TABLE 3

NADPH-dependent NNK α-hydroxylation and NNAL formation by human lung microsomes with varying expression levels of CYP2A6 and CYP2A13 proteins

Complete reaction mixtures contained 100 mM phosphate buffer, pH 7.4, 10 μM [5-³H]NNK, 1.0 mM EDTA, 3.0 mM MgCl₂, 5.0 mM glucose-6-phosphate, 1.0 mM NADPH, 0.15 units of glucose-6-phosphate dehydrogenase, and 62.5 μg of human lung microsomal protein, in a final volume of 50 μl. The NADPH and glucose-6-phosphate dehydrogenase were omitted in no-NADPH controls. Reactions were carried out for 20 min, a length of time that afforded a determination of initial rates. NNK metabolites were detected as described under Materials and Methods. All experiments were performed in duplicate. The rates of NADPH-dependent NNK α-hydroxylation were calculated by subtracting the values for no-NADPH controls from the values for the corresponding complete reactions. Values presented are means ± S.D. Available microsomal samples were assayed in groups according to their levels of CYP2A6 and CYP2A13 proteins: CYP2A6-High (30–220 fmol/mg microsomal protein), CYP2A6-Low (<2–10 fmol/mg microsomal protein), CYP2A13-High (3–20 fmol/mg microsomal protein), and CYP2A13-Low (<2 fmol/mg microsomal protein).