慢性染砷大鼠肝脏线粒体DNA及相关酶活性变化研究

Study of changes in mitochondrial DNA and activity of the correlative enzymes of hepatic cell in chronic arsenism rats

目的 探讨慢性染砷大鼠肝脏线粒体DNA（mtDNA）及线粒体相关酶，特别是mtDNA编码酶活性的变化。方法 Wistar大鼠经饮水给75mg／L三氧化二砷6个月。用分光光度法测定肝脏线粒体呼吸酶活性：碱变性法提取大鼠肝脏mtDNA；以PCR大片段扩增法扩增长片段mtDNA；用HindⅢ、EcoRⅠ、SalⅠ、BamHⅠ、MspⅠ、MboⅠ、StuⅠ7种限制性内切酶对扩增片段进行酶切分析。结果 染砷大鼠肝脏线粒体细胞色素C氧化酶（CytCOX）和ATP酶活性下降；而经mtDNA扩增片段凝胶电泳分析和7种限制性内切酶酶切分析，未发现染砷大鼠肝脏参与编码上述2种线粒体酶的4151bp片段有碱基缺失、重复和突变等异常表现。结论 慢性染砷能够导致肝细胞mtDNA参与编码的CytCOX和ATP酶活性下降，进一步证实了染砷对肝细胞及其线粒体的损害作用。但其活性下降的机制，目前尚不能以砷致mtDNA片段的变化来解释，需进一步进行DNA序列分析研究。

Objective To explore the changes of mitochondrial DNA（mtDNA） and activity of the correlative enzymes, especially those coded by mtDNA, in livers of chronic arsenism rats. Methods 75 mg/L arsenic trioxide was administered to Wistar rats in drinking water for 6 months. The activities of respiratory enzymes of hepatic mitoehondria were determined by speetrophotometer, mtDNA was abstracted from hepatic cell using alkaline denaturation method, and the amplification of the long segment of mtDNA was performed with long-extension PCR. The PCR segments of mtDNA amplified were analyzed with digestion of restriction endonucleases： Hind Ⅲ, EcoR Ⅰ , Sal Ⅰ, BamH Ⅰ , Msp Ⅰ , Mbo Ⅰ , Stu Ⅰ , respectively. Results The activities of hepatic mitochondrial cytochrome C oxidase （CytC OX） and adenosine triphosphatease （ATPase） were decreased. However, no mutation and deletion and duplication had been detected in the amplifying segments of mtDNA 4151 bp containing the coding regions of the CytC OX and ATPase. Conclusions The chronic arsenism could result in the declined activities of the mitochondrial CytC OX and ATPase coded partly by mtDNA, which further demonstrate the damage to hepatic cell and its mitochondria from arsenism. Since the declined activities could not be explained by the changes of mtDNA segments caused by arsenic, further DNA sequence analysis is needed.