Kuo M L, Jee S H, Chou M H, Ueng T H
Institute of Toxicology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.
Mutat Res. 1998 Mar 16;413(2):143-50. doi: 10.1016/s1383-5718(98)00020-5.
In this study, we investigated the involvement of reactive oxygen species (ROS) in the motorcycle exhaust particle (MEP)-induced genotoxic and non-genotoxic activity in mammalian cell systems. Initially, the capability of MEP to induce ROS was evaluated by using 2',7'-dichlorofluorescin diacetate (DCFH-DA) to detect hydrogen peroxide (H2O2). A five-fold increase in H2O2 was observed in Chinese hamster lung V79 and human lung carcinoma Calu-1 cells treated with 100 microg/ml MEP for 2 h. Under the same experimental conditions, only a two-fold elevation in H2O2 was detected in hepatic cell systems such as BNL.Cl.2, HepG2, and Hep3B. Treatment of the V79 cells with varying concentrations of MEP caused a dose-dependent increase in sister chromatid exchanges (SCEs), which are effectively inhibited by addition of antioxidants, N-acetyl-l-cysteine (NAC) and ascorbic acid. Furthermore, we determined the oxidized bases in the V79 cells after exposure to MEP. The result showed that 500 microg/ml MEP induced a 3.7-fold increase in thymine glycol (TG) and a seven-fold increase in 8-hydroxy-guanosine (8-OHGua) as compared to untreated cells. We subsequently examined whether MEP would affect gap junctional intercellular communication (GJIC), a tumor promotion process, in V79 cells. We found that MEP inhibited GJIC in a dose-response fashion. Maximal inhibition occurred at 500 microg/ml. The concentration that inhibited at 0.5 of the fraction of the control was 200 microg/ml. Interestingly, when cells were pretreated with NAC or ascorbic acid, they could abolish the MEP-mediated inhibition of GJIC. In addition, a moderate decrease of glutathione was observed in the V79 cells during exposure to MEP. Taken together, our findings suggest that MEP can induce oxidative stress in a broad range of cell lines, especially in lung cell systems. The MEP-induced oxidative stress was critically involved in both genotoxic and non-genotoxic activity.
在本研究中，我们调查了活性氧（ROS）在摩托车尾气颗粒（MEP）诱导的哺乳动物细胞系统遗传毒性和非遗传毒性活性中的作用。最初，通过使用二氯荧光素二乙酸酯（DCFH-DA）检测过氧化氢（H2O2）来评估MEP诱导ROS的能力。在用100μg/ml MEP处理2小时的中国仓鼠肺V79细胞和人肺癌Calu-1细胞中，观察到H2O2增加了五倍。在相同实验条件下，在诸如BNL.Cl.2、HepG2和Hep3B等肝细胞系统中，仅检测到H2O2升高了两倍。用不同浓度的MEP处理V79细胞导致姐妹染色单体交换（SCE）呈剂量依赖性增加，而添加抗氧化剂N-乙酰-L-半胱氨酸（NAC）和抗坏血酸可有效抑制这种增加。此外，我们测定了V79细胞暴露于MEP后的氧化碱基。结果表明，与未处理细胞相比，500μg/ml MEP诱导胸腺嘧啶二醇（TG）增加了3.7倍，8-羟基鸟苷（8-OHGua）增加了7倍。我们随后检查了MEP是否会影响V79细胞中的间隙连接细胞间通讯（GJIC），这是一个肿瘤促进过程。我们发现MEP以剂量反应方式抑制GJIC。最大抑制发生在500μg/ml。抑制对照分数0.5时的浓度为200μg/ml。有趣的是，当细胞用NAC或抗坏血酸预处理时，它们可以消除MEP介导的GJIC抑制。此外，在暴露于MEP期间，V79细胞中的谷胱甘肽有适度下降。综上所述，我们的研究结果表明，MEP可在广泛的细胞系中诱导氧化应激，尤其是在肺细胞系统中。MEP诱导的氧化应激与遗传毒性和非遗传毒性活性均密切相关。
