Covalent binding of leukotriene A4 to DNA and RNA.

Leukotriene A(4) (LTA(4)) is a highly reactive electrophilic intermediate formed during the biosynthesis of the lipid mediators leukotriene B(4) and leukotriene C(4). Deoxynucleosides were found to react as nucleophiles with LTA(4) in aqueous solutions as assessed by UV spectroscopy and electrospray ionization mass spectrometry. Aqueous solutions of native DNA and RNA were also found to react with LTA(4) as assessed by mass spectrometric analysis of the constituent nucleosides derived from enzymatic hydrolysis of the nucleic acids. The most abundant adducts were observed for guanine- and adenine-containing deoxynucleosides and nucleosides. At neutral pH, these reactions led to an overall modification of deoxyguanosine/guanosine residues in DNA and RNA at 15 +/- 1 adducts/10(7) bases and 230 +/- 20 adducts/10(7) bases, respectively, determined by quantitative assay using stable isotope-labeled LTA(4)-nucleoside adduct. An estimation of the relative reactivity of LTA(4) with each of the purine and pyrimidine bases in DNA and RNA was carried out by comparisons of the mass spectral ion abundance of the different adducts (LTA(4)-dAdo, LTA(4)-dCyd, LTA(4)-Thd, LTA(4)-Ado, LTA(4)-Cyd, and LTA(4)-Urd) to the ion signal of known amounts of LTA(4)-dGuo and LTA(4)-Guo standards. The data were corrected for different mass spectrometric response factors that were experimentally determined for each adduct product. The structures of the two most abundant LTA(4)-Guo products were determined by NMR, UV spectroscopy, and mass spectrometry to be 5-hydroxy,12-[Guo-N(2)-yl]-6,8,11,14-eicosatetraenoic acid. Stimulation of human neutrophils with calcium ionophore led to the covalent modification of DNA within the cell as determined by mass spectrometric analysis of lipophilic nucleosides obtained after hydrolysis of extracted DNA. These observations, combined with the intracellular site of 5-lipoxygenase translocation and LTA(4) biosynthesis at the nuclear envelope, suggest that LTA(4) may have access to DNA and RNA within cells and furthermore modify nucleic acids in situ following the activation of 5-lipoxygenase and initiation of LTA(4) biosynthesis.