Quantitation of Four Guanine Oxidation Products from Reaction of DNA with Varying Doses of Peroxynitrite

Abstract

The oxidation products obtained from the reaction of peroxynitrite (ONOO^-) with dG includeamong others8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxodG), 2,2-diamino-4[(2-deoxy-β-d-erythro-pentafuranosyl)amino]-5(2H)-oxazolone (oxazolone), spiroiminodihydantoin, and N¹-(β-d-erythro-pentofuranosyl)-5-guanidinohydantoin (guanidinohydantoin). In the present work, the formation of these products from the treatment of calf thymus DNA with varying amounts of ONOO^- was studied quantitatively in vitro. ¹³C-, ¹⁵N-labeled standards were synthesized for the nucleosides of interest, and calf thymus DNA was reacted with ONOO^- and digested enzymatically down to the nucleoside level. Specific modifications in the DNA were measured by HPLC separation followed by electrospray ionization tandem mass spectrometric analysis in the selected reaction-monitoring mode. Artifacts of the above four oxidation products, arising from oxidation of dG and/or 8-oxodG during DNA digestion and subsequent workup, were evaluated with 7-¹⁵N-dG and/or stable-isotope-labeled 8-oxodG as internal standards. Levels of artifactual 8-oxodG were about 5/10⁶ nucleosides. The artifacts of spiroiminodihydantoin and guanidinohydantoin, arising from 8-oxodG, were 3.7% and 0.6% of the measured 8-oxodG values, respectively. No artifacts of oxazolone were detected. 8-OxodG and oxazolone were formed dose-dependently in DNA treated with ONOO^-, while the levels of spiroiminodihydantoin and guanidinohydantoin increased significantly at low ONOO^- doses, and then dropped off at higher ONOO^- doses. The complexity of these dose−response relationships is likely due to the dual role of peroxynitrite as both an oxidant and a nucleophile in competition with water.