抗氧化酶基因作为多环麝香污染分子标志物研究

Antioxidant Enzyme Gene Expression as Molecular Biomarkers of Exposure to Polycyclic Musks

为研究土壤中低剂量合成麝香暴露的分子毒理效应,以蚯蚓超氧化物歧化酶(SOD)和过氧化氢酶(CAT)基因为供试基因,建立mRNA表达水平的Sybr GreenⅠ荧光定量PCR检测方法;并且采用自然土壤染毒实验,检测了吐纳麝香(AHTN)或佳乐麝香(HHCB)胁迫诱导SOD、CAT mRNA的表达水平变化.序列同源性比较与熔解曲线分析表明设计的引物适合供试基因mRNA的检测.SOD与CAT基因构建的荧光定量标准曲线的线性关系分别为0.997和0.994,且PCR扩增效率均接近于100%,故可实现两基因mRNA表达的相对定量分析.土壤染毒暴露28 d后,丙二醛(MDA)含量水平显著升高,表明自由基诱导的细胞氧化损伤是AHTN或HHCB毒性效应的主要途径方式.SOD、CAT mRNA总体表现为上调表达趋势,且表达量与MDA含量呈显著正相关,表明抗氧化酶基因的诱导表达与氧化应激水平有关联.而且,SOD、CAT mRNA表达水平与AHTN或HHCB浓度存在正相关剂量-效应关系.综上表明,SOD与CAT基因可成为潜在的分子生物标志物,用于表征合成麝香污染暴露水平及其毒理效应.

The objective of this study was to investigate the molecular toxicological effects of low level synthetic musks exposure on the earthworm Eisenia fetida.The method of Sybr Green Ⅰ real time quantitative PCR（RT-qPCR） for detecting gene expression level was established.SOD（superoxide dismutase） and CAT（catalase） mRNA expression levels were measured after 28 days of AHTN and HHCB exposure.The analysis results of both sequence alignment and melting curve demonstrated that the selected primers were suitable for mRNA quantification.The liner correlation coefficients of SOD and CAT standard curves were 0.997 and 0.994,respectively,and the PCR amplification efficiencies were both close to 100%.Therefore,relative quantification method could be applied to analyze the gene expression levels.The significant elevation of malondialdehyde（MDA） levels indicated that the reactive oxygen species-induced cellular oxidative injury might be one of the main toxic effects of AHTN and HHCB.Besides,a significant positive correlation was observed between the up-regulation of SOD,CAT mRNA and the MDA levels,suggesting that possible changes in the transcript expression of antioxidant enzyme genes were associated with the oxidative stress.Furthermore,the dose-response correlation between SOD,CAT mRNA levels and the exposure concentrations was also found.The overall results indicated that SOD and CAT genes might be potential molecular biomarkers for the evaluation of the pollution stress and toxicological effects of synthetic musks in the environment.