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 in volved in Zn and Cu homeostasis as well as in heavy metal detoxication . It is also believed that when MT is overexpressed, it can confer res istance against alkylating agents. However, the mechanisms involved ar e still poorly understood. The purpose of the present work was to inve stigate whether metal treatment, which induces MT synthesis, could pro tect isolated rat hepatocytes against the cytotoxic effects of the alk ylating agents methyl meth-anesulfonate (MMS) and N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). Exposure to 12.5 mu M ZnSO4 for 18 hr raised MT levels approximate to 15-fold (as measured by the Cd-109-heme assay ). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydro genase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 mu M vs 362 +/- 13 mu M for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. I n addition, the synthesis of graded amounts of MT, achieved by incubat ion 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 incubatin g Cd-109-labeled MT with MNNG and by analyzing the mixture using Sepha dex G-75 chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of Cd-109 bound to MT. Simi lar results were obtained with Cd-109-exposed hepatocytes, Cd-109 bein g redistributed from MT to high-molecular-weight proteins after incuba tion with MNNG. None of the metals used to induce MT modulated glutath ione (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 th at Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might r eside in the antioxidant properties of MT and on its ability to scaven ge electrophilic species. (C) 1996 Academic Press, Inc.