Glutathione depletion in a liver microsomal assay as an in vitro biomarkerfor reactive metabolite formation

Glutathione (GSH) plays a major role in cytoprotection, acting as a nucleop hile trap for reactive species derived from xenobiotics. This has led to th e development of an assay for the detection of reactive species generated b y liver microsomal metabolism of xenobiotics. This assay has been used exte nsively to study reactive metabolites which initiate toxicity through a dir ect (non-immunological) mechanism, but there are few data on its ability to detect reactive metabolites that initiate toxicity through neo-antigen for mation, or to detect xenobiotics that cause GSH loss by oxidation mediated by a redox cycling process. Accordingly, the ability of rat and human liver microsomes to metabolize xenobiotics to GSH-depleting metabolites has been investigated further. Of the five neo-antigen-forming xenobiotics tested, four (amodiaquine, phenobarbitone, procainamide, and sulphanilamide) displa yed GSH reactivity that was either dependent or independent (amodiaquine) o n metabolism. The other neo-antigen-forming xenobiotic (carbamazepine) was inactive in all microsomal samples tested. Four quinones believed to exert toxcity through arylation (1,4-benzoquinone) and/or redox cycling (duroquin one, menadione, mitomycin c) displayed GSH reactivity, as did nitrofurantoi n and diquat, two other redox cycling xenobiotics. Induction of the mixed f unction oxidase system with Aroclor afforded little advantage when using ra t liver microsomes, whilst there was considerable inter-individual variatio n in the ability of human liver microsomes to mediate metabolism-dependent GSH depletion. It is concluded that the liver microsome GSH depletion assay may be of general utility as a screen for a number of xenobiotic-derived r eactive species.