Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

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Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H 2O 2 generation, followed by significant changes in the mitochondrial membrane potential (ΔΨm). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H 2O 2-resistant clone of HL-60, confirming the involvement of H 2O 2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited ΔΨm change and DNA ladder formation but not H 2O 2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H 2O 2 generation and DNA ladder formation. These results suggest that rotenone induces O 2 −-derived H 2O 2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H 2O 2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.