THEOPHYLLINE N-DEMETHYLATIONS AS PROBES FOR P4501A1 AND P4501A2

Polycyclic aromatic hydrocarbons present in cigarette smoke induce cyt ochromes P4501A1 and P4501A2. These isozymes are of toxicological impo rtance because they convert several environmental pollutants to reacti ve intermediates that form covalent adducts with cellular DNA resultin g in mutations and/or malignant transformations. The aim of our resear ch was to investigate whether theophylline metabolites could be used a s probes for P4501A1 and P4501A2. It is known that at least two human P450 isozymes are involved in theophylline metabolism. The N-demethyla tions of theophylline to 3-methylxanthine (3-MX) and 1-methylxanthine (I-MX) appear to be mediated by P4501A1 and/or P4501A2 and the 3-hydro xylation by different isozymes. Theophylline metabolism was measured i n liver microsomes from control, benzo(a)pyrene (BP)-, and isosafrole (ISO)-induced rats. Theophylline was also incubated in microsomes prep ared from cells expressing high levels of human P4501A1 and P4501A2. A plot of v vs. v/S was linear for 3-MX in the ISO-induced microsomes, but nonlinear for 1-MX, indicating that P4501A2 mainly forms 3-MX, whe reas P4501A1 and 1A2 probably mediate 1-MX. A similar nonlinear relati on was also obtained in the BP-induced microsomes. Incubation of theop hylline in the microsomes from the cells indicated that only 1-MX coul d be measured in cells expressing P4501A1, and both 1-MX and 3-MX were formed in the P4501A2 cell microsomes. Therefore 1-MX seems to be med iated by P4501A1/ P4501A2 and 3-MX specifically by P4501A2, and theoph ylline N-demethylations can be used as probes for P4501A1 and P4501A2. Measurement of 3-MX formation would be specific for P4501A2, and the ratio of the rates of formation of (1-MX - 3-MX)/1,3-dimethyl uric aci d would be a good measure of P4501A1 activity.