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 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 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, numax 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 mmol/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.