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 and/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are linked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductase (GR), although the mechanism by which this inhibition occurs remains unknown. In the study presented here, rats were injected with TOLB (200 mg/kg ip), and bile was collected for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarbamoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl isocyanate, 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 presence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentration-dependent manner. No inhibition was observed when NADPH was omitted from incubations, or when the natural substrate for the enzyme, glutathione disulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over the concentration range of 0.8-2.0 mM. It is concluded that metabolic activation of TOLB in vivo leads to the generation of reactive intermediates (n-butyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At critical periods of organogenesis, the resulting perturbation of GSH homeostasis in exposed tissues may play a key role in the teratogenic and/or embryotoxic effects of TOLB.