Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein

Cadmium is carcinogenic in humans and rodents. Although extensive evidence indicates that the toxicity and genotoxicity of Cd is ameliorated by binding to cysteine clusters in metallothionein (MT), the factors governing Cd release at intracellular target sites remain unknown. Nitric oxide is a pollutant gas and an important intercellular messenger in the inflammatory immune response. When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdC12 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In experiments designed to determine whether NO acts by displacing Cd from cellular MT, cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% reductions, respectively, in the amount of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were observed at DEA/NO concentrations between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concentrations. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiology of Cd-induced carcinogenesis in human populations.