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  

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

作　　者：谭庆平[1,2] 胡建安[1] 黄云[1] 武越[1] 熊敏如[1] 

机构地区：[1]中南大学公共卫生学院劳动卫生与环境卫生学系,长沙410078 [2]深圳市宝安区大浪卫生监督所

出　　处：《中华劳动卫生职业病杂志》2009年第1期25-29,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：教育部高等学校博士学科点专项科研基金（200605330210）

摘　　要：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、30．0μmol／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1．42nmol／（min·nmol）SLO，BZD的米氏常数（Km）值为1．48mmol/L。LOX抑制剂去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，产生亲电子自由基，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。Objective To investigate the effect of intracellular 5-lipoxygenase on oxidation of benzidine in HBE cells and to provide further evidence that lipoxygenase is an alternative pathway for the oxidation of xenobioties 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- lipoxygcnase in HBE cells were assessed by Western-blot, and the DNA damage by the single cell gel electrophoresis. At last,the effect of the specific inhibitor of 5-lipoxygenase （AA861） on 5-lipoxygenase 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 value of the oxidation of benzidine catalyzed by SLO was 1.42 nmol·min^-1 SLO, and the Km value of benzidine was 1.48 mmo/L. EGCG could inhibit the oxidation of benzidine by SLO. Benzidine could induce 5-lipoxygenase 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-oxidation of benzidine by 5-LOX could produce into electrophile 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.

关 键 词：5-脂氧合酶 上皮细胞 联苯胺类 DNA损伤 

分 类 号：R686[医药卫生—骨科学]