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.     

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.     

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.     

Response of the mixed-function oxidase system to toxicant dose, food and acclimation temperature in the bluegill sunfish

The degree of induction of hepatic mixed function oxidase (MFO) enzymes in fish is modulated by environmental conditions. This study was designed to investigate the influence of water temperature, presence or absence of food, and exposure to benzo(a]pyrene (BaP) on the inductive response of bluegill sunfish (Lepomis macrochirus). The results show an increase in 7-ethoxyresorufin O-deethylase (EROD) activity with an increase in acclimation temperature and dose. This activity appears to be associated with a very small fraction of the total cytochrome P-450 induced. Major changes were observed in the 53- and 57-kilodaltons (kDa) electrophoretic bands.     

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.     

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.     

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.     

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.     

Responses of the mixed function oxidase system of some bivalve and gastropod molluscs to exposure to polynuclear aromatic and other hydrocarbons

Bivalve and gastropod molluscs readily accumulate polynuclear aromatic (PNAH) and other hydrocarbons from the environment and are widely used in environmental monitoring programmes.¹ The response of the cytochrome P-450 monooxygenase or mixed function oxidase (MFO) system to organic xenobiotics is of interest from the comparative viewpoint and in biological effects monitoring.² We have studied the bivalves Mytilus edulis (mussel) and Cardium edule (cockle) and the gastropod Littorina littorea (periwinkle) exposed to hydrocarbons, experimentally or naturally in the field. The general trend of response in digestive gland microsomes was an increase in cytochrome P-450 content and NADPH-cytochrome c reductase (NADPH-CYTCRED) activity but no increase in benzo[a]pyrene hydrolyase (BPH) activity. Sex and seasonal interactions were evident. We conclude that aspects of the responses may be peculiar to the Mollusc and that NADPH-CTYCRED possibly offers potential for this phylogenetic group as a specific indicator of biological impact by organic pollution.     

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.     

Induction of cytochrome p-450 monooxygenase activities in plaice by ‘model’ inducers and drilling muds

The hepatic cytochrome P-450-dependent monooxygenase system of the marine fish plaice has been characterised by i.p. injection with chemical inducing agents, followed by measurement of a variety of monooxygenase activities. The results suggest that more than one isozyme is present in these fish. The response to oil-based drilling fluids, a major contaminant of the North Sea, has also been examined and indicates that more than one isozyme is responsive to these products.     

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.     

Metabolic activation of polycyclic aromatic hydrocarbons by fish liver cytochrome P-450

The incidence of hepatoma, epidermal and other forms of cancerous growths in fish is well documented (Halver, 1967; Matsushima & Sugimura, 1976; Dawe et al., 1964). In fish, as in mammals, cancer may be a result of metabolically activated carcinogens. In mammals the primary enzyme system involved in the activation of environmental carcinogens is the cytochrome P-450 linked mixed-function oxidase (MFO). The hepatic microsomes of the species offish studied contain variable levels of cytochrome P-450. Liver microsomes of the trout Salmo trutta lacustris are surprisingly active in metabolising benzo-[a]pyrene (BP) and other compounds preferentially metabolised by polycyclic aromatic hydrocarbon (PAH)-specific cytochrome P-450. This finding may be significant, because it is apparent that the detrimental effects of PAHs require metabolic activation.We have studied the production of reactive intermediates of BP by following their binding to DNA and by measuring the specific nucleotide-BP metabolite complexes by chromatography. Untreated S. trutta liver microsomes catalyse the production of reactive intermediates of BP which bind to nucleotides of DNA at a rate that is 3–4 times higher than that catalysed by control rat liver microsomes. The most prominent DNA-BP metabolite adducts produced by trout liver microsomes are the nucleoside adduct of BP-7, 8-diol-9,10-epoxide and 9-OH-BP-4,5-oxide and other phenol oxides. In contrast to the trout, another fish species, roach (Rutilus rutilus), has extremely low activity. Although the in vitro binding of BP to DNA is not strictly correlated to in vivo binding or biological effects, our results show that reactive intermediates are formed by trout liver and thus the prerequisite for chemical carcinogenesis or mutagenesis is ful filled. This is further supported by the fact that in Ames's test, trout liver preparations produce mutagenic products from promutagens.     

Perturbation of gene expression and steroidogenesis with in vitro exposure of fathead minnow ovaries to ketoconazole

Ketoconazole is a fungicidal drug that inhibits function of cytochrome P450s in the synthesis of steroids. To examine if inhibition of P450 function affects gene expression in a dynamic manner, we conducted in vitro exposures of ovary tissue from fathead minnows (Pimephales promelas) to 0.5 microM ketoconazole to investigate effects on steroid production and gene expression over time. Expression of four key steroidogenesis genes was examined at 1, 6, and 12h of exposure. 11 beta- and 20 beta-hydroxysteroid dehydrogenases were down regulated at 1h and Cytochrome P450 17 was down-regulated at 12h, consistent with the absence of steroid production. In contrast, cytochrome P450 19A was up-regulated at 6h, indicating feedback regulation. Microarray analysis of 12h exposures indicated enrichment of biological processes involved in neurotransmitter secretion, lymphocyte cell activation, sodium ion transport, and embryonic development. These data suggest that, with the exception of cytochrome P450 19A, these steroid metabolic genes are regulated in a feed forward manner and that the effects of ketoconazole may be broader than anticipated based on the mechanism of action alone.     

Toxicity of sediments from Bahía de Chetumal, México, as assessed by hepatic EROD induction and histology in nile tilapia Oreochromis niloticus.

The effect of environmental pollutants present in sediments obtained from Bahía de Chetumal, a bay on the border between Mexico and Belize, was studied in nile tilapia (Oreochromis niloticus) intraperitoneally injected with sediment extracts from six different sites of the Bay. Sediment samples used for the study contained a variety of organic chemicals such as organochlorine pesticides, polychlorinated biphenyls (PCBs) and polynuclear aromatic hydrocarbons (PAHs). Total cytochrome P-450 and EROD activity were measured in fish liver. Haematological and histological analyses were also carried out. Hepatic P-450 content in treated fish increased from 43 to 240%, and EROD activity from 85 to 160% compared to controls. Extracts from two sampling sites inhibited EROD activity. There were positive significant correlations between P-450 content and the levels of PCBs 44 and 128. EROD activity correlated to HCB, op'-DDE, pp'-DDE, pp'-DDD, mirex and PCB 18 concentrations. Blood examination showed cell degeneration and binucleated leukocytes with abnormal chromatin. Extract treatment also resulted in foci of hyperplasia on the basement of gill lamellae, hypertrophy and oedema in gills and liver necrosis. Control fish showed no abnormalities. The results demonstrate that sediments from Bahía of Chetumal have the potential to cause histopathological, haematological and biochemical alterations in fish. The administration of sediment extracts to fish may serve as a useful test to screen the toxicity of sediments from different areas.     

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.     

Metabolism of the S-triazine herbizide atrazine in early life stages of the zebrafish, Danio rerio

Increasing anthropogenic pollution of the environment can have adverse consequences for organisms which are more subtle than direct toxicity. Detectable levels of persistent pollutants remain even if the substance is no longer used. An example is atrazine, a herbicide in common use throughout the world but one which has been banned in Germany since 1992. As a lipophilic substance, atrazine is bioconcentrated which may lead to chronic intoxication or physiological stress. In order to withstand chemical stressors, many organisms possess detoxication enzymes, for example certain P-450 monoxygenases and glutathione S-transferases. But detoxication demands cellular energy, and in developing organisms, such as fish embryos which have particularly high energy needs for sustaining growth and organogenesis, the additional energy needs of detoxication may present additional stress. In this study, uptake of atrazine, cytochrome P-450 binding spectra, effects on microsomal and soluble glutathione S-transferase activities, and the initial detoxication steps of atrazine via microsomal and soluble glutathione S-transferases were studied using early life stages of zebrafish.     

Gene expression in caged fish as indicators of contaminants exposure in tropical karstic water bodies

Karstic areas in Yucatan are very permeable, which allows contaminants to move rapidly into the aquifer. In the present study, we evaluated gene expression of vitellogenin (VTG) and cytochrome P-450 1A (CYP1A) in caged juvenile zebrafish deployed for 15 days in 13 different water bodies, cenotes and aguadas, throughout karstic region of the Yucatan peninsula. Gene expression was evaluated using qRT-PCR. Results indicated induction of VTG in 7 water bodies with respect to reference cage. The highest relative VTG expression, about 3000 times higher than reference cage, was found in an aguada close to a cattle farm. CYP1A induction with respect to reference cage was observed in 3 water bodies, all of them located near villages or used for tourist activities. Pollutants and biomarkers of effect should be monitored in these water bodies in order to have a better understanding of the actual levels of pollutants that are present at Yucatan’s aquifer and the potential risk to human and environmental health.     

Metabolism of tributyltin oxide by crabs, oysters and fish

Bis(tributyltin) oxide (TBTO) is widely used as an antifouling agent in various antifouling paints. Thus, some marinas have TBTO concentrations as high as 2 μg/liter.¹ These concentrations can be toxic to zooplankton.² The objectives of the present study were to determine the ability of a number of marine animals, including crabs, oysters and fish, to metabolize TBTO. Earlier work showed that extracts of rat liver were able to metabolize TBTO to a variety of metabolites, e.g. β-hydroxybutyldibutylin.³ The role of the cytochrome P-450 dependent mixed-function oxygenase system in oxidizing TBTO in marine animals was also of interest. Both in vivo (uptake of ¹⁴C-TBTO from food or water) and in vitro studies demonstrated that all the animals were able to metabolize TBTO. The oysters metabolized TBTO at a much slower rate than the other animals. The mixed-function oxygenase system from hepatic tissues of the various animals was able to metabolize TBTO by forming a number of hydroxylated metabolites.     

Effects of the inhibitor ellipticine on cytochrome P450-reductase and cytochrome P450 (1A) function in hepatic microsomes of flounder (Platichthys flesus)

The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b₅ reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe³⁺-P450 and P450R.