Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1)

Abstract

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, β-naphthoflavone (β-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b 5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.