FORMATION OF GLUTATHIONYL SPIRONOLACTONE DISULFIDE BY RAT-LIVER CYTOCHROMES P450 OR HOG LIVER FLAVIN-CONTAINING MONOOXYGENASES - A FUNCTIONAL PROBE OF 2-ELECTRON OXIDATIONS OF THE THIOSTEROID

We have previously reported that the diuretic thiosteroid spironolactone (SPL) inactivates rat liver microsomal cytochromes P450 [P450 (P450 3A and P450 2C11)] in a mechanism-based fashion, and we have identified two polar SPL metabolites (SPL-sulfinic acid and -sulfonic acid), formed in a partition ratio of almost-equal-to 20:1 in such rat liver microsomal incubations [Decker et al. (1989) Biochemistry 28, 5128-5136]. We proposed at the time that these metabolites were most likely derived from further enzymatic (or nonenzymatic) oxidants of the one-electron oxidation product [SPL-thiyl radical (SPL-S.)] and/or the two-electron-oxidized species [SPL-sulfenic acid (SPL-SOH)]. In those studies, glutathione (GSH) was found to attenuate both SPL-mediated P450 loss as well as polar metabolite formation by almost-equal-to 40%. We have now reexamined this in greater detail and report that it is due to GSH trapping of an electrophilic oxidized SPL species to form an adduct that we have isolated and unambiguously characterized by mass spectral analyses as the glutathionyl-SPL adduct (SPL-SSG). Moreover, we have found not only that rat liver microsomal formation of this adduct is enhanced at pH 9.0, the pH optimum for flavin-containing monooxygenase (FMO), but also that such adduct formation was indeed efficiently catalyzed by purified hog liver FMO. Because FMO oxidations of thiols are thought to entail a two-electron process to form the corresponding sulfenic acids, we infer that such a SPL-SSG adduct most likely reflects FMO-catalyzed oxidation of SPL to SPL-SOH, which on leaving the FMO active site is then trapped by GSH. Moreover, if not intercepted by external nucleophiles, FMO-generated SPL-SOH may attack rat liver microsomal P450s, thus accounting for the GSH-inhibitable component of SPL-mediated P450 loss. This possibility is strengthened by finding that GSH-mediated attenuation of such a SPL-mediated P450 loss was diminished by thermal inactivation of rat liver microsomal FMO. The isolation and characterization of the SPL-SSG adduct, we believe, not only rationalize the previously reported GSH-mediated attenuation of P450 loss but also provide the first direct evidence for the intermediacy of GSH conjugates in reduction of FMO-generated sulfenic acids. Furthermore, because we detected no polar oxidized SPL metabolites in purified FMO-catalyzed systems even in the absence of GSH, it appears that these metabolites must be derived from the one-electron-oxidized SPL-S. species.