4,6-DINITRO-O-CRESOL UNCOUPLES OXIDATIVE-PHOSPHORYLATION AND INDUCES MEMBRANE-PERMEABILITY TRANSITION IN RAT-LIVER MITOCHONDRIA

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioen ergetics and inner membrane permeability of isolated rat liver mitocho ndria was studied. We observed that DNOC (10-50 mu M) acts as a classi cal uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respir ation in the presence or absence of ADP and a decrease in transmembran e potential. The protonophoric activity of DNOC was evidenced by the i nduction of mitochondrial swelling: in hyposmotic Kf-acetate medium, i n the presence of valinomycin. At higher concentrations (> 50 mu M), D NOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be obse rved. The addition of uncoupling concentrations of DNOC to Ca2+-loaded mitochondria treated with Ruthenium Red results in non-specific membr ane permeabilization, as evidenced by mitochondrial swelling in isosmo tic sucrose medium. Cyclosporin A, which inhibits mitochondrial permea bility transition, prevented DNOC-induced mitochondrial swelling in th e presence of Ca2+, which was accompanied by a decrease in mitochondri al membrane protein thiol content, owing to protein thiol oxidation. C atalase partially inhibits mitochondrial swelling and protein thiol ox idation, indicating the participation of mitochondrial-generated react ive oxygen species in this process. It is concluded that DNOC is a pot ent protonophore acting as a classical uncoupler of oxidative phosphor ylation in rat liver mitochondria by dissipating the proton electroche mical gradient. Treatment of Ca2+-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transitio n, associated with membrane protein thiol oxidation by reactive oxygen species. Copyright (C) 1997 Elsevier Science Ltd.