HUMAN LIVER OXIDATIVE-METABOLISM OF O-6-BENZYLGUANINE

The oxidation of O-6-benzylguanine, an inactivator of O-6-alkylguanine -DNA alkyltransferase, was examined using human liver cytosol, microso mes, and several P450 isoforms. Incubation of O-6-benzylguanine with h uman liver cytosol resulted in the formation of O-6-benzyl-8-oxoguanin e, which was inhibited by menadione, a potent inhibitor of aldehyde ox idase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was le ss dramatic. Oxidation of O-6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and trole andomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Hu man P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 mu M O-6-benzylguanine. At 10 mu M, O-6-benzylguanine w as oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. Howe ver, an appreciable increase in CYP3A4 contribution was noted at 200 m u M. CYP1A2 exhibited a more than 200-fold higher relative catalytic a ctivity (V-max/K-m) compared with CYP3A4. Therefore, at therapeuticall y relevant concentrations of O-6-benzylguanine, CYP1A2 could be primar ily involved in its oxidation since it shows a much lower K-m value (1 .3 mu M) than CYP3A4 (52.2 mu M) and cytosol (81.5 mu M). However, one would expect interindividual variation in the extent of oxidation of O-6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.