Institute of Environmental Pollution and Health, Shanghai University, Shanghai, People's Republic of China.
Mutat Res. 2011 Nov 1;716(1-2):84-91. doi: 10.1016/j.mrfmmm.2011.08.007. Epub 2011 Aug 27.
1,3-Butadiene (BD) is an air pollutant and a known carcinogen. 1,2,3,4-Diepoxybutane (DEB), one of the major in vivo metabolites of BD, is considered the ultimate culprit of BD mutagenicity/carcinogenicity. DEB is a bifunctional alkylating agent, being capable of inducing the formation of monoalkylated DNA adducts and DNA cross-links, including DNA-DNA and DNA-protein cross-links (DPC). In the present study, we investigated DEB-caused DNA cross-links and breaks in human hepatocyte L02 cells using comet assay. With alkaline comet assay, it was observed that DNA migration increased with the increase of DEB concentration at lower concentrations (10-200μM); however, at higher concentrations (200-1000μM), DNA migration decreased with the increase of DEB concentration. This result indicated the presence of cross-links at >200μM, which was confirmed by the co-treatment experiments using the second genotoxic agents, tert-butyl hydroperoxide and methyl methanesulfonate. At 200μM, which appeared as a threshold, the DNA migration-retarding effect of cross-links was just observable by the co-treatment experiments. At <200μM, the effect of cross-links was too weak to be detected. The DEB-induced cross-links were determined to be DNA-DNA ones rather than DPC through incubating the liberated DNA with proteinase K prior to unwinding and electrophoresis. However, at the highest DEB concentration tested (1000μM), a small proportion of DPC could be formed. In addition, the experiments using neutral and weakly alkaline comet assays showed that DEB did not cause double-strand breaks, but did induce single-strand breaks (SSB) and alkali-labile sites (ALS). Since SSB and ALS are repaired more rapidly than cross-links, the results suggested that DNA-DNA cross-links, rather than DPC, were probably responsible for mutagenicity/carcinogenicity of DEB.
1,3-丁二烯（BD）是一种空气污染物和已知的致癌物。1,2,3,4-环氧丁烷（DEB）是 BD 的主要体内代谢物之一，被认为是 BD 致突变/致癌性的罪魁祸首。DEB 是一种双功能烷化剂，能够诱导单烷基化 DNA 加合物和 DNA 交联的形成，包括 DNA-DNA 和 DNA-蛋白质交联（DPC）。在本研究中，我们使用彗星试验研究了 DEB 对人肝细胞 L02 细胞造成的 DNA 交联和断裂。通过碱性彗星试验，观察到在较低浓度（10-200μM）下，随着 DEB 浓度的增加，DNA 迁移增加；然而，在较高浓度（200-1000μM）下，随着 DEB 浓度的增加，DNA 迁移减少。该结果表明，在>200μM 时存在交联，这通过用第二遗传毒性剂叔丁基过氧化物和甲基甲烷磺酸盐进行的共同处理实验得到证实。在 200μM 时，作为一个阈值，只有通过共同处理实验才能观察到交联的 DNA 迁移抑制作用。在<200μM 时，交联的作用太弱而无法检测到。通过在解旋和电泳之前用蛋白酶 K 孵育释放的 DNA，确定 DEB 诱导的交联是 DNA-DNA 交联而不是 DPC。然而，在所测试的最高 DEB 浓度（1000μM）下，可能形成少量的 DPC。此外，使用中性和弱碱性彗星试验的实验表明，DEB 不会引起双链断裂，但会诱导单链断裂（SSB）和碱不稳定位点（ALS）。由于 SSB 和 ALS 的修复速度比交联快，因此结果表明，DNA-DNA 交联而不是 DPC，可能是 DEB 致突变/致癌性的原因。
