Molecular mechanisms of butylated hydroxylanisole-induced toxicity: Induction of apoptosis through direct release of cytochrome c

Butylated hydroxyanisole (BHA), a commonly used food preservative, is repor ted to have anticarcinogenic properties in some animal models. However, the use of BHA as a chemopreventive agent against cancer in human has been cha llenged by the observation that BHA may exert toxic effect in some tissues of animals. Therefore, it is of great significance to understand the mechan ism of BHA-induced toxicity. Here, we report that BHA induces apoptosis in freshly isolated rat hepatocytes. Treatment of hepatocytes with BHA also in duced loss of mitochondrial transmembrane potential (Delta psi(m)), cytochr ome c, and activation of caspase-3, -8, and -9 but not caspase-1. Pretreatm ent with cyclosporin A, an agent that stabilizes mitochondrial permeability transition pore, inhibited BHA-induced loss of Delta psi(m), cytochrome c release, caspase activation, and apoptosis. Interestingly, benzyloxycarbony l-Val-Ala-Asp-fluoromethyl ketone failed to prevent these mitochondrial eve nts, although it blocked caspase activation and apoptosis. Furthermore, BHA -induced apoptosis appeared to be independent of formation of reactive inte rmediates, as evidenced by the lack of effects of antioxidants N-acetyl-L-c ysteine and ascorbic acid. Indeed, direct incubation of BHA with isolated m itochondria triggered cytochrome c release. Thus, these results indicate th at the cytotoxicity of BHA is due to the induction of apoptosis that is med iated by the direct release of cytochrome c and the subsequent activation o f caspases.