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1.
Chronic exposure to organic contaminants such as polychlorinated biphenyls (PCBs) can lead to the development of resistance to these chemicals, a condition associated with reduced response of CYP1A1, a pollutant-inducible biomarker. We measured CYP1A activity (ethoxyresorufin o-deethylase, EROD) and PCB concentrations in feral fish from the Town Branch/Mud River system (Logan County, KY), a stream historically contaminated with PCBs and partially remediated. As a first step in evaluating the possible development of resistant populations in this system, we measured CYP1A expression and PCB body burdens in resident fish from sites we previously characterized as containing biologically significant levels of CYP1A inducing compounds. Mean PCB concentrations in edible flesh ranged from 75.2 to 16.7 microg/g in fish collected from Town Branch remediated sites and were relatively low (1.23 microg/g) in Town Branch reference site fish. However, hepatic CYP1A activity was similar among individuals of most species collected from reference and contaminated/remediated sites. The absence of elevated CYP1A levels in resident fish species despite the presence of significant PCB body burdens may indicate these fish have developed reduced sensitivity to CYP1A induction, a condition associated with acquired resistance to toxicants.  相似文献   

2.
Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 micro(micro)M) with or without 0.2 micro(micro)g/l of the CYP1A inducer 3,3',4,4',5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.  相似文献   

3.
Three biomarkers of hydrocarbon exposure, CYP1A in liver vascular endothelium, liver ethoxyresorufin O-deethylase (EROD), and biliary fluorescent aromatic compounds (FACs), were examined in the nearshore fishes, masked greenling (Hexagrammos octogrammus) and crescent gunnel (Pholis laeta), collected in Prince William Sound, Alaska, 7-10 years after the Exxon Valdez oil spill (EVOS). All biomarkers were elevated in fish collected from sites originally oiled, in comparison to fish from unoiled sites. In 1998, endothelial CYP1A in masked greenling from sites that were heavily oiled in 1989 was significantly higher than in fish collected outside the spill trajectory. In 1999, fishes collected from sites adjacent to intertidal mussel beds containing lingering Exxon Valdez oil had elevated endothelial CYP1A and EROD, and high concentrations of biliary FACs. Fishes from sites near unoiled mussel beds, but within the original spill trajectory, also showed evidence of hydrocarbon exposure, although there were no correlations between sediment petroleum hydrocarbon and any of the biomarkers. Our data show that 10 years after the spill, nearshore fishes within the original spill zone were still exposed to residual EVOS hydrocarbons.  相似文献   

4.
Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Port Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P < 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).  相似文献   

5.
Newark Bay (NB) killifish (Fundulus heteroclitus) have been chronically exposed to environmental contaminants that activate the aryl hydrocarbon receptor (AHR) and are tolerant to toxic effects and CYP1A induction provoked by AHR ligands. Resistance to CYP1A induction could be due to an epigenetic mechanism such as DNA methylation. We measured in-ovo CYP1A catalytic activity (ethoxyresorufin-O-deethylase, EROD) in NB and reference site killifish embryos aqueously exposed to various concentrations of the de-methylating agent 5-azacytidine, 5-AC (5, 50 and 500 μ(micro)M) with or without 0.2 μ(micro)g/l of the CYP1A inducer 3,3′,4,4′,5 pentachlorobiphenyl (IUPAC PCB126). Neither PCB126 alone, nor PCB126 plus 5-AC, induced EROD above levels in vehicle treated Newark Bay fish. In reference site fish, the same PCB126 dose provoked a 7.4-fold EROD induction relative to controls. We conclude that Newark Bay killifish are resistant to CYP1A induction by co-planar PCBs during early embryological development and our data suggests that DNA methylation does not play a critical role in resistance to CYP1A induction in this model.  相似文献   

6.
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   

7.
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.  相似文献   

8.
Aim of this study was to determine the extent of pollution in the West Black Sea Coast of Turkey by measuring CYP1A associated EROD activity, phase II enzyme, glutathione S-transferase and antioxidant enzymes, catalase and glutathione reductase activities and immunochemical detection of CYP1A protein level in the liver of mullet. The fish samples were caught from six locations having a varying degree of pollution in the West Black Sea Region of Turkey in August 2005, 2006 and 2007. Mullets caught from Zonguldak Harbour, Ere?li Harbour and Gülüç Stream’s Mouth displayed 6–9-fold higher EROD, 2–4-fold higher glutathione S-transferase and 2–3-fold higher catalase activities than the reference site, Amasra. Total polyaromatic hydrocarbon levels in mullets caught from these locations were also significantly higher (2–4-fold) than Amasra. The results of this study indicate that Zonguldak Harbour, Ere?li Harbour and Gülüç Stream are highly polluted by polycyclic aromatic hydrocarbons and related contaminants.  相似文献   

9.
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   

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.
The prevalence of toxicopathic liver lesions in English sole (Pleuronectes vetulus) was determined along a presumed gradient of chemical contamination in Vancouver Harbour, Canada. Fish were captured from five sites in or near Vancouver Harbour, British Columbia, Canada. No toxicopathic lesions were observed in fish examined at the reference site (Howe Sound outside Vancouver Harbour), or at the outer harbour site. In contrast, 20-23% of the fish from three sites located in the central harbour, Indian Arm and Port Moody Arm had one or more types of toxicopathic lesions. Likewise, aromatic hydrocarbon (AH) metabolites measured in bile exhibited a gradient in levels from lower concentrations at the reference site to significantly higher levels in fish from Indian Arm and Port Moody Arm harbour sites. The occurrence of toxicopathic liver lesions was statistically associated with concentrations of AHs measured in sediment and AH metabolite levels measured in bile.  相似文献   

12.
为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。  相似文献   

13.
The initial sampling in the Marine Monitoring Program (MOMAM), coordinated by the Ministry of Marine Affairs (IEAPM), was performed along the southeast coast of Brazil. Orthopristis ruber samples were collected at Guanabara, Sepetiba and Ilha Grande Bays. Microsomal CYP1A levels and cytosolic cholinesterase (ChE), catalase (CAT) and glutathione S-transferase (GST) activities were measured in the liver of these fish according to established procedures. CAT activity and CYP1A content were significantly higher (P < or = 0.05) in fish caught at Guanabara Bay, which might be due to higher levels of peroxisome proliferators and Ah receptor agonists, respectively, at this site compared to the other sites. Also, lower GST activity was observed in fish from this site, which may possibly be related to the presence of oxidative-stress inducing compounds.  相似文献   

14.
The use of fish in environmental monitoring has become increasingly important in recent years as anthropogenic substances, many of which function as prooxidants, are accumulating in aquatic environments. We have measured a battery of antioxidant defenses as a measure of oxidative status, as well as protein carbonylation as a measure of oxidative damage, in corkwing wrasse (Symphodus melops) captured near a disused copper mine, where water and sediment are contaminated with heavy metals, and an aluminum smelter, a site contaminated with PAHs. Results were compared to two different reference sites. Fish at the heavy metal site had lower glucose-6-phosphate dehydrogenase activity and elevated protein carbonyls (1.8 times) compared to fish from the reference site. At the PAH site, EROD was increased 2-fold, while total glutathione and methemoglobin reductase concentration, were decreased. No differences were seen in protein carbonyl levels at the PAH site. Measures of both antioxidant defenses and oxidative damage should be used when assessing effects of xenobiotics on oxidative stress in fish species.  相似文献   

15.
Hepatic levels of the pollutant inducible enzyme, CYP1A, are strongly suppressed in spawning female fish, a phenomenon attributed to high plasma levels of the female sex steroid hormone, estradiol. To evaluate the contribution of estrogen metabolites to estradiol-mediated CYP1A regulation, we treated primary hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss) with vehicle, 17beta-estradiol, or the estrogen metabolite, estriol, alone and in combination with each other and with the potent CYP1A inducer, benzo[a]pyrene (B[a]P). We found dose-dependent suppression of B[a]P-induced CYP1A activity by both steroids relative to controls. At 10(-7) M doses, estradiol and estriol suppressed B[a]P-induced CYP1A activity by 3- and 2-fold, respectively. Although not statistically significant, mean basal CYP1A activity levels were 15- and 13-fold lower in estradiol and estriol treated hepatocytes, respectively, relative to vehicle treated controls. Combining doses of estradiol and estriol failed to produce synergistic suppression of either basal or B[a]P-induced CYP1A activity relative to treatment with either steroid alone. The observed suppression is well below the often strong suppression observed in spawning female fish. We conclude that factors in addition to estradiol and estriol are likely involved in producing sexual dimorphism in CYP1A expression observed in spawning fish.  相似文献   

16.
Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.  相似文献   

17.
Gonadally mature fish display strong sex-related differences in the content and activity of P4501A, the major polynuclear aromatic hydrocarbon-inducible P450 form in teleosts. Such differences appear related to plasma levels of the female sex steroid, estradiol (E2); however, neither the mechanism of estradiol suppression of P4501A nor the capacity for hormonal regulation to overcome P4501A induction by high concentrations of potent inducers are known. Gonadally mature flounder (Pseudopleuronectes americanus) were collected from Fox Island (FI), Rhode Island, a reference site, and New Bedford Harbor (NB), Massachusetts, a site highly contaminated with polychlorinated biphenyls (PCBs). Differences in flounder P4501A expression were determined at the level P4501A catalytic activity (measured as ethoxyresorufin-O-deethylase, EROD), P4501A protein content (immunoquantitated), and P4501A mRNA content (by Northern blot) as they relate to sex, reproductive status, and hepatic PCB content. Our results confirm that suppression of P4501A in gonadally mature female fish is probably due, at least in part, to elevated E2 titers, and demonstrate that such suppression occurs at a pretranslational level and, further, that endogenous regulation of P4501A expression can ‘override’ exogenous regulation by even high concentrations of P4501A inducers.  相似文献   

18.
Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.  相似文献   

19.
The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.  相似文献   

20.
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.  相似文献   

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