Debenzylation of O-6-benzyl-8-oxoguanine in human liver: implications for O-6-benzylguanine metabolism

O-6-Benzylguanine (BG) effectively inactivates the DNA repair protein O-6-alklylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and tumor-bearing animals. BG is presently in phase II clinical trials. In humans, BG is converted to O-6- benzyl-8-oxoguanine (8-oxoBG), a longer-lived, yet equally potent inactivator. We have isolated and identified the debenzylated product, 8-oxoguanine, in plasma and urine of patients following administration of BG. The purpose of this work was to determine the human liver enzymes responsible for the debenzylation of 8-oxoBG. Therefore, 8-oxoBG was incubated with human liver microsomes and cytosol, and the concentration of 8-oxoguanine was determined. No appreciable product was formed in the cytosol; however, increasing amounts of 8- oxoguanine were formed with increasing concentrations of pooled human liver microsomes. The amount of 8-oxoguanine formed increased with time and substrate concentration. Go-incubation of human liver microsomes with 8-oxoBG and various cytochrome P450 isoform-selective inhibitors suggested the possible involvement of CYP1A2, 2E1, and/or 2A6 in this reaction. Incubation of 8-oxoBG with baculovirus cDNA-overexpressed CYP1A2, 2E1, 2A6, and 3A4 demonstrated that formation of 8- oxoguanine was due mainly to CYP1A2. Debenzylation of 8-oxoBG complied with Michaelis-Menten kinetics with K-m and V-max values of 35.9 muM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver. (C) 2001 Elsevier Science Inc. All rights reserved.

See More