Rat hepatocytes with elevated metallothionein expression are resistant to N-methyl-N'-nitro-N-nitrosoguanidine cytotoxicity

Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.