title: 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, M1G, undergoes oxidative metabolism in vitro in rat liver cytosol (RLC, Km = 105 microM and vmax/Km = 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-M1G, and then at the 2-position of the imidazole ring, yielding 2,6-dioxo-M1G. Authentic 6-oxo-M1G was chemically synthesized and observed to undergo metabolism to 2,6-dioxo-M1G in RLC (Km = 210 microM and vmax/Km = 0.005 min-1 mg-1). Allopurinol partially inhibited M1G metabolism (75%) and completely inhibited 6-oxo-M1G metabolism in RLC. These inhibition studies suggest that xanthine oxidase is the principal enzyme acting on M1G in RLC and the only enzyme that converts 6-oxo-M1G to 2,6-dioxo-M1G. Both M1G and 6-oxo-M1G are better substrates (5-fold) for oxidative metabolism in RLC than the deoxynucleoside, M1dG. Alternative repair pathways or biological processing of M1dG makes the fate of M1G of interest as a potential marker of oxidative damage in vivo.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Charles G
authors.initials: CG
authors.lastname: Knutson
authors.forename: Dapo
authors.initials: D
authors.lastname: Akingbade
authors.forename: Brenda C
authors.initials: BC
authors.lastname: Crews
authors.forename: Markus
authors.initials: M
authors.lastname: Voehler
authors.forename: Donald F
authors.initials: DF
authors.lastname: Stec
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000493:Allopurinol / Q000097:blood; D000818:Animals; D001711:Biotransformation; D003600:Cytosol / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism* / Q000493:pharmacokinetics / Q000652:urine; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D011687:Purines / Q000378:metabolism* / Q000493:pharmacokinetics / Q000652:urine; D011743:Pyrimidines / Q000378:metabolism* / Q000493:pharmacokinetics / Q000652:urine; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018736:DNA Adducts; D007202:Indicators and Reagents; D011687:Purines; D011743:Pyrimidines; C107643:pyrimido(1,2-a)purin-10(3H)-one; D000493:Allopurinol