Cytochromes P-450 in cod (Gadus morhua): Effects of induces on regiospecificity of phenanthrene metabolism and immunochemical properties

The metabolism of some xenobiotics can lead to the formation of reactive intermediates with mutagenic/carcinogenic properties. With the carcinogenic PAH these have been identified as bay-region diol-epoxides.¹ Phenanthrene, a non-carcinogenic, bay-region containing model PAH, is metabolised in vivo by bony fish at the proximate bay-region position, whereas mammals and other marine organisms mainly form the K-region metabolite 9,10-dihydro-9,10-dihydroxy-phenanthrene.² We wanted to investigate this difference more closely by studying the regiospecificity of phenanthrene metabolism in vitro both with microsomes from differently pretreated cod and with isolated cytochrome P-450 isozymes from BNF-induced cod.³ Secondly, by preparing antibodies to the major isozyme isolated (called cod P-450c), we investigated the immunochemical properties of the variously treated cod liver microsomes.     

Characterization and induction of cytochrome P-450 in the rainbow trout kidney

Cytochrome P-450 monooxygenases catalyze the biotransformation of a great variety of foreign, as well as endogenous, lipid-soluble compounds to more water-soluble products. As in mammals, highest concentration of cytochrome P-450 in fish is found in the liver. However, previous studies have indicated that fish kidney contains relatively high cytochrome P-450-mediated activities.^1,2 We have therefore prepared and characterized subcellular fractions from the kidney of rainbow trout suitable for studies on cytochrome P-450-dependent reactions. Furthermore, as in the liver, several cytochrome P-450-mediated reactions in the kidney were induced following treatment of the fish with β-naphthoflavone.     

Cytochrome P-450 isozymes in salmonids determined with antibodies to purified forms of P-450 from rainbow trout

Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM_4b)^* appears to be a P-448 type of cytochrome. A minor form (LM_4a), having properties very similar to LM_4b, was also obtained. In addition, a P-450 form (LM₂) was isolated, with properties quite different from LM_4a or LM_4b, including a high rate of aflatoxin B₁ (AFB₁) metabolism (Williams & Buhler, 1983c). Antibodies to all three forms were obtained from rabbits. The IgGs prepared against LM_4a and LM_4b both cross-reacted (forming lines of identity) equally well with both antigens on Ouchterlony plates. Rat P-448 cross-reacted (without lines of identity) with both LM_4a- and LM_4b-IgG. LM_4b-IgG was much more effective than LM_4a-IgG for inhibition of LM_4a or LM_4b reconstituted benzo[a]pyrene (BP) hydroxylase, suggesting that these two antibodies recognize different antigenic sites. The LM₂-IgG did not cross-react with any of the other rat or trout cytochromes P-450 examined. Levels of LM₂ and LM_4b in microsomes from untreated, polychlorinated biphenyl (PCB), phenobarbital (PB) or BNF-treated trout were estimated with an immunological technique involving electrophoresis on SDS-PAGE, followed by transfer to nitrocellulose and staining with either LM ₂ - or LM_4b -IgG. The ratio of in microsomes from PCB- or BNF-treated rainbow trout was much higher than 1, whereas the reverse was true with microsomes from untreated rainbow trout. These results are consistent with previous observations (Vodicnik et al., 1982) that pretreatment with BNF induced the synthesis of a P-448 type cyytochrome, presumably responsible for the great increase in the metabolism and activation of BP seen in these fish. Conversely, pretreatment with PB did not affect the levels of either LM₂ or LM_4b. This specific immunological technique should make it possible to assay the levels of these P-450 and P-448 isozymes in various strains of rainbow trout and other species of fish. In addition, the effect of age, sex, diet and exposure to P-450 and P-448 inducers could be examined and, perhaps, utilized to predict the relative risk of certain populations to pollutants activated by these different isozymes.     

Turnover of hepatic hemoproteins in rainbow trout

The hepatic mixed function oxidase system in the fish differs from that in mammals in its responses to the two classic mammalian inducers. The cytochrome P-448-type inducers (polycyclic aromatic hydrocarbons) stimulate monooxygenase activity, but phenobarbital, a P-450-type inducer, does not.¹ We have compared the effects of phenobarbital (PB) and polychlorinated biphenyls (PCB) on the turnover of hepatic microsomal hemoproteins in trout (PCB's are P-448- and P-450-type inducers in mammals, which in fish induce only cytochrome P-448). We show here that neither PCB nor PB treatment changed the turnover rate. However, both the rates of synthesis and degradation were much slower than in the rat.     

Immunohistochemical localization of cytochrome P-450E in liver, gill and heart of scup (Stenotomus chrysops) and rainbow trout (Salmo gairdneri)

Monoclonal antibody directed against a major β-naphthoflavone (BNF)-induced form of teleost cytochrome P-450, P-450E (equivalent to P-450c in rat) was used to immunolocalize this enzyme in liver, gill and heart of scup and trout. Liver sections from both species showed P-450E in the cytoplasm of hepatocytes. No regional differences were observed which might indicate zonation of cytochrome P-450E within subpopulations of hepatocytes. Scup exocrine pancreatic cells were only weakly positive. In the gill of both fish, cytochrome P-450E was restricted to the endothelium (pillar cells) of secondary lamellae, where fluorescence appeared as a chain in longitudinal sections through lamellae and as star-shaped clusters in en face views. Sections of ventricular wall in both species revealed P-450E was restricted to endothelium at margins of muscle bands limiting heart ventricular lumen. Localization in the specific cells of these and other organs may be fundamentally important in understanding the role of cytochrome P-450E.     

Catalytic properties of cytochrome P-450 in hepatopancreas of the spiny lobster, Panulirus argus

These studies were designed to determine the metabolic capability of the microsomal cytochrome(s) P-450 in spiny lobster hepatopancreas, and to determine how chemicals which selectively modify mammalian monooxygenase activity catalyzed by different cytochrome P-450 isozymes affect the spiny lobster cytochrome P-450. We used a washing procedure to concentrate the hepatopancreas microsomal cytochrome P-450 and remove the inhibitors of monooxygenase activity which are normally present in microsomes. The resulting reparation (MI fraction) was used to determine monooxygenase activity towards benzo[a]pyrene, benz-phetamine, 7-ethoxyresorufin in the presence of either cumene hydroperoxide or NADPH and vertebrate liver cytochrome P-450 reductase. Benzphetamine was the best substrate for the lobster cytochrome P-450, whereas 7-ethoxyresorufin was metabolized very slowly. Studies with chemical modifiers showed that the responses of the lobster cytochrome(s) P-450 were not similar to those of any of the well-characterized cytochrome P-450 isozymes purified from mammalian liver.     

The use of polyclonal antibodies raised against rat and trout cytochrome P450 CYP1A1 orthologues to monitor environmental induction in the channel catfish (Ictalurus punctatus)

Induction of hepatic cytochrome P450-dependent microsomal mono-oxygenase by xenobiotics is a well-established phenomenon in teleost fish. As in laboratory mammals, fish possess multiple forms of cytochrome P450 that display overlapping substrate specificity. One such isoform, CYP1A1, which has been cloned and sequenced from rainbow trout, has been shown to be orthologous to rat CYP1A1 and, as in mammals, is inducible up to several hundred-fold by planar aromatic hydrocarbons, PCBs and dioxins. It has been suggested that induction of CYP1A1 orthologues might provide a sensitive biomonitor for environmental pollution by mixtures of such compounds. In the current study, polyclonal antibodies directed against CYP1A1 purified from rat and trout liver were used to monitor induction of the CYP1A1 orthologue in hepatic microsomes from the fresh water species, the channel catfish (Ictalurus punctatus). Catfish from a local fish farm were induced in the laboratory by three daily injections of 50 mg/kg of the PCB mixture Aroclor 1254 and compared with fish taken from a site in central Arkansas—the Bayou Meto, known to be polluted with dioxin. Hepatic microsomal activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD) were measured and Western blot analysis carried out with the two antibodies. EROD was elevated in both the Aroclor-treated fish and in the Bayou Meto fish compared with untreated fish farm controls; smaller but significant increases were observed in PROD. Spearman's rank correlations of 0·74 and 0·89 were observed between EROD and immunoquantified cross-reactivity towards the rat CYP1A1 and trout CYP1A1 antibodies.     

Individual variation in patterns of benzo[a]pyrene metabolism in the marine fish scup (Stenotomus chrysops)

The metabolism at specific sites on carcinogenic hydrocarbons such as benzo[a]pyrene (BP) is responsible for activation to the ultimate mutagens and carcinogens, and patterns of metabolism can thus influence the biological effect of such compounds. Marine fish are known to efficiently metabolize BP at the benzo-ring, forming high percentages of the 9,10-dihydrodiol (DHD) and 7,8-DHD, the latter including the penultimate carcinogen.^1,2 Hydrocarbon-induced cytochrome P-450 in fish is responsible for initiating metabolism on the benzo-ring, but epoxide hydrolase (EH) activity is required for DHD formation.^3,4 Both factors could influence formation of the DHD leading to the ultimate carcinogenic diol-epoxide. In the present study, patterns of BP metabolism were evaluated in a number of individual scup Stenotomus chrysops sampled from local Woods Hole waters, and a correlation is described between variation in the DHD formation and EH activity in these feral fish.     

The role of cytochrome P-450 in the metabolism of pollutants

The monooxygenase cytochrome P-450 is a ubiquitous enzyme well known for its role in the detoxication of drugs and xenobiotics and the biosynthesis of the steroid hormones. The toxicity and carcinogenicity of many chemicals is frequently due to the formation of oxygenated reactive intermediates, which are formed by the same enzymes that effect the detoxication of these chemicals, namely the cytochromes P-450. In mammalia the cytochromes P-450 exist as a group of isoenzymes with overlapping substrate affinities, selectively and differentially induced by their specific substrates or inhibitors. As few enzyme assays have been shown to be specific for a particular cytochrome P-450 isoenzyme the characterisation of these isoenzymes has been dependent on their isolation and purification. It is therefore important to know whether one or more isoenzyme of cytochrome P-450 is specifically concerned with the activation of carcinogens and chemicals, rather than their detoxication, and to develop a specific enzymic assay for this activating enzyme.     

Quantification of cytochrome P4501A1 and catalytic activities in liver microsomes of isosafrol- and β-Naphthoflavone-treated rainbow trout (Oncorhynchus mykiss)

The effects of isosafrol (ISF) or β-naphthoflavone (βNF) treatments on cytochrome P450 (P450) levels in rainbow trout liver were investigated using immunochemical and catalytic techniques. The discrepancies in catalytic activities and ELISA quantification of rainbow trout P4501A1 protein levels between ISF- and βNF-treated fish indicate that important differences exist between the responses induced by βNF and ISF treatments in the rainbow trout liver.     

7-ethylresorufin O-deethylase activity and level of DNA-adducts in trout treated with benzo(a)pyrene

In fish, as well as in mammals, it is well known that the cytochrome P450-dependent oxidative metabolism of xenobiotics can generate DNA-reactive species. Moreover, this metabolism is known to be inducible by several compounds of environmental significance, such as polychlorobiphenyls, polycyclic aromatic hydrocarbons (PAHs) and dioxins. Consequently, we studied the relationship between the degree of induction of the cytochrome P4501A, expressed as that of 7-ethylresorufin O-deethylase (EROD) activity, and the level of DNA-adducts, using the post-labelling assay, in the liver of rainbow trout exposed to benzo(a)pyrene (a representative PAH). The results showed a significant 2- to 4-fold increase in EROD activity 2, 4 and 8 days after treatment, paralleled by an increase in DNA-adduct levels. This work further emphasizes the involvement of cytochrome P4501A in the metabolism of benzo(a)pyrene into genotoxic metabolites in rainbow trout.     

Microsomal cytochrome P-450 function in fish evaluated with polyclonal and monoclonal antibodies to cytochrome P-450E from scup (Stenotomus chrysops)

Monooxygenase reactions catalyzed by cytochromes P-450 are paramount in the oxidative metabolism of many xenobiotics, determining both the persistence and effects of numerous types of compounds. Immunological probes are proving useful in evaluating the functions of P-450 isozymes in microsomal preparations from many species. The regulation of specific isozymes by endogenous and exogenous factors can also be evaluated with such probes. Here we describe studies on the activities apparently catalyzed by induced P-450 in fish, evaluated with both polyclonal and monoclonal antibodies to cytochrome P-450E, the apparent major β-naphthoflavone(BNF) or methylcholanthrene(MC)-inducible isozyme purified from scup (S. chrysops) liver.     

Influence of biological and environmental factors on hepatic steroid and xenobiotic metabolism in fish: interaction with PCB and β-naphthoflavone

The degree of induction of hepatic cytochrome P-450 monooxygenases in fish by various chemicals may vary owing to many factors such as sex, sexual maturity, age, season and environmental temperature. In the present investigation the influences of gonadal steroids and water temperature on the inductive response were studied. The data indicate that gonadal steroids and water temperature modulate the response of the cytochrome P-450 system in rainbow trout to PCB and β-naphthoflavone.     

Growth hormone effect on xenobiotic-metabolizing activities of rainbow trout

In recent years several studies reported the regulation by growth hormone (GH) of the expression of a variety of P450 forms in mammals. However the effect of GH on the xenobiotic-metabolizing enzymes of fish are still unknown. The aim of this work was to investigate the effects of ovine GH—a growth hormone known to be efficient in trout—on the cytochrome P450 level and on aryl hydrocarbon hydroxylase, aminopyrine-N-demethylase, glucuronyl transferase and glutathione transferase activities in trout. The GH-implanted trout (n = 50) each received a single cholesterol pellet containing ovine GH and were compared to control animals (n = 50) receiving a single cholesterol pellet without GH. After 15 days fish were killed and the liver and gills were excised for the measurement of xenobiotic-metabolizing enzyme activities. GH treatment significantly decreased the level of hepatic cytochrome P450 and the activities of cytochrome P450 dependent monooxygenases. In contrast, no significant effect of the treatment was observed on the glutathione transferase and UDP-glucuronyl transferase activities. Moreover, GH treatment had no effect on branchial phase I and phase II enzyme activities. This study provides evidence that GH level significantly affects the expression of several members of the hepatic cytochrome P450 family in trout.     

Microsomal estrogen metabolism in channel catfish

Our goal was to study the involvement of cytochrome P450 genes in estrogen metabolism and the extent to which the potentially carcinogenic 4-hydroxyestradiol metabolite is formed by channel catfish (Ictalurus punctatus; CC). Estradiol metabolism and ethoxyresorufin-O-deethylase (EROD) activity were assessed in several tissues from fish collected from three variably contaminated sites in the Mississippi River Delta, from laboratory control fish, and from fish exposed to 20 mg/kg benzo(a)pyrene (BaP) i.p. for 4 days. Liver EROD activity was induced by BaP, but Delta fish EROD activities were not statistically higher than activities in control fish. Gill microsomal EROD activity was also induced by BaP, but activities were 8- to 77-fold lower than those from liver. The predominant estrogen metabolites formed by CC liver, gill and gonad microsomes were 2-hydroxyestradiol and estrone as detected by GC/MS. Liver and gill 2-hydroxyestradiol formation was induced in BaP-dosed fish. The trends in hydroxyestradiol formation in field collected fish were more variable. In all fish liver microsomes there was more 2- compared to 4-hydroxyestradiol formed. However, BaP-treatment increased the 4:2 hydroxyestradiol ratio from 0.04 in control fish to 0.2 in BaP-exposed fish, suggesting that BaP induces the formation of the potentially genotoxic estrogen metabolite. No detectable 4-hydroxyestradiol was produced by gill and gonad microsomes. These results will ultimately help in determining which fish P450 genes are susceptible to environmental contamination and may be involved in estrogen genotoxicity.     

Preparation and characterization of subcellular fractions suitable for studies of xenobiotic metabolism from leaf sheaths of a marine seagrass: Posidonia oceanica (Linnaeus) Delile

The capacity of the mammalian liver microsomal P-450-dependent systems to metabolize a wide variety of endogenous and exogenous compounds is thought to reflect the presence of multiple forms of P-450 haemoproteins with broad and overlapping substrate specificity. In plants, the functions and specificity of cytochrome P-450 systems are less well known.This study was designed to prepare and characterize subcellular fractions from fresh sheaths (basal parts of leaves) of a mediterranean seagrass Posidonia oceanica (Linnaeus) Delile, the aim being the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by centrifugation, as well as the recovery of different organelles, was determined using enzyme markers (cytochrome c oxidase, alkaline phosphatase, glucose-6-phosphatase) and morphological examination by transmission electron microscopy. Some assays of enzymes involved in xenobiotic metabolism (cytochrome c reductase, laurate hydroxylase, ethoxyresorufin O-deethylase and glutathione-S-transferase) were also performed on different fractions of the preparation. The subcellular distribution for drug metabolism and marker enzymes showed a loss of endoplasmic reticulum in the pellet obtained after the first centrifugation, but the microsomal fraction was relatively free of mitochondria and fragments of the plasma membrane.Some assays are still being performed to avoid the small loss of endoplasmic reticulum experienced with the first pellet. However, the microsomes prepared in this study from sheaths of Posidonia oceanica appear suitable for further investigation of xenobiotic metabolism.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Detection of mRNA sequences homologous to the human glutathione peroxidase and rat cytochrome P-450IVA1 genes in Mytilus edulis

In this study we have used cloned gene probes for human glutathione peroxidase (GPX), rat cytochrome P-450IVA1 and rat cytochrome P-450IIE1 to detect homologous sequences in RNA from the hepato-pancreas of Mytilus edulis. The presence of sequences hybridising to the GPX and P-450IVA1 probes, but not to the P-450IIE1 probe, confirms the ancient origin of the former genes and indicates that conserved-sequence DNA probes from higher organisms can be used to examine the structure and function of genes of environmental interest in marine organisms.     

Comparison of cytochrome P450 in three butterflyfish species: Ecological implications of elevated CYP2B and CYP3A in Chaetodon capistratus

The relationship between cytochrome P450 and feeding on terpenoid-rich gorgonian corals was investigated in a species of tropical butterflyfish and compared with two other sympatric congeners that do not feed on gorgonians. Fish were collected from non-polluted waters in Belize and the levels of two cytochrome P450 isozymes (CYP2B and CYP3A) were immunoquantitated in addition to quantification of total P450. Chaetodon capistratus regularly feeds on gorgonian corals and has higher levels of total hepatic microsomal cytochrome P450 than C. ocellatus or C. striatus. The content of hepatic P450 (0.588–0.794 nmol mg⁻¹) in C. capistratus is among the highest ever reported in teleosts from non-polluted waters and is significantly greater than detected in C. ocellatus or C. striatus. Chaetodon capistratus also had a larger hepatic index (g liver per g fish) and more microsomal protein (mg protein per g liver), factors that translate into 3.3- to 8-fold more total P450 per g fish. Sexual differences in total P450 were observed between male and female C. capistratus, but not among the other species. The contents of proteins detected by immunoassay with polyclonal anti-scup P450B (CYP2B) and anti-human P4503A (CYP3A) were 2- to 10-fold and 2- to 20-fold greater, respectively, in C. capistratus than in the congeneric species. CYP2 and CYP3 gene families in mammals are thought to have evolved partially in response to dietary allelochemicals. These results suggest that these P450 isozymes may also be important in marine teleosts that feed on terpenoid-rich prey.     

Unique monooxygenation pattern indicates novel flavin-containing monooxygenase in liver of rainbow trout

Vertebrate flavin-containing monooxygenases (FMOs) have only been isolated from mammalian organisms. However, many FMO substrates include pesticides which may adversely affect fish and other aquatic organisms residing in adjacent waterways to treated fields. Although FMO activities have been identified in fish, the exact isoform profile is uncertain. Utilizing prochiral methyl tolyl sulfides (MTS) and isoform-selective antibodies, an attempt was made to identify specific FMO isoforms which may be involved in sulfoxidation reactions which have been shown to bioactivate thioether pesticides, such as aldicarb. Rainbow trout hepatic microsomes treated with detergent to eliminate cytochrome P450 contributions catalyzed the formation of the sulfoxide of MTS in 75% S enantiomeric excess. These catalytic results contrast activities of the five other FMO isoforms including FMO1 (> 98% R) and FMO3 (50% R). Benzydamine N-oxidation was also observed as were methimazole, thiourea, and aldicarb sulfoxidation reactions. Antibodies to FMO1 recognized a single protein of 60 kDa in trout liver microsomes, while anti-FMO3 antibodies only slightly reacted with a 55-kDa microsomal protein. These results indicate a novel isoform profile in rainbow trout liver implicating either a mixture of competing FMO isoforms or a FMO1-like isoform displaying unique catalytic activity.