Metabolism in vitro and in vivo of the DNA base adduct, M1G

Oxidative damage is considered a major contributing factor to genetic diseases including cancer. Our laboratory is evaluating endogenously formed DNA adducts as genomic biomarkers of oxidative injury. Recent efforts have focused on investigating the metabolic stability of adducts in vitro and in vivo. Here, we demonstrate that the base adduct, M(1)G, undergoes oxidative metabolism in vitro in rat liver cytosol (RLC, K-m = 105 mu M and v(max)/K-m = 0.005 min(-1) mg(-1)) and in vivo when administered intravenously to male Sprague Dawley rats. LC-MS analysis revealed two metabolites containing successive additions of 16 amu. One- and two-dimensional NMR experiments showed that oxidation occurred first at the 6-position of the pyrimido ring, forming 6-oxo-M(1)G, and then at the 2-position of the imidazole ring, yielding 2,6-dioxo-M(1)G. Authentic 6-oxo-M(1)G was chemically synthesized and observed to undergo metabolism to 2,6-dioxo-M(1)G in RLC (K-m = 210 mu M and v(max) /K-m = 0.005 min(-1) mg(-1)). Allopurinol partially inhibited M(1)G metabolism (75%) and completely inhibited 6-oxo-M(1)G metabolism in RLC. These inhibition studies suggest that xanthine oxidase is the principal enzyme acting on M(1)G in RLC and the only enzyme that converts 6-oxo-M(1)G to 2,6-dioxo-M(1)G. Both M(1)G and 6-oxo-M(1)G are better substrates (5-fold) for oxidative metabolism in RLC than the deoxynucleoside, M(1)dG. Alternative repair pathways or biological processing of M(1)dG makes the fate of M(1)G of interest as a potential marker of oxidative damage in vivo.