Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B-1

Accumulating evidence demonstrates that oxidative damage is one of the underlying mechanisms to the cytotoxicity and carcinogenicity of AFB(1). The main objective of this study is to show that AFB(1) increases reactive oxygen species (ROS) formation in hepatocytes. The ROS level was detected using a fluorescence probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS. It was found that AFB(1) exposure significantly enhanced DCF fluorescence formation in cultured rat hepatocytes. A dose-response of AFB(1) was also observed within the range of 10 nM to 1000 nM. Catalase (CAT) was able to completely prevent the increase of DCF fluorescence in AFB(1)-treated cells in a dose-dependent manner (from 500 to 2000 U/ml). Moreover, the significant inhibitory effects of desferrioxamine (DFO) and dimethyl sulfoxide (DMSO) on DCF fluorescence formation were also observed in both control and AFB(1)-treated hepatocytes. Therefore, results from the present study provide in vitro evidence indicating the generation of ROS in cultured rat hepatocytes caused by AFB(1) exposure. It is postulated that the metabolic process of AFB(1) by cytochrome P450 might be the possible source of the elevated ROS level in AFB(1)-treated hepatocytes. The enhanced level of ROS may be responsible for the oxidative damage caused by AFB(1), which may ultimately contribute to the cytotoxic and carcinogenic effects of AFB(1).