposure of aquatic resources to environmental chemical contamination in tropical waters.
Abstract: Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study. Copyright (C) 1996 Elsevier Science Ltd.
Abstract: Polyclonal and monoclonal antibodies against immunoaffinity-purified cod liver cytochrome P450 1A (CYP1A) have been developed. The monoclonal antibody (denoted MAb-NP7) and polyclonal antibody (PAb-Dy) recognized inducible CYP1A orthologues (55-58 kDa) in hepatic microsomes and 12,000 X g supernatants from several fish species exposed to P-naphthoflavone and aromatic hydrocarbon in Western blots, with some differences in cross-reactivity. The antibodies worked in enzyme-linked immunosorbent assay (ELISA) and immunohistochemical analyses, but were noninhibitory in analytical assays. The results indicate that anti-cod CYP1A PAb-Dy and MAb-NP7 recognize different epitopes on the CYP1A protein, and that the crossreacting determinants are only partially conserved over the wide range of fish phylogeny.
Abstract: Flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) were subjected to caging at polluted sediments in a Norwegian fjord (Sorfjorden) for a period of 3 months. Three caging sites were located close to metal smelters, whereas a fourth site was located 30 km away as a reference. In sediment samples, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were elevated at the innermost sites (1, 2 and 3) compared with the reference location (site 4). In fish, the biliary levels of fluorescent aromatic compounds (FACs) were elevated 5-20 fold in both species at the polluted sites. A two-fold difference in heavy metal levels was observed in cod (site 2 vs. 4), whereas no differences were seen in flounder. Pesticides bioaccumulated in a diffuse manner at all sites. In flounder at the innermost sites, plasma aspartate aminotransferase (AST) and hepatic cytochrome P450 1A (CYP1A) dependent 7-ethoxyresorufin O-deethylase (EROD) activity were elevated 4-5 and 5-10 fold, respectively, compared with the reference site. Both of these biomarkers were significantly correlated with FACs levels. For other biomarkers, the site effect was more marginal. The biomarkers seemed in general more responsive in flounder than in cod. The present study demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of pollutants in marine teleosts.
Abstract: Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of the CYP3 gene family and probably the CYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.
Abstract: Tilapia (Oreochromis niloticus) and mudfish (Clarias anguillaris) were given a single intraperitoneal injection of beta-naphthoflavone (BNF, 50 mg/kg) and Clophen A50 (Clo A50, 20 mg/kg). Hepatic levels of CYP1A protein, 7-ethoxyresorufin O-deethylase (EROD), glutathione S transferase (GST) and UDP-glucuronasyl transferase (UDP-GT) activities were determined at days 3 and 10 after treatment. Highest levels of EROD activity (5-6-fold) and CYP1A protein (3-5-fold) were observed in both species at day 3 on treatment with BNF. The same treatment also increased GST and UDP-GT activities in mudfish at day 3, but only UDP-GT activity was induced in tilapia at day 10. In tilapia treated with Clo A50, EROD activity was highest at day 3, whereas CYP1A protein level, GST and UDP-GT activities were highest at day 10. In mudfish, Clo A50 appeared to inhibit EROD activity, but CYP1A protein level was increased at day 10, whereas GST and UDP-GT activities reached highest levels at day 3. The control EROD levels did not differ significantly between the species, hut control GST levels were three to five times higher in tilapia, whereas those of UDP-GT were six times higher in mudfish. Although there are many common features in the hepatic xenobiotic biotransformation systems of the two freshwater teleosts, a number of species characteristics exist; most importantly, the differences in response to polychlorinated biphenyls and the different temporal pattern of induction response. These factors should be taken into account when using these species in studies of xenobiotic biotransformation and biomarker responses in tropical freshwater fish.
Abstract: Immunohistochemical and histopathological studies were conducted in two marine teleosts, Atlantic cod (Gadus morhua) and European flounder (Platichthys flesus), caged for three months on contaminated sediments in a Norwegian fjord, Cellular localization of CYP1A was analyzed immunohistochemically in liver, intestine, heart, gill and kidney, as part of an extensive study that included a number of chemical and biological measurements. Both species exhibited marked CYP1A induction when caged at contaminated sites. CYP1A induction was observed in liver as well as in several extrahepatic organs, and with increased expression at the more contaminated sites, Staining was particularly strong in vascular en endothelium. Induction responses were also observed in epithelial cells, including hepatocytes, biliary epithelial cells, mucosal epithelial cells in the intestine, and renal tubular epithelial cells, Histopathological examination of the liver did not reveal major cellular abnormalities, The immunohistochemical data indicate a strong relationship between CYP1A induction and exposure to sediment-associated industrial contaminants (polycyclic aromatic hydrocarbons). Interesting species differences in localization of CYP1A expression in Various cell types in cod and flounder were demonstrated immunohistochemically.
Abstract: Primary cultures of salmon (Salmo salar L.) hepatocytes were analysed using (35)s-methionine/cysteine incorporation and SDS-PAGE gel electrophoresis (1 and 2-D) and Western blotting after treatment with representative environmental pollutants (benzo(a)pyrene (BaP), 2,3,3',4,4'-pentachlorobiphenyl (PCB-105), arsenite (AsO2-) and cadmium (Cd)). The results demonstrated striking similarities in changes in protein expression after treatment with the different pollutants. Hsp70 (Hsp72/73) proteins were induced after treatment with all the compounds as shown by S-35- methionine/cysteine labelling, However, high background levels of these proteins were shown with Western blotting and an anti-Hsp70 antibody, indicating a slow turnover of these proteins, The Hsp70s in salmon hepatocytes were extremely susceptible to degradation in urea used in 2-D electrophoresis, resulting in peptide fragments of 45-46 kDa, In addition to these Hsp70 fragments, arsenite induced several proteins of 42, 38, and in the 30-32 kDa range, CYP1A (58 kDa) and an unidentified protein of 16 kDa were furthermore induced after treatment with the organic xenobiotics (BaP, PCB and the model compound beta-naphthoflavone, BNF). CYP1A was expressed in a dose-dependent manner, and was resolved into several protein spots in 2-D Western blotting, Elevated levels of metallothionein and haem oxygenase (HO) were indicated in Western blots after treatment with cadmium or arsenite (only HO), The hepatocytes showed cytoplasmic protrusions after treatment with 35 mu M arsenite and 100 mu M Cd, indicative of cells entering apoptosis.
Abstract: A study was conducted over the course of a year to determine the induction of hepatic cytochrome P4501A (CYP1A) in three species of benthic fish collected from a contaminated site compared to fish sampled from a less-contaminated site. Juvenile fish were used to minimize effects of reproductive status and migration. CYP1A was determined by two catalytic assays [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD)I and by an immunoassay (ELISA) utilizing polyclonal antibodies raised against purified CYP1A from cod. AHH activities were measured by a standard method (AHH(std)) and by two variations of the standard method. All three primary CYP1A measures (AHH(std), EROD, and ELISA) showed consistent between-site differences, indicating that induction of CYP1A can be a reliable biomarker of contaminant exposure in fish if appropriate biological variables are controlled for in field studies. Multiple ANOVA demonstrated that the AHH(std) and ELISA data showed less variability due to species or temporal differences, and less unexplained variability, compared to the data from the EROD assay or either variation of the AHH assay. For all measures, variability associated with site differences far outweighed species or temporal variability. Immunoassay, while less sensitive than the AHH(std) assay, is nonetheless recommended to be used in conjunction with catalytic assays because of the potential for samples to lose catalytic activity if not handled properly. The current results suggest that the lower noncontaminant-related variability of AHH(std) makes this CYP1A measure potentially more useful for monitoring programs in which analysis of trends is a primary goal.
Abstract: Mature specimens (n = 686) of male dab (Limanda limanda) were collected at several stations from the southern North Sea during two surveys in 1991 and 1992. Levels of CYP1A protein and 7-ethoxyresorufin O-deethylase (EROD) activity were measured in liver and heart. Elevated CYP1A levels were observed in dab collected from off-shore stations with low bottom water temperatures due to stratification of the water column. Considerably lower CYP1A levels were observed at stations with higher water temperatures. Multiple regression analyses with PCB concentrations in fish and water temperature as independent variables influencing EROD activity or CYP1A protein levels demonstrated significant correlations with both parameters. The water temperature was inversely related to CYP1A levels, whereas PCB concentrations showed a positive relation with CYP1A levels. The effect of water temperature dominated over the effect of PCB contamination. The relationship between water temperature and CYP1A levels was also examined in a laboratory study, where dab were acclimated to 8, 12 and 16 degrees C for 4 weeks. A three-fold increase in EROD activity in the group acclimated to 8 degrees C compared to the group acclimated to 16 degrees C was observed, whereas no differences were observed for CYP1A protein levels. Multiple regression analyses with PCB concentrations and condition factor of fish as independent variables influencing CYP1A levels also demonstrated significant correlations in the field. Thus, differences in water temperature and nutritional status of dab between sampling locations obscured the effects of contamination with polyhalogenated aromatic compounds on CYP1A levels, The results indicate that these factors have to be taken into account when employing CYP1A responses in dab as biomarkers for environmental contamination in the North Sea.
Abstract: The flatfish dab (Limanda limanda) serves as an indicator species in pollution monitoring programmes in the North Sea. The present study investigated the induction response of the monooxygenase system and haematological changes in female dab after multiple administrations of a technical mixture of polychlorinated biphenyls (PCBs). Mature female dab were dosed with 1 mg of the PCB mixture Clophen A40 (Clo A40) in sunflower oil every 6 weeks, with a maximum of three doses per fish. In all PCB-administered groups, levels of cytochrome P4501A (CYP1A) protein, measured with a semi-quantitative ELISA method, and 7-ethoxyresorufin O-deethylase (EROD) activity showed a three- to ninefold induction 14 d after dosing compared with control groups, smaller but also significant increases were observed in total cytochrome P450 (Sigma P450) levels. Although the PCB concentrations and the corresponding toxic equivalent (TEQ) value in muscle tissue still increased after administration of the second and third dose of Clo A40, maximum responses of the EROD activity were already reached after the first dose at a TEQ value for chlorinated biphenyls (CB-TEQ) of 2 ng/g lipid. The PCB patterns of liver and muscle tissue of female dab from the central North Sea were found to be virtually identical. Hence, the use of PCB concentrations in muscle as a qualitative model for changes in the liver appears legitimate. Haemoglobin concentrations were elevated after the third dose of Clo A40, whereas haematocrit values and the mean corpuscular haemoglobin concentration (MCHC) between treated and control groups did not differ.
Abstract: Variations in the expression of cytochrome P450 (CYP) isozymes in cells may affect the response of and consequent toxic effects in those cells during xenobiotic exposure. The expression of cytochrome P4501A (CYP1A), a major inducible CYP in teleosts, was examined immunohistochemically in multiple toxicopathic hepatic lesion types, including neoplasms and several types of preneoplastic foci of cellular alteration (FCA), in English sole (Pleuronectes vetulus) captured from the Duwamish Waterway, a highly contaminated estuary of Puget Sound, WA. Formalin or Bouin's-fixed, paraffin-embedded tissue sections were stained with polyclonal rabbit anticod CYP1A IgG by the avidin-biotin peroxidase complex method; this antibody binds to both Atlantic cod (Gadus morhua) and English sole CYP1A in Western blotting and ELISA, and immunohistochemically-localizes CYP1A in hepatic and other tissues of cod exposed to beta-naphthoflavone. CYP1A-associated staining intensity was lower in hepatocellular and cholangiocellular neoplasms, areas of biliary hyperplasia, and normal intrahepatic exocrine pancreas and bile ducts, as compared to histologically normal hepatocellular parenchyma. FCA of basophilic, eosinophilic and clear cell types showed variable staining intensities in comparison to background parenchymal staining, but generally showed reduced CYP1A-associated staining. Staining intensity for a single hemangiopericytic sarcoma showed highly reduced staining for CYP1A. These results, in fish naturally exposed to environmental toxicants, suggest a parallel response between teleosts and mammals with respect to CYP1A expression and resistance to cytotoxicity in cells composing several types of hepatic neoplasms and foci of cellular alteration.
Abstract: The sensitivity of cytochrome P450 1A (CYPIA) induction as a biomarker for environmental contaminants in the flatfish dab (Limanda limanda) was evaluated by studying fish of different age and sex from the southern North Sea. Mature and juvenile dab from both sexes were collected in autumn and winter during two surveys from four different stations with varying levels of polychlorinated biphenyls (PCBs) contamination in the southern North Sea. All groups of fish exhibited highest muscle PCB concentrations near the Dutch coast. CB153 was always the dominant congener. Since the concentrations of the other congeners measured covaried to a large degree with CB153, this congener appears to be a good marker for general differences in PCB concentrations. In summer, bottom water temperature differences of up to 10 degrees C can occur between stratified and vertically mixed areas. This was previously shown to have a strong effect on CYPIA expression. In autumn and winter, stratification has disappeared, resulting in almost equal water temperatures between stations of the same survey. CYPIA levels were measured as 7-ethoxyresorufin of O-deethylase (EROD) activity and immunoquantitated CYPIA protein concentrations. Highest levels were also found close to the Dutch coast for mature fish from both sexes in October and for juvenile female and mature male fish in February during the spawning season. During this season, gravid female fish had significantly lowered contents of CYPIA protein and EROD activity compared to mature males and juveniles of both sexes. The sensitivity of CYPIA induction in dab as a biomarker for halogenated aromatic hydrocarbons is highest in mature males when stratification during autumn is lacking.
Abstract: Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by beta-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.
Abstract: The cellular localization of inducible CYP1A and constitutive CYP3A-like forms in different organ systems of Atlantic cod (Gadus morhua) was determined in control fish and fish exposed to beta-naphthoflavone (BNF). Paraffin-embedded sections were stained with polyclonal rabbit anti-cod P450 1A IgG or rabbit anti-rainbow trout P450con (a putative CYP3A form which cross-reacts with purified cod P450b) serum by the avidin-biotin peroxidase complex method. Following BNF-exposure of cod, CYP1A induction was immunohistochemically demonstrated in hepatocytes and endothelial cells of liver, the endocardium and vascular endothelium in the atrium and ventricle, and epithelial cells of the proximal tubular segment, endothelial cells, and interrenal cells in kidney. The vascular endothelium was the main site of induction of CYP1A in gills, spleen, gut, pyloric caecae, and gonads. The CYP3A-like isozyme P450b was mainly localized to hepatocytes, renal tubular epithelium, and epithelial cells of the mucosa in the intestine. Furthermore, the distribution of P450b was not affected by BNF exposure. The localization of P450b bears interesting similarities to the localization of CYP3A in mammals supporting the CYP3A-like identity of cod P450b. Simultaneous localization of inducible CYP1A and a constitutively expressed CYP isoenzyme has not previously been reported in fish. This is also the first presentation of cellular distribution of a CYP3-like isozyme in fish. Staining of CYP1A in endothelial cells supports previous observations that endothelium is a major site of CYP1A induction following xenobiotic exposure in fish. The observation of CYP1A induction in interrenal cells has important implications for possible endocrine effects of xenobiotic exposure.
Abstract: Juvenile Atlantic cod were placed in net cages on the bottom at 20-30 m depth at four sites from the inner end of Sorfjorden (Hardanger, Western Norway), to the northern mouth of the fjord in October 1990. After 4 weeks the fish were killed, and liver samples were analyzed for aromatic and chlorinated hydrocarbons (PAHs and PCBs). Liver and gill were also analyzed for cytochrome P450 1A induction, a biomarker for exposure and effects of these contaminants, using catalytic measurements (7-ethoxyresorufin-O-deethylase, EROD) and immunodetection (P450 1A1-ELISA). The caging resulted in the accumulation of a number of PAHs, but very little PCBs, in the liver of the cod, with an inward gradient in the fjord. The gradient, although not dramatic in absolute terms, was parallelled by elevated levels of P450 1A1 when measured by EROD and, partly, by ELISA. The caging strategy seems to be a promising way to approach ecotoxicologically relevant problems, such as bioavailability of contaminants, biomarker responses in the field, and dose-response relationships, also under mixed contaminant situations.
Abstract: Groups of Atlantic salmon (Salmo salar) parr were fed a basal diet containing 27 mg Fe-kg feed-1 supplemented with different iron levels (0, 80, and 160 mg.kg-1, respectively) for 8 wk. After this period, a subgroup of each iron regime was treated intraperitoneally with beta-naphthoflavone (BNF), a potent inducer of cytochrome P450 1A1 in fish. Differences between the iron regimes were observed in hepatic iron content, which was higher in the high-iron group, and in P450 1A1 levels and monooxygenase activity, measured as 7-ethoxyresorufin 0-deethylase (EROD), which were lower in the high-iron group. Differences were also observed in the induction response of P450 1A1 and EROD to BNF. Levels of a constitutive isozyme denoted P450con, belonging to the P450 3A subfamily, did not respond to the dietary iron in the same manner, demonstrating selective regulatory effects of iron on different P450 isozymes.
Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was administered intragastrically twice with 4 days interval, to juvenile cod and rainbow trout, total dose 8 mug/kg body weight. Fish were killed after 9 and 17 days and the effects on hepatic xenobiotic metabolizing enzymes were determined by examining aldrin epoxidase (AE), glutathione-S-transferase (GST) against 1-chloro-2,4-dinitrobenzene (CDNB) and the cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD) activities, and by immunoquantitating cytochrome P4501A1 using an indirect enzyme-linked immunosorbent assay (ELISA). AE and GST activities were not induced. However, 2,3,7,8-TCDD significantly induced EROD activities in rainbow trout and cod to 1450% and 415%, respectively, of the corresponding controls 9 days after the first treatment. The increase in EROD activity was supported by induction of the main catalyst P4501A1 as revealed by ELISA analyses. The distribution pattern of C-14-labelled 2,3,7,8-TCDD was studied by whole-body autoradiography. The liver concentration of radiolabelled compound in cod exceeded that of the rainbow trout. However, the degree of hepatic enzyme induction did not correspond to the concentration of radiolabelled 2,3,7,8-TCDD in the liver. Despite of the substantially higher level of 2,3,7,8-TCDD in the liver of cod, the EROD induction was lower in this species compared to rainbow trout.
Abstract: Samples from nine fish species caught at different sites in the Gulf during leg IV of the Mt Mitchell cruise in the spring of 1992 were analysed for levels of cytochrome P450 1A enzymes. P450 monooxygenases catalyse the first step in the biotransformation of lipophilic xenobiotics. The P450 1A enzyme subfamily is induced by planar, organic compounds, and is used as a biomarker for exposure to oil compounds and PAH-type xenobiotics. The P450 1A analyses of the samples included both a catalytic method (EROD activity measurements) and immunochemical methods (Western blotting and an indirect ELISA). Polyclonal antibodies against P450 1A1 from cod (Gadus morhua) were used in the immunochemical assays. The Western blotting results demonstrated strong cross-reaction between polyclonal anti-cod P450 1A1 IgG and a single protein band with an M(r) of approximately 58 kDa in eight species examined. Elevated EROD levels were found in fish caught at the northern stations compared to individuals of the same species caught further south. The results from the P450 1A1 ELISA analyses supported these findings, although with some differences between species. Overall, conclusive statements about the P450 1A status in fish from the survey area are difficult to make from this investigation. This is mainly due to diverse species composition and the small size of the catches at the different fishing locations.
Abstract: Mirror carp were exposed to Rotterdam Harbor sediment, highly contaminated with polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorobiphenyls (PCBs) (0.5 mug 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] equivalents per kilogram dry weight). In two additional separate experiments rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) received a single intraperitoneal injection of approximately 0.01, 0.03, 0.06, 0.3, 0.6, or 3.0 mug TCDD per kilogram body weight. In all three experiments induction of hepatic P450 IA was measured with immunochemical and enzymatic methods. The polyclonal antibodies anti-cod (Gadus morhua), anti-perch (Perca fluviatilis), and anti-rainbow trout P450 1A all cross-reacted with the P450 1A orthologue of the carp and rainbow trout. In most cases high correlations were found between 7-ethoxyresorufin O-deethylation (EROD) activity and cytochrome P450 1A protein contents, the latter measured by the enzyme-linked immunosorbent assay (ELISA) and protein blot methods. However, the correlations between EROD activity and P450 1A protein levels were higher within the separate sampling periods (i.e., 3, 6, and 12 weeks after dosage) than with the total data set, especially in the dose-effect study with the rainbow trout. This was probably caused by a difference in time-dependent relationships between P450 1A protein content and EROD enzyme activity: 12 weeks after dosage the P450 1A protein was still increased, although EROD activity had returned to background level. In addition, there were higher correlations of the EROD activity and P450 1A protein content with total P450 content in rainbow trout and carp treated with a single dose of TCDD, than with total P450 content in carp exposed to contaminated sediment. In our study, the ELISA method appeared to be more useful than the protein blot technique, because the ELISA is faster and has higher reproducibility. In addition, in all our experiments EROD activity showed a higher induction than the P450 IA protein, indicating a higher sensitivity of the EROD assay. Our results strongly indicated that determination of the P450 1A protein content and EROD activity provides complementary information. Thus we recommended the use of both the ELISA and the EROD activity assay in order to understand the nature of P450 1A induction,
Abstract: Cytochrome P450 monooxygenase activities, reflecting the expression of various subfamilies of P450, were measured in liver samples of harp and hooded seal. Differences in some of the investigated parameters were observed between the two species, between sexes, and between pups and adults. Treatment of single female pups from each species with phenobarbital (i.v., 45 mg/kg), resulted in increased levels of EROD and estradiol 2-hydroxylase activities in both species, whereas MCOD, ECOD and PROD activities were induced only in the harp seal sample. Antibodies against a dog P450 2B form (anti-dog PBD-2 IgG) gave a single band around 52 kD in both species, strongest in male pups. This band seemed elevated in the PB-treated harp seal pup. Based on the single treated pup of each species, the results suggest that seals respond to PB-type treatment with a weaker response, and with different enzyme patterns, than most terrestrial mammals. Antibodies against cod P450 1A1 cross-reacted with two bands in liver samples from adult seals of both species (about 54 and 52 kD), but this was strongest in the hooded seal. The intensity of the bands reflected the EROD activities in the samples, suggesting the application of immunodetection in screening marine mammals for effects of environmental contamination.
Abstract: 1. Larvae of yellow mealworm (Tenebrio molitor L.), European rhinoceros beetle (Oryctes nasicornis L.), garden chafer (Phyllopertha horticola L.) and summer chafer (Amphimallon solstitiale) were kept in semi-natural substrates which had been mixed with varying amounts of Thiodan 35 (endosulfan). 2. The microsomal fractions from midgut and/or gut-free soft tissues were assayed for NADPH cytochrome c reductase (NccR) and aldrin epoxidase (AE), and the corresponding postmicrosomal supernatants were assayed for glutathione S-transferase (GST) using 1 -chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as second substrates. 3. In gut-free soft tissues, activities of GST towards CDNB were 30-fold higher in garden chafer and summer chafer, and 5-fold higher in European rhinoceros beetle, compared to the corresponding value in yellow mealworm. 4. In midgut of European rhinoceros beetle, activities of NccR, AE and GST with DCNB as substrate were all 4-5 times higher than in gut-free soft tissues, while the GST activity with CDNB as substrate was similar. 5. Commercial endosulfan was an inducer of microsomal activities in gut-free soft tissues of European rhinoceros beetle and garden chafer, and in microsomes from gut-free soft tissues of yellow mealworm treated with technical endosulfan, elevated levels of a protein of 52 kDa was observed. This protein was suggested to be a cytochrome P450 isoenzyme.
Abstract: Aquatic toxicology has been defined as the qualitative and quantitative study of adverse or toxic effects of chemicals and other anthropogenic materials on aquatic organisms. The subject also includes the study of transport, distribution, transformation and ultimate fate of chemicals in the aquatic environment. Within this multidisciplinary field of science, studies of the biochemistry and function of biotransformation enzymes in aquatic organisms hold a central role. Metabolism or biotransformation through the phase I (cytochrome P-450 monooxygenase enzymes) and phase II (conjugating enzymes) pathway is a requisite for detoxification and excretion of lipophilic chemicals. In addition, such a transformation is also responsible for the activation of foreign chemicals to the intermediates that ultimately result in toxicity, carcinogenicity, and other adverse effects. The dual role of many of these enzyme systems, being involved in both xenobiotic and endogenous metabolism, furthermore makes interactions between foreign chemicals and physiological processes possible. Lastly, the response of some of these enzyme systems, in particular the cytochrome P-450 IAI subfamily, to organic xenobiotics such as oil hydrocarbons, PCBs, dioxins and dibenzofurans, makes analysis of enzyme levels by catalytic or immunochemical methods a potent way to monitor pollution effects at the molecular level. Several of these aspects will be discussed with special reference to fish.
Abstract: Polyclonal antibodies, anti-perch P4501A and anti-cod P4501A, both show cross-reactions with the P4501A orthologue in rainbow trout and carp. These fish were treated either with a single intraperitoneal dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or exposed to Rotterdam harbour sediment, heavily contaminated with polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs) and polychlorobiphenyls (PCBs). In the fish exposed to a single TCDD dose the total P450 content was well correlated with both 7-ethoxyresorufin 0-deethylase (EROD) and P4501A protein content. For the sediment-exposed fish, the correlation between EROD and P4501A protein was lower. High correlations were found between EROD activity and the cytochrome P4501A content, as measured with enzyme-linked immunosorbent assay (ELISA) in both rainbow trout and carp. The higher correlations for the individual sampling periods, i.e. 3, 6 and 12 weeks, indicate a difference in time course between the EROD activity and P4501A protein content. Based on these results it is concluded that EROD, ELISA and, even a nonspecific parameter such as total P450 content, give supplemental information about the induction process.
Abstract: We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAA's Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for 11 species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.
Abstract: Na+,K+-ATPase activity, ouabain binding, metallothionein and P450 1A1 were measured in gill tissue from the dab Limanda limanda caught from a contaminant gradient in the German Bight during the Bremerhaven Workshop. Metallothionein levels were highest at the most contaminated sites closest to the mouth of the Elbe and lowest at the outermost station on the Dogger Bank. Tissue levels of zinc and cadmium were also elevated at these sites but copper levels did not change along the gradient. P450 1A1 levels, measured with dn antibody probe, showed a complex pattern of induction which suggests an inhibitory effect at the most contaminated stations. Na+,K+-ATPase was similar in fish from all of the stations but measurement of ouabain binding show that levels of the enzyme are elevated at the innermost and outer stations. Calculation of activity per binding site indicates that the turnover rates of the enzyme were lower at the most contaminated stations. The results show that the role of adaptive responses in the face of inhibition by contaminants is an important consideration in identifying biological effects. The data were analysed with respect to the contaminant burdens in the total and fine particulate fraction (< 63 mum) of the sediment and in the liver of dab caught at each station. The utility of biological effects measurement in the gill as an indicator of environmental contamination is discussed.
Abstract: A group of Atlantic salmon (Salmo salar) was followed through their first year of maturation and spawning. At monthly intervals, starting with juvenile fish in December, 5-7 fish of each sex were killed, and liver and plasma were sampled. The last sampling point was of spawning fish in November a year later. Variables in the cytochrome P450 (P450) system were studied in hepatic microsomes, and estradiol 17beta was measured in the plasma of females to assess the maturational status. The P450 1A1-mediated 7-ethoxyresorufin O-deethylase (EROD) started at high levels in winter, but decreased to non-detectable activities in pre-spawning females. Decreases, but not to the same extent, were also observed during this period in total cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and in the content of two immunochemically determined P450 isozymes. At the same time, LSI levels increased in maturing females (starting in July), and GSI levels increased in both sexes (starting in May). Sex specific differences were observed in pre-spawning fish in September and October, with levels of total P450, b5, NADPH-cytochrome P450 reductase, EROD and P450 isozymes significantly lower in females. At the same time, plasma estradiol-17beta levels reached peak values in females. The results point to the important role of sex steroids such as estradiol-17beta as major factors in the regulation of final sexual maturation. However, this study also indicates that there may be estradiol-17beta independent events of equal importance in the early stages of gonadal maturation that may involve the P450 system. The changes observed in the P450 system (as a major drug and steroid metabolizing system) of Atlantic salmon during sexual maturation may be of importance both in the endogenous transduction of hormonal signals, and as a pharmacological basis for designing therapeutic treatment of diseases in the aquaculture industry.
Abstract: We have developed polyclonal and, recently, monoclonal antibodies against the aromatic hydrocarbon-inducible P450 1A1 form purified from Atlantic cod (Gadus morhua). A simple, indirect enzyme-linked immunosorbent assay (ELISA) based on these antibodies has been developed in our laboratory and tested in numerous experiments with both field-collected and laboratory-exposed fish of different species. The exposure situations studied to date include complex mixtures such as polychlorinated biphenyls (PCBs), dioxins and water-soluble oil fractions, as well as defined compounds such as CB congeners, TCDD, and pesticides. Factors affecting the induction response, including sexual maturation and dietary factors, have also been investigated. The ELISA technique generally shows good correlation with contaminant exposure and catalytic measurements, but has also given new and important information in certain cases where catalytic measurements failed to reveal effects.
Abstract: Primary cultures of rainbow trout hepatocytes were used to study the expression of CYP1A1 mRNA, its protein product (P4501A1), and catalytic activities (7-ethoxy-resorufin-O-deethylase, EROD) during a 96-h period after exposure of cells to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes were isolated from immature rainbow trout by a two-step perfusion method and incubated at 10 degrees C. Cells were exposed to the inducers for 48 h after 24 h of preculturing. The EROD activity of BNF-treated hepatocytes was higher than that of the control hepatocytes 12 h after addition of the inducer. Activities peaked after 48 h and had declined to control levels after 72 h. EROD activity in TCDD-treated cells was significantly elevated after 12 h, but in contrast to BNF-exposure, activities continued to increase during the experimental period. The content of P4501A1 protein, measured with an indirect ELISA technique (using anti-codP4501A1 IgG), increased linearly during the first 12 h and remained constant thereafter. In TCDD-exposed cells the immunochemically determined P4501A1 levels changed in parallel with EROD activity. CYP1A1 mRNA levels, determined by Northern blot and slot-blot analyses (using the trout P4501A1 cDNA pSg15 probe), were hardly detectable in control cells. In BNF- and TCDD-treated cells a 2.8-kb mRNA band was detected by the probe 6 h before the protein and catalytic activities became detectable. The elevated levels of CYP1A1 mRNA were sustained more effectively by TCDD than by BNF. In addition, a second mRNA band at 1.9 kb was seen. The results suggest that transcriptional activation is probably the prime factor even though post-transcriptional events may be involved in the regulation of P4501A1 induction by PAHs in rainbow trout hepatocytes.
Abstract: Liver and kidney tissue of dab Limanda limanda were analyzed during the Bremerhaven Workshop by immunochemical and catalytic assays for cytochrome P450 1A1 induction. Antibodies to cod P450 1A1 cross-reacted with a protein of M(r) = 58 000 daltons in the dab liver samples, suggested to be the dab P450 1A1 protein. By employing a recently developed indirect ELISA with these antibodies, we observed elevated levels of P450 1A1 protein in the innermost stations of the German Bight transect in both liver and kidney (head and trunk kidney) samples. Ethoxyresorufin O-deethylase (EROD) activities in trunk kidney were also elevated in the innermost stations. Along the drilling station gradient no induction could be detected. Immuno-detection of the cytochrome P450 1A1 induction response should provide a convenient indicator for biological effects of several classes of organic contaminants.
Abstract: Female plaice (Pleuronectes platessa) were orally dosed with a gelatin capsule containing a solution of the technical PCB mixture Clophen A40 in sunflower oil. They were compared to plaice injected with a gelatin capsule containing only the sunflower oil at 10 and 16 days after injection. Even at 16 days after injection, the increase in concentrations of individual CB congeners in muscle was proportional to their contribution in Clophen A40. Biochemical effects are related to increases in concentrations of well-separable CB congeners in muscle, which increased by factors between 1.6 and 64 compared to the reference group of fish. Of both sampling points, total cytochrome P-450 levels were higher than the control groups, but surprisingly ethoxyresorufin-O-deethylase (EROD) activities did not differ between the groups. Enzyme-linked immunosorbent assay (ELISA) showed increased concentrations of the inducible cytochrome P-450IA1 in PCB-treated fish. The apparent lack of EROD induction may be due to competitive substrate inhibition by certain CB congeners present in the sample. The activity of glutathione-S-transferase (GST) (with CDNB as model substrate) was significantly elevated by PCB-treatment at day 16, but not at day 10. A longer time interval between injection with PCBs and induction of GST compared to P-450 monooxygenase activities has been reported earlier and may indicate that in fish both groups of enzymes are regulated individually and not as an [Arylhydrocarbon] gene battery as appears to be the case in mammals. Haemoglobin concentrations and MCHC were decreased in fish treated with Clophen A40. Haematocrit values did not differ between groups of fish.
Abstract: The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The K(m) for 7-ethoxyresorufin was 0.4-mu-M, and V(max) 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20-30 pmol/min/mg protein). Treatment with the P450 1A1 inducer beta-naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30-40 fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (M(r) = 58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at -80-degrees-C in 20% glycerol without losing more than 20-40% of its catalytic activity.
Abstract: Using a flow-through biotest exposure system, cod eggs, larvae and juveniles were exposed to the water-soluble fraction (WSF) of North Sea (Statfjord B) crude oil at concentrations ranging from 40-300-mu-g-1-1 (ppb) for 1-6 weeks. The WSF was analysed by GC/MS, and was shown to be dominated by low-aromatic compounds such as benzenes, toluene and xylenes (80-90%). Cytochrome P450IA1 levels were measured by immunochemical techniques in 10 000 x g supernatants of whole larvae or juvenile gill homogenates, and in juvenile liver microsomal fractions. With rabbit anti-cod P450IA1 IgG as primary antibody in an indirect ELISA, a clear induction response was observed in the exposed groups of both larvae and juveniles. The response was dose-dependent, and recovery in clean sea-water resulted in normalization of the induced P450IA1 levels. In larvae exposed during the egg stage, the induction process seemed restricted until the time of hatching. In juvenile cod, the response was observed in the gill in addition to the liver. The study demonstrates the usefulness of the cytochrome P450IA1 ELISA in detecting sublethal biological effects of pollutants in small sample sizes where enzyme activity is difficult to measure.
Abstract: An indirect enzyme-linked immunosorbent assay (ELISA) for detection of the aromatic and chlorinated hydrocarbon-inducible cytochrome P450IA1 isozyme in fish samples is presented. The method can be utilized in monitoring programs for screening large series of samples simultaneously, and may be especially suited to show induction responses in samples where catalytic measurement is difficult or impossible due to (a) small sample or organ size, (b) loss of activities in bad storage conditions, or (c) inhibiting compounds present in the sample.
Abstract: A single i.v. injection (75-mu-g/kg body weight) of the insecticide endosulfan was administered to gonadally immature rainbow trout (Oncorhynchus mykiss). Experiments were done with both technical and analytical grade endosulfan. In liver microsomes prepared from fish killed 24 h after administration, the cytochrome P-450-dependent monooxygenase activities of 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), and aldrin epoxidase (AE) were measured. In addition, NADPH-cytochrome c reductase (NCCR) was analyzed, and the content of a specific cytochrome P-450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anti-cod P-4501Al lgG. Technical grade endosulfan significantly induced EROD and AE to 160 and 144%, respectively, of the corresponding control values, while analytical grade endosulfan significantly increased only AE activity to 147% and tended to induce EROD to 162%. AHH and NCCR were not affected. The antibodies to cod P-4501A1 recognized a single protein band (M(r) = 58 Da) in the rainbow trout liver microsomes. The ELISA absorbances of this protein in the technical and analytical grade endosulfan treated fish were 151 and 138%, respectively, of the corresponding values in the controls. These results were supported by corresponding results from a 14 days study, where rainbow trout were exposed to technical grade endosulfan (8.3-mu-g/l water). In this study glutathione S-transferase (GST) activity towards 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also included, however, no effect on GST was observed.
Abstract: Antibodies prepared against the major beta-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNF-treated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000-59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin O-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr = 54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50-200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P450IA gene family).
Abstract: The levels of several environmental contaminants, including selected polyaromatic hydrocarbons (PAH), organochlorines (DDT/DDE, hexachlorobenzene), 15 polychlorinated biphenyl (PCB) congeners, and polychlorinated dibenzofurans and dibenzo-p-dioxins, PCDF/PCDD), and heavy metals (Cd, Hg, Pb, and As) were analyzed in muscle and liver of three different flatfish species (dab, Limanda limanda; flounder, Platichthys flesus; plaice, Pleuronectes platessa) caught by gill netting at different sites in the Hvaler Archipelago. Indices of biochemical effects in liver S9-fractions were studied by measuring cytochrome P450-dependent monooxygenase and UDP-glucuronyl transferase activities, and by immunoquantitating cytochrome P450 1A1 using an indirect enzyme-linked immunosorbent assay (ELISA). Only low levels of PCDD/PCDF, Cd, and Pb were observed, whereas PCB levels were significantly elevated in fish from the inner sites of the Archipelago compared to a reference site. The contaminant gradient toward the Glomma estuary was correlated with increased cytochrome P450 1A1 activity, measured as 7-ethoxyresorufin O-deethylase (EROD), and with immunoquantitated P450 1A1. In contrast, fish from the site at Idefjorden, although containing elevated contaminant levels, did not show elevated EROD activity, but apparently elevated P450 1A1 protein. These findings may reflect different pollution histories of the sites, and indicate the applicability of biochemical effect indices (i.e., EROD and P450 1A1 immunoquantitation) to monitoring studies. The integrated chemical-biochemical approach employed in this study can obviously be expanded to give fruitful information about cause-effect relationships in other contaminant situations.
Abstract: 1. An enzyme-linked immunosorbent assay (ELISA) based on polyclonal rabbit anti-cod cytochrome P-450 IA1 IgG has been developed in our laboratory. 2. The antibodies employed in the ELISA demonstrate good cross-reactivity with the homologous protein in a number of other fish species, giving cross-reacting protein bands o 54-59 kDa in liver microsomes, as determined by Western blotting. 3. The ELISA technique has been used in numerous experiments with both field collected and laboratory exposed fish of different species, showing good correlation with contaminant exposure. 4. In some instances of PCB exposure where the classical P-450 IA1 monooxygenase assay 7-ethoxyresorufin O-deethylase (EROD) failed to reveal any induction, the ELISA technique demonstrated increased levels of P-450 IA1 protein, indicating inhibiting effects of the PCBs on EROD measurement. 5. In tissues like gill filaments, and in whole cod larvae, where EROD activity is barely detectable, if at all, the ELISA technique showed induction after exposure to a water soluble fraction (WSF) of North Sea crude oil. 6. The results reviewed indicate the usefulness of the ELISA technique to allow rapid screening of a large number of samples, and especially when catalytic measurement is difficult or impossible due to (a) small sample or tissue size, (b) loss of activities in bad storage conditions, or (c) presence of compounds inhibiting activity.
Abstract: 1. A procedure was developed for isolating and purifying cytochrome P-450 from hepatic microsomes of BNF-treated perch, using modified versions of the methods of Williams and Buhler (1982. Biochim. biophys. Acta 717, 398-404) and Goksøyr (1985. Biochim. biophys. Acta 850, 409-417). 2. Following chromatography on phenyl-Sepharose CL 4B and DEAE-Sepharose CL-6B, the major peaks, fractions b and c, were resolved into five fractions, possibly representing different isoenzymes, by a FPLC with a strong anion exchange column (Mono Q). 3. These fractions have been characterized on the basis of their spectral, electrophoretic and immunological properties. 4. The purified form of cytochrome P-450 in fraction V from perch liver showed a number of similarities to cytochrome P-450c, the major BNF-inducible cytochrome P-450 in cod liver. 5. Therefore we suggest that this purified form of cytochrome P-450 is a BNF-induced form in perch and that it is closely related to the gene subfamily cytochrome P-450 IA1.
Abstract: The mono-ortho-substituted polychlorinated PCB congener 2,3',4,4',5'-pentachlorobiphenyl (PCB-118) was administered i.p. (30 mg/kg body weight) to gonadally immature rainbow trout (Oncorhynchus mykiss), of both sexes. In liver microsomes prepared from fish killed 4 days after administration, the cytochrome P450-dependent monooxygenase activities of 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), and aldrin epoxidase (AE) were measured. In addition, NADPH-cytochrome c reductase (NCCR) was analyzed, and the content of a specific cytochrome P450 isozyme was determined with Western blotting and an enzyme-linked immunosorbent assay (ELISA) using rabbit anticod P450IA1 IgG. The monooxygenase parameters EROD and AHH were significantly induced to 558 and 268%, respectively, of the corresponding control values, while NCCR and AE activities were not affected. The antibodies to cod P450IA1 recognized a single protein band (Mr = 58,000 D) in the rainbow trout liver microsomes. The ELISA absorbance of this protein in the PCB-118 treated fish was 401% of the corresponding value in the controls. These results demonstrate that PCB-118 is an effective inducer of the subfamily cytochrome P450IA1 in rainbow trout liver microsomes.
Abstract: Groups of Atlantic cod and rainbow trout were treated (ip) with beta-naphthoflavone (BNF), phenobarbital, or peanut oil (controls), and properties of the hepatic xenobiotic and steroid metabolizing enzyme systems were evaluated. In both species, BNF treatment resulted in significant induction of microsomal 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase, biphenyl 4-hydroxylase, and phenanthrene oxidation, especially at the 1,2-position. Immunochemical studies with rabbit IgG prepared against the major BNF-inducible cytochrome P-450 in cod, P-450c, revealed increased amounts of immunoreactive protein in liver slices from both species after BNF treatment. The molecular weight of the induced protein was approximately 58,000 Da, as shown by Western blotting. When titrating biphenyl 4-hydroxylation, however, the antibodies distinguished between the two species, inhibiting the activity of BNF-induced cod 90% and that of rainbow trout 40% at 10 mg IgG/nmol P-450. Furthermore, cytochrome b5 content and UDP-glucuronyltransferase activity were significantly induced only in rainbow trout, whereas the specific content of cytochrome P-450 was significantly increased only in cod. Differences between the two species were observed in the levels of constitutive activities, the amount of induction, and in the regioselectivity of phenanthrene oxidation and androstenedione metabolism. Treatment with phenobarbital showed no effect on any of the parameters investigated in either species. The results show that although there are many common features of the hepatic xenobiotic and steroid biotransformation systems of the two teleosts, certain species characteristics exist in constitutive properties and induction responses.
Abstract: The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (greater than 90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography-mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16 alpha-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with beta-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55-60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.
Abstract: Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with beta-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with beta-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (Mr 55000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identity with cod cytochrome P-450b (Mr 54000), possibly as a result of contaminating cytochrome P-450c in this fraction.
