Determination of metallothionein content in hepatoma cells by differential pulse polarography

Differential pulse polarography (DPP) was applied to measure metallothionein (MT) content in the RH-35 rat hepatoma cell. The method was sensitive in measuring MT at nanogram concentrations. The MT measurement was not interfered by small molecular weight proteins. Cellular MT content increased upon incubated in medium containing different concentrations of CdCl2 or ZnCl2. The increase was concentration-dependent. In the case of CdCl2, significant increase in cellular MT content was observed at a level as low as 0.18 mg/l. The change in MT occurred when there was no significant change in cell viability or cellular protein content. The concentration of ZnCl2 needed to induce the same amount of MT as that with 0.18 mg/l CdCl2 was 12 mg/l. At high concentrations (> 12 mg/l), ZnCl2 also caused an increase in cellular protein content. Therefore, when calculation was based on cellular protein content, ZnCl2 did not caused further increase in MT. L-ascorbic caused significant increase in cellular MT content when it was presented at levels above 100 mg/l but not at 50 mg/l. The effect of L-ascorbic acid on MT induction was not dose-dependent. At 150 mg/l, the amount of MT induced was not different from that with 100 mg/l. Thus, L-ascorbic might not directly influence cellular MT content. Cysteine at 4 and 40 mg/l did not increase cellular MT content. The results of this study showed that cellular MT content could be measured by DPP. Our finding that CdCl2 at sub-lethal concentrations can effectively induce MT suggests that MT may serve as an early warning signal prior to serve as an early warning signal prior to the occurrence of cell death.