CCl4对建鲤肝细胞DNA的毒性作用及对CYP3A基因表达的影响  

Toxicity effects of CCl_4 on DNA and expression of CYP3A gene in hepatocytes of Cyprinus carpio var. Jian

作　　者：曹丽萍[1,2] 贾睿[1,2] 杜金梁[1,2] 丁炜东[1] 殷国俊[1,2] 

机构地区：[1]中国水产科学研究院淡水渔业研究中心,农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心,鱼类免疫药理学国际联合实验室,江苏无锡214081

出　　处：《水产学报》2013年第10期1452-1459,共8页Journal of Fisheries of China

基　　金：国家自然科学基金项目[青年科学基金(31200918,31202002)];江苏省自然科学基金项目(BK2011184)

摘　　要：四氯化碳(CCl4)作为一种经典的肝脏毒物,被广泛应用于哺乳动物的肝损伤模型构建及保肝药物筛选。低、中、高浓度的CCl4-橄榄油溶液(6.25%、12.50%和15.00%,0.01 mL/g)腹腔注射建鲤72 h后,采用单细胞凝胶电泳和肝组织病理切片技术,测定血清中肝损伤生化酶来考察CCl4对肝细胞DNA的毒性作用;同时采用实时荧光定量PCR法测定肝组织中CYP3A表达量的变化。结果显示,6.25%CCl4作用建鲤72 h后肝组织切片检查没有发生明显的改变,对血清中酶学指标无显著影响,CYP3A的mRNA表达量与空白对照组相比也没有显著变化,而彗星实验结果显示该浓度的CCl4作用肝细胞后,尾长、TDNA、尾矩及Olive尾矩等DNA损伤指标与对照组相比,显著增大;随着CCl4作用浓度的增加,肝细胞肿胀、广泛空泡变性,出现核固缩和核溶解等组织学的变化,12.50%和15.00%CCl4均能显著引起血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)水平升高,15.00%CCl4能显著促进乳酸脱氢酶(LDH)和丙二醛(MDA)大量生成;彗星实验中各项损伤指标也随着染毒剂量增加而极显著增大;同时,12.50%和15.00%CCl4组的CYP3A mRNA表达量显著降低。结果表明,CCl4对建鲤肝细胞DNA具有毒性作用,在实验的浓度范围内,CCl4能抑制CYP3A mRNA表达;随着CCl4作用浓度的增大,血清酶、病理切片和彗星实验结果表现出剂量效应,并有一定的一致性,彗星实验表现出更高的灵敏性。Carbon tetrachloride（CCl4）has been widely used as a kind of classic liver poison in constructing mammalian liver injury model and screening liver protecting drug.To investigate toxicity effects of CCl4 on hepatocyte DNA of Cyprinus carpio var.Jian,the alkaline single-cell gel electrophoresis（comet assay）,histopathological examination of liver and the measuring of biomarkers of hepatocyte damage were performed respectively after low,middle and high concentrations（6.25% and 12.50% and 15.00% CCl4 in olive oil,v/v）of CCl4 at a volume of 0.01 mL/g of body weight being injected intraperitoneally into C.carpio var.Jian for 72 h.Meanwhile,the Real-time PCR was carried out to elevate the effects of CCl4 on CYP3A mRNA expression.Results showed that the group injected with 6.25% of CCl4 for 72 h has no obvious change（P〉0.05）on histopathological examination,serum hepatase activities and mRNA expression of CYP3A,compared to the control,however,the comet assay results of hepatic cells showed that tail length,tail DNA（%）,tail moment and olive tail moment increased significantly（P〈0.01）.With the increase of concentrations,histological changes such as cell swelling,extensive vacuoles degeneration,karyopyknosis and karyolysis were observed.The groups injected with 12.5% and 15% of CCl4 could significantly elevate the serum levels of glutamate oxalate transaminase（GOT）and glutamate pyruvate transaminase（GPT）（P〈0.01 or P〈0.05）,and the group with 15.00% of CCl4 produced a significant effect in the case of lactate dehydrogenas（LDH）and malondialdehyde（MDA）（P〈0.05）.All damage indexes of comet assay also increased with the increase of CCl4 concentrations（P〈0.01）.At the same time,the expressions of CYP3A of the groups injected with 12.5% and 15% of CCl4 were significantly reduced（P〈0.05）.Overall results proved the toxicity effects of CCl4 on hepatocyte DNA of C.carpio var.Jian.Within the range of experimental concentrations,CCl4 could inhibit CYP3A mRNA expression.And

关 键 词：建鲤 四氯化碳 CYP3A 彗星实验 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]