Anders Goksøyr

The cytochrome P-450 system in fish, aquatic toxicology and environmental monitoring

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.

Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption

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-17β 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.

Biomonitoring of aquatic pollution with feral eel (Anguilla anguilla). II. Biomarkers: pollution-induced biochemical responses.

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 b5 (Cyt b5), 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 sites, 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 (UDPGT) in eel may, however, be a useful biomarker, while glutathione S-transferase (GST) 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 b5, CYP1A, EROD, EROD/P450, UDPGT and DNA adducts.

Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed by caging to polluted sediments in Sørfjorden, Norway

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.

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish : an evaluation of compound specificity, inner filter effect and signal interpretation

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.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.

A semi-quantitative cytochrome P450IA1 ELISA: A simple method for studying the monooxygenase induction response in environmental monitoring and ecotoxicological testing of fish

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.

Immunochemical cross-reactivity ofβ-naphthoflavone-inducible cytochrome P450 (P450IA) in liver microsomes from different fish species and rat.

Antibodies prepared against the major β-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).

Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol

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.

Endocrine Disruptors in the Marine Environment: Mechanisms of Toxicity and their Influence on Reproductive Processes in Fish

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.

Purification of hepatic microsomal cytochromes P-450 from β-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

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.