Inhibition of palmityl carnitine oxidation in rat liver mitochondria by tert-butyl hydroperoxide

Mitochondria as an energy generating cell device are very sensitive to oxidative damage. Our previous findings obtained in hepatocytes demonstrated that Complex I of the respiratory chain is more sensitive to oxidative damage than other respiratory chain complexes. We present additional data on isolated mitochondria showing that palmityl carnitine oxidation is strongly depressed at a low (200 microM) tert-butyl hydroperoxide (tBHP) concentration, while oxidation of the flavoprotein-dependent substrate - succinate is not affected and neither is ATP synthesis inhibited by tBHP. In the presence of tBHP, the respiratory control index for palmityl carnitine oxidation is strongly depressed, but when succinate is oxidized the respiratory control index remains unaffected. Our findings thus indicate that flavoprotein-dependent substrates could be an important nutritional factor for the regeneration process in the necrotic liver damaged by oxidative stress.     

Tetrandrine concentrations not affecting oxidative phosphorylation protect rat liver mitochondria from oxidative stress

The effects of tetrandrine (6,6', 7,12-tetramethoxy-2, 2'-dimethyl-berbaman) on the mitochondrial function were assessed on oxidative stress, mitochondrial permeability transition (MPT), and bioenergetics of rat liver mitochondria. At concentrations lower than 100nmol/mg protein, tetrandrine decreased the hydrogen peroxide formation, the extent of lipid peroxidation, the susceptibility to Ca(2+)-induced opening of MPT pore, and inhibited the inner membrane anion channel activity, not significantly affecting the mitochondrial bioenergetics. High tetrandrine concentrations (100-300nmol/mg protein) stimulated succinate-dependent state 4 respiration, while some inhibition was observed for state 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations. The respiratory control ratio and the transmembrane potential were depressed but the adenosine diphosphate to oxygen (ADP/O) ratio was less affected. A slight increase of the inner mitochondrial membrane permeability to H(+) and K(+) by tetrandrine was also observed. It was concluded that low concentrations of tetrandrine afford protection against liver mitochondria injury promoted by oxidative-stress events, such as hydrogen peroxide production, lipid peroxidation, and induction of MPT. Conversely, high tetrandrine concentrations revealed toxicological effects expressed by interference with mitochondrial bioenergetics, as a consequence of some inner membrane permeability to H(+) and K(+) and inhibition of the electron flux in the respiratory chain. The direct immediate protective role of tetrandrine against mitochondrial oxidative stress may be relevant to clarify the mechanisms responsible for its multiple pharmacological actions.     

Free radical involvement in the oxidative phenomena induced by tert-butyl hydroperoxide in erythrocytes

Free radical involvement in the oxidative events induced by tert-butyl hydroperoxide in erythrocytes has been demonstrated by the use of the electron spin resonance technique of spin trapping with the spin trap 5.5-dimethyl-1-pyrroline-N-oxide (DMPO). The reactions of tert-butyl hydroperoxide with haemoglobins and intact cell systems were studied. Oxyhaemoglobin-containing system showed exclusive production of the t-butyloxy radical spin adduct of DMPO (DMPO-OBut), indicating t-butyloxy radical production. Methaemoglobin-containing systems showed the production of an oxidised derivative of DMPO, 5,5-dimethyl-2-ketopyrrolidino-1-oxyl (DMPOX)-previously associated with the generation of highly oxidised haem-iron. Carbon monoxyhaemoglobin-containing systems show the production of both DMPO-OBut and DMPOX but markedly slower than in either of the other haemoglobin systems. Generally, free radical production in haemoglobin systems was faster than in intact cell systems, indicating a membrane transport rate-limiting step for the tert-butyl hydroperoxide-mediated effects. Data from the use of free radical scavengers to inhibit DMPO-OBut production was consistent with the known reactivities of the scavengers toward t-butyloxy radicals. These and previously reported results (Trotta, R. J., Sullivan, S. G. and Stern, A. (1981) Biochim. Biophys. Acta 679, 230-237 and (1982) Biochem. J. 204, 405-415) implicate important roles for t-butyloxy radicals and haem intermediates in tert-butyl hydroperoxide-induced lipid peroxidation and haemoglobin oxidation in erythrocytes, respectively.     

Mechanism of nitrofurantoin toxicity and oxidative stress in mitochondria

5-Nitrofuran derivatives change the inner mitochondrial membrane permeability as indicated by the transmembrane potential, the rate of spontaneous K+ efflux and the basal respiratory rate: (a) at low concentrations nitrofurantoin prevents the increase of inner membrane permeability due to hydroperoxides or to diamide; (b) at higher concentrations or after longer times of incubation, nitrofurantoin enhances the membrane damage due to hydroperoxides or to diamide; the damage due Ca2+ plus Pi is enhanced by nitrofurantoin at all concentrations; (c) higher nitrofurantoin concentrations cause membrane damage independently of the presence of hydroperoxides or of diamide. The effect of nitrofurantoin is cancelled by the addition of free-radical scavengers. The above effects of nitrofurantoin are compatible with the observations of Mason and colleagues that nitrofurantoin is reduced by a NADPH nitroreductase to a nitro anion radical which can then undergo subsequent reactions, among which are (a) initiation of a free-radical reaction chain and (b) reduction of hydroperoxides and diamide.     

Cyclosporin A modifies cytoplasmic calcium levels in isolated hepatocytes exposed to oxidative stress due to tert-butyl hydroperoxide

Within the framework of our studies on hypertension in various rat strains, we have examined the effect of cyclosporin A (CsA) on intracellular calcium signaling under conditions of oxidative stress. For these preliminary experiments, we have chosen isolated hepatocytes of normotensive rats as a model system for the study of the role of intracellular calcium. We used tert-butyl hydroperoxide (t-BHP, 1 mmol x l(-1)) as an prooxidant agent. When compared to the controls, we found increased levels of cytosolic free calcium concentration (Ca2+i) during 120 min incubation. The preincubation of hepatocytes with CsA in the concentration of 0.5 micromol x l(-1)] did not change the physiological level of cytosolic calcium. However, a dual action of CsA on elevated Ca2+i was observed during oxidative injury of hepatocytes: while in the first period of incubation CsA increased Ca2+i, CsA reduced the effect of t-BHP on Ca2+i during the next period of incubation. This indicates the ability of CsA to modify oxidative stress, but further studies are necessary to explain these findings.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Catalase takes part in rat liver mitochondria oxidative stress defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.     

The effects of linoleate hydroperoxide on respiration and oxidative phosphorylation of rat liver mitochondria.

Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.     

Heme oxygenase induction by menadione bisulfite adduct-generated oxidative stress in rat liver

The in vivo effect of menadione bisulfite adduct on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 h after menadione bisulfite adduct administration. To evaluate liver antioxidant enzymatic defenses, superoxide dismutase, catalase and glutathione peroxidase activities were determined. Antioxidant enzymes significantly decreased 3 h after menadione bisulfite adduct injection. Heme oxygenase activity appeared 6 h after treatment, peaking 9 h after menadione bisulfite adduct administration. Such induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady-state concentration, both effects taking place some hours before induction of heme oxygenase. Iron ferritin levels and ferritin content began to increase 6 h after heme oxygenase induction, and these increases were significantly higher 15 h after treatment and remained high for at least 24 h after menadione bisulfite adduct injection. Administration of bilirubin entirely prevented heme oxygenase induction as well as the decrease in hepatic GSH and the increase in lipid peroxidation when administered 2 h before menadione bisulfite adduct treatment. These results indicate that the induction of heme oxygenase by menadione bisulfite adduct may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.     

Susceptibility of cultured rat hepatocytes to oxidative stress by peroxides and iron. The extracellular matrix affects the toxicity of tert-butyl hydroperoxide

The aim of this study was to set up an in vitro model for studying the importance of an altered extra-cellular matrix composition and its importance for the resistance to oxidative stress, in hepatocytes from normal and iron loaded rats. Primary cultures of hepatocytes from iron loaded and normal rats were plated on a laminin rich extracellular matrix or on collagen type I, and incubated with tert-butyl hydroperoxide (TBH). Malon dialdehyde (MDA) and the activities of lactate dehydrogenase (LDH) in cell culture medium were analyzed. The protein synthesis, the concentrations of glutathione and the expression of manganese-superoxide dismutase and ferritin genes were measured. All hepatocytes contained lower concentrations of glutathione when plated on collagen than on EHS. Ferritin H and Mn-SOD gene expression showed no difference. The rate of lipid peroxidation in iron loaded hepatocytes exposed to TBH was higher on collagen than in those plated on EHS (0.95 +/- 0.28 microM MDA vs. 1.62 +/- 0.22 microM MDA, p < 0.05). Iron loaded cells were in general more susceptible to TBH than were normal hepatocytes (MDA, LDH, protein synthesis and glutathione content). Lipid peroxidation could be prevented by adding desferrioxamine. In conclusion, we show that the combination of iron overload and collagen matrix in rat hepatocytes leads to an increased susceptibility to oxidative stress. These findings may be of interest for the further studies on effects of iron overload and the altered matrix composition in liver fibrosis.     

Oxidative stress is responsible for mitochondrial permeability transition induction by salicylate in liver mitochondria

The interaction of salicylate with the respiratory chain of liver mitochondria generates hydrogen peroxide and, most probably, other reactive oxygen species, which in turn oxidize thiol groups and glutathione. This oxidative stress, confirmed by the prevention of action by antioxidant agents, leads to the induction of the mitochondrial permeability transition in the presence of Ca2+. This phenomenon induces further increase of oxidative damage resulting in impairment of oxidative phosphorylation and beta-oxidation, cardinal features of Reye's syndrome in the liver. Mitochondrial permeability transition induction also induces the release of cytochrome c and apoptotic inducing factor from mitochondria, suggesting that salicylate also behaves as a pro-apoptotic agent. The reactive group of salicylate for inducing oxidative stress is the hydroxyl group which, by interacting with a Fe-S cluster of mitochondrial Complex I, the so-called N-2(Fe-S) center, produces reactive oxygen species.     

The effect of low concentrations of sodium nitrites and nitrates on respiration and oxidative phosphorylation in rat liver mitochondria]

Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.     

Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment

The addition of tert-butyl hydroperoxide (t-BuOOH) to isolated mitochondria resulted in oxidation of approximately 80% of the mitochondrial reduced glutathione (GSH) independently of the dose of t-BuOOH (1-5 mM). Concomitant with the oxidation of GSH inside the mitochondria was the formation of GSH-protein mixed disulfides (protein-SSG), with approximately 1% of the mitochondrial protein thiols involved. A dose-dependent rate of GSH recovery was observed, via the reduction of oxidized GSH (GSSG) and a slower reduction of protein-SSG. Although t-BuOOH administration affected the respiratory control ratio, the mitochondria remained coupled and loss of the matrix enzyme, citrate synthase, was not increased over the control and was less than 3% over 60 min. A slow loss of GSH out of the coupled non-treated mitochondria was not increased by t-BuOOH treatment, in fact, a dose-dependent drop of GSH levels occurred in the medium. However, no GSSG was found outside the mitochondria, indicating the necessary involvement of enzymes in the t-BuOOH-induced conversion of GSH to GSSG. The absence of GSSG in the medium also suggests that, unlike the plasma membrane, the mitochondrial membranes do not have the ability to export GSSG as a response to oxidative stress. Our results demonstrate the inability of mitochondria to export GSSG during oxidative stress and may explain the protective role of mitochondrial GSH in cytotoxicity.     

Chronic ethanol feeding causes oxidative stress in rat liver mitochondria. Prevention by S-adenosyl methionine

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5–5-tetramethyl-1-H-pyridoyl-1-yloxy (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 μg ml^?1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 μmol/1 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.     

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress

Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold‐stress response was reproduced in gilthead sea bream acclimated to 20°C (Warm group) when the water temperature was lowered to 8°C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2‐DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine‐homocysteine‐methyl transferase, glutathione‐S‐transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin‐methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen‐like protein indicated an increase in proteolysis. Increases in elongation factor‐1α, the GAPDH oxidative form, tubulin and Raf‐kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.