摘要
根据鱼类的生态习性选择了暖温性近岸底层岩礁鱼类褐菖鲉和浅海底层鱼类黄鳍鲷和黑鲷作为实验动物,系统地研究了0#柴油WSF诱导组和对照组褐菖鲉肝微粒体EROD动力学反应系统的测定条件,确定了最适测定条件为:pH值7.60、温度15℃、底物7-乙氧基异吩恶唑浓度0.5μmol/L、样品蛋白量100—800μg、还原型辅酶II NADPH 70 nmol/mL。诱导组和对照组的最适反应条件没有差别,但是诱导组的酶活性比对照组的高;对照组和诱导组EROD的Km值分别为0.144μmol/L和0.357μmol/L,Vmax值分别为0.466 nmol min-1mg-1蛋白和2.360 nmol min-1mg-1蛋白。首次在我国建立了褐菖鲉肝微粒体EROD活性检测技术。此外,比较了上述3种实验鱼类肝微粒体EROD反应系统的最适pH值和最适温度。结果表明,与褐菖鲉不同,黄鳍鲷和黑鲷肝EROD反应最适pH值都是7.80,最适温度都是25℃。
Cytochrome P450 （CYP） enzymes are a large and ubiquitous family of hemecontaining proteins found in vertebrates, invertebrates, plants, and microorganisms that catalyze the oxidative biotransformation of diverse lipophilic xenobiotic and endogenous compounds including steroids, fatty acids, drugs, and organic pollutants. CYP enzymes play a role both in the detoxification and activation of halogenated and non-halogenated hydrocarbons found in the food chain. Because CYP enzymes are central to metabolism, levels of individual CYP enzymes are important determinants of bioaccumulation and ultimately of susceptibility to environmental contaminant exposure. A toxicologically relevant aspect of CYP regulation is the observation that several substrates can increase expression of specific CYP enzymes involved in their metabolism. For example, induction of hepatic CYP1A enzymes is a common and characteristic biochemical response in vertebrates of exposure to certain planar halogenated and non-halogenated hydrocarbons, such as polychlorinated dibenzo-p- dioxins （PCDDs）, polychlorinated biphenyls （PCBs） and polynuclear aromatic hydrocarbons （PAHs）, that often precedes toxicity. In mammals, the CYPIA subfamily contains two related forms, CYP1A1 and CYP1A2, that can be separated by chromatographic, electrophoretic and immunochemical means. Expression of both CYP1A1 and CYP1A2 is regulated in a differential and tissue-specific manner by the aryl hydrocarbon （Ah） receptor. Although several different CYP enzymes have been identified in fish, CYP1A has received the most attention as the major hydrocarbon inducible form and as a possible biomarker of exposure to marine hydrocarbon contaminants. CYP1A enzyme induction in fish populations is generally recognized as a useful and sensitive biochemical marker of contaminant exposure, and by inference, of marine ecosystem health. Usually, induction of CYP1A was determined by measurement of an associated enzyme activity, namely ethoxyresorufin O-deethylase （EROD）. The EROD activity is measured by following the increment in fluorescence of the reaction product resonffin. But the optimal conditions for the EROD activity vary from marine fish species. According to ecological features, Sebastiscus marmoratus, Sparus latus and Sparus macrocephalus were selected as experimental animals in the present study. A systematic study had been performed on optimal conditions of hepatic microsomal EROD kinetic reaction system for the control and the induced groups of Sebastiscus marmoratus with the water soluble fraction （WSF） of 0# diesel fuel. The optimal conditions for the microsomal EROD activity of the Sebastiscus marmoratus were pH 7.60, 15℃, 0.5 μmol/L 7-ethoxyresorufin （ substrate ）, 100--800 μg microsomal protein and 70 nmol/mL NADPH, which showed no difference between the control and the induced groups. However, the EROD activity was apparently higher in the induced groups than the control ones. The optimal conditions for the hepatic microsomal EROD activity from Sebastiscus marmoratus were firstly established in this study. Moreover, Km values for the control and the induced groups were 0.144 μmol/L and 0. 357μmol/L, and Vmax values for the two groups were 0.466 nmol min^-1mg^-1 protein and 2.360 nmol min^-1 mg^-1 protein, respectively. Furthermore, the comparison of optimal pH values and temperatures （two major factors） for the hepatic microsomal EROD activity among the three species of fish were conducted in the study. The results indicated that optimal pH values and temperatures for the EROD activity of both Sparus latus and Sparus macrocephalus were pH 7.80 and 25 ℃, which were different from those of Sebastiscus marmoratus.
出处
《生态学报》
CAS
CSCD
北大核心
2014年第19期5416-5424,共9页
Acta Ecologica Sinica
基金
国家自然科学基金(40476042)
国家海洋局青年海洋科学基金(2007111)
海洋公益性行业科研专项经费(201005016)
关键词
褐菖鲉
黄鳍鲷
黑鲷
肝微粒体EROD
0#柴油WSF
原油WSF
Sebastiscus marmoratus
Sparus latus
Sparus macrocephalus
EROD activity
O# diesel oil WSF
crude oil WSF.
作者简介
通讯作者Corresponding author．E-mail：zhangyusheng@tio．org．cn
