Fábio E. Mingatto

Effect of Naturally Occurring Flavonoids on Lipid Peroxidation and Membrane Permeability Transition in Mitochondria

The ability of eight structurally related naturally occurring flavonoids in inhibiting lipid peroxidation and mitochondrial membrane permeability transition (MMPT), as well as respiration and protein sulfhydryl oxidation in rat liver mitochondria, was evaluated. The flavonoids tested exhibited the following order of potency to inhibit ADP/ Fe(II)-induced lipid peroxidation, estimated with the thiobarbituric acid assay: 3-O-methyl-quercetin > quercetin > 3,5,7,3,4-penta-O-methyl-quercetin > 3,7,3,4-tetra-O-methyl-quercetin > pinobanksin > 7-O-methyl-pinocembrin > pinocembrin > 3-O-acyl-pinobanksin. MMPT was estimated by the extent of mitochondrial swelling induced by 10 microM CaCl2 plus 1.5 mM inorganic phosphate or 30 microM mefenamic acid. The most potent inhibitors of MMPT were quercetin, 7-O-methyl-pinocembrin, pinocembrin, and 3,5,7,3,4-penta-O-methyl-quercetin. The first two inhibited in parallel the oxidation of mitochondrial protein sulfhydryl involved in the MMPT mechanism. The most potent inhibitors of mitochondrial respiration were 7-O-methyl-pinocembrin, quercetin, and 3-O-methyl-quercetin while the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin. In contrast 3,7,3,4-tetra-O-methyl-quercetin and 3,5,7,3,4-penta-O-methyl-quercetin showed the lowest ability to affect mitochondrial respiration. We conclude that, in general, the flavonoids tested are able to inhibit lipid peroxidation on the mitochondrial membrane and/or MMPT. Multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration, and therefore, increases the pharmacological potential of these compounds against pathological processes related to oxidative stress.

A proposed sequence of events for cadmium-induced mitochondrial impairment.

Cadmium is a very important environmental toxicant, the cytotoxicity mechanism of which is likely to involve mitochondria as a target. In the present study we addressed the cause/effect relationship between the multiple cadmium-induced responses involving the mitochondrial energetic and oxidative status. Assays were performed with succinate-energized rat liver mitochondria incubated with 5 microM CdCl(2) for 0-25 min, in the absence or presence, respectively, of N-ethylmaleimide (NEM), butylhydroxytoluene (BHT), ruthenium red (RR), and cyclosporine A+ADP. A sequence of events accounting for cadmium-induced mitochondrial impairment is proposed, beginning with an apparent interaction of Cd(2+) with specific protein thiols in the mitochondrial membrane, which stimulates the cations uptake via the Ca(2+) uniporter, and is followed by the onset of mitochondrial permeability transition (MPT); both effects dissipate the transmembrane electrical potential (Deltapsi), causing uncoupling, followed by an early depression of mitochondrial ATP levels. The respiratory chain subsequently undergoes inhibition, generating reactive oxygen species which together with iron mobilized by the cation, cause late, gradual mitochondrial membrane lipid peroxidation.

Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis – potentially implicated mechanisms

We evaluated the effects 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 effects. Within the 0u2003–u2003100u2003μM range thioridazine did not reduce the free radical 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) nor did it chelate iron. However, at 10u2003μM thioridazine showed important antioxidant activity on mitochondria, characterized by inhibition of accumulation of mitochondria‐generated O2•−, assayed as lucigenin‐derived chemiluminescence, inhibition of Fe2+/citrate‐mediated lipid peroxidation of the mitochondrial membrane (LPO), assayed as malondialdehyde generation, and inhibition of Ca2+/t‐butyl hydroperoxide (t‐BOOH)‐induced mitochondrial permeability transition (MPT)/protein‐thiol oxidation, assayed as mitochondrial swelling. Thioridazine respectively increased and decreased the fluorescence responses of mitochondria labelled with 1‐aniline‐8‐naphthalene sulfonate (ANS) and 1‐(4‐trimethylammonium phenyl)‐6 phenyl 1,3,5‐hexatriene (TMA‐DPH). The inhibition of LPO and MPT onset correlated well with the inhibition of cytochrome c release from mitochondria. 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 O2•− accumulation, as well as LPO, MPT and associated release of cytochrome c.

Effects of nimesulide and its reduced metabolite on mitochondria

We investigated the effects of nimesulide, a recently developed non‐steroidal anti‐inflammatory drug, and of a metabolite resulting from reduction of the nitro group to an amine derivative, on succinate‐energized isolated rat liver mitochondria incubated in the absence or presence of 20u2003μM Ca2+, 1u2003μM cyclosporin A (CsA) or 5u2003μM ruthenium red. Nimesulide uncoupled mitochondria through a protonophoretic mechanism and oxidized mitochondrial NAD(P)H, both effects presenting an EC50 of approximately 5u2003μM. Within the same concentration range nimesulide induced mitochondrial Ca2+ efflux in a partly ruthenium red‐sensitive manner, and induced mitochondrial permeability transition (MPT) when ruthenium red was added after Ca2+ uptake by mitochondria. Nimesulide induced MPT even in de‐energized mitochondria incubated with 0.5u2003mM Ca2+. Both Ca2+ efflux and MPT were prevented to a similar extent by CsA, Mg2+, ADP, ATP and butylhydroxytoluene, whereas dithiothreitol and N‐ethylmaleimide, which markedly prevented MPT, had only a partial or no effect on Ca2+ efflux, respectively. The reduction of the nitro group of nimesulide to an amine derivative completely suppressed the above mitochondrial responses, indicating that the nitro group determines both the protonophoretic and NAD(P)H oxidant properties of the drug. The nimesulide reduction product demonstrated a partial protective effect against accumulation of reactive oxygen species derived from mitochondria under conditions of oxidative stress like those resulting from the presence of t‐butyl hydroperoxide. The main conclusion is that nimesulide, on account of its nitro group, acts as a potent protonophoretic uncoupler and NAD(P)H oxidant on isolated rat liver mitochondria, inducing Ca2+ efflux or MPT within a concentration range which can be reached in vivo, thus presenting the potential ability to interfere with the energy and Ca2+ homeostasis in the liver cell.

Antioxidant activity of flavonoids in isolated mitochondria

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 µmol/L) on Fe2+/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 (IC50 = 1.23 ± 0.27 and 2.39 ± 0.79 µmol/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. Copyright

Fluoxetine interacts with the lipid bilayer of the inner membrane in isolated rat brain mitochondria, inhibiting electron transport and F1F0-ATPase activity.

The effects of fluoxetine on the oxidative phosphorylation of mitochondria isolated from rat brain and on the kinetic properties of submitochondrial particle F1F0-ATPase were evaluated. The state 3 respiration rate supported by pyruvate + malate, succinate, or ascorbate + tetramethyl-p-phenylenediamine (TMPD) was substantially decreased by fluoxetine. The IC50 for pyruvate + malate oxidation was ∼ 0.15 mM and the pattern of inhibition was the typical one of the electron-transport inhibitors, in that the drug inhibited both ADP- and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-stimulated respirations and the former inhibition was not released by the uncoupler. Fluoxetine also decreased the activity of submitochondrial particle F1F0-ATPase (IC50 ∼ 0.08 mM) even though K0.5 and activity of Triton X-100 solubilized enzyme were not changed substantially. As a consequence of these effects, fluoxetine decreased the rate of ATP synthesis and depressed the phosphorylation potential of mitochondria. Incubation of mitochondria or submitochondrial particles with fluoxetine under the conditions of respiration or F1F0-ATPase assays, respectively, caused a dose-dependent enhancement of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. These results show that fluoxetine indirectly and nonspecifically affects electron transport and F1F0)-ATPase activity inhibiting oxidative phosphorylation in isolated rat brain mitochondria. They suggest, in addition, that these effects are mediated by the drug interference with the physical state of lipid bilayer of inner mitochondrial membrane.

Hg(II)-induced renal cytotoxicity: In vitro and in vivo implications for the bioenergetic and oxidative status of mitochondria

The effects of Hg(II) on bioenergetic and oxidative status of rat renal cortex mitochondria were evaluated both in vitro, and in vivo 1 and 24 h after treatment of animals with 5 mg HgCl2/kg ip. The parameters assessed were mitochondrial respiration, ATP synthesis and hydrolysis, glutathione content, lipid peroxidation, protein oxidation, and activity of antioxidant enzymes. At low concentration (5 µM) and during a short incubation time, Hg(II) uncoupled oxidative phosphorylation while at slightly higher concentration or longer incubation time the ion impaired the respiratory chain. The rate of ATP synthesis and the phosphorylation potential of mitochondria were depressed, although inhibition of ATP synthesis did not exceed 50%. In vivo, respiration and ATP synthesis were not affected 1 h post-treatment, but were markedly depressed 24 h later. ATP hydrolysis by submitochondrial particle FoF1-ATPase was inhibited (also by no more than 50%) both in vitro, and in vivo 1 and 24 h post-treatment. Hg(II) induced maximum ATPase inhibition at about 1 uM concentration but did not have a strong inhibitory effect in the presence of Triton X-100. Oxidative stress was not observed in mitochondria 1 h post-treatment. However, 24 h later Hg(II) reduced the GSH/GSSG ratio and increased mitochondrial lipid peroxidation and protein oxidation, as well as inhibited GSH-peroxidase and GSSG-reductase activities. These results suggest that the following sequence of events may be involved in Hg(II) toxicity in the kidney: (1) inhibition of FoFl-ATPase, (2) uncoupling of oxidative phosphorylation, (3) oxidative stress-associated impairment of the respiratory chain, and (4) inhibition of ATP synthesis.

Vitamin K1 Prevents the Effect of Hypoxia on Phenylephrine-Induced Contraction in the Carotid Artery

The purpose of the present study was to investigate the effects of vitamin K₁ on vascular smooth muscle contractility in response to phenylephrine (Phe) during hypoxia. Rat carotid rings were placed in an organ chamber containing Krebs’ solution. The rings were subjected to hypoxia by changing the gas from 95% O₂:5% CO₂ to a mixture containing 95% N₂:5% CO₂. Concentration response curves for Phe were determined before, during, and after exposure to hypoxia. Endothelium-intact rings were incubated with vitamin K₁ for 10 min in normoxic conditions before being subjected to hypoxia. In another set of experiments, endothelium-intact rings were incubated with N^G-nitro-L-arginine methyl ester (L-NAME), indomethacin or a combination of these drugs for 30 min. In endothelium-intact rings, hypoxia caused significant reductions in E_max (from 0.97 ± 0.03 to 0.61 ± 0.04 g/mg; mean ± SEM) and pD₂ values (from 8.26 ± 0.07 to 7.67 ± 0.10). Removal of a functional endothelium effectively prevented the hypoxia-induced reduction in E_max values, but not in pD₂ values (from 9.14 ± 0.10 to 8.70 ± 0.11). Pretreatment with vitamin K₁ at 3 concentrations (5 × 10^–8, 5 × 10^–7, 5 × 10^–6 mol/l) prevented the inhibitory effect of hypoxia in intact rings. Exposure of endothelium-intact rings to L-NAME plus indomethacin also inhibited the hypoxic effect. Our results show that vitamin K₁ prevents the deleterious vascular effects induced by hypoxia, probably due to its action on endothelial cells.

Vitamin K1 attenuates hypoxia-induced relaxation of rat carotid artery

Vascular responses to hypoxia are heterogeneous and involve the release of vasodilators substances such as nitric oxide (NO) and prostacyclin (PGI(2)). In vitro studies have shown that Vitamin K(1) modulates the release of arachidonic acid (AA) in vascular cells, and thus inhibits the capacity of blood vessels to synthesise vasodilator AA metabolites. The aim of our work was to investigate the effects of Vitamin K(1) on the hypoxia-induced vasorelaxation. Hypoxia was induced by changing the gas from 95% O(2)/5% CO(2) to a mixture containing 95% N(2)/5% CO(2). Rat carotid arteries were pre-contracted with phenylephrine (Phe, 10(-8)mol/l) and when the contraction reached a plateau, the bath was bubbled with 95% N(2)/5% CO(2) for 15 min. In intact rings, there was a total relaxation after 15 min of exposure to hypoxia. Removal of the endothelium strongly reduced hypoxia-induced relaxation. In intact rings, indomethacin and L-NAME reduced the hypoxic relaxation after 5 min of exposure but not after 10 or 15 min. Exposure of endothelium-intact rings to Vitamin K(1) (5 x 10(-6) and 5 x 10(-5)mol/l), L-NAME+indomethacin as well as the combination of L-NAME+indomethacin+Vitamin K(1) reduced the hypoxic relaxation after 5 and 10 min of exposure but not after 15 min. At 5 x 10(-7)mol/l Vitamin K(1) did not attenuate hypoxia-induced relaxation. It was also found that Vitamin K(1) (5 x 10(-6) and 5 x 10(-5)mol/l) inhibited ACh-induced relaxation in normoxic conditions. These results show that the effect of Vitamin K(1) on attenuating hypoxia-induced vasorelaxation is concentration-dependent and probably related to its action on endothelial cells.