Interaction of nitromethane with reduced hepatic microsomal cytochrome P-450

Abstract

Nitromethane interacts with sodium dithionite reduced rabbit liver microsomes to generate a difference spectrum characterized by maxima at 388, 423, 454 and 520 nm and minima at 405, 436, 480 and 550 nm. Spectral binding constants ( K s ) of 0.306 ± 0.082 mM ( A max = 0.032 ± 0.004), 0.178 ± 0.029 mM ( A max = 0.040 ± 0.002), and 1.168 ± 0.250 mM ( A max = 0.035 ± 0.006) were calculated for the 388, 423 and 454 nm peaks respectively. These difference spectra are qualitatively different from those previously reported for aromatic nitro compounds [L. A. Sternson and R. E. Gammans, Drug Metab. Dispos. 3, 266 (1975)]. Interaction of nitromethane with microsomes from rabbits pretreated with phenobarbital produced absorbance maxima and minima within 2 nm of the controls. Interaction of nitromethane with reduced microsomes from 3-methyl-cholanthrene (3-MC)-pretreated animals produced ferrohemochromes in which maximal absorbance changes were shifted to maxima at 384, 422, 451 and 514 nm and minima at 407, 433, 473 and 552nm. Peak-height ratios derived from difference spectra generated by the addition of 1 mM nitromethane to the sample cuvette were considerably different depending on whether the microsomes were obtained from control, phenobarbital- or 3-MC-pretreated rabbits and may indicate that phenobarbital, like 3-MC, induces qualitative changes in cytochrome P-450. Nitromethane apparently competes with carbon monoxide for a common binding site. Addition of nitromethane to CO-saturated microsomes reduced the magnitude of the 450 nm peak with a concomitant increase in the 423 nm peak of nitromethane. Similarly, addition of CO to reduced microsomes containing nitromethane caused reduction of the 423 nm peak of nitromethane with a corresponding increase of the 450 nm peak of CO. Nitromethane does not generate difference spectra with oxidized microsomes nor does it alter the K s or A max of aminopyrine, hexobarbital, aniline or zoxazolamine binding spectra. Nitromethane does inhibit the binding of the type II compound, nicotinamide. Addition of nitromethane to incubation flasks enhanced the metabolism of aniline while tending to inhibit the oxidative demethylation of ethylmorphine.