Molecular mechanism of cell death induced by the antioxidant tert-butylhydroxyanisole in human monocytic leukemia U937 cells

A phenolic antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) is a widely used food additive. BHA had cytotoxicity in human monocytic leukemia U937 cells. BHA at 0.75 mm caused nuclear condensation and fragmentation, structural damage in mitochondria, decrease in mitochondrial transmembrane potential, and internucleosomal DNA cleavage. It induced the activities of caspase-3 and/or -7, -6, -8 and -9, especially high when DEVD-MCA was the substrate (caspase-3 and/or -7). DEVDase activity increased in time- and dose-dependent manner and high activity was observed in lysates of cells treated for 3 h at 0.75 mm. Addition of GSH (reduced glutathione) during the treatment of cells with BHA inhibited the induction of DEVDase activity, and the intracellular GSH level decreased as the concentration of BHA was raised. Intracellular ATP levels decreased in time- and dose-dependent manner when the cells were treated with BHA in the presence or absence of glucose. Enzyme activities involved in the respiratory chain were assayed with the mitochondrial fraction prepared from U937 cells. BHA distinctly inhibited NADH-ubiquinone oxidoreductase (complex I) and cytochrome c oxidase (complex IV) at low concentrations. Suceinate-ubiquinone oxidoreductase (complex II) was also inhibited, but to somewhat less extent. Without mitochondrial enzymes, BHA stimulated the ubiquinol-dependent reduction of cytochrome c (complex III), but it might have some detrimental effects on the mitochondrial enzyme reaction of complex III. The inhibition of mitochondrial oxidative phosphorylation might corroborate the mechanistic evidence for apoptosis of leukemia cells by BHA. Cell death induced by BHA is primarily ascribable to apoptosis.