Lars Förlin

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.

Ethinyloestradiol — an undesired fish contraceptive?

Environmental oestrogens are natural or synthetic substances present in the environment, which imitate the effects of endogenous oestrogen. Oestrogenic substances were identified by gas chromatogra ...

Regulation of the cytochrome P450 enzyme system in fish

Abstract The cytochrome P450 monooxygenase system metabolizes a large number of xenobiotic compounds including many environmental pollutants. This metabolism can lead to detoxification, or in some cases, activation to reactive intermediates with toxic and carcinogenic effects. Among animals living in the aquatic environment the cytochromes P450 have been studied best in fish. The catalytic properties and factors regulating their activity have been an important field for aquatic toxicology investigations. A major concern is the mechanisms associated with the induction of cytochrome P450 by organic hydrocarbons. Several variables such as development stage, sex, and ambient temperature may influence the P450 enzyme system and its inductive response. The objective of this review is to discuss recent progress in studies of regulation of the cytochrome P450 enzyme system in fish.

Biochemical indicators of pollution exposure in shorthorn sculpin (Myoxocephalus scorpius), caught in four harbours on the southwest coast of Iceland

Shorthorn sculpins (Myoxocephalus scorpius) were caught in four Icelandic harbours, differing in size, use and traffic. Biochemical responses in liver were measured and chemicals analysed in bile. Eyrarbakki harbour, which has not been in use for many years was chosen as a control site. Njar partial differentialvík harbour is a small fishing harbour and a marina, Sandger partial differentiali harbour is a large fishing harbour, and Reykjavík harbour is a large fishing harbour and an international transport harbour. Higher levels of DNA-adducts and cytochrome P4501A (CYP1A) in the fish from the harbours in Sandger partial differentiali, Njar partial differentialvík and Reykjavík, compared to Eyrarbakki harbour, indicate PAH exposure. This was confirmed by PAH analysis in bile. The higher activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in fish caught in Sandger partial differentiali, than in fish caught in the other harbours, indicate exposure of sculpin to prooxidative compounds in Sandger partial differentiali harbour. Shorthorn sculpin seems to be a convenient species for monitoring pollution in northern coastal areas.

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.

Assessment of the impact of organic pollutants on goby (Zosterisessor ophiocephalus) and mussel (Mytilus galloprovincialis) from the Venice Lagoon, Italy: Biochemical studies

The use of cytochrome P4501A (CYP1A) and other measurements as biomarkers was investigated in liver of goby (Z. ophiocephalus) and digestive gland of mussel (M. galloprovincialis) from several sites in the Venice lagoon as part of the UNESCO-MURST Venice Lagoon Ecosystem Project. Most tissue contaminants (PAHs, PCBs, DDTs) and biochemical measurements varied seasonally. Elevated 7-ethoxyresorufin O-deethylase activity and CYP1A-protein levels in goby were correlated with high tissue contaminant levels at the industrial Porto Marghera site. On occasions, activities of the antioxidant enzymes catalase and putative DT-diaphorase (resorufin reductase activity) in male but not female goby were also higher at Porto Marghera than other sites, but no differences were seen in Superoxide dismutase (SOD) activity. A range of measurements (SOD, catalase, NADPH-cytochrome c reductase and glutathione S-transferase activities, P450 and ‘418-peak’ contents) in mussel showed little difference between sites. However, indications were obtained of elevated levels of CYP1A1-like mRNA, CYP1A-like protein and metabolism of benzo[a]pyrene to free metabolites in mussels from the Venice lagoon compared to a site in the Adriatic Sea. The studies demonstrate the usefulness of CYP1A as a biomarker for organic pollution in fish and indicate some potential for its application in molluscs.

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).

Responses of hepatic biotransformation and antioxidant enzymes to CYP1A-inducers (3-methylcholanthrene, β-naphthoflavone) in sea bass (Dicentrarchus labrax), dab (Limanda limanda) and rainbow trout (Oncorhynchus mykiss)

Abstract The time-course responses of hepatic biotransformation and antioxidant enzymes to cytochrome P4501A-inducers in fish were examined using marine and freshwater species, viz. respectively, sea bass (Dicentrarchus labrax) and dab (Limanda limanda) exposed up to 5 to 11 days to 3-methylcholanthrene (3MC; intraperitoneal (i.p.) 20 mg kg−1), and rainbow trout (Oncorhynchus mykiss) exposed up to 15 days to β-naphthoflavone (BNF; i.p. 5 mg kg−1). The enzymes included those which are part of the mammalian [Ah]-gene locus, viz. cytochrome P4501A (CYP1A), DT-diaphorase (DTD; EC 1.6.99.2), aldehyde dehydrogenase (ADH; EC 1.2.1.3), glutathione S-transferase (GST; EC 2.5.1.18) and UDP-glucuronyltransferase (UDPGT). DTD was assayed as dicoumarol-inhibitable menadione reductase, dicoumarol-inhibitable dichlorophenolindophenol (DCPIP) reductase and resorufin reductase activities; and ADH as benzaldehyde dehydrogenase (BADH) and propionaldehyde dehydrogenase (PADH) activities. The responses varied considerably with enzyme, species and treatment. CYP1A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity was markedly inducible, more so in BNF-treated O. mykiss (maximal 284-fold increase after 2 days) than the 3MC-treated species (maximal 24–30-fold). Modest increases (up to 2-fold) and/or inhibition were seen in UDPGT and GST activities. DTD activities increased about 2-fold after 8 to 15 days in BNF-treated O. mykiss, but were not elevated in the other species. Changes in dicoumarol-inhibitable DCPIP and menadione reductase activities were more similar than resorufin reductase activity. No marked increases were evident in BADH, PADH, superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione peroxidase (GPX; EC 1.11.1.9) activities in any species. Overall, some co-induction of CYP1A and DTD, over different time-courses, was indicated for BNF-treated O. mykiss.

Species characteristics of the hepatic xenobiotic and steroid biotransformation systems of two teleost fish, Atlantic cod (Gadus morhua) and rainbow trout (Salmo gairdneri)

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.

Variations in hepatic metallothionen, zinc and copper levels during an annual reproductive cycle in rainbow trout,Salmo gairdneri

The normal variations in hepatic levels of metallothionein, zinc and copper were studied during an annual reproductive cycle in rainbow trout of both sexes. In female fish, the total hepatic zinc levels closely followed the estradiol-17β and the LSI levels. Hence, the zinc levels rose in September, peaked in December and dropped in January. No distinct peaks were, however, observed in the whole-liver copper content. The hepatic metallothionein levels in female fish began to increase at the onset of exogenous vitellogenesis. Maximum levels were reached after estradiol-17β and LSI levels had dropped in January. In male fish no distinct peaks in either zinc or copper levels were observed. The metallothionein levels increased somewhat during the time of spermatogenesis. It is suggested that metallothionein may regulate the hepatic zinc distribution during the annual reproductive cycle in female rainbow trout, thereby ensuring the organism of a control mechanism to keep the pool of available zinc at an appropriate level.