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)-alkylguanine-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. Co-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 microM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver.
Long
L
L
Section of Hematology-Oncology, Department of Medicine and Cancer Research Center, Committee on Clinical Pharmacology, University of Chicago, IL 60637, USA.
Moschel
R C
RC
Dolan
M E
ME
D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; C097065:O(6)-benzyl-8-oxoguanine; C064976:O(6)-benzylguanine; C024829:8-hydroxyguanine; D006147:Guanine; D019388:Cytochrome P-450 CYP1A2