The Activation And Cytotoxicity Of Benzo(a) Pyrene Mediated By 5-lipoxygenase In Human Bronchial Epithelial Cell

[Objectives] The effects of benzo(a)pyrene to 5-lipoxygenase (5-LOX) protein expression and cytotoxicity and DNA damage were been investigated in HBE cells, with the experiment that can affirm the co-oxidation of benzo (a) pyrene mediated by SLO in the vitro-enzymic system, in order to approach the metabolism of xenobiotic by 5-LOX and 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 (benzo[a]pyrene) and other component react in the enzymic system, and followed by detecting the reaction product in the reaction medium by spectrophotometry. The oxidative rate of benzo(a)pyrene catalyzed by SLO and the inhibition ratio of the GTP for the oxidation are calculated, respectively. At the same time, in vitro detect of benzo(a)pyrene-DNA adducts with a UV spectrophotometer and HPLC.Cellular experiment:After HBE cells exposure to different concentrations of benzo(a)pyrene, the effect of benzo(a)-pyrene on cell survival rate were assessed by reductions of tetrazolium dye(MTT) and flow cytometry in cultured HBE cells,and 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 last, the effect of the specific inhibitor of 5-lipoxygenase (AA-861) on 5-lipoxygenase protein expression and DNA damage in the cells are detected.[Results] SLO can catalyze the co-oxidation of benzo(a)pyrene to generate benzo(a)pyrene-7,8-epoxide in the presence of hydrogen peroxide. GTP can inhibit the oxidation of benzo(a)pyrene by SLO, which appears to concentration-dependent manner in special range. SLO can catalyze the co-oxidation of benzo(a)pyrene, but fail to form DNA adducts in vitro. Benzo(a)pyrene can induce 5-lipoxygenase protein expression, but AA-861 can not in HBE. Benzo(a)pyrene causes toxic action and DNA damage in HBE, which can significantly inhibit by AA-861 and naproxen.[Conclusions] In the presence of hydrogen peroxide, SLO can mediate benzo (a) pyrene co-oxidation, which can be inhibited by GTP. In vitro, only with SLO, B(a)P metabolites cannot adduct with DNA. 5-LOX protein expression in HBE can be regulated by benzo (a) pyrene, and specific inhibitor of 5-LOX (AA-861) has no obvious effect on the expression. The co-oxidate of benzo(a)pyrene by 5-LOX turns into electrophiles that covalently bind to DNA and induce DNA damage, which can be significantly inhibited by AA-861.