Mechanism of cadmium-induced cytotoxicity in rat hepatocytes: cadmium-induced active oxygen-related permeability changes of the plasma membrane.

The present study was performed to further elucidate the mechanism of cadmium (Cd)-induced cytotoxicity in rat hepatocytes focusing on the effects of Cd-induced acidification on cellular production of H2O2 and the integrity of the plasma membrane. Exposure of cells of Cd levels < 50 microM stimulated cellular production of H2O2 in a dose-dependent manner. In cells exposed to 50 microM Cd, generation of the toxic oxygen increased from 5 min after exposure, and reached a plateau at 15 min. The acidic medium at pH 6.5, a value which is corresponding to the cellular pH at maximal acidification induced by Cd, also enhanced production of the active oxygen at almost the same level as 25 microM Cd. These treatments affected permeability barrier of plasma membranes as assessed by nuclear staining with propidium iodide (PI, MW 668) and release of intracellular lactic dehydrogenase (LDH) into surrounding medium. Cd at 50 microM caused nuclear staining by the fluorescent probe, beginning from 15 min at exposure, reaching a peak at 60 min. LDH leakage likewise started from 60 min of Cd exposure onward. The acidic partially prevented by L-ascorbic acid pretreatment. H2O2-induced nuclear staining increased with the increasing pH values from 6.7 to 7.1 Cd at 50 microM lowered the cellular pH within 5 min, but the decreased cellular pH returned to a value near physiological levels 25 min later. Pretreatment with Amiloride, an inhibitor of the Na+/H+ exchange, partially blocked this pH recovery after acidification. The results indicate that Cd caused H2O2 accumulation and H+, Cd and H2O2-related permeability changes of the plasma membrane. This may link to subsequent extensive membrane damage occurring at near physiological cellular pH.