Vol. 61, Issue 4, 870-878, April 2002

Delayed Formation of Hydrogen Peroxide Mediates the Lethal Response Evoked by Peroxynitrite in U937 Cells

Ilaria Tommasini, Piero Sestili, and Orazio Cantoni

Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino, Urbino, Italy

The toxicity paradigm used in the present study involves exposure of U937 cells to a concentration of authentic peroxynitrite, leading to a rapid necrotic response mediated by mitochondrial permeability transition. We found that addition of catalase after treatment with peroxynitrite specifically prevents the loss of mitochondrial membrane potential and the ensuing lethal response. The protective effects of catalase were mimicked by the cocktail glutathione peroxidase/reduced glutathione. A defensive role of intracellular catalase was implied by experiments showing that catalase-depleted cells are hypersensitive to peroxynitrite and that cells with an increased catalase content, selected for their resistance to H₂O₂, are cross-resistant to peroxynitrite. Further experiments demonstrated that H₂O₂ formation takes place after peroxynitrite exposure. Various approaches using inhibitors of the mitochondrial respiratory chain as well as respiration-deficient cells revealed that the oxidant is produced upon dismutation of superoxides generated at the level of complex III. Interestingly, respiration-deficient cells were found to be resistant to peroxynitrite toxicity, and all those treatments increasing formation of H₂O₂ produced a parallel increase in toxicity. In conclusion, the results presented in this study indicate that peroxynitrite-induced impairment of electron transport from cytochrome b to cytochrome c1 leads to delayed formation of hydrogen peroxide, which plays a pivotal role in the ensuing necrotic response.