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1.
Effects on the hepatic cytochrome P450 1A1 system were investigated in juvenile rainbow trout i.p. injected with three different aromatic containing fractions: kerosene, light gas oil or heavy gas oil, originated from distilled North Sea crude oil. Kerosene treatment resulted in no effect on the P450 1A1 system, light gas oil injection caused a weak induction of EROD activities and heavy gas oil treatment resulted in a prominent induction of EROD activities as well as accumulation of CYP1A1 mRNA and P450 1A1 protein levels. The effects of heavy gas oil were compared with effects of β-napthoflavone (β-NF) on the P450 1A1 system. It was obvious that important discrepancies seemed to exist between EROD activities and corresponding CYP1A1 mRNA and P450 1A1 levels in rainbow trout treated with either heavy gas oil or β-NF i.e. heavy gas oil treatment resulted in higher specific EROD activities (EROD/P450 1A1) compared to β-NF. GC-MS analyses revealed that liver and bile from heavy gas oil treated rainbow trout in addition to naphthalene also contained polycyclic aromatic hydrocarbons such as phenanthrenes, anthracene, pyrenes, fluoranthene benz(a)anthracene and chrysene, while none of these compounds were detected in control trout.  相似文献   

2.
To assess chemical contaminant stress in the marine environment, ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P450 1A (CYP1A) expression were measured in 88 English Sole (Pleuronectes vetulus) collected during May and June 1999 from four sites in Vancouver Harbour and at an expected reference site outside the harbour. Hepatic microsomes were prepared from the fish and analyzed for total CYP content, EROD activity, and CYP1A protein levels. Hepatic EROD activity and CYP1A protein levels were elevated in fish from two sites in the inner harbour. A comparison with sediment chemistry data showed that fish with increased EROD activity and CYP1A levels came from sites containing relatively high levels of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Unexpectedly high levels of EROD activity and CYP1A protein were also found in fish from a reference site near Gibsons, in Howe Sound. The elevated EROD activity and CYP1A expression in fish from this site cannot be explained by the chemical analysis data collected.  相似文献   

3.
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   

4.
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.  相似文献   

5.
Extracts from semi-permeable membrane devices (SPMDs) deployed on beaches in Prince William Sound (PWS), Alaska, were used to evaluate if complex contaminant mixtures from different sources can be distinguished by the resulting cytochrome P450 1A (CYP1A) activity in exposed test animals. Deployment sites included canneries, salmon hatcheries, and beaches where lingering oil remains from discharges during the 1964 earthquake or the 1989 Exxon Valdez oil spill. Other sites were selected at random to evaluate region-wide contaminant inputs or were located in salmon streams to evaluate contaminants carried and released by migrating salmon carcasses following reproduction. Following standard deployments of approximately 28 d, an aliquot of the accumulated contaminants was intraperitoneally injected without cleanup into juvenile rainbow trout (Oncorhynchus mykiss). After 2 d and 7 d, the activity of CYP1A was measured by the ethoxyresorufin-o-deethylase (EROD) assay. Exposure to extracts from the oiled sites and one hatchery site with numerous creosote pilings elicited strong EROD responses, whereas fish exposed to salmon stream extracts elicited weak but significant responses during late summer compared to late spring. Responses from the other sites were not significant, indicating contaminants from these sources are unlikely to cause CYP1A induction in resident biota. Rather than simply assessing extant contaminants, this method evaluates the potency of the different sites for bringing about aryl hydrocarbon receptor responses in resident biota.  相似文献   

6.
Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.  相似文献   

7.
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   

8.
Cytochrome P4501A (CYP1A) metabolizes a wide array of lipophilic xenobiotics. In fish liver, CYP1A is constitutively expressed at low levels, but xenobiotics can strongly induce CYP1A expression via a receptor-mediated pathway. While induction of hepatic CYP1A in teleosts by xenobiotics is well investigated, very little is known on the regulation of constitutive CYP1A expression and its induction by factors other than xenobiotics. In the present study we show that in the rainbow trout liver cell line, RTL-W1, CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity can be induced by a change of the culture medium, in the absence of xenobiotics. The increase in cellular EROD levels is of transient nature. Experiments with cell incubation solutions supplemented with various medium components indicate that photooxidized tryptophan is the agent causing the increase of EROD activity after medium change.  相似文献   

9.
Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.  相似文献   

10.
Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.  相似文献   

11.
Recent studies demonstrating feminization of effluent-exposed wild-caught male fish in the UK have prompted much research regarding the estrogenic activity of effluent from municipal sewage treatment plants (MSTPs). To investigate the estrogenicity and cytochrome P450 1A (CYP1A) induction potency of MSTP effluent, two species of fish, adult male mummichogs, Fundulus heteroclitus, and juvenile sunshine bass, Morone saxatilis x Morone chrysops, were exposed to un-chlorinated effluent (75% effluent, 25% seawater) from a large MSTP in Yonkers, NY, USA. After a 21-day static-daily (75%) renewal exposure, significant elevations over controls were observed in levels of vitellogenin (VtG) in plasma (1730%) and liver (131%) in effluent-exposed sunshine bass. In contrast, hepatic VtG was not elevated in mummichogs; plasma VtG was not measured in this species. Effluent exposure elevated hepatic CYP1A protein (140-145%) and ethoxyresorufin-O-deethylase (EROD) activity (408-598%) in both species. These findings suggest ontogenetic and/or species differences in response to estrogenic compounds in MSTP effluent. Furthermore, the elevation of CYP1A in response to sewage effluent exposure indicates the presence of additional compounds that may alter xenobiotic and/or steroid biotransformation in fish.  相似文献   

12.
To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.  相似文献   

13.
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   

14.
Hepatic monooxygenase enzyme activities and relative cytochrome P4501A protein content were measured to evaluate the time-course alterations in rainbow trout after change in living habitat. Fish were transferred from one fish farm to the tanks of another hatchery and/or into cages kept in a lake. In the new habitats, cytochrome P450-dependent enzyme activities in rainbow trout decreased, and were at their lowest levels after two or three weeks in the summer. Later the activities partly reversed. The immunodetection of cytochrome P4501A protein expressed a similar trend as for catalytic monooxygenase activities.  相似文献   

15.
The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system. Recombinant CYP1A4 and 1A5 proteins from common cormorant (Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that Vmax value for ethoxyresorufin-O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin-O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin-O-demethylase activity. The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nanomolar TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.  相似文献   

16.
The cytochromes P450 (CYP) constitute a multigene family of enzymes playing a critical role in the oxidation of many endogenous and xenobiotic substrates. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and aryl amines. Three members of the CYP1 family, CYP1A1, CYP1A2, and CYP1B1, have been identified in mammals. We report here on the identification and cloning of cytochrome P4501B-like sequences from two teleost fish species and a marine mammal. Sequences clustering with CYP1B1 in phylogenetic analysis were obtained from liver cDNA of scup (Stenotomus chrysops), genomic DNA of plaice (Pleuronectes platessa), and liver cDNA of striped dolphin (Stenella coeruleoalba).  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
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20.
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   

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