A Schiff base is a major DNA adduct of crotonaldehyde

Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in hu man tissues as well as tissues of untreated laboratory animals. A second cl ass of crotonaldehyde-DNA adducts releases 2-(2-hydroxypropyl)-4-hydroxy-6- methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are qu antitatively more significant than the Michael addition adducts in vitro. I n this study, we demonstrate that the major source of the paraldol-releasin g DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyd e with DNA, followed by treatment with NaBH3CN and enzyme hydrolysis, resul ted in the formation of N-2-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonst rated that the Schiff base precursor to N-2-(3-hydroxybutyl)dG is N2-(3-hyd roxybutylidene)dG (7), identified by UV, LC-APCI-MS, and MS/MS. Four isomer s of N2-(3-hydroxybutylidene)dG were observed. The (R)- and (S)-isomers wer e identified by reactions of chiral paraldol with dG; each existed as a pai r of interconverting (E)- and (Z)-isomers. These data indicate that the str ucture of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N-2-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotona ldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.