The Expression Of 5-lipoxygenase In Human Bronchial Epithelial Cell And The Activation And Cytotoxicity Of Benzidine

[Objectives] The enzymic effect of intracellular 5-lipoxygenase on co-oxidation of benzidine and effects of benzidine to 5-lipoxygenase protein expression and DNA damage were been investigated in HBE cells, with the study that can affirm the co-oxidation of benzidine mediated by SLO in the enzymic system, inorder to provide further proof that lipoxygenase is the alternative pathway for the oxidation of xenobiotics mediated by cytochrome P450.[Methods] Enzymic experiment: In vitro suitable reaction conditions soybean lipoxygenase (SLO), substrate (benzidine) and other component react in the enzymic system, and followed by detecting the reaction product and free radical intermediate in the reaction medium by spectrophotometry. The oxidative rate of benzidine catalyzed by SLO and the inhibitory rate of the EGCG for the oxidation are calculated, respectively.Cellular experiment: After HBE cells exposure to different concentrations of benzidine for 24 hours, the protein expressions of 5-lipoxygenase in the cells are tested by western-blot, and the DNA damages by the single cell gel electrophoresis. At the last, the effect of the specific inhibitor of 5-lipoxygenase(AA861) on 5-lipoxygenase protein expression and DNA damage in the cells are detected.[Results] SLO can catalyze the co-oxidation of bezidine to generate benzidine diimine in the presence of hydrogen peroxide. Under optimal condition, V_max value of the oxidation of bezidien catalyzed by SLO was 1.42nmolmin^-1 nmor^-1 SLO, And the Km value of bezidine was 1.48mmo·L^-1. EGCG can inhibit the oxidation of benzidine by SLO, which appears to concentration-dependent manner in special range. Benzidine can induce 5-lipoxygenase protein expression, but AA861 can not in HBE. Benzidine causes DNA damage in HBE, which can significantly inhibit by AA861.[Conclusions] In the presence of hydrogen peroxide, SLO can mediate benzidine co-oxidation, which can be inhibited by EGCG. 5-LOX protein expression in HBE can be regulated by benzidine, and specific inhibitor of 5-LOX (AA861) has no obvious effect on the expression. The co-oxidate of benzidine by 5-LOX turns into electrophiles that covalently bind to DNA and induce DNA damage, which can be significantly inhibited by AA861.