Importance of protein thiols during N-methyl-N'-nitro-N-nitrosoguanidine toxicity in primary rat hepatocytes.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent toxicant in isolated rat hepatocytes, was evaluated for its mechanism of cytotoxicity. This direct acting toxicant generates an alkylating carbonium ion that covalently binds to cell macromolecules, depletes nonprotein thiols (NPT), and subsequently kills cells. In this study MNNG depleted protein thiols (PT) in a two-phase process. The first phase (about 30% depletion) occurred rapidly, in parallel with the depletion of NPT. After a plateau, a second phase of PT depletion occurred 5-8 min prior to cell death. Indole-3-carbinol (I-3-C), added prior to MNNG, did not alter the depletion of NPT nor the first phase of PT depletion. However, cell killing was substantially retarded and was still immediately preceded by the second phase of PT depletion. The addition of o-phenanthroline or 5,10-dihydroindeno[1,2-b]indole (DHII) prior to MNNG did not alter the first phase of PT depletion, but partially protected (about 30%) against the depletion of NPT. However, o-phenanthroline or DHII completely protected against the MNNG-induced loss of cell viability and the second-phase depletion of PT. When DHII was added after MNNG and prior to the expected second phase of PT depletion, that depletion was markedly depressed, as was the subsequent loss of cell viability. We conclude that MNNG covalent binding, depletion of NPT, and first-phase depletion of PT may be necessary, but insufficient to kill cells. We propose that rapid depletion of cellular antioxidants predisposes the cell to oxidative stress and that oxygen toxicity is responsible for the second-phase depletion of PT and the final cytotoxic events. The fact that the second-phase depletion of PT is required for and immediately precedes cell death suggests the importance of critical but as yet unidentified target thiol proteins in MNNG hepatotoxicity.