Cytotoxic effects of cadmium and zinc co-exposure in PC12 cells and the underlying mechanism.

Cadmium (Cd(2+)) is a well studied inducer of cellular necrosis and apoptosis. Zinc (Zn(2+)) is known to inhibit apoptosis induced by toxicants including Cd(2+) both in vitro and in vivo. The mechanism of Zn(2+)-mediated protection from Cd(2+)-induced cytotoxicity is not established. In this study, we aimed to understand the effects of Zn(2+) on Cd(2+)-induced cytotoxicity and apoptosis using PC12 cells. Cell viability and DNA fragmentation assays in PC12 cells exposed to Cd(2+) and/or Zn(2+) revealed that Cd(2+) (5 and 10 μmol/L) alone induced significant cell death, and co-exposure to Zn(2+) (5, 10, and 100 μmol/L) for 48 h had a protective effect. Assessment of intracellular free sulfhydryl levels and lactate dehydrogenase activity suggested that Cd(2+) (10 μmol/L) induced oxidative stress and disrupted cell membrane integrity. Addition of Zn(2+) (10 and 100 μmol/L) reduced Cd(2+)-mediated cytotoxicity. Changes in expression of the apoptotic factors Bax, Bcl-2, Bcl-x, and cytochrome c were measured via western blot and expression of caspase 9 was detected via reverse transcriptase polymerase chain reaction. Western blots showed that Zn(2+) (10 and 100 μmol/L) suppressed Cd(2+)-induced apoptosis (10 μmol/L) by reducing cytochrome c release into the cytosol, and downregulating the proapoptotic protein, Bax. In addition, expression of caspase 9 was lower in Cd(2+) (5 μmol/L)-treated PC12 cells when co-treated with Zn(2+) (2 and 5 μmol/L). These findings suggest that the effective inhibition of Cd(2+)-induced apoptosis in PC12 cells by Zn(2+) might be due to suppression of mitochondrial apoptosis pathway and inhibition of Cd(2+)-induced production of reactive oxygen species.