【摘要】 【目的】研究辛硫磷暴露对鲫鱼肝微粒体中细胞色素P4501a(CYP1A)活性及其mRNA和蛋白表达的影响,明确其剂量—效应及时间—效应关系,为揭示有机磷农药对水生生物的代谢调节机制提供理论依据。【方法】设辛硫磷低、中、高浓度组(0.0825、0.165和0.33 mg/L)和丙酮(助溶剂)对照组,采用半静态染毒法染毒鲫鱼,分别于染毒24、48、72和96 h后取样并迅速剖解取出鲫鱼肝胰脏,以CYP1A相关酶7-乙氧基-3-异吩唑酮-脱乙基酶(EROD)活性反映CYP1A活性,采用实时荧光定量PCR和Western blotting分别测定鲫鱼肝微粒体中CYP1A基因mRNA及其蛋白的表达情况。【结果】辛硫磷对鲫鱼肝微粒体中CYP1A活性产生抑制作用,辛硫磷染毒24 h时CYP1A活性与辛硫磷存在明显的剂量—效应关系;各辛硫磷浓度组间均存在明显的时间—效应关系,至染毒96 h时低、中、高浓度组鲫鱼肝微粒体中CYP1A活性分别较对照组下降54.7%、30.4%和65.5%,且差异极显著(P<0.01,下同)。辛硫磷染毒后,鲫鱼肝微粒体中CYP1A基因m RNA的表达整体上呈明显下调趋势,各染毒时间组间均存在较强的剂量—效应关系,除染毒48 h时CYP1A基因mRNA相对表达量与辛硫磷浓度呈正相关外,其他染毒时间点均呈负相关。辛硫磷对鲫鱼肝微粒体中CYP1A蛋白表达的影响均达极显著水平,且呈明显的剂量—效应及时间—效应关系,至染毒96 h时低、中、高浓度组的表达量分别较对照组降低74.6%、82.6%和85.7%。【结论】辛硫磷能抑制鲫鱼肝微粒体中CYP1A活性并下调CYP1A基因mRNA及其蛋白的表达;相对于辛硫磷产生的剂量—效应影响,其对CYP1A活性及其蛋白表达的时间—效应影响更明显。更多还原

【Abstract】 【Objective】The effects of phoxim on the activity,mRNA and protein expression of cytochrome P4501 a(CYP1 A)in crucian carp liver microsomes were investigated,and the dose-dependent and time-dependent relations were studied to provide basis for further exploring the regulatory effects and mechanisms of organophosphorus pesticide on hydrobios.【Method】Semi-static exposure method was used in the experiment to infect crucian carp. Low-,mid-and highdose groups of phoxim(0.0825、0.165、0.33 mg/L)and acetone(solvent)control group,were set up. The crucian carps were sampled and dissected after 24,48,72 and 96 h of infection,then hepatopancreas was taken out. CYP1 A activity was reflected by the activity of CYP1 A-related enzyme 7-ethoxy-3-ethoxyresorufin-O-deethylase(EROD). The mRNA and protein expression of CYP1 A gene in crucian carp liver microsomes were detected by real-time fluorescence quantitative PCR and Western blotting,respectively.【Result】Phoxim had inhibitory effect on the activity of CYP1 A,it had obvious dose-dependent manner exposed to phoxim after 24 h and had obvious time-dependent manner in different concentration groups. The activities of CYP1 A decreased by 54.7%,30.4% and 65.5% in low-,mid-and high-dose groups respectively compared with the control group after 96 h and the difference was extremely significant(P<0.01,the same below). The mRNA expression of CYP1 A was significantly down-regulated in crucian carp liver microsomes after infection,and there was a obvious dose-dependent manner at different exposure times. The mRNA expression level of CYP1 A was positively correlated with the phoxim concentration after 48 h but negatively correlated with concentration in other exposure times.The effects of phoxim on protein expression of CYP1 A in crucian carp liver microsomes all reached extremely significant level,and presented dose-dependent and time-dependent relations. The expression levels after 96 h of exposure at low-,mid-and high-dose groups were decreased by 74.6%,82.6% and 85.7% respectively compared with the control.【Conclusion】Phoxim can inhibit the activity of CYP1 A in the liver microsomes of crucian carp and down-regulate the mRNA and protein expression of CYP1 A. Compared with the dose-dependent effect,phoxim has a more obvious time-dependent manner on the activity and protein expression of CYP1 A.更多还原