An Evaluation of the Metabolism of 1-Nitro[¹⁴C]Pyrene by Rabbit Tracheal Epithelial Cells: Kinetic Analysis

Abstract

The metabolism of 1-nitro[¹⁴C]pyrene (¹⁴C-1-NP) by freshly isolated rabbit tracheal cells has been investigated in order to determine the kinetic parameters К_M amd V_Max. Experiments to optimize the cell number and the incubation time yielding maximum metabolite formation following incubations with ¹⁴C-1-NP were also performed. Metabolites from the incubation medium and cell lysates at each stage of this investigation were extracted, analyzed and quantitated by HPLC and liquid scintillation spectrophotometry. 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, experiments were performed to assess the kinetics of the metabolism of ¹⁴C-1-NP over a concentration range of (0.1 to 50µM). The apparent К_M ranged from 2.43 ± 1.81 x 10^-1 µM (S.E.) to 3.89 ± 7.31 x 10 ¹ µМ (S.E.). V_Max was determined to range from 5.62 x 10^-3 ± 3.9 x 10^-4 µmoles/mg/hr (S.E.) to 7.65 x 10^-3 ± 8.7 x 10^-4 µmoles/mg/hr (S.E.). The apparent К_М and V_Max were determined using three linear transformation models (Lineweaver-Burk, Hofstee, Woolf and a nonlinear model). Scatchard analysis of the kinetic data resulted in a curvilinear plot indicating the presence of two enzymes, one with a low affinity (K_M = 0.584µM) and high capacity (V_Max = 2.38 x 10^-3 µmoles/mg/hr) and another with a high affinity (К_M = 0.062µM) and low capacity V_Max= 8.1 x 10^-4 µmoles/mg/hr). The results of this study provide information needed to optimize experimental conditions in evaluating the comparative metabolism and genotoxicty of ¹⁴C-1-NP by respiratory tract cells from various species.