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

ABSTRACT

Cadmium (Cd2+) is a well studied inducer of cellular necrosis and apoptosis. Zinc (Zn2+) is known to inhibit apoptosis induced by toxicants including Cd2+ both in vitro and in vivo. The mechanism of Zn2+-mediated protection from Cd2+-induced cytotoxicity is not established. In this study, we aimed to understand the effects of Zn2+ on Cd2+-induced cytotoxicity and apoptosis using PC12 cells. Cell viability and DNA fragmentation assays in PC12 cells exposed to Cd2+ and/or Zn2+ revealed that Cd2+ (5 and 10 µmol/L) alone induced significant cell death, and co-exposure to Zn2+ (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 Cd2+ (10 µmol/L) induced oxidative stress and disrupted cell membrane integrity. Addition of Zn2+ (10 and 100 µmol/L) reduced Cd2+-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 Zn2+ (10 and 100 µmol/L) suppressed Cd2+-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 Cd2+ (5 µmol/L)-treated PC12 cells when co-treated with Zn2+ (2 and 5 µmol/L). These findings suggest that the effective inhibition of Cd2+-induced apoptosis in PC12 cells by Zn2+ might be due to suppression of mitochondrial apoptosis pathway and inhibition of Cd2+-induced production of reactive oxygen species.