Synthesis and Biochemical Characterization of N1-, N2-, and N7-Guanosine Adducts of Butadiene Monoxide

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Abstract
1,3-Butadiene is a known rodent carcinogen, but the molecular mechanisms of its carcinogenicity are poorly understood. Butadiene monoxide (BM), a known mutagenic metabolite of 1,3-butadiene, was previously shown to react with guanosine to yield two N7-guanine adducts. In the present study, eight guanosine adducts of BM were purified and characterized as diastereomeric pairs of N7-(2-hydroxy-3-buten-1-yl)guanosine (G-1 and G-3), N7-(1-hydroxy-3-buten-2-yl)guanosine (G-2 and G-5), N2-(1-hydroxy-3-buten-2-yl)guanosine (G-4 and G-7), and N1-(1-hydroxy-3-buten-2-yl)guanosine (G-6 and G-8) on the basis of stability studies and analyses by UV, 1H NMR, and fast atom bombardment mass spectrometry. While the N7-adducts exhibited half-lives of approximately 50 (G-1 and G-3) and 90 h (G-2 and G-5) upon incubation for 192 h in 100 mM phosphate buffer (pH 7.4) at 37 °C, the N1- and N2-adducts remained stable. When guanosine was reacted with excess BM in phosphate buffer (pH 7.4) at 37 °C, adduct formation exhibited pseudo-first-order kinetics, with the N7-adducts being formed approximately 10-fold more favorably than the N1- and N2-adducts. When incubations were carried out at lower BM concentrations, the N7-adducts remained the major detectable adducts, but the N2-adducts were also detectable at equimolar BM and guanosine concentrations, and the N1-adducts were detectable at a 5-fold molar excess of BM. These results, which provide clear evidence that guanosine can be alkylated at multiple sites following 1,3-butadiene exposure, may aid in the development of useful biomarkers for exposure to 1,3-butadiene. The results may also contribute to a better understanding of the molecular mechanisms of 1,3-butadiene-induced carcinogenicity.