Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats and mice.

1,3-Butadiene (BD), a widely used monomer in the production of synthetic rubber and other resins, is one of the 189 hazardous air pollutants identified in the 1990 Clean Air Act Amendments. BD induces tumors at multiple organ sites in B6C3F1 mice and Sprague-Dawley rats; mice are much more susceptible to the carcinogenic action of BD than are rats. Previous in vivo studies have indicated higher circulating blood levels of butadiene monoepoxide (BMO), a potential carcinogenic metabolite of BD, in mice compared to rats, suggesting that species differences in the metabolism of BD may be responsible for the observed differences in carcinogenic susceptibility. The metabolic fate of BD in humans is unknown. The objective of these studies was to quantitate in vitro species differences in the oxidation of BD and BMO by cytochrome P450-dependent monooxygenases and the inactivation of BMO by epoxide hydrolases and glutathione S-transferases using microsomal and cytosolic preparations of livers and lungs obtained from Sprague-Dawley rats, B6C3F1 mice and humans. Maximum rates for BD oxidation (Vmax) were highest for mouse liver microsomes (2.6 nmol/ mg protein/min) compared to humans (1.2) and rats (0.6). The Vmax for BD oxidation by mouse lung microsomes was similar to that of mouse liver but > 10-fold higher than the Vmax for the reaction in human or rat lung microsomes. Correlation analysis revealed that P450 2E1 is the major P450 enzyme responsible for oxidation of BD to BMO. Only mouse liver microsomes displayed quantifiable rates for metabolism of BMO to butadiene diepoxide (Vmax = 0.2 nmol/mg protein/min), a known rodent carcinogen. Human liver microsomes displayed the highest rate of BMO hydrolysis by epoxide hydrolases. The Vmax in human liver microsomes ranged from 9 to 58 nmol/mg protein/min and was at least 2-fold higher than the Vmax observed in mouse and rat liver microsomes. The Vmax for glutathione S-transferase-catalyzed conjugation of BMO with glutathione was highest for mouse liver cytosol (500 nmol/mg protein/min) compared to human (45) or rat (241) liver cytosol. In general, the KMs for the detoxication reactions were 1000-fold higher than the KMs for the oxidation reaction. Because of the low solubility of the BD and the relatively high KM for oxidation, it is likely that the Vmax/KM, ratio will be important for BD and BMO metabolism in vivo. In vivo clearance constants were calculated from in vitro data for BD oxidation and BMO oxidation, hydrolysis and GSH conjugation. The overall activation/detoxication ratio was markedly different for mice (72), rats (5.8) and humans (5.9) and suggest that at concentrations below the KM for the reactions, mice have a significantly higher ratio of activation/detoxication than do either rats or humans. The differences in the activation/ detoxication ratios between mice and rats correlate with the higher carcinogenic sensitivity of mice than rats to BD

Human liver debrisoquine 4-hydroxylase: Test for specifity toward various monooxygenase substrates and model of the active site.

Polyclonal antibodies raised toward a debrisoquine 4-hydroxylating cytochrome P-450 species purified from rat liver (P-450UTA) were used to determine which monooxygenase reactions are linked to debrisoquine hydroxylation in human liver. Anti P-450UTA did not inhibit the oxidation of dimethylnitrosamine, morphine, diazepam, vinylidene chloride, trichloroethylene, benzo(a)pyrene and its 7.8-dihydrodiol, but was inhibitory for the hydroxylation of debrisoquine, (±)-bufuralol, lasiocarpine and monocrotaline. A model interpreting the substrate specificity of the human liver enzyme is presented.

Development of a Pharmacophore for Inhibition of Human Liver Cytochrome P-450 2D6: Molecular Modeling and Inhibition Studies.

To gain insight into the specificity of cytochrome P-450 2D6 toward inhibitors, a preliminary pharmacophore model was built up using strong competitive inhibitors. Ajmalicine (1), the strongest inhibitor known (Ki = 3 nM) was selected as template because of its rigid structure. The preliminary pharmacophore model was validated by performing inhibition studies with derivatives of ajmalicine (1) and quinidine (9). Bufuralol (18) was chosen as substrate and the metabolite 1′-hydroxybufuralol (19) was separated by high performance liquid chromatography. All incubations were carried out using human liver microsomes after demonstration that the Ki values obtained with microsomes were in accordance with those obtained with a reconstituted monooxygenase system containing purified cytochrome P-450 2D6. Large differences of Ki values ranging between 0.005 and 100 μM were observed. Low-energy conformers of tested compounds were fit within the preliminary pharmacophore model. The analysis of steric and electronic properties of these compounds led to the definition of a final pharmacophore model. Characteristic properties are a positive charge on a nitrogen atom and a flat hydrophobic region, the plane of which is almost perpendicular to the N-H axis and maximally extends up to a distance of 7.5 Å from the nitrogen atom. Compounds with high inhibitory potency had additional functional groups with negative molecular electrostatic potential and hydrogen bond acceptor properties on the opposite side at respective distances of 4.8-5.5 Å and 6.6-7.5 Å from the nitrogen atom. The superposition of strong and weak inhibitors led to the definition of an excluded volume map. Compounds that required additional space were not inhibitors. This is apparently the first pharmacophore model for inhibitors of a cytochrome P-450 enzyme and offers the opportunity to classify compounds according to their potency of inhibition. Adverse drug interactions which occur when both substrates and inhibitors of cytochrome P-450 2D6 are applied may be predicted