砷对NIH 3T3细胞谷胱甘肽水平、相关酶活性及其基因表达的影响

Effect of Arsenic on GSH level:Its Related Enzyme Activity and Gene Expression in NIH 3T3 Cells

[目的]探讨急性、慢性砷暴露对小鼠成纤维细胞 (NIH3T3细胞 )氧化还原状态的影响 ,进而探讨其对细胞谷胱甘肽 (GSH)水平、相关酶活性及其基因表达的影响。[方法]将经过不同浓度砷处理的细胞 ,抽提细胞总蛋白。采用经过改进的Clark法测定细胞中谷胱甘肽 ;采用Thomas法测定谷胱甘肽还原酶 (GR)活性 ;使用分光光度法测定谷胱甘肽与1 氯 2 ,4 二硝基苯 (CDNB)的结合以确定谷胱甘肽转移酶 (GST)活性。采用SDS聚丙烯酰胺凝胶电泳和固定化蛋白质的免疫学测定法测定血红素氧化酶 (HO 1)蛋白表达 ;采用RNA印迹法测定谷胱甘肽还原酶 (GR)和谷胱甘肽过氧化物酶(GSH Px)的基因表达。[结果]急性砷处理细胞在较低浓度范围可以引起GSH水平的增加 ;而当砷浓度高于25μmol/L时 ,GSH水平降低 ;在0.1和0.5 μmol/L慢性染毒之后细胞GSH水平明显降低。然而 ,在急性和慢性砷处理组GST和GR的活性均有升高 ,而在高浓度24h处理组则显著降低 ;GR的mRNA水平与GR的活性相一致 ,在急、慢性染毒组中均表现为GR的mRNA表达上调 ;砷可以引起血红素氧化酶 1(HO 1)蛋白水平的增加。急性、慢性砷处理均引起GSH Px mRNA表达下调。[结论]三价砷可以引起氧化应激、GSH水平改变、以及相关酶活性及其基因表达的改变 ,急性与慢性砷接触对GSH系统?

ObjectiveTo investigate the arsenic effect on glutathione(GSH)level and related gene expression in NIH3T3cells treated with arsenic both in short term as well as long term.[Methods]Cell extracts were obtained after treatment for certain time for enzyme activityassay.Glutathione concentrationwas determined byusingClarke method.Glutathione transferase(GST)activitywas deter-mined by assessing the formation of conjugated glutathione and CDNB.Glutathione reductase(GR)was assayed using protocols described by Styblo and Thomas.Protein expression ofglutathione transferase(GST)and hemeoxygenase(HO-1)were determined by Western Blot Assay;The gene expression of GR,GST-P x ,were determined using Northern Blot Assay.[Results] Acute treatment with0.1～10μmol/Lof As(Ⅲ)increased GSHlevel in NIH3T3cells,but decreased in25μmole/Lof As(Ⅲ)group;while chronic treatˉment with0.1and0.5μmol/L of As(Ⅲ)decreased GSH level.However,GSTand GR activity increased both in acute and chronic treatment with arsenic.GSTand GR activity at high dose(25μmole/L As(Ⅲ))with short termtreatment decreased which was consistent with GSH level;mRNA level of GR was also consistent with its enzyme activity,increased both after acute and chronic treatment,while mRNA level of GSHˉP x decreased both after short term and long-term treatment with arsenic.This was the first term to investigate GSTand GR activity both in short-termand longˉtermtreatment with arsenic.Western Blot assay showed that arsenic induced HO_1.[Conclusion]Arsenic may alter GSHlevel and related enzyme activityboth after shotˉtermtreatmentand longˉtermtreatmentwith arsenic,butwithdifferentmanneraccordingtodifferentdoseandtimecourse.ThealterationofGSHlevel,itsrelatedenzymes activityandgeneexpresˉsionafterlowdose andlongˉtermtreatmentwitharsenic mightplayanimportantroleinthe mechanismofarsenicˉinducedtoxicityand carcinoˉgenesis.