CAJ | 학술논문

目的探讨细胞内5-脂氧合酶(5-LOX)对苯并(a)芘(B[a]P)的代谢活化及DNA损伤的影响,为脂氧合酶(LOX)作为外源化学物氧化代谢的替代途径提供进一步依据.方法体外酶系统实验:B(a)P在含有大豆脂氧合酶(SLO)的酶体系中反应,用分光光度法检测其氧化产物;酶系统中加入DNA,用高效液相色谱仪检测其DNA加合物.细胞实验:人支气管上皮细胞(HBE)染毒24 h,B[a]P剂量分别为4、8、16、32、64、128 μmol/L,抑制剂(AA-861)、萘普生和α-萘黄酮分别为0.1、1、10 μmol/L,用免疫印迹法(western-blot)法检测各组5-LOX蛋白表达情况;用流式细胞计数及单细胞凝胶电泳实验检测各组的细胞毒性及DNA损伤情况;同时,检测特异性5-LOX抑制剂(AA-861)及其他特异性酶抑制剂(萘普生和α-萘黄酮)对5-LOX蛋白表达和对DNA损伤的影响.结果在过氧化氢(H2O2)参与下,SLO可以协同氧化B[a]P,氧化生成B[a]P-7,8-环氧化物,在一定范围内呈剂量效应关系.GTP能够抑制SLO介导的B[a]P氧化,其IC50为0.46 mg/L.SLO介导B[a]P活化后,生成新的产物,出现一明显新增峰,但本次在体外未能检测到与DNA形成加合物.随着B[a]P染毒浓度的增加,HBE细胞存活率逐渐下降,AA-861和萘普生抑制后,细胞存活率上升,差异均有统计学意义(P＜0.05).B[a]P可以使HBE内5-LOX蛋白表达增加,且表达量随B[a]P浓度增加而增加,5-LOX抑制剂AA-861对5-LOX蛋白表达基本无影响;流式细胞计数及单细胞凝胶电泳实验显示,各B[a]P染毒组DNA损伤指标随着B[a]P浓度增加而加重,与阴性对照组比较,差异有统计学意义(P＜0.05).与64 μmol/L B[a]P组比较,加入抑制剂AA-861、萘普生和α-萘黄酮后DNA损伤程度逐渐减轻,差异均有统计学意义(P＜0.05).结论在HBE内,B[a]P可能主要通过5-LOX介导的协同氧化,形成亲电子物质与DNA结合,使HBE产生DNA损伤,AA-861可以明显抑制该反应.
목적 탐토세포내5-지양합매(5-LOX)대분병(a)비(B[a]P)적대사활화급DNA손상적영향,위지양합매(LOX)작위외원화학물양화대사적체대도경제공진일보의거.방법 체외매계통실험:B(a)P재함유대두지양합매(SLO)적매체계중반응,용분광광도법검측기양화산물;매계통중가입DNA,용고효액상색보의검측기DNA가합물.세포실험:인지기관상피세포(HBE)염독24 h,B[a]P제량분별위4、8、16、32、64、128 μmol/L,억제제(AA-861)、내보생화α-내황동분별위0.1、1、10 μmol/L,용면역인적법(western-blot)법검측각조5-LOX단백표체정황;용류식세포계수급단세포응효전영실험검측각조적세포독성급DNA손상정황;동시,검측특이성5-LOX억제제(AA-861)급기타특이성매억제제(내보생화α-내황동)대5-LOX단백표체화대DNA손상적영향.결과 재과양화경(H2O2)삼여하,SLO가이협동양화B[a]P,양화생성B[a]P-7,8-배양화물,재일정범위내정제량효응관계.GTP능구억제SLO개도적B[a]P양화,기IC50위0.46 mg/L.SLO개도B[a]P활화후,생성신적산물,출현일명현신증봉,단본차재체외미능검측도여DNA형성가합물.수착B[a]P염독농도적증가,HBE세포존활솔축점하강,AA-861화내보생억제후,세포존활솔상승,차이균유통계학의의(P＜0.05).B[a]P가이사HBE내5-LOX단백표체증가,차표체량수B[a]P농도증가이증가,5-LOX억제제AA-861대5-LOX단백표체기본무영향;류식세포계수급단세포응효전영실험현시,각B[a]P염독조DNA손상지표수착B[a]P농도증가이가중,여음성대조조비교,차이유통계학의의(P＜0.05).여64 μmol/L B[a]P조비교,가입억제제AA-861、내보생화α-내황동후DNA손상정도축점감경,차이균유통계학의의(P＜0.05).결론 재HBE내,B[a]P가능주요통과5-LOX개도적협동양화,형성친전자물질여DNA결합,사HBE산생DNA손상,AA-861가이명현억제해반응.
Objective The oxidation of benzo(a) pyrene mediated by 5-lipoxygenase (5-LOX) were investigated in HBE ceils in order to provide further proof that lipoxygenase is the alternative pathway for the oxidation of xenobiotics.Methods Enzymic experiment:Soybean lipoxygenase (SLO),substrate (benzo[a] pyrene) and other component react in the enzymic system and the reaction product are detected by spectrophotometry.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 poison (B[a]P 4、8、16、32、64、128μmol/L,AA-861、naproxen or α-naphthoflavone 0.1、1、10 μmol/L) for 24 hours,the effect of benzo (a)-pyrene on cell survival rate were assessed by reductions of tetrazolium dye (MTT) and flow cytornetry 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.And then,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 reaction,the IC50 value is 0.46 mg/L,the model equation is Probit (P) =0.8985 +2.6824Log (dose).SLO can catalyze the co-oxidation of benzo(a) pyrene to generate a new product,but fail to form DNA adducts in vitro.HBE cell viability decreased with the benzo(a) pyrene concentration increased,but AA-861 and naproxen can inhibit it.Flow cytometry and single cell gel electrophoresis experiments show,Benzo(a)pyrene can induce 5-lipoxygenase protein expression,but AA-861 cannot in HBE.Benzo(a)pyrene causes toxic action and DNA damage in HBE,which can significantly inhibit by AA-861,the difference is statistically significant(P＜0.05).Conclusions 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.