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1.
The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species. 相似文献
2.
Studies of xenobiotic metabolism and the regulation of enzyme systems for their metabolism (Phase I and II enzyme systems) require large numbers of animals, intensive use of experimental aquarium systems and in some instances can pose major problems when the compounds are scarce, expensive or too toxic to be disposed of easily. Previous studies have demonstrated the usefulness of isolated primary hepatocytes of fish for metabolic and enzyme induction studies. Functionally and structurally competent hepatocytes were isolated from juvenile plaice and after overnight acclimation to culture conditions, were exposed to a variety of PAHs for 24h. The levels of CYP1A1, phenol UDPGT and GST-A mRNAs were then estimated by slot blotting and hybridisation to their cDNA probes. The method enabled rapid and easy determination of the structure/activity relationships of these compounds as inducers of these key Phase I and II enzymes, and is potentially useful for screening large numbers of compounds both as an invitro toxicity test and for mechanistic studies. 相似文献
3.
4.
cDNA's coding for cytochrome P4501A1 (CYP1A1), phenol UDP-glucuronosyltransferase (UDPGT) and glutathione S-transferase (GST-A) were cloned and sequenced from an expression library prepared from the liver of a 3-methylcholanthrene (3-MC) induced plaice. Plaice CYP1A1 and Phenol UDPGT display a high degree of structural conservation with homologous mammalian isoforms and their mRNAs were shown to be highly induced in liver after 3-MC treatment of fish. Expression of plaice GST-A, which displays closer homology to GSTs from plants and invertebrates than those of mammals, is repressed after 3-MC treatment. 相似文献
5.
A cDNA clone for glutathione S-transferaseA (GSTA) from plaice (Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min−1 mg−1. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min−1 mg−1. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products. 相似文献
6.
The phthalate diesters DEHP and DBP do not induce lauric acid hydroxylase activity in rainbow trout 总被引：1，自引：0，他引：1
Aquatic organisms are extensively exposed to phthalate esters. We have investigated in trout the effects of diethylhexylphthalate (DEHP) and dibutylphthalate (DBP) on xenobiotic metabolizing enzymes which have been suggested as possible environmental biomarkers. Rainbow trout (Oncorhynchus mykiss) were waterborne exposed to DEHP (1 mg/l) or DBP (0.1 or 1 mg/l) for 72 h. Another group of rainbow trout received daily for 3 days an intraperitoneal injection of 50 mg/kg of DEHP or DBP. Laurate hydroxylation, ethoxyresorufin-o-deethylation, UDP-glucuronyltransferase activity and glutathione-S-transferase activity were measured in liver and extrahepatic tissues. The phthalate esters have been found not to induce these enzymes; in particular, the results do not support the previously described induction of lauric acid hydroxylase in sea bass treated with DEHP [Comp. Biochem. Physiol. B122 (1999) 253.]. 相似文献
7.
A. Khler B. Lauritzen S. Banns S. G. George L. Frlin C. J. F. Van Noorden 《Marine environmental research》1998,46(1-5)
Progression from eosinophilic foci to persistent basophilic foci and carcinomas was observed in pollution-induced hepatocellular carcinogenesis in European flounder (Platichthys flesus L.) in a similar sequence as in chemically induced liver cancer in mammals. Image analysis was used to quantify enzyme activity and protein expression as visualised by enzyme and immunohistochemistry in various stages of toxipathic lesions and carcinogenesis. In eosinophilic foci, growth advantage was achieved by increased production of NADPH and pentoses for biosynthesis and cell proliferation due to increased G6PDH capacity during the first steps of clonal adaptation. Simultaneously, oxyradical production by CYP450, phase I was reduced. Overexpression of P-gp mediating multi-xenobiotic resistance was noted in basophilic cell types which persisted during progression towards carcinomas. This was accompanied by increased protein levels of oxyradical scavenging GST-A. These changes are consistent with adaptation and phenotypic expression of the multidrug or xenobiotic resistance (MDR/MXR) type in hepatocarcinomas of European flounder. 相似文献
8.
Short-term variability of multiple biomarker response in fish from estuaries: Influence of environmental dynamics 总被引：1，自引：0，他引：1
Fonseca VF França S Vasconcelos RP Serafim A Company R Lopes B Bebianno MJ Cabral HN 《Marine environmental research》2011,72(4):172-178
Short-term variability in biomarker responses and the effects of temperature and salinity variation were explored in three fish species (Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps) occurring in the Tejo estuary. Short-term variability in biomarkers was observed in all species although no pattern was discerned over time (days to weeks). Antioxidant enzymes activity (superoxide dismutase (SOD) and catalase (CAT)) showed low temporal variability, indicating some constancy or baseline level in antioxidant responses. Only CAT activity in S.?senegalensis was correlated with temperature, suggesting that exposure to contaminants triggered antioxidant acclimation. Higher short-term variability was observed in xenobiotic biotransformation enzymes activity (phase I ethoxyresorufin O-deethylase (EROD) and phase II glutathione S-transferase (GST)). Yet a significant correlation between EROD and GST in D.?labrax and S.?senegalensis suggests a concomitant response to contaminants. Moreover the lack of correlation between xenobiotic biotransformation enzymes and abiotic variables on concordant time scales, suggest a high specificity of these biomarkers to chemical exposure, rather than high variability due to environmental dynamics. 相似文献
9.
In the present study, long-term exposure to PCB resulted in marked induction of the xenobiotic biotransformation enzymes UDP glucuronosyl transferase, glutathione transferase and DT-diaphorase (measured as menadione reductase and DCPIP reductase) in rainbow trout 15 or 20 weeks post-injection during the experimental period. The increase of the phase II enzymes were paralleled by a marked increase of the glutathione reductase activity. These results indicate that prolonged exposure to PCB results in increased production of organo-radicals. 相似文献
10.
We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to beta-naphthoflavone (beta-NF, 66 mg/kg, i.p.) elicited a 7-9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to beta-NF, and generally tracked the minimal changes observed in GST-CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by beta-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to beta-NF. 相似文献
11.
The present study aimed to obtain additional data on the effect of long-term depuration on the levels of oxidative stress biomarkers, and to clarify the role of mullets for monitoring pollution in River Douro estuary. Mullets chronically exposed to a mixture of contaminants in Douro estuary were captured in Spring of 2001, 2002 and 2003. The activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX); and oxidative damages in lipids (lipid peroxidation) and in proteins (protein carbonyl content) were assessed at capture day and after transfer to unpolluted seawater for 1, 4 and 8 months. An overall decrease in the activities of the antioxidant enzymes was detected, except for the GPX after 4 months depuration. CAT activity exhibited the more significant decrease at the end of the long-term depuration. The decrease in SOD activity after 1 month of depuration was then maintained during the remaining depuration period. Regarding oxidative damages, a decrease in lipid peroxidation as well as the content of oxidised proteins was observed during depuration. Indeed, at capture the activities of antioxidant defences were higher as a result of the formation of reactive oxygen species (ROS) from the metabolism of pollutants. The oxidative damaged molecules were repaired or degraded during the depuration period, supporting the use of such damages as indicators of exposure to pro-oxidant pollutants. 相似文献
12.
黄斑篮子鱼去毒相关基因的克隆与肝脏组成型表达分析 总被引：1，自引：0，他引：1
从基因水平探讨海洋鱼类对海洋藻毒素的去毒分子机理。采用RT-PCR法成功克隆了黄斑篮子鱼Siganus oramin肝脏I时相代谢酶细胞色素P450 1A(CYP1A)、II时相代谢酶alpha型谷胱甘肽S-转移酶(GSTA)和rho型谷胱甘肽S-转移酶(GSTR)、热休克蛋白70 (HSP70)、alpha 1型钠钾ATP酶(ATP1A1)及β-肌动蛋白(beta-actin, ACT)基因cDNA核心序列，序列分别长879 bp、582 bp、588 bp、660 bp、749 bp和554 bp。序列同源性分析发现，属鲈形目的黄斑篮子鱼CYP1A、GSTA和GSTR与同属鲈形目的牙鲆Paralichthys olivaceus、欧洲鲽Pleuronectes platessa、真鲷Pagrus major、鲤形目的斑马鱼Brachydanio rerio 相应氨基酸序列同源性较高，CYP1A和GSTA与非洲爪蟾(两栖类)、鸡(鸟类)、小鼠、大鼠和人(哺乳类)相应氨基酸序列同源性低，这可能与鱼类I、II时相去毒酶基因承担水环境毒素去毒代谢的特殊功能有关；而HSP70、ATP1A和β-肌动蛋白在鱼类、两栖类、鸟类、哺乳类中均有较高的同源性，这可能与这些基因在机体中承担的最基本的生命功能相关。应用半定量RT-PCR的方法，以β-肌动蛋白作为外参照，在指数期增长范围内分别得到了CYP1A、GSTA、GSTR、HSP70和ATP1A1 mRNA与β-肌动蛋白mRNA (%)的比值，确定黄斑篮子鱼肝脏去毒相关基因的组成型表达水平。其中，黄斑篮子鱼肝脏CYP1A、GSTA和GSTR基因组成型表达相对较高，HSP70和ATP1A1基因组成型表达相对较低，这可能与不同基因在黄斑篮子鱼海洋藻毒素去毒分子机理中承担的作用相关，为海洋藻毒素在海洋鱼类中的积聚及代谢去毒分子机制的研究提供了相关数据。 相似文献
13.
Almli B Egaas E Christiansen A Eklo OM Lode O Källqvist T 《Marine environmental research》2002,54(3-5):237-240
In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole [(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole] and fenpropimorph [(+/-)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc] were administrated in the water separately and together in a static system (80 microg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole[2-(thiazol-4'-yl)benzimidazole] in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment. 相似文献
14.
Baseline data for hepatic xenobiotic metabolizing biomarker enzyme activities were obtained for artificially reared tilapia Oreochromis niloticus, and were compared with those of the plaice (Pleuronectes platessa) and rainbow trout (Onchorynchus mykiss). Basal activities exhibited species variations with notably higher CYP1A and phenol UGT activities and lower GST activity in plaice than the freshwater species. Interspecies relationships between gene families determined by immunoblotting and substrate-activity profiles demonstrated the presence of homologous CYP1A and CYP3A enzymes in all three species, alpha class GSTs in plaice and trout, mu and pi class GSTs in trout and theta class GSTs in plaice and tilapia. CYP1A of tilapia was induced by 3-MC or PBO treatment, whilst CYP3A was induced by PCN treatment. 相似文献
15.
The aim of this work was to investigate the activity of antioxidant enzymes and oxidative damage in the digestive gland of the limpet Nacella concinna, and their suitability as biomarkers for hydrocarbon pollution in Antarctic coasts. Three groups of 30 individuals each were kept in seawater containing 0%, 0.05% or 0.1% diesel. Superoxide dismutase, catalase, glutathione S transferase and glutathione peroxidase activities, as well as lipid peroxidation and protein oxidation were studied in 18 animals of each group after 24, 48 and 168 h of exposure. The activity levels of most enzymes were increased by diesel in a dose-dependent manner. Glutathione peroxidase showed the most clear effect; its activity significantly increased in the 0.1% diesel group respect to the control. Lipid peroxidation and protein oxidation were significantly increased by diesel after 168 h. Both variables were higher in the group exposed to the lowest dose. 相似文献
16.
Paralytic shellfish poisoning (PSP) toxins have been implicated as the causative agent of a number of fish kills. Exposure experiments indicate that fish are susceptible to PSPs by intraperitoneal (i.p.) and oral administration, while sampling of fish affected by toxic blooms reveals that these toxins can be accumulated. In spite of the potential impact to marine fisheries, little research has been conducted on the potential metabolism and detoxification of PSPs in marine fishes. Previous work by this group has shown that the xenobiotic metabolising enzyme (XME) cytochrome P-450 (CYP1A) is induced in Atlantic salmon (Salmo salar) following i.p. exposure to saxitoxin (STX). Salmon injected i.p. with sub-lethal doses of STX show a four- to eight-fold induction of hepatic CYP1A (as shown by ethoxyresorufin-O-deethylase activity) over controls after 96 h. Results presented here show that the phase II XME glutathione S-transferase (GST) is also induced in salmon following PSP exposure. Post smolts were exposed to three injections of PSPs (2 micrograms STXeq/kg) over 21 days. Injection of both STX and PSPs extracted from a toxic strain of dinoflagellate (Alexandrium fundyense, CCMP 1719) resulted in induction of hepatic GST, as measured by activity for 1-chloro 2,4-dinitrobenzene. Such inductions indicate a potential role for XMEs in PSP metabolism. Possible roles for other enzymes are also discussed. 相似文献
17.
壬基酚、辛基酚、二氯酚和双酚A是水环境中普遍存在的酚类污染物,由于其内分泌干扰活性和致癌性近年来受到广泛的关注.本研究以菲律宾蛤仔(Ruditapes philippinarum)为受试生物,研究4种典型酚类污染物暴露对其内脏组织和鳃组织中抗氧化酶包括超氧化物歧化酶(SOD)和过氧化氢酶(CAT)以及解毒酶谷胱甘肽硫转移酶(GST)活性的影响.研究结果表明:壬基酚低浓度处理组(0.05～0.2 mg/L)3种酶活性无显著变化,高浓度处理组(0.4 mg/L),SOD、CAT和GST 3种酶活性均受到显著抑制,抑制率分别为56.6％,59.0％和35.5％.在实验浓度范围(0.05～0.4 mg/L)内,二氯酚能够显著抑制内脏组织CAT活性,双酚A能够显著抑制鳃组织GST活性.3种酶活性变化对于酚类污染物的响应呈现较好的规律性,因此可以作为生物标志物联合指示水体中的酚类污染. 相似文献
18.
The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver. 相似文献
19.
《Deep Sea Research Part I: Oceanographic Research Papers》2006,53(7):1101-1116
Hydrothermal vent mussels are exposed continually to toxic compounds, including high metal concentrations and other substances like dissolved sulphide, methane and natural radioactivity. Fluctuations in these parameters appear to be common because of the characteristic instability of the hydrothermal environment. Temporal variation in the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidases (Total GPx), selenium dependent glutathione peroxidases (Se-GPx)), metallothioneins and lipid peroxidation (LPO) in the gills and mantle of the mussel Bathymodiolus azoricus from Menez–Gwen hydrothermal vent site was evaluated and related to the accumulated metal concentrations (Ag, Cu, Cd, Fe, Mn and Zn) in the tissues. Maximum antioxidant enzyme activities in the gills were detected in the beginning of summer, followed by a gradual decrease throughout the following months. One year after, the levels of antioxidant enzyme activities were similar to those reported one year before. LPO in this tissue exhibited a similar temporal variation trend. A different pattern of temporal variation in antioxidant enzyme activities was observed in the mantle, with a gradual increase from summer to the end of autumn (November). LPO in the mantle exhibited an almost reverse trend of temporal variation to that of antioxidant enzyme activities in this tissue. Antioxidant defences in the gills of B. azoricus were significantly enhanced with increasing concentrations of Ag, Cu and Mn, while negative relationships between antioxidant enzymes and Cd, Cu, Mn and Zn concentrations in the mantle were observed, suggesting different pathways of reactive oxygen species (ROS) production and that these tissues responded differently to the metal accumulation. However, temporal variation in biomarkers of defence and damage were in general similar to coastal bivalve species and can be associated with temporal variations of the physiological status due to reproduction. These variations might also be linked to the highly unstable nature of the hydrothermal environment. 相似文献
20.
We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF. 相似文献