Effect of carbamate thioester derivatives of methyl- and 2-chloroethyl isocyanate on glutathione levels and glutathione reductase activity in isolated rat hepatocytes.

The present study examined the effects of S-(N-methylcarbamoyl)glutathione (SMG), S-(N-methylcarbamoyl)-L-cysteine (L-SMC) and some analogs of these S-linked conjugates of methyl isocyanate (MIC) on the activity of glutathione reductase (GR) in freshly isolated rat hepatocytes and on the levels of reduced and oxidized glutathione (GSH and GSSG) in exposed cells. Both SMG and its monoethyl ester (0.5 mM) were found to inhibit GR weakly, although L-SMC proved to be an effective inhibitor of the enzyme (60 +/- 4% activity remaining after a 4-hr incubation at 0.5 mM). The cysteine adduct (SCC) of 2-chloroethyl isocyanate (CEIC) was a strong inhibitor of GR (27 +/- 1% activity remaining after a 1-hr incubation at 0.1 mM) and was essentially equipotent with the antitumor agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). L-SMC depleted intracellular GSH in a time- and concentration-dependent manner up to 2 hr of incubation, beyond which time GSH levels began to recover. Exposure of cells to the enantiomeric conjugate, D-SMC, led to a similar concentration- and time-dependent inhibition of GR and fall in intracellular GSH, but in this case the depletion of GSH was extensive and was sustained throughout the 5-hr incubation period. Only a small amount (less than 10%) of the GSH that was lost from cells exposed to SMC was recovered in the medium, indicating that SMC did not cause efflux of GSH (most of the free cysteine released during breakdown of SMC was recovered in the medium). Experiments with hepatocytes exposed for 5 hr to SCC (0.1 mM) demonstrated that GSSG levels were elevated by 32 +/- 5% relative to controls. Collectively, these results indicate that carbamate thioester conjugates of MIC and CEIC inhibit GR, probably via release of the free isocyanate at the cell surface, which then penetrates the hepatocyte. The inhibitory effects of the isocyanates on GR, coupled with their propensity to react spontaneously with GSH, combine to deplete significantly intracellular stores of GSH.