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mRNA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。
关键词： 鲫鱼;辛硫磷;肝微粒体;细胞色素P4501a（CYP1A）;活性;表达
中图分类号： S965.117 文献标志码： A 文章编号：2095-1191（2019）10-2329-06
Effects of phoxim on the activity and expression of CYP1A in liver microsomes of Carassius auratus gibebio（crucian carp）
LI Si1， LIU Xiao-yu1，2*
（1College of Food Science and Technology， Huazhong Agricultural University， Wuhan 430070， China;
2Key Laboratory of Environment Correlative Dietology， Ministry of Education， Wuhan 430070， China）
Abstract：【Objective】The effects of phoxim on the activity， mRNA and protein expression of cytochrome P4501a（CYP1A） 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 high-dose 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. CYP1A activity was reflected by the activity of CYP1A-related enzyme 7-ethoxy-3-ethoxyresorufin-O-deethylase（EROD）. The mRNA and protein expression of CYP1A 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 CYP1A， 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 CYP1A 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 CYP1A 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 CYP1A 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 CYP1A 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 CYP1A in the liver microsomes of crucian carp and down-regulate the mRNA and protein expression of CYP1A. Compared with the dose-dependent effect， phoxim has a more obvious time-dependent manner on the activity and protein expression of CYP1A.
推荐访问： 微粒 鲫鱼 活性 表达 水平