title: Diepoxybutane induces the formation of DNA-DNA rather than DNA-protein cross-links, and single-strand breaks and alkali-labile sites in human hepatocyte L02 cells.
abstract: 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.
authors: Institute of Environmental Pollution and Health, Shanghai University, Shanghai, People's Republic of China.
authors: Ying
authors: Y
authors: Wen
authors: Pan-Pan
authors: PP
authors: Zhang
authors: Jing
authors: J
authors: An
authors: Ying-Xin
authors: YX
authors: Yu
authors: Ming-Hong
authors: MH
authors: Wu
authors: Guo-Ying
authors: GY
authors: Sheng
authors: Jia-Mo
authors: JM
authors: Fu
authors: Xin-Yu
authors: XY
authors: Zhang
mesh_terms: D000393:Air Pollutants / Q000633:toxicity*; D002273:Carcinogens / Q000633:toxicity*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D020552:Comet Assay; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects*; D004852:Epoxy Compounds / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans
chemical_list: D000393:Air Pollutants; D002273:Carcinogens; D004852:Epoxy Compounds; C007366:diepoxybutane; D004247:DNA