人支气管上皮细胞5-脂氧合酶介导4-氨基联苯的活化及DNA损伤

目的探讨人支气管上皮(HBE)细胞内5-脂氧合酶(5-LOX)对4-氨基联苯(4-ABP)的氧化活化及所致DNA损伤,为LOX作为前致癌物氧化活化的代谢途径提供依据。方法①体外酶系统实验:4-ABP在含有大豆脂氧合酶(SLO)的体外酶体系中反应,用分光光度法检测体系中反应产物生成。②细胞实验:4-ABP 100～800μmol.L-1染毒HBE细胞,MTT法检测HBE细胞存活率;Western印迹法检测5-LOX蛋白表达;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蛋白表达和多种酶抑制剂对细胞存活率和DNA损伤的影响。结果在过氧化氢参与下,SLO可以协同氧化4-ABP,LOX抑制剂去甲二氢愈创木酸可抑制该协同氧化作用。4-ABP可以使HBE细胞内5-LOX蛋白表达增加,AA861对5-LOX蛋白表达没有影响;4-ABP 400μmol.L-1可以使HBE细胞产生明显的DNA损伤,彗星细胞的阳性率达47.7%(P<0.01),AA861和萘普生可以抑制该浓度4-ABP所致的DNA损伤,最大保护率分别为58.1%和21.7%。结论 4-ABP上调HBE的5-LOX蛋白表达。5-LOX可能通过介导4-ABP协同氧化,导致DNA损伤,这可能是4-ABP致癌的机制之一。

Activation of 4-aminobiphenyl mediated by 5-lipoxygenase and DNA damage in human bronchial epithelial cells

OBJECTIVE To investigate the effect of 4-aminobiphenyl(4-ABP) on 5-lipoxygenase(5- LOX) protein expression, cytotoxicity and DNA damage in human bronchial epithelial (HBE) cells, and to provide envidence that LOX is a pathway for oxidation and activation of precarcinogens. METHODS ① Enzymatic experiment: soybean lipoxygenase (SLO)，substrate (4-ABP) and other components reacted in an enzymic system; the product was detected with spectrophotometry. ② Cellular experiment: The effect of 4-ABP on the cellular survival rate was assessed by reduction of tetrazolium dye(MTT) in cultured HBE cells. After HBE cells were exposed to 4-ABP 100-800 μmol·L^-1 for 4 h, the protein expression of 5-LOX in HEB cells was tested by Western blotting, and DNA damage by single cell gel electrophoresis. Finally the effect of a specific inhibitors of 5-LOX, AA861, on 5- LOX protein expression and DNA damage in the cells was detected. RESULTSSLO catalyzed the co-oxidation of 4-ABP in the presence of hydrogen peroxide. Nordihydroguaiaretic acid(NDGA) inhibited the oxidation of 4-ABP by SLO, seemingly in a concentration- dependent manner and with in a special range. 4-ABP induced 5-LOX protein expression, but AA861 was invalid in HBE. 4-ABP caused toxic action and DNA damage in HBE, as the positive rate comet cells was increased to 47.7% by 4-ABP at the concentration of 400 μmol·L^-1. Such damage could be significantly inhibited by AA861 and naproxen with a maximum rate of protection of 58.1% and 21.7%, respectively. CONCLUSION 4-ABP can regulate 5-LOX protein expression in HBE cells. The co-oxidation of 4-ABP with 5-LOX could induce DNA damage, which could be one of the mechanisms for carcinogenesis of 4-ABP.