中华劳动卫生职业病杂志
中華勞動衛生職業病雜誌
중화노동위생직업병잡지
CHINESE JOURNAL OF INDUSTRIAL HYGIENE AND OCCUPATIONAL DISEASES
2009年
1期
25-29
,共5页
谭庆幸%胡建安%黄云%武越%熊敏如
譚慶倖%鬍建安%黃雲%武越%熊敏如
담경행%호건안%황운%무월%웅민여
5-脂氧合酶%上皮细胞%联苯胺类%DNA损伤
5-脂氧閤酶%上皮細胞%聯苯胺類%DNA損傷
5-지양합매%상피세포%련분알류%DNA손상
5-1ipoxygenase%Epithelial cells%Benzidines%DNA damage
目的 研究人支气管上皮(HBE)细胞内5-脂氧合酶(5-LOX)对联苯胺(BZD)的氧化代谢,为LOX作为细胞色素P450氧化代谢外源化合物的替代途径提供进一步的依据.方法 体外酶系统实验:BZD在含有大豆脂氧合酶(SLO)的酶体系中反应,用分光光度法检测体系中反应产物.细胞实验:HBE细胞染毒24h(BZO剂量分别为100、200、500μmol/L,AA861剂量分别为0.3、3.0、30.0 μmol/L),用Western-blot检测5-LOX蛋白表达;用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蛋白表达和对DNA损伤的影响.结果 在过氧化氢参与下,SLO可以介导BZD氧化生成二亚胺联苯胺.最大反应速度(Vmax)值为1.42 nmol/(min·nmol)SLO,BZD的米氏常数(Km)值为1.48 mmol/L.LOX抑制剂上甲二氢愈创木酸可抑制该协同氧化作用.BZD可以使HBE细胞5-LOX蛋白表达增加,AA861对5-LOX蛋白表达无影响;BZO可以使HBE细胞产生明显的DNA损伤,AA861可以明显抑制BZD所致的DNA损伤,且有剂量-效应关系.结论 5-LOX在HBE细胞内的蛋白表达可以受BZD的影响.HBE细胞的5-LOX可能通过介导BZD协同氧化,产生亲电子白南基,而与DNA结合,使HBE细胞产生DNA损伤,这可能是BZD致癌的机制之一;AA861可以明显抑制这种作用.
目的 研究人支氣管上皮(HBE)細胞內5-脂氧閤酶(5-LOX)對聯苯胺(BZD)的氧化代謝,為LOX作為細胞色素P450氧化代謝外源化閤物的替代途徑提供進一步的依據.方法 體外酶繫統實驗:BZD在含有大豆脂氧閤酶(SLO)的酶體繫中反應,用分光光度法檢測體繫中反應產物.細胞實驗:HBE細胞染毒24h(BZO劑量分彆為100、200、500μmol/L,AA861劑量分彆為0.3、3.0、30.0 μmol/L),用Western-blot檢測5-LOX蛋白錶達;用單細胞凝膠電泳檢測DNA損傷.同時,檢測特異性5-LOX抑製劑(AA861)對5-LOX蛋白錶達和對DNA損傷的影響.結果 在過氧化氫參與下,SLO可以介導BZD氧化生成二亞胺聯苯胺.最大反應速度(Vmax)值為1.42 nmol/(min·nmol)SLO,BZD的米氏常數(Km)值為1.48 mmol/L.LOX抑製劑上甲二氫愈創木痠可抑製該協同氧化作用.BZD可以使HBE細胞5-LOX蛋白錶達增加,AA861對5-LOX蛋白錶達無影響;BZO可以使HBE細胞產生明顯的DNA損傷,AA861可以明顯抑製BZD所緻的DNA損傷,且有劑量-效應關繫.結論 5-LOX在HBE細胞內的蛋白錶達可以受BZD的影響.HBE細胞的5-LOX可能通過介導BZD協同氧化,產生親電子白南基,而與DNA結閤,使HBE細胞產生DNA損傷,這可能是BZD緻癌的機製之一;AA861可以明顯抑製這種作用.
목적 연구인지기관상피(HBE)세포내5-지양합매(5-LOX)대련분알(BZD)적양화대사,위LOX작위세포색소P450양화대사외원화합물적체대도경제공진일보적의거.방법 체외매계통실험:BZD재함유대두지양합매(SLO)적매체계중반응,용분광광도법검측체계중반응산물.세포실험:HBE세포염독24h(BZO제량분별위100、200、500μmol/L,AA861제량분별위0.3、3.0、30.0 μmol/L),용Western-blot검측5-LOX단백표체;용단세포응효전영검측DNA손상.동시,검측특이성5-LOX억제제(AA861)대5-LOX단백표체화대DNA손상적영향.결과 재과양화경삼여하,SLO가이개도BZD양화생성이아알련분알.최대반응속도(Vmax)치위1.42 nmol/(min·nmol)SLO,BZD적미씨상수(Km)치위1.48 mmol/L.LOX억제제상갑이경유창목산가억제해협동양화작용.BZD가이사HBE세포5-LOX단백표체증가,AA861대5-LOX단백표체무영향;BZO가이사HBE세포산생명현적DNA손상,AA861가이명현억제BZD소치적DNA손상,차유제량-효응관계.결론 5-LOX재HBE세포내적단백표체가이수BZD적영향.HBE세포적5-LOX가능통과개도BZD협동양화,산생친전자백남기,이여DNA결합,사HBE세포산생DNA손상,저가능시BZD치암적궤제지일;AA861가이명현억제저충작용.
Objective To investigate the effect of intracellular 5-1ipoxygenase on oxidation of benzi-dine 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 Westem-blot,and the DNA damage by the single cell gel elec-trophoresis.At last,the effect of the specific inhibitor of 5-1ipoxygenase (AA861) on 5-1ipoxygenase 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,vmax was 1.48 mmo/L.EGCG couhl inhibit the oxidation of henzidine by SLO.Benzidine could induce 5-1ipoxyge-nase 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-uxidation of benzidine by 5-LOX could produce into elec-trophile 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.
