Many xenobiotics are metabolized by formation of reactive electrophilic intermediates and subsequent conjugation with glutathione, yielding readily excreted products. This detoxification route may be compromised if conjugation to glutathione is inefficient or if the glutathione conjugates are themselves metabolized to electrophilic products. In these instances, toxic effects may result from binding of xenobiotics to cellular protein. Characterization of glutathione and protein conjugate metabolites can provide information on the structure of electrophilic intermediates, facilitating the understanding of structure-toxicity relationships. This is a particularly demanding analytical problem because formation of the electrophilic species may represent a quantitatively minor metabolic route and because structural characterization must be conducted in the presence of an excess of other components coextracted from the biological sample. We have developed a strategy for the screening and characterization of glutathione conjugates based on the use of stable isotope labelling and multiple scan modes in fast atom bombardment/tandem mass spectrometry (MS/MS and MS/MS/MS). This approach permits the direct analysis of complex biological extracts, with minimal sample preparation and correspondingly low risk of failure to observe metabolites of interest. Coupled liquid chromatography-electrospray ionization/tandem mass spectrometry provides a complementary approach of particular value for the recognition and characterization of quantitatively minor glutathione conjugate metabolites. These techniques will be applied to the analysis of glutathione conjugates (formed in vivo and in vitro) of several substituted furans, a class of compounds which shows marked toxicological variability with respect to the protective effect of glutathione. We will assess the hypothesis that these differences in properties may be attributed to structural differences between the reactive electrophilic metabolites of the substituted furans. Preliminary tandem mass spectrometric studies of the metabolism of 2- furamide have indicated that the principal glutathione metabolite in the rat incorporates the unmodified substituted furan. The characterization of xenobiotic-derived moieties conjugated to protein supplements the identification of glutathione conjugates because of the possibility of further metabolism of glutathione conjugates to reactive electrophilic species. Tandem mass spectrometric methods will be applied to the characterization of protein conjugates after hydrolysis to the single amino acid level. Subsequent work will explore characterization at the levels of intact protein or derived oligopeptides. The significance of the proposed research derives from the development of advanced analytical strategies of general importance to studies of xenobiotic metabolism, together with the elucidation of structure/toxicity relationships among xenobiotics yielding reactive electrophilic metabolites.
