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1.
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.  相似文献   

2.
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.  相似文献   

3.
Purification of cytochromes P-450 from liver microsomes of β-naphthoflavone (BNF)-fed rainbow trout yielded three apparently homogeneous forms. The major form (LM4b)* appears to be a P-448 type of cytochrome. A minor form (LM4a), having properties very similar to LM4b, was also obtained. In addition, a P-450 form (LM2) was isolated, with properties quite different from LM4a or LM4b, 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 LM4a and LM4b 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 LM4a- and LM4b-IgG. LM4b-IgG was much more effective than LM4a-IgG for inhibition of LM4a or LM4b 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 LM4b 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 LM4b -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 LM4b. 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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
The hepatic microsomal monooxygenase system of fish-eating sea birds has received little attention considering the vulnerability of this group of animals to environmental pollution. Knight et al.1 measured monooxygenase activities of six species of fish-eating sea birds towards the organochlorine pesticide aldrin and the dieledrin analogue HCE (1,2,3,4,9,9-hexachloro-1,4,4a,5,6,7,8,8a-octohydro-exo-7,8-epoxy-1,4-methanonaphaline) and found very low activities in all species except the puffin (Fratercula arctica), activities more comparable to those of the fish than those of terrestrial birds. The aims of the present study were to compare the aldrin epoxidase and HCE hydroxylase systems of the razorbill (Alca torda) and the puffin with those of the male rat. Kinetics of the two reactions were compared at substrate concentrations down to environmentally realistic levels and the forms of cytochrome P-450 of the birds were partially characterised and compared with forms equivalently from untreated male rat liver.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
The whales are an interesting group in several aspects of science, including evolutionary biology, marine ecology and toxicology. Some of the whale species, including the minke whale, graze at the top of the food chains, where they become susceptible to the accumulation of marine pollutants ingested by organisms at lower levels. The northern stock of the minke whale grazes in Arctic waters in the summer, an area of increasing interest in the development of new oil fields. Studies on the metabolism, disposition and effects of xenobiotics in marine mammals are few. The only reports in the open literature so far have dealt with these questions in seals.1–3 Although the marine mammals comprise a particularly interesting group in marine ecology and toxicology, no data have to our knowledge been presented on the xenobiotic metabolising enzyme systems of the whales. In this study, components of the microsomal cytochrome P-450 system were measured in liver samples from several females, one male, and two foetal minke whales, caught in the Norwegian whaling season of 1983. Because of the limited number of samples studied we have treated the samples individually and not performed any statistical analysis of the data. The trends discussed below must therefore be considered with this background.  相似文献   

13.
A growing number of studies concerning organic chemical pollutant induction of cytochrome P450 mixed function oxidases in fish stimulate a need for common terminology. Similar names for proteins might be based on similarities in function, in regulation, and in their structure. Under the current guidelines for P450 proteins, classification is based on measured or inferred amino acid sequence. At present, a single teleost hydrocarbon-inducible P450 (rainbow trout) can conclusively be termed 1A1: a second (scup P450E) can be so termed on the basis of partial sequence. The lack of primary sequence information makes it difficult to assign a specific identity to hydrocarbon inducible P450s in other fish. Nevertheless, the sum of information on catalytic activities, introduction and immunological cross-reactivities provides a weight of evidence sufficient to assume (but not prove) gene family (I) and subfamily (A) identities for hydrocarbon-inducible proteins in many species. Reference to these proteins as P4501 or IA (or CYPI or IA) in fish species where sequence data are lacking is suggested as more appropriate than reference to them as P450IAI (or CYPIAI). Additional primary sequence data are required to further define orthologous relationships among P450IA genes in fish, and to determine whether IAI is a correct designation for a hydrocarbon-inducible P450 in many species.  相似文献   

14.
Cytochrome P450 from the digestive gland of M. edulis was partially purified by sodium cholate solubilization, 4–15% polyethylene glycol fractionation, and octyl-Sepharose affinity, DEAE-Sephacel ion-exchange and hydroxylapatite chromatography (yields of up to 7–10%). Three peaks were resolved by DEAE-Sephacel chromatography (termed peaks 1–3). P450 specific content was increased from 26 to 800 pmol per mg protein, and the ratio of P450 content to NADPH-cytochrome c (P450) reductase activity reduced by a factor of 250. Oxidised spectrum λmax of P450 was 410.5 ± 1.5 nm. Type II difference spectra were seen with both type II (clotrimazole, metyrapone) and type I (α-naphthoflavone, 7-ethoxycoumarin) compounds. Western blotting with polyclonal anti-P4501A from perch (Perca fluviatilis) gave a single band of approximately 54 kDa molecular weight. A reconstituted system containing peak 2 or 3, rat liver P450 reductase, lipid and NADPH metabolised benzo[a]pyrene to diones, diols, phenols and putative protein adducts. Peak 2 plus cumene hydroperoxide was indicated to produce diones and protein adducts only. Peak 2 alone was indicated to produce diones and phenols. The major free metabolites in all cases were diones (53–100%). The results indicate the existence of a P4501A-like enzyme in M. edulis, possibly with unusual properties as indicated by the difference spectra, metabolism in absence of NADPH and added P450 reductase, and predominance of diones.  相似文献   

15.
试验研究了不同质量浓度下(5、20、50 mg/L)三组柴油分散液,对马粪海胆的肠体、性腺、体液三个部位的抗氧化还原系统细胞色素P450活性变化的影响。结果表明,随着污染暴露时间的增加,三个部位的细胞色素P450活性均呈现出先被诱导、后被抑制的规律,并且油浓度越高,出现诱导和抑制效应的时间越早,活性变化的幅度也越大。海胆P450活性变化在一定程度上能够反映油污染的强度及其对海洋生物的毒性效应,可作为海洋石油烃污染监测的毒理学指标。  相似文献   

16.
This study was conducted to test whether live cells from the liver of fish could be used to detect early changes that are indicative of pollutant-induced liver damage. Low-molecular-weight fluorescent molecular probes were inserted into isolated hepatocytes from dab (Limanda limanda) from a contaminated site and a reference site in the North Sea. These included bioprobes for endoplasmic reticulum (ER), cytochrome P-450-associated 7-ethoxyresorufin-O-deethylase (EROD), oxyradicals, reduced glutathione (GSH) and microtubules (MT). Endocytosis of Texas Red-albumin was used as an integrated indicator of hepatocyte performance. Findings showed increases in ER-associated fluorescence, EROD and oxyradical generation, with a marked decrease in endocytosis in hepatocytes from fish caught at the contaminated site. These results indicate that fish from the historically contaminated site were impacted by organic xenobiotics which induced the biotransformation system, radical production and cell injury.  相似文献   

17.
Cytochrome P450 monooxygenases constitute an enzyme superfamily, with at least 74 known families. Members of CYP families 1–4 are important in the phase 1 metabolism of lipophilic xenobiotics, such as those found in contaminated marine environments. Previous studies (James et al. Arch. Biochem. Biophys. (1996) 329, 31–38) showed that a major form of P450 in spiny lobster, Panulirus argus, hepatopancreas was CYP2L1, a new sub-family, and that there was evidence for other P450 forms in hepatopancreas. We now report the sequence of a second member of this subfamily, named CYP2L2, present in P. Argus hepatopancreas. The deduced amino acid sequences of CYP2L1 and CYP2L2 share 54.7% sequence identity and an additional 13.6% of the sequences show conservative substitutions. Analysis of the sequences of CYP2L1, CYP2L2 and other representative CYP2 family members (from rat and mouse sub-families 2A, 2B, 2D and 2E) showed that the crustacean sequences clustered together. In addition to CYP2L2, cDNA clones of 66 to 117 base pairs from the 5′ coding region of two more P450 isoforms were isolated from the spiny lobster cDNA library. The deduced amino acid sequence of one of these additional cDNA clones was identical to the first 22 amino acids of the N-terminal sequence of a P450 protein previously isolated from hepatopancreas microsomes. These studies confirm earlier biochemical evidence that the hepatopancreas contains multiple forms of cytochrome P450.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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