Synthesis, characterization, and identification of N7-guanine adducts of isoprene monoepoxides in vitro

Isoprene (IP, 2-methylbuta-1,3-diene) is ubiquitous in the environment through emission by plants, combustion processes, and endogenous formation and exhalation by mammals, including humans. IP is also an industrial chemical, widely used in the manufacture of synthetic rubber and plastics. Like butadiene, IP is metabolized to reactive epoxides, which form adducts with macromolecules, and is a demonstrated carcinogen in mice. To date, DNA adducts of IP monoepoxides have not been reported. We report here on the formation of N7-guanine (N7-Gua) adducts of isoprene-1,2-oxide (IP-1,2-O, 2-ethenyl-2-methyloxirane) and isoprene-3,4-oxide (IP-3,4-O, propen-2-yloxirane). DNA adducts are useful as biomarkers to estimate exposure, as well as to investigate mechanisms of IP carcinogenesis. Incubation of 2'-deoxyguanosine with the monoepoxides followed by deglycosylation gave four N7-Gua adducts that were isolated by HPLC and characterized by high-resolution FAB(+)-MS, ESI+-MS, ESI+-MS/MS, and H-1 NMR and two-dimensional heteronuclear H-1, C-13 correlation NMR spectrometry. IP-1,2-O and IP-3,4-O reacted at both terminal and internal oxirane carbons to form the following regioisomeric adducts at Gua N7: N7-(2'-hydroxy-2'-methyl-3'-buten-1'-yl)guanine, N7-(1'-hydroxy-2'-methyl-3'-buten-2'-yl)guanine, N7-(1'-hydroxy-3'-methyl-3'-buten-2'-yl)guanine, and N7-(2'-hydroxy-3'-methyl-3'-buten-1'-yl)guanine. The same adducts were identified by UV spectra, HPLC retention times, and LC/ESI+-MS in the neutral thermal hydrolysates of single- and double-stranded calf thymus DNA after incubation with IP monoepoxides. Characterization of the N7-Gua adducts identified in incubations of DNA with IP monoepoxides represents the first step toward establishing biomarkers of IP metabolism and exposure.