Spin trapping of free radical intermediates produced during the metabolism of isoniazid and iproniazid in isolated hepatocytes

Abstract

By the use of spin trapping agents phenyl-t-butyl nitrone (PBN) and 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) free radical species were detected in isolated hepatocytes incubated with either isoniazid, iproniazid and their respective metabolites acetyl-hydrazine and isopropyl-hydrazine. The addition of bis-nitrophenyl phosphate, an inhibitor of the acylamidase enzymes, to isolated hepatocytes decreased the free radical activation of isoniazid and iproniazid, but not that of acetyl- and isopropyl-hydrazine, confirming that the radical species were originating from the biotransformation of these latter compounds. The ESR spectra were ascribed to the trapping of, respectively, acetyl and isopropyl free radicals on the basis of the analogies of the spectral feature with those of chemically-prepared spin adducts. Comparable ESR spectra were also observed during the metabolism of acetyl- and isopropyl-hydrazines by liver microsomes and their formation was inhibited by the omission of NADP+, anaerobic incubation and enzyme denaturation. In the microsomal preparations inhibitors of the mixed function oxidase system decreased to various extents the free radical formation and a similar effect was also observed following the destruction of cytochrome P-450 induced by pretreating the rats with CoCl2. The addition of reduced glutathione also decreased the radical trapping indicating that GSH can effectively compete with the spin traps for the reaction with the free radicals. The incubation of isolated hepatocytes with isoniazid or acetyl-hydrazine reduced by 20-25% the intracellular GSH content, while a 50% decrease in GSH was present in the cells exposed to iproniazid and isopropyl-hydrazine. In the same hepatocyte preparations stimulation of lipid peroxidation and leakage of LDH were also observed during cell incubation with iproniazid and isopropyl-hydrazine, but not with isoniazid or acetyl-hydrazine and the extent of both phenomena correlated with the susceptibility of the above compounds to the free radical activation.