Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1
Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1
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DOI：
10.1093/carcin/20.6.1121
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发表时间：
1999-06-01
期刊：
影响因子：
4.7
通讯作者：
Townsend, AJ
中科院分区：
文献类型：
--
作者：
Fields, WR;Morrow, CS;Townsend, AJ
黄曲霉毒素B(1)对黄曲霉毒素B(1)的一种强致突变和致癌代谢产物黄曲霉毒素B(1)-8，9-氧化物(AFB(1)-8，9-Oxate，AFB(1))激活。在小鼠体内，研究了GST对α-谷胱甘肽S转移酶(GST)mGSTA3-3(MGST-YC)的有效解毒作用。将GST转染大鼠细胞色素P450-2B1的仓鼠V79细胞(V79MZr2B1，原名V79/SD1)，研究了GST对AFBO细胞毒性或遗传毒性的化学预防作用。免疫印迹显示，在mGSTA3-3转染组细胞中有较强的α-类GST蛋白表达，而对照组(空载体转染组)细胞中未检测到α-类GST蛋白的表达。以往对V79MZr2B1细胞系的研究表明，它可以通过稳定表达的大鼠P450-2B1蛋白将AFB(1)激活为诱变代谢物。我们检测了表达的mGSTA3-3对黄曲霉毒素B(1)诱导的细胞毒性或细胞核酸中[H-3]共价加合物形成的保护作用。空白对照细胞和表达mGSTA3-3的细胞暴露于600 nM的[H-3]-AFB(1)表明，mGSTA3-3的表达使DNA和RNA加合物减少了70%-80%。克隆存活实验表明，mGSTA3-3细胞对AFB(1)细胞毒的抗性是空载体对照组SD1细胞的4.6倍，IC50值分别为69和15mU M。这些研究的结果表明，在这个转基因细胞模型系统中，mGSTA3-3对AFB(1)的核酸共价修饰和细胞毒性具有实质性的保护作用。
Aflatoxin B-1 (AFB(1)) is activated to AFB(1)-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB(1), In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc), A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO, Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells, Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB(1) to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB(1)-induced cytotoxicity or [H-3]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [H-3]-AFB(1) indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB(1) cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 mu M, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB(1) in this transgenic cell model system.