Coincubation of 0.4 mM [3H]
glutathione with 4 mM
isaxonine , an
NADPH-generating system,
glutathione S-transferase and mouse liver microsomes, followed by thin-layer chromatography of the incubation mixture, resulted in the appearance of a 3H-labeled peak with characteristics consistent with a
glutathione-
isaxonine metabolite adduct: this peak was absent if either
isaxonine or the
NADPH-generating system was omitted and was decreased if the
transferase was omitted. In vivo, the concentrations of hepatic
glutathione and
glutathione disulfide were markedly decreased 2.5 hr after administration of
isaxonine (4 mmol X kg-1 i.p.); this depletion of
glutathione was prevented essentially by pretreatment with
piperonyl butoxide. In vitro, addition of 4 mM
glutathione decreased markedly the amount of [14C]
isaxonine metabolite that bound to microsomal
proteins during incubation of 1 mM [2-14C]
isaxonine with hepatic microsomes and an
NADPH-generating system. In vivo, pretreatment with
diethylmaleate decreased further hepatic
glutathione concentration and markedly increased the amount of [14C]
isaxonine metabolite covalently bound to hepatic
proteins, 2.5 hr after administration of [2-14C]
isaxonine (4 mmol X kg-1 i.p.). Administration of
isaxonine (4 mmol X kg-1 i.p.) decreased hepatic
cytochrome P-450 concentration, but failed to produce liver cell
necrosis, even in mice pretreated with
phenobarbital,
3-methylcholanthrene or
diethylmaleate, despite high levels of in vivo covalent binding in pretreated animals. We conclude that the reactive metabolite of
isaxonine may be conjugated with
glutathione or may covalently bind to hepatic
proteins. The metabolite, however, has limited hepatotoxic potential in mice.