4,6-Dinitro- o-cresol uncouples oxidative phosphorylation and induces membrane permeability transition in rat liver mitochondria

Abstract

The effect of the herbicide 4,6-dinitro- o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10–50 μM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K +-acetate medium, in the presence of valinomycin. At higher concentrations ( > 50 μM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca 2+-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca 2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca 2+-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.