Cytochrome P-450 3A4 (nifedipine oxidase) is responsible for the C-oxidative metabolism of 1-nitropyrene in human liver microsomal samples.

The nitrated polycyclic aromatic hydrocarbon 1-nitropyrene is a ubiquitous environmental pollutant. The role of cytochromes P-450 in the human metabolism of [3H]-1-nitropyrene was investigated using human liver microsomes. The range of microsomal metabolism from 16 individual liver specimens was 0.13 to 0.99 nmol/min/mg protein. Using 3 microsomal samples exhibiting different maximal velocities, the Km of 1-nitropyrene metabolism was 3.3 +/- 0.5 microM, indicating that perhaps a single or similar cytochromes P-450 was involved in the metabolism of 1-nitropyrene in these samples. The P-450 3A inhibitor triacetyloleandomycin inhibited 86 +/- 8% of the microsomal metabolism of 1-nitropyrene. Further evidence for the role of P-450 3A in human microsomal metabolism of 1-nitropyrene was gained using inhibitory anti-P-450 3A antibodies. Using 3 separate microsomal samples, antibody conditions that inhibited approximately 90% of the metabolism of the P-450 3A4-specific substrate nifedipine inhibited approximately 60-70% of the metabolism of 1-nitropyrene. Human liver microsomes demonstrated a preference for 1-nitropyren-3-ol formation over 1-nitropyren-6-ol or 1-nitropyren-8-ol, which is in contrast to that noted in rodents where the 6-ol and 8-ol are preferentially formed over the 3-ol, yet in agreement with earlier studies on the metabolism of 1-nitropyrene using Vaccinia-expressed human cytochromes P-450. These results indicate that the human hepatic metabolism of 1-nitropyrene is carried out by at least two or more P-450s including those in the P-450 3A subfamily. These studies also suggest that the metabolism of this compound by humans may differ from that in rodents in both the cytochromes that are involved and the specific metabolites that are formed.

The kinetics of 1-nitropyrene and 3-nitrofluoranthene metabolism using bovine liver xanthine oxidase.

The cytosolic molybdoflavoprotein xanthine oxidase has been shown to catalyze the reduction of exocyclic nitro groups to the corresponding nitroso, hydroxylamino and amino derivatives for a wide variety of xenobiotics including the nitrated polycyclic aromatic hydrocarbons 1-nitropyrene and 3-nitrofluoranthene. Using commercially available bovine liver xanthine oxidase, we have studied the kinetics of the metabolism of 1-nitropyrene and 3-nitrofluoranthene. The nitroreduction of these nitro compounds in the presence of xanthine oxidase is dependent on the presence of hypoxanthine or xanthine and the absence of oxygen. This nitroreduction is independent of added flavins (FMN and FAD), unlike the related molybdoflavoprotein aldehyde oxidase. Xanthine oxidase has a Km of 0.7 microM and Vmax of 0.06 nmol/min per unit enzyme for 1-nitropyrene and a Km of 8.6 microM and Vmax of 0.7 nmol/min per unit enzyme for 3-nitrofluoranthene. The importance of these kinetic constants in evaluating the cytosolic metabolism of 1-nitropyrene and 3-nitrofluoranthene are discussed.

Kinetics and cofactor requirements for the nitroreductive metabolism of 1-nitropyrene and 3-nitrofluoranthene by rabbit liver aldehyde oxidase.

Nitrated polycyclic aromatic hydrocarbons are environmental pollutants that have been shown to arise from a variety of sources, including diesel exhaust emissions and urban air. Most of these compounds are mutagenic in in vitro tests, and several have been shown to be carcinogenic in animals. We have investigated the kinetics of the metabolism of two of these compounds, 1-nitropyrene and 3-nitrofluoranthene, using rabbit liver aldehyde oxidase, an enzyme that has been shown to catalyze the bioactivation of 1-nitropyrene. The nitro-reduction of 20 microM [4,5,9,10-3H]1-nitropyrene or 20 microM [4-3H]3-nitrofluoranthene by aldehyde oxidase required the presence of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), and was inhibited by oxygen in a concentration-dependent manner. In contrast, the aldehyde oxidase oxidation of the electron donor 1-methylnicotinamide did not require FMN or FAD, indicating that the aldehyde oxidase was not isolated as an apoenzyme. The aldehyde oxidase Km and Vmax for 1-nitropyrene were 4.2 microM and 16.3 pmol/min/unit enzyme, while the respective values for 3-nitrofluoranthene nitroreduction were 1.9 microM and 5.4 pmol/min/unit enzyme. The requirement for flavins in the nitroreduction of 1-nitropyrene and 3-nitrofluoranthene suggests that reduced free flavins may be required in cytosolic nitroreduction of 1-nitropyrene and 3-nitrofluoranthene. More importantly, the inhibition of nitroreduction by concentrations of oxygen that are representative of intracellular concentrations strongly suggests that the reasons for the apparent lack of 1-nitropyrene nitroreduction in vivo may be due to oxygen-mediated oxidation of a reduced metabolite of 1-nitropyrene.

The metabolism of 1-nitropyrene by human cytochromes P450.

The metabolism of [3H]1-nitropyrene by specific forms of human cytochrome P450 was investigated in vitro using Vaccinia virus expression of P450 cDNAs in HepG2 cells. Cell lysates were injected individually with recombinant Vaccinia virus containing human P450 cDNA (P450 IA2, IIA3, IIB7, IIC8, IIC9, IID6, IIE1, IIF1, IIIA3, IIIA4, IIIA5 AND IVB1). Only IIIA3 and IIIA4 demonstrated significant activity in the C-oxidation of 1-nitropyrene. The principal metabolite from both P450 forms was 1-nitropyren-3-ol, produced in at least 4-fold greater abundance than the mixture of 1-nitropyren-6-ol and 1-nitropyren-8-ol, or the K-region dihydrodiols. This is in contrast to the metabolism in many species where 6-ol and 8-ol formation predominate over 3-ol formation. In fact, in rats and rabbits, P450 forms quite distinct from the IIIA P450s catalyze the majority of the metabolism of this pollutant. This is the first demonstration of the role of individual human P450 forms in the metabolism of a representative chemical in this important class of environmental pollutants. The importance of these observations in the overall carcinogenic risk of humans to these chemicals remains to be established. These studies furthermore establish a marked species difference in the metabolism of nitrated polycyclic aromatic hydrocarbons.