人支气管上皮细胞5-脂氧合酶表达与联苯胺的活化及细胞毒性的研究

目的 研究人支气管上皮(HBE)细胞内5-脂氧合酶(5-LOX)对联苯胺(BZD)的氧化代谢,为LOX作为细胞色素P450氧化代谢外源化合物的替代途径提供进一步的依据.方法 体外酶系统实验:BZD在含有大豆脂氧合酶(SLO)的酶体系中反应,用分光光度法检测体系中反应产物.细胞实验:HBE细胞染毒24h(BZO剂量分别为100、200、500μmol/L,AA861剂量分别为0.3、3.0、30.0 μmol/L),用Western-blot检测5-LOX蛋白表达;用单细胞凝胶电泳检测DNA损伤.同时,检测特异性5-LOX抑制剂(AA861)对5-LOX蛋白表达和对DNA损伤的影响.结果 在过氧化氢参与下,SLO可以介导BZD氧化生成二亚胺联苯胺.最大反应速度(Vmax)值为1.42 nmol/(min·nmol)SLO,BZD的米氏常数(Km)值为1.48 mmol/L.LOX抑制剂上甲二氢愈创木酸可抑制该协同氧化作用.BZD可以使HBE细胞5-LOX蛋白表达增加,AA861对5-LOX蛋白表达无影响;BZO可以使HBE细胞产生明显的DNA损伤,AA861可以明显抑制BZD所致的DNA损伤,且有剂量-效应关系.结论 5-LOX在HBE细胞内的蛋白表达可以受BZD的影响.HBE细胞的5-LOX可能通过介导BZD协同氧化,产生亲电子白南基,而与DNA结合,使HBE细胞产生DNA损伤,这可能是BZD致癌的机制之一;AA861可以明显抑制这种作用.

Protein expression of 5-1ipoxygenase and activation and cytotoxicity of Benzidine in human bronchial epithelial cells

Objective To investigate the effect of intracellular 5-1ipoxygenase on oxidation of benzi-dine in HBE cells and to provide further evidence that lipoxygenase is an alternative pathway for the oxidation of xenobiotics mediated by cytochrome P450.Methods Enzyme system test:Soybean lipoxygenase (SLO),substrate(benzidine) and other components reacted in the enzyme system,followed by detection of the reaction products by spectrophotometry.In vitro test:After HBE cells were exposed to benzidine,the protein levels of 5-lipoxygenase in HBE cells were assessed by Westem-blot,and the DNA damage by the single cell gel elec-trophoresis.At last,the effect of the specific inhibitor of 5-1ipoxygenase (AA861) on 5-1ipoxygenase protein expression and DNA damage in HBE cells were detected.Results SLO could catalyze the co-oxidation of benzidine to generate benzidine diimine in the presence of hydrogen peroxide.Under optimal condition,vmax was 1.48 mmo/L.EGCG couhl inhibit the oxidation of henzidine by SLO.Benzidine could induce 5-1ipoxyge-nase protein expression in HBE cells,but AA861 was invalid.Benzidine caused DNA damage in HBE cells,which could be significantly inhibited by AA861.Conclusion 5-LOX protein expression in HBE cells can be regulated by benzidine,which suggests that the co-uxidation of benzidine by 5-LOX could produce into elec-trophile that could covalently bind to DNA and induce DNA damage,which could be one of the mechanisms for carcinogenesis of BZD.5-LOX inhibitor AA861 can inhibit this effect.