Metabolism in Vitro and in Vivo of the DNA Base Adduct, M₁G

Abstract

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 µM 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 µM 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.

Monitoring in Vivo Metabolism and Elimination of the Endogenous DNA Adduct, M1dG {3-(2-Deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one}, by Accelerator Mass Spectrometry

“…Purine-based therapeutics such as acyclovir, penciclovir, zaleplon, and others readily undergo oxidative metabolism and active transport (26)(27)(28)(29)(30)(31)(32)(33)(34). Our results on the oxidative metabolism of M 1 dG and M 1 G suggest that metabolism and alternate routes of elimination may also contribute to the complexity of DNA adduct analysis in biological matrixes (18,19).…”

Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

“…However, it would seem that most of these studies have examined the oxidation of 8-oxoGua in situ in DNA (84), rather than as a postexcision product of DNA repair, which may alter the likelihood of oxidation and its ''oxidizability.'' In contrast, M 1 G and 3-(2-deoxy-h-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one both seem to undergo further oxidative metabolism in rat liver cytosol, with the base adduct being a better substrate for such enzymic oxidation than the deoxyribonucleoside adduct (85,86). There is also some evidence to suggest that M 1 G is further oxidized when given i.v., although 3-(2-deoxyh-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one was not examined (85).…”

Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates

“…With M 1 dG, metabolic and pharmacokinetic studies in rats revealed a biphasic elimination from plasma with M 1 dG found in the urine for more than 24 hr after dosing 150. Analysis of urine revealed a metabolite of M 1 dG, 6-oxo-M 1 dG, likely derived from hepatic xanthine oxidase activity,150 with evidence for further oxidation of 6-oxo-M 1 dG on the imidazole ring to give 2,6-dioxo-M 1 G 151. Both of these studies raise the possibility that urinary biomarker studies may be underestimating the true level of adducts as a result of loss of the parent forms.…”