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1 We evaluated the eects of the phenothiazine derivative thioridazine on mechanisms of mitochondria potentially implicated in apoptosis, such as those involving reactive oxygen species (ROS) and cytochrome c release, as well as the involvement of drug interaction with mitochondrial membrane in these eects. 2 Within the 0 ± 100 mM range thioridazine did not reduce the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) nor did it chelate iron. 3 However, at 10 mM thioridazine showed important antioxidant activity on mitochondria, characterized by inhibition of accumulation of mitochondria-generated O 2 .7, assayed as lucigeninderived chemiluminescence, inhibition of Fe 2+ /citrate-mediated lipid peroxidation of the mitochondrial membrane (LPO), assayed as malondialdehyde generation, and inhibition of Ca 2+ /t-butyl hydroperoxide (t-BOOH)-induced mitochondrial permeability transition (MPT)/protein-thiol oxidation, assayed as mitochondrial swelling. 4 Thioridazine respectively increased and decreased the¯uorescence responses of mitochondria labelled with 1-aniline-8-naphthalene sulfonate (ANS) and 1-(4-trimethylammonium phenyl)-6 phenyl 1,3,5-hexatriene (TMA-DPH). 5 The inhibition of LPO and MPT onset correlated well with the inhibition of cytochrome c release from mitochondria. 6 We conclude that thioridazine interacts with the inner membrane of mitochondria, more likely close to its surface, acquiring antioxidant activity toward processes with potential implications in apoptosis such as O 2 .7 accumulation, as well as LPO, MPT and associated release of cytochrome c.

We described the effects of nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) and its reduced metabolite in isolated rat hepatocytes. Nimesulide stimulated the succinatesupported state 4 respiration of mitochondria, indicating an uncoupling effect of the drug. Incubation of hepatocytes with nimesulide (0.1-1 mM) elicited a concentration-and time-dependent decrease in cell viability as assessed by lactate dehydrogenase leakage, a decrease of mitochondrial membrane potential as assessed by rhodamine 123 retention, and cell ATP depression. Nimesulide also decreased the levels of NAD(P)H and glutathione in hepatocytes, but the extent of the effects was less pronounced in relation to the energetic parameters; in addition, these effects did not imply the peroxidation of membrane lipids. The decrease in the viability of hepatocytes was prevented by fructose and, to a larger extent, by fructose plus oligomycin; it was stimulated by proadifen, a cytochrome P450 inhibitor. In contrast, the reduced metabolite of nimesulide did not present any of the effects observed for the parent drug. These results indicate that: 1) nimesulide causes injury to the isolated rat liver cells, 2) this effect is mainly mediated by impairment of ATP production by mitochondria due to uncoupling, and 3) on account of the activity of its nitro group, the parent drug by itself is the main factor responsible for its toxicity to the hepatocytes.

Mitochondria are important intracellular sources and targets of reactive oxygen species (ROS), while flavonoids, a large group of secondary plant metabolites, are important antioxidants. Following our previous study on the energetics of mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, the present work addressed the antioxidant activity of these compounds (1-50 micromol/L) on Fe(2+)/citrate-mediated membrane lipid peroxidation (LPO) in isolated rat liver mitochondria, running in parallel studies of their antioxidant activity in non-organelle systems. Only quercetin inhibited the respiratory chain of mitochondria and only galangin caused uncoupling. Quercetin and galangin were far more potent than taxifolin and catechin in affording protection against LPO (IC(50) = 1.23 +/- 0.27 and 2.39 +/- 0.79 micromol/L, respectively), although only quercetin was an effective scavenger of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals. These results, together with the previous study, suggest that the 2,3-double bond in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids in mitochondria, the presence of an o-di-OH structure on the B-ring, as occurs in quercetin, favours this activity via superoxide scavenging, while the absence of this structural feature in galangin, favours it via a decrease in membrane fluidity and/or mitochondrial uncoupling.