EFFECT OF CARBAMATE THIOESTER DERIVATIVES OF METHYL AND 2-CHLOROETHYLISOCYANATE ON GLUTATHIONE LEVELS AND GLUTATHIONE-REDUCTASE ACTIVITY IN ISOLATED RAT HEPATOCYTES

The present study examined the effects of S-(N-methylcarbamoyl)glutath ione (SMG), S-(N-methylcarbamoyl)-L-cysteine (L-SMC) and some analogs of these S-linked conjugates of methyl isocyanate (MIC) on the activit y of glutathione reductase (GR) in freshly isolated rat hepatocytes an d 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 a t 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 anti tumor agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). L-SMC deplet ed 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 simil ar concentration- and time-dependent inhibition of GR and fall in intr acellular GSH, but in this case the depletion of GSH was extensive and was sustained throughout the 5-hr incubation period. Only a small amo unt (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 wa s 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 v ia release of the free isocyanate at the cell surface, which then pene trates the hepatocyte. The inhibitory effects of the isocyanates on GR , coupled with their propensity to react spontaneously with GSH, combi ne to deplete significantly intracellular stores of GSH.