An evaluation of the metabolism of 1-nitro[14C]pyrene by rabbit tracheal epithelial cells: Kinetic analysis

Kinetic parameters (apparent KM and VMax) have been determined for the metabolism of 1-nitro[14C]pyrene by rabbit tracheal cells. Metabolites were extracted, analyzed and quantitated by HPLC and liquid scintillation spectrophotometry. The maximum rate of metabolite production was attained at 2 x 106 cells and 4 hours incubation, with no significant increase at 20 hours. Using these optimized conditions, kinetic studies were conducted over a concentration range of (0.1 to 50μM). The apparent KM ranged from 2.43 ±1.81 x 10-1μM (S.E.) to 3.89 ± 7.31 x 10-1μM (S.E.). VMax was determined to range from 5.62 x 10-3 ±3.9 x 10-4μmoles/mg/hr (S.E.) to 7.65 x 10-3 ± 8.7 x 10-4 μmoles/mg/hr (S.E.). Scatchard analysis of the kinetic data resulted in a curvilinear plot indicating the involvement of two enzymes in the metabolism of 1-nitropyrene, one with a low affinity (KM = 0.58μM) and high capacity (Vmax= 2.38 x 10-3μmoles/mg/hr) and another with a high affinity (KM = 0.062μM) and low capacity (Vmax= 8.1 x 10-4μmoles/mg/hr). This high rats of metabolism suggest that tracheal cells may be a target tissue for tumor induction by nitroaromatic compounds