Identification of S-(n-butylcarbamoyl) glutathione, a reactive carbamoylating metabolite of tolbutamide in the rat, and evaluation of its inhibitory effects on glutathione reductase in vitro

Tolbutamide (TOLB), a widely used hypoglycemic agent in the therapy of non- insulin-dependent diabetes mellitus, has been reported to be teratogenic an d/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are licked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductas e (GR), although the mechanism by which this inhibition occurs remains unkn own. In the study presented here, rats were injected with TOLB (200 mg/kg i p), and bile was collected for analysis by liquid chromatography/tandem mas s spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarb amoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl iso cyanate, as a minor metabolite of TOLB in bile. Upon incubation of SBuG (0. 25-1.0 mM) with GR from either yeast or bovine intestinal mucosa in the pre sence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentra tion-dependent manner. No inhibition was observed when NADPH was omitted fr om incubations, or when the natural substrate for the enzyme, glutathione d isulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over th e concentration range of 0.8-2.0 mM. It is concluded that metabolic activat ion of TOLB in vivo leads to the generation of reactive intermediates (n-bu tyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At crit ical periods of organogenesis, the resulting perturbation of GSH homeostasi s in exposed tissues may play a key role in the teratogenic and/or embryoto xic effects of TOLB.