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.

Structure of the 1,4-Bis(2‘-deoxyadenosin-N6-yl)-2S,3S-butanediol Intrastrand DNA Cross-Link Arising from Butadiene Diepoxide in the Human N-ras Codon 61 Sequence

“…The genotoxicity is probably due to its potential to form DNA-DNA (43)(44)(45)(46) and DNA-protein cross-links (47,48), the latter having been observed in mice (49,50). Mice possess greater sensitivity to butadiene exposure than rats, and this is attributed to their efficient oxidation of BD to BDO 2 (51,52), presumably facilitating DNA cross-linking.…”

“…The predominant DNA cross-link induced by BDO 2 , isolated from DNA and characterized by mass spectrometry (46), involves inter-strand bis-alkylation at N7-dG, particularly in 5′-GNC-3′ sequences (45). The formation of N7,N7-dG cross-links is dependent upon stereochemistry of the BDO 2 ; with S,S BDO 2 > R,R BDO 2 > meso BDO 2 (56,57).…”

The 5‘-GNC Site for DNA Interstrand Cross-Linking Is Conserved for Diepoxybutane Stereoisomers

“…The isolation of N7-linked guanineguanine conjugates from DEB-treated DNA (9) originally led to the proposal of interstrand cross-linking at 5'-GC sites, which have the minimal N7-to-N7 distance within canonical B-DNA (10). The structural assignment of the DEB-DNA conjugate as 1,4-bis(guan-7-yl)-2,3-butanediol was later confirmed independently (11). However, the preferential site of crosslinking was found to be at 5'-GNC sequences (where N is any base) for racemic DEB within both DNA oligomers and defined sequence nucleosomal core particles (12,13).…”

Characterization of 1,2,3,4-Diepoxybutane−2‘-Deoxyguanosine Cross-Linking Products Formed at Physiological and Nonphysiological Conditions

“…Unexpectedly, bis-N7G-BD was not among the major products detected from the reaction of DEB and dG in glacial acetic acid at 60 °C for 4 h despite the fact that bis-N7G-BD was prepared through the reaction of guanosine with DEB in glacial acetic acid at 80 °C (37). This is probably caused by the large excess of DEB in our experiment (DEB:dG = 5.7:1) in comparison with the conditions used to prepare bis-N7G-BD (DEB:guanosine = 1.1:1).…”