低氧下苯醌类药物对人肝癌细胞的毒性及其机制

Effects of hypoxia on the toxicities of benzoquinone drugs onhuman hepatocarcinoma cells and its mechanism

目的 为阐明低氧下生物还原剂类药物 (两种苯醌 )对人肝癌细胞的毒性和DNA损伤及其机制。方法 人肝癌细胞SMMC 772 1在低氧和常氧下培养不同时间 ,用稍加改良的微孔板直接法检测还原型辅酶Ⅰ /Ⅱ依赖醌氧化还原酶 1 (NQO1 )比活性 ;并用不同浓度的地吖醌 (AZQ)和 2 ,5 双 (氮杂苯) 1 ,4 苯醌 (DZQ)在低氧和常氧下分别处理 1和 6h ,用MTT法测药物细胞毒性 ;用碱性单细胞凝胶电泳(ASCGE)及改良法 (MSCGE)测DNA链断裂和链交联。结果 低氧处理 6h肝癌细胞的NQO1 比活性降低 ,更长时间处理后升高 (P <0 .0 1 )。低氧下AZQ与DZQ(1 ,6h)的细胞毒性升高。在ASCGE实验中 ,常氧下AZQ处理导致DNA链断裂 ;低氧下AZQ处理后则检测不到DNA链断裂。而无论低氧和常氧下DZQ均未导致明显DNA链断裂。在MSCGE实验中 ,低氧下AZQ使H2 O2 所致DNA链断裂减轻至明显低于空白 /常氧组 (P <0 .0 1 ) ;而低氧和常氧下DZQ均能减轻H2 O2 所致DNA链断裂。结论 低氧短时 (6h后 )可诱导NQO1 比活性升高。短时低氧下(6h内 )AZQ和DZQ对人肝癌细胞的毒性上升。DZQ致DNA损伤主要形成DNA链交联 ,低氧加重此效应 ;AZQ主要造成DNA链断裂 (常氧 )和链交联 (低氧 )。

AIM To investigate whether hypoxia can induce changes in NAD(P)H: quinone oxidoreductase 1(NQO 1) activity and its effect on the cytotoxicity and DNA damage in SMMC 7721 cells induced by two bioreductive drugs 〔diaziquone, 2,5 bis(aziridinyl) 3,6 bis(carboethoxyamino) 1,4 benzoquinone, AZQ; 2,5 bis(aziridinyl) 1,4 benzoquinone,DZQ〕 and the underlying mechanism. METHODS Human hepatocarcinoma cells SMMC 7721 were grown under normoxic and hypoxic exposure for different periods (6-36 h). Specific activity of NQO 1 was dynamically detected and calculated by spectrophotometric assays using slightly modified direct measurement in 96 well plates. Cytotoxicities of two benzoquinone drugs(AZQ, DZQ) were determined by MTT assay; DNA strand break and cross link were detected by alkaline single cell gel electrophoresis (ASCGE) and modified SCGE(MSCGE) on AZQ and DZQ treated cells under normoxia or hypoxia(1, 6 h) respectively. RESULTS Six hour hypoxic exposure can inhibit specific activity of cellular NQO 1, while longer hypoxic exposure at each time point can increase specific activity of NQO 1. The cytotoxicities of AZQ and DZQ were both enhanced under hypoxic exposure (1, 6 h). ASCGE assay showed DNA strand break induced by AZQ under normoxia but not hypoxia, meanwhile no DNA strand break was found after DZQ treatment under either oxygen condition. MSCGE assay observed DZQ induced DNA strand cross link under both oxygen condition, and the crosslink effect of AZQ was only seen under hypoxia. CONCLUSION Increase in NQO 1 specific activity in hepatocarcinoma cells SMMC 7721 can be induced by a short period (6 h) of hypoxia. Hypoxia can enhance both cytotoxicities of AZQ and DZQ. DZQ induces DNA damage mainly via DNA strand cross link; while AZQ causes DNA strand break under normoxia and strand cross link under hypoxia.