Reduced cadmium-induced cytotoxicity in cultured liver cells following 5-azacytidine pretreatment

Recent work indicated that administration of the pyrimidine analog 5-azacytidine (AZA), either to cells in culture or to rats, results in an enhancement of expression of the metallothionein (MT) gene. Since MT is thought to play a central role in the detoxification of cadmium, the present study was designed to assess the effect of AZA pretreatment on cadmium cytotoxicity. Cultured rat liver cells (TRL 1215) in log phase of growth were first exposed to AZA (8 microM). Forty-eight hours later, cadmium (10 microM) was added. MT concentrations were then measured 24 hr after the addition of cadmium. A modest increase in MT amounts over control (1.7-fold) was detected after AZA treatment alone. Cadmium alone resulted in a 10-fold increase in MT concentrations. The combination of AZA pretreatment followed by cadmium exposure caused a 23-fold increase in MT concentrations over control. Treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of AZA pretreatment on cadmium induction of MT, indicating that cell division is required. AZA-pretreated cells were also harvested and incubated in suspension with cadmium (250 microM, 37 degrees C) for 0 to 90 min. After incubation intracellular and extracellular fluids were separated by centrifugation through an oil layer. AZA-pretreated cells showed marked reductions in cadmium-induced cytotoxicity as reflected by reduced intracellular potassium loss, glutamic-oxaloacetic transaminase loss, and lipid peroxidation (assessed by thiobarbituric acid reactants) following cadmium exposure. AZA pretreatment had no effect on the cellular uptake of cadmium. Results suggest that AZA pretreatment induces tolerance to cadmium cytotoxicity which appears to be due to an increased capacity to synthesize MT rather than high quantities of preexisting MT at the time of cadmium exposure.