Proteção do dano oxidativo hepático induzido por ferro pelo extrato aquoso da planta Plectranthus barbatus / Protection against hepatic oxidative damage induced by iron by the aqueous extract of Plectranthus barbatus

Plectranthus barbatus Andrews (Lamiaceae) é uma planta muita utilizada na medicina popular para o tratamento de doenças gastrointestinais e hepáticas. O objetivo do presente trabalho foi estudar o efeito protetor do extrato aquoso de P. barbatus (EAPB) sobre os danos hepáticos causados pela sobrecarga de ferro provocada pelo ferro-dextran em ratos. O tratamento com ferro-dextran induziu uma redução significativa na concentração de glutationa reduzida nos animais tratados em relação ao grupo controle e o tratamento prévio dos animais com o EAPB protegeu o fígado do efeito provocado pelo ferro neste parâmetro. Com relação à lipoperoxidação, houve aumento significativo na concentração de malondialdeído (MDA) nos animais tratados em relação ao controle, entretanto, quando os animais receberam o tratamento prévio com o EAPB, houve redução significativa na concentração do MDA. A análise histopatológica mostrou que o grupo tratado com ferro-dextran apresentou grânulos de ferro no citoplasma das células de Kupffer com alargamento das mesmas e algumas com os núcleos hipertróficos. O tratamento prévio com EAPB resultou no desaparecimento dos sinais de danos às células de Kupffer sem nenhum núcleo hipertrófico, mas com a presença de grânulos de ferro totalmente fagocitados, o que demonstra uma aparência morfológica normal. Portanto, o EAPB pode ser útil na prevenção de danos hepáticos induzidos por sobrecarga de ferro.

The Plectranthus barbatus Andrews (Lamiaceae) is a plant largely used in folk medicine to treat gastrointestinal and liver diseases. The objective of this work was to study the protective effect of the aqueous extract of P. barbatus (EAPB) against damage caused by iron overload induced by iron dextran in rat liver. Treatment with iron-dextran induced a significant reduction in the glutathione levels in treated animals compared to control group, and the pretreatment of animals with EAPB protected the liver from the effects caused by iron in this parameter. With respect to lipid peroxidation, a significant increase in the malondialdehyde (MDA) levels in treated animals compared to control was observed; however, when the animals were pretreated with EAPB, there was a significant reduction in the MDA levels. Histopathological analysis showed that the group treated with iron-dextran showed iron granules in the cytoplasm of the Kupffer cells and some of them presented enlarged nuclei. The group previously treated with EAPB showed the disappearance of the signs of damage to the Kupffer cells with no nucleus hypertrophy but with the presence of iron granules completely phagocytosed by these cells, which showed a normal morphological appearance. Therefore, the EAPB may be useful in the prevention of liver damage induced by iron overload.

Aromatic antiepileptic drugs and mitochondrial toxicity: effects on mitochondria isolated from rat liver.

Idiosyncratic hepatotoxicity is a well-known complication associated with aromatic antiepileptic drugs (AAED), and it has been suggested to occur due to the accumulation of toxic arene oxide metabolites. Although there is clear evidence of the participation of an immune process, a direct toxic effect involving mitochondria dysfunction is also possible. The effects of AAED on mitochondrial function have not been studied yet. Therefore, we investigated, in vitro, the cytotoxic mechanism of carbamazepine (CB), phenytoin (PT) and phenobarbital (PB), unaltered and bioactivated, in the hepatic mitochondrial function. The murine hepatic microsomal system was used to produce the anticonvulsant metabolites. All the bioactivated drugs (CB-B, PB-B, PT-B) affected mitochondrial function causing decrease in state three respiration, RCR, ATP synthesis and membrane potential, increase in state four respiration as well as impairment of Ca2+ uptake/release and inhibition of calcium-induced swelling. As an unaltered drug, only PB, was able to affect mitochondrial respiration (except state four respiration) ATP synthesis and membrane potential; however, Ca2+ uptake/release as well as swelling induction were not affected. The potential to induce mitochondrial dysfunction was PT-B>PB-B>CB-B>PB. Results suggest the involvement of mitochondrial toxicity in the pathogenesis of AAED-induced hepatotoxicity.

Effects of nimesulide and its reduced metabolite on mitochondria.

1. 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 20 microM Ca(2+), 1 microM cyclosporin A (CsA) or 5 microM ruthenium red. 2. Nimesulide uncoupled mitochondria through a protonophoretic mechanism and oxidized mitochondrial NAD(P)H, both effects presenting an EC(50) of approximately 5 microM. 3. Within the same concentration range nimesulide induced mitochondrial Ca(2+) efflux in a partly ruthenium red-sensitive manner, and induced mitochondrial permeability transition (MPT) when ruthenium red was added after Ca(2+) uptake by mitochondria. Nimesulide induced MPT even in de-energized mitochondria incubated with 0.5 mM Ca(2+). 4. Both Ca(2+) efflux and MPT were prevented to a similar extent by CsA, Mg(2+), ADP, ATP and butylhydroxytoluene, whereas dithiothreitol and N-ethylmaleimide, which markedly prevented MPT, had only a partial or no effect on Ca(2+) efflux, respectively. 5. 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. 6. 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. 7. 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 Ca(2+) efflux or MPT within a concentration range which can be reached in vivo, thus presenting the potential ability to interfere with the energy and Ca(2+) homeostasis in the liver cell.

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 approximately 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 approximately 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.

Influence of nonsteroidal anti-inflammatory drugs on calcium efflux in isolated rat renal cortex mitochondria and aspects of the mechanisms involved.

In the present study we investigated the influence of several nonsteroidal anti-inflammatory drugs on calcium efflux in isolated rat renal cortex mitochondria in order to assess their potential to disrupt cell calcium homeostasis, as well as aspects of the mechanisms associated with oxidation of mitochondrial pyridine nucleotides (NAD(P)H) and with inhibition of the process by cyclosporin A (CsA). Calcium efflux was estimated with arsenazo III as an indicator and the redox state of NAD(P)H was monitored fluorimetrically at the 366/450 nm excitation/emission wavelength pair. Dipyrone, paracetamol and ibuprofen did not induce calcium efflux even at 1 mM, piroxicam and salicylate were poor inducers, while diclofenac sodium and mefenamic acid were potent inducers releasing calcium even at 20 microM and 10 microM, respectively. In the presence of 10 microM calcium, CsA had no appreciable effect while in the presence of 30 microM calcium it delayed calcium efflux. Oxidation of mitochondrial NAD(P)H, concomitant with calcium efflux and inhibited by CsA, was observed only in the presence of 30 microM calcium. The results suggest that diclofenac sodium and mefenamic acid induce calcium efflux in mitochondria through both a mechanism intrinsic to the mitochondrial membrane permeability transition and a mechanism including the electroneutral Ca2+/nH+ porter.

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.

Diclofenac sodium and mefenamic acid: potent inducers of the membrane permeability transition in renal cortex mitochondria.

The ability of nonsteroidal anti-inflammatory drugs (NSAIDs) to induce Ca(2+)-mediated/cyclosporin A-sensitive mitochondrial membrane permeability transition (MMPT) was evaluated by monitoring swelling of isolated rat renal cortex mitochondria in the presence of 20 microM CaCl2. Dipyrone and paracetamol did not induce MMPT, while piroxicam and acetylsalicylic acid (and its metabolite salicylate) were poor inducers. In contrast, diclofenac sodium and mefenamic acid were potent triggering agents, inducing MMPT at 2 microM, a concentration below those previously shown to uncouple and/or inhibit oxidative phosphorylation. When compared to salicylate, a classical uncoupler and inducer of MMPT, the potency of diclofenac sodium and mefenamic acid was about 50-fold greater. Swelling was completely prevented by EGTA, cyclosporin A, or MgCl2, and only partially by ADP or dithiothreitol. Under the same experimental conditions as for the swelling assays, the drugs depressed the membrane potential of mitochondria, an effect prevented by cyclosporin A and restored by EGTA. Also, the drugs did not induce membrane lipid peroxidation or changes in GSSG levels, but led to a small decrease in protein thiol content, as well as to a substantial decrease in the NADPH levels of mitochondria. Hence, membrane depolarization and pyridine nucleotide oxidation seem to be involved in MMPT induction by these NSAIDs. The potency in eliciting the process, like the uncoupling activity, seems to be influenced by the lipophilic character of the molecules.

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 i.p. 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 microM) 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 microM 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 FoF1-ATPase, (2) uncoupling of oxidative phosphorylation, (3) oxidative stress-associated impairment of the respiratory chain, and (4) inhibition of ATP synthesis.

In vitro interaction of nonsteroidal anti-inflammatory drugs on oxidative phosphorylation of rat kidney mitochondria: respiration and ATP synthesis.

The in vitro interference of some of most important nonsteroidal anti-inflammatory drugs (NSAIDs) with the respiration of rat kidney (renal cortex) mitochondria and ATP synthesis was evaluated. Acetylsalicylic acid, diclofenac sodium, mefenamic acid, and piroxicam both uncoupled and inhibited oxidative phosphorylation in mitochondria energized with glutamate plus malate or with succinate, while dipyrone only uncoupled and paracetamol only inhibited it. The drug concentrations affecting mitochondrial respiration were in the low to middle micromolar range for diclofenac, mefenamic acid, and piroxicam, and in the low millimolar range for acetylsalicylic acid, dipyrone, and paracetamol. The pattern of inhibition, except for the paracetamol, was similar to that expressed by the respiratory chain inhibitors. NSAIDs also inhibited the rate of ATP synthesis in mitochondria energized with glutamate plus malate, as well as the phosphorylation potential of mitochondria. The IC50 values for rate of ATP synthesis, using 2 mM ADP, were about 0.1 mM for diclofenac sodium and mefenamic acid, 0.7 mM for piroxicam, and in the range of 5-8 mM for acetylsalicylic acid, dipyrone, and paracetamol. The potential for renal energetic cytotoxicity of NSAIDs is discussed considering their ability to interact with the oxidative phosphorylation in rat renal cortex mitochondria. A comparison is made with the interference of salicylate, the main metabolite of acetylsalicylic acid, and a classical uncoupler of oxidative phosphorylation.