摘要
目的:1,4-苯醌(BQ)又称对苯醌,是一种有刺激性气味的黄色晶体物质,是有机合成工业的重要原料,广泛应用于医药、农药、化工、染料等工业。研究表明,BQ是苯在体内毒性最强的一种代谢物。在许多实验系统中均可引起遗传损伤,近年来对其遗传毒性机制的研究取得一些进展。动物实验研究证明,苯醌是一类确认的致白血病物质,但其毒作用机理并不是很清楚。本研究旨在了解BQ的细胞毒性、周期阻滞和DNA损伤及修复情况,试图探讨BQ可能的遗传损伤机制和防护剂,为提出有效保护苯和BQ职业暴露人群健康的措施提供依据。
方法:本研究通过MTT比色法检测不同浓度的BQ对V79细胞(中国仓鼠肺成纤维细胞)作用后细胞存活率的变化,了解浓度-毒性效应之间的关系;同时采用碘化丙啶( PI)染色观察细胞周期分布状况;用单细胞凝胶电泳技术研究不同浓度BQ对V79细胞的DNA损伤程度及类型,并观察不同时间段细胞的自身修复情况;用DCFH-DA法检测不同浓度BQ处理V79细胞2h后活性氧(ROS)的含量并用激光共聚焦进行形态观察;分别用不同浓度维生素C与BQ共同作用于V79细胞后,用单细胞凝胶电泳技术(SCGE)检测DNA损伤的变化情况,观察维生素C对BQ所致DNA损伤的拮抗作用。
结果:在2h处理过程中,当BQ终浓度50μmol/L及以上时,各染毒组吸光值与阴性对照组相比,有统计学意义(P < 0.01),并且随着浓度和时间的增加,吸光值逐渐下降,细胞存活率也呈时间和浓度依赖性降低。25～100μmol /L范围内的BQ作用24 h后,V79细胞的细胞周期也发生了明显的改变,表现为S期细胞比例明显增加,G1期细胞的比例下降。单细胞凝胶电泳结果显示,在12.5～100μmol/LBQ作用后,彗星细胞拖尾率随着浓度的增加而增加,且有统计学意义(P < 0.05)。其他分析指标彗星尾长、Olive尾矩和彗尾DAN%也明显高于阴性对照组( P < 0.05) ,且随着BQ浓度的增加,损伤呈上升趋势。经流式细胞仪检测,当BQ终浓度在50～100μmol/L时细胞内ROS含量显著升高,有统计学意义(P < 0.01)。BQ致DNA损伤程度的变化与ROS含量变化存在线性相关(p<0.01)。在本研究中,40、200μmol/L维生素C和BQ同时作用使彗星拖尾率、彗星尾长、Olive尾矩和彗尾DAN%明显低于相应BQ剂量组( P < 0.01)。而1000μmol/L维生素C则加重了BQ的DNA损伤。
结论:本研究结果认为,①在体外培养条件下,BQ能明显抑制V79细胞的增殖,100μmol/L BQ浓度作用2h,25μmol/L BQ浓度作用24h就对细胞产生明显的毒作用,并呈浓度依赖关系;②流式细胞仪检测发现,25μmol/L的BQ作用24 h后,可引起V79细胞周期也发生明显的改变,表现为S期细胞比例明显增加, G1期细胞的比例下降;③单细胞凝胶电泳实验表明,6.25μmol/L BQ就能引起DNA损伤,主要是单链断裂,这种损伤部分可自身修复;④流式细胞术及激光共聚焦技术检测结果显示,在体外培养条件下,50μmol/L的BQ可引起V79细胞内ROS生成明显增多;BQ致细胞DNA损伤与引起ROS生成增多有关;⑤单细胞凝胶电泳结果显示,一定剂量的维生素C对BQ诱导的V79细胞DNA氧化损伤具有抑制作用。
Objective: 1,4-benzoquinone(BQ) is a yellow crystal-solid, which was an important row material in organic synthesie and was used in medical、chemical and dye industry widely. BQ is one of the main active metabolites of benzene.Some studies demonstrate clearly that BQ is the most potent metabolite in vitro. In recent years, some progresses have been made in the research on genotoxicity mechanism of BQ. Although BQ is a known leukemogen, the basis of its toxicity is not well defined.
The aim of research is to examine the effect of BQ on cell viability、cell cycle and genetic damage, to explore possible genotoxicity mechanism from DNA damage and DNA repair in order to provide bases of effective protectional measures for occupational exposure benzene and BQ.
Methods:In this study,the viability of V79 cells was measured by MTT method and the dose-effect relation could be decided;The cell cycle distribution of V79 cells treated with BQ were detected by flowcytometry;Alkli single cell gel electrophoresis (SCGE) was used to study the degree and type of DNA damage induced by BQ in V79 cells, and subsequent self-repair effect; DCFH-DA assay was used to study the content of reactive oxygen species(ROS) after V79 cells stained by the fluorescent probes , which had been treated for 2 hour by BQ; after co-cultured by BQ with various concentration of VitC , SCGE was used to study the DNA damage of V79 cells in order to observe the influence of VitC on BQ induced DNA damage and repair.
Results: As showed in the MTT assay, V79 cell proliferation was inhibited obviously by BQ (treated 2 hours, from the concentration of 100μmol/L), and concentration-dependent relationship was show. Treated with BQ (25- 100μmol /L) for 24 h, the distribution of cell cycle was changed. Cells in S stage were increased, while the cells in G1 stage were decreased. The comet cells ratio elevated as the concentration increased in SCGE in the range of 12.5～100μmol/L showing significant statisitic differences (P < 0.05). All the analysis indexes including tail length, Olive tail moment and tail DNA % increased in a dose-dependent pattern.
Levels of ROS induced by 50 and 100μmol/L p-BQ were significantly increased as evidenced by elevated fluorescence intensity (P<0.01). The comet cells ratio ,tail length , Olive tail moment elevated as the concentration increased in SCGE in the range of 12.5-100μmol/L showing significant statisitic differences (P<0.05). There was a linear correlation between the DNA damage and the ROS intensity(P<0.01). Combined treatment of vitamin C(40、200μmol/L)and BQ,the levels of the comet cells ratio ,tail length and Olive tail moment were significant lower than those of correspondent BQ alone treatment(P<0.05). However, 1000μmol/L vitamin C has an aggravation effect on the BQ induced DNA damage.
Conclusion: The results of this study showed that:the first,conditions in vitro, low concentration BQ could inhibit proliferation of V79 cells and had a concentration-dependent mode; the second,the distribution of cell cycle could be altered by BQ in V79 cell, cells in S stage increased, while the cells in G1 stage decreased ; the third,DNA single strand breaks could induced by BQ and the damage could be self-repaired partly; the fourth,by flow cytometry and confocal laser scanning technology test results showed that, BQ at the concentrations of 50、100μmol/L induced enhanced ROS production. And it indicated that excess production of ROS may be a possible mechanism of the DNA damage induced by BQ; the fifth,Vitamin C at certain concentration(40、200μmol/L)has the antagonistic effects on oxidative stress and DNA damage induced by BQ.
引文
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