Disruption of metallothionein expression with antisense oligonucleotides abolishes protection against cadmium cytotoxicity in molluscan hemocytes

The relationship between metallothionein (MT) induction and cytotoxicity was examined in isolated oyster hemocytes exposed in vitro to cadmium, copper, and zinc. In all cases MT induction increased to peak levels with increased metal dose, then declined with continued increases in dose. The effectiveness of these metals as inducers of MT was cadmium > copper > zinc, with cadmium the most effective inducer in magnitude of induction and sensitivity to dose. Cytotoxicity of the metals, based on decreases in lysosomal neutral red retention times, was copper > cadmium > zinc. Zinc was relatively nontoxic and a poor inducer of MT. Cadmium, which was intermediate in toxicity, required concentrations in excess of that causing peak induction of MT to have substantial effects on lysosomal membrane integrity. In contrast, copper was highly cytotoxic at concentrations below that which resulted in peak MT induction. This reversal in the rank order of cadmium and copper suggested that the toxicity of cadmium was tempered by the protective effects of MT. This was verified by disrupting MT expression with antisense phosphorothioate-substituted oligodeoxynucleotides. Administration of 2.5 microM CdCl(2), which induced MT but had no effect on lysosomal membrane stability when administered alone, reduced neutral red retention time to 41% of control levels when administered in the presence of antisense. The resulting toxicity was greater than that caused by a comparable concentration of copper. The findings represented evidence that expression of MT protects against cadmium toxicity in an estuarine mollusc.