Characterization of Covalent Adducts of Nucleosides and DNA Formed by Reaction with Levuglandin

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Abstract
Enhanced expression of cyclooxygenase-2 (COX-2) is associated with development of several cancers. The product of COX-2, prostaglandin H2 (PGH2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form the highly reactive levuglandin E2 (LGE2) or D2 (LGD2). Incubation with LGE2 causes DNA−protein cross-linking in cultured cells, suggesting that levuglandins can directly react with DNA. We report the identification by liquid chromatography−tandem mass spectrometry of a stable levuglandin−deoxycytidine (LG−dC) adduct that forms upon reaction of levuglandin with DNA. We found that LGE2 reacted with deoxycytidine, deoxyadenosine, or deoxyguanosine in vitro to form covalent adducts with a dihydroxypyrrolidine structure, as deduced from selective ion fragmentation. For LG−deoxycytidine adducts, the initial dihydroxypyrrolidine structure converted to a pyrrole structure over time. Reaction of LG with DNA yielded a stable LG−dC adduct with a pyrrole structure. These results describe the first structure of levuglandinyl−DNA adducts and provide the tools with which to evaluate the potential for LG−DNA adduct formation in vivo.

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