Structural characterization of the major DNA-DNA cross-link of 1,2,3,4-diepoxybutane

Abstract

1,2,3,4-Diepoxybutane (DEB) is a bifunctional alkylating agent that exhibits both cytotoxic and promutagenic properties. DEB is the ultimate carcinogenic species of the major industrial chemical 1,3-butadiene (BD), as well as the active form of the antitumor prodrug treosulfan. DEB is tumorigenic in laboratory animals and is capable of inducing a variety of genotoxic outcomes, including point mutations, large deletions, and chromosomal aberrations. These potent biological effects are thought to result from the ability of DEB to form DNA-DNA cross-links by consecutive alkylation of two nucleobases within a DNA duplex. Earlier studies have provided evidence for the formation of interstrand DNA-DEB lesions involving guanine nucleobases, but the covalent structure of DEB-induced DNA cross-link has not been previously elucidated. In the present work, the major DNA-DNA cross-link of DEB has been identified as 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD). The DNA-derived N7-N7 guanine DEB cross-link was characterized by comparing its mass spectra, UV spectra, and chromatographic properties to an authentic standard prepared by an independent synthesis. Calf thymus DNA treated with relatively low concentrations of DEB (5-50 microM) contained similar numbers of bis-N7G-BD and the corresponding monoadducts (N7-trihydroxybutyl-guanine), while higher DEB exposures produced predominantly monoalkylated lesions. Although both lesions spontaneously depurinate at physiological conditions giving rise to abasic sites in DNA, bis-N7G-BD lesions have a longer half-life in double-stranded DNA than the N7-guanine monoadducts. These studies provide the first rigorous characterization of the covalent structure and hydrolytic stability of the major DEB-induced DNA-DNA cross-link.