Effect of lead exposure on copper metabolism in glioma cells and its mechanism / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To study the roles of copper transporter 1 (CTR1 )and Cu2 + transporting ATPase αpolypeptide (ATP7A)in lead exposure-induced copper accu mulation and oxidative stress in rat C6 glio ma cells.METHODS Methyl thiazolyl tetrazoliu m (MTT)assay was performed to determine the proper Pb doses (without affecting cell viability)by treated the cells with lead acetate 0 -100 μmol·L -1 for 24 h and 48 h.Superoxide dis mutase (SOD)activity or malondialdehyde(MDA)level were detected respectively by xanthine oxidase technique and thiobarbituric acid (TBA) method.Ato mic absorption spectrophoto metry was e mployed to determine the intracellular levels of Pb and Cu ions.Real-ti me quan-titative PCR and Western blot were used to detect the mRNA and protein levels of CTR1 and ATP7A, respectively.RESULTS The cell viability significantly decreased when the doses of Pb treat ment was higher than 10 μmol·L -1 ,so 10 μmol·L -1 was chosen as a working concentration of Pb exposure in this study.Co mpared with those in the normal controls,a moderately decreased T-SOD activity and an increased MDA level was determined in the cells treated with Pb 10 μmol·L -1 or Cu 5 μmol·L -1 alone, while a significant drop of T-SOD activity and a re markable increase of MDA level was found in the cells co-exposed to Pb and Cu (P<0.01 ).Pb exposure for 24 and 48 h increased the cellular Cu uptake by 1 .2 and 2.5 fold,respectively (P <0.01 ).Evidences fro m RT-PCR showed that Pb exposure for 24 and 48 h upregulated the CTR1 mRNA level by 23.2% and 58.7%,and downregulated the ATP7A mRNA level by 58.1 % and 50.0%,respectively.Results fro m Western blot confirmed that Pb exposure also resulted in an increased CTR1 expression and a decreased ATP7A expression at protein level (P<0.01 ).CONCLUSION Pb exposure lead to Cu accu mulation,by affecting the expression levels of CTR1 and ATP7A,and increased oxidative stress in C6 cells.