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Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress. [ABSTRACT FROM AUTHOR]

In the article, rat liver of Pb2+ and Cd2+ ionsthe effect on mitoKATP-channel activity in mitochondria was studied.Inhibition of mitoKATF-channel activity was observed experimentally in the presence of 200 μM concentration of ATP in the incubation medium of mitochondria isolated from rat liver.In the absence of ATP in the incubation medium, 8.0 µM concentration of Pb2+ and Cd2+ salts was noted to reduce the permeability of liver mitoKATP-channel activity in both energized and de-energized states compared to the control. [ABSTRACT FROM AUTHOR]

Recent data have shown that the mitochondrial permeability transition pore (MPTP) is the complex of the Ca2+-modified adenine nucleotide translocase (ANT) and the Ca2+-modified ATP synthase. We found in a previous study that ANT conformational changes may be involved in Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat liver mitochondria. In this study, the effects of thiol-modifying agents (eosin-5-maleimide (EMA), fluorescein isothiocyanate (FITC), Cu(o-phenanthroline)2 (Cu(OP)2), and embelin (Emb)), and MPTP inhibitors (ADP, cyclosporine A (CsA), n-ethylmaleimide (NEM), and trifluoperazine (TFP)) on MPTP opening were tested simultaneously with increases in swelling, membrane potential (ΔΨmito) decline, decreases in state 3, 4, and 3UDNP (2,4-dinitrophenol-uncoupled) respiration, and changes in the inner membrane free thiol group content. The effects of these thiol-modifying agents on the studied mitochondrial characteristics were multidirectional and showed a clear dependence on their concentration. This research suggests that Tl+-induced MPTP opening in the inner membrane of calcium-loaded mitochondria may be caused by the interaction of used reagents (EMA, FITC, Emb, Cu(OP)2) with active groups of ANT, the mitochondrial phosphate carrier (PiC) and the mitochondrial respiratory chain complexes. This study provides further insight into the causes of thallium toxicity and may be useful in the development of new treatments for thallium poisoning. [ABSTRACT FROM AUTHOR]

Permeability transition pore (mPTP) opening was studied under energized and deenergized conditions in rat liver mitochondria, and the effect of membrane depolarization on mPTP activity was evaluated. To assess mPTP activity, cyclosporine-sensitive swelling and cyclosporine sensitive Ca2+ efflux from mitochondria was studied using light absorbance techniques. In energized mitochondria, mPTP opening in sub-conductance states, at [Ca2+] ≤ Ka, contributed positively to the rate of respiration, without affecting ΔΨm. Threshold Ca2+ concentrations were found, which excess resulted in fast mitochondrial depolarization upon mPTP opening. An estimate of mPTP activity by cyclosporine-sensitive Ca2+ transport under energized and deenergized conditions have shown that membrane depolarization by protonophore CCCP essentially increased initial rate (V0), at simultaneous decrease of the half-time (t1/2) of Ca2+ efflux, which indicated mPTP activation, as compared to energized mitochondria. However, only partial release of Ca2+ via mPTP upon membrane depolarization was observed. With the use of selective blockers of Ca2+ uniporter and mPTP, ruthenium red (RR) and cyclosporine A (CsA), partial contribution of Ca2+ uniporter and mPTP in Ca2+ transport was found. “Titration” of Ca2+ transport by adding RR at different times from the onset of depolarization showed that depolarization dramatically reduced “life span” of mPTP as compared to energized mitochondria, which agreed with the kinetic characteristics of CsA-sensitive Ca2+ transport after the abolition of ΔΨm. Ca2+ added from the outer side of mitochondrial membrane produced dual effect on mPTP activity: activation at the onset of depolarization, but consequent promotion of mPTP closure. Based on the experiments, it was concluded that mitochondrial energization was required for prolonged mPTP functioning in sub-conductance states, whereas membrane depolarization promoted the transition of mPTP to inactive state during calcium release from mitochondria.

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to 17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals. [ABSTRACT FROM AUTHOR]

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4′-diisothiocyanostilbene-2,2′-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups. [ABSTRACT FROM AUTHOR]

Since thymoquinone (2-isopropyl-5-methylbenzoquinone) isolation from Nigella sativa in 1963, various studies have reported on its diverse pharmacological properties. However, despite its versatile healing abilities, clinical trials involving the use of thymoquinone have not been initiated due to its poor bioavailability. Many attempts have been made to improve the therapeutic efficacy of thymoquinone by synthesizing analogs, as well as by developing nanotechnology-based delivery systems. We hypothesized that some of the issues with thymoquinone delivery and bioavailability could be resolved by targeted delivery to mitochondria of thymoquinone derivatives conjugated to the penetrating lipophilic cationic triphenylphosphonium fragment. As mitochondria are the major site of reactive oxygen species generation in the cell, such a membranotropic thymoquinone derivative can act as an efficient antioxidant or prooxidant depending on the concentration used. Based on these theoretical considerations, a novel mitochondria-targeted compound, SkQThy, was synthesized and its effects on rat liver mitochondria and yeast cells were examined. SkQThy was found to exhibit pronounced antioxidant activity in mammalian mitochondria and yeast cells, decreasing hydrogen peroxide production in mitochondria, as well as preventing prooxidant-induced oxidative stress and mitochondrial fragmentation in yeast cells and increasing cell viability. Moreover, SkQThy proved itself to be the most efficient mitochondria-targeted antioxidant within the SkQs family, showing good therapeutic potential. • SkQThy exhibits pronounced antioxidant activity in mammalian mitochondria and yeast cells. • SkQThy prevents prooxidant-induced mitochondrial fragmentation in yeast cells and increases cell viability. • SkQThy usage might offer a new strategy for alleviating neurodegenerative pathologies related to oxidative stress. [ABSTRACT FROM AUTHOR]

Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called “mild uncoupling” and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parame- ters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and “opener” of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria. [ABSTRACT FROM AUTHOR]

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO 3 and KNO 3 resulted in the Tl + -induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca 2+ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl + -induced MPTP opening in the inner membrane of Ca 2+ -loaded rat liver mitochondria. [ABSTRACT FROM AUTHOR]

The process of aging is considered to be tightly related to mitochondrial dysfunction. One of the causes of aging is an increased sensitivity to the induction of mitochondrial permeability transition pore (mPTP) opening in the inner membrane of mitochondria. Melatonin, a natural antioxidant, is a hormone produced by the pineal gland. The role of melatonin whose level decreases with aging is well understood. In the present study, we demonstrated that long-term treatment of aged rats with melatonin improved the functional state of mitochondria; thus, the Ca 2 + capacity was enhanced and mitochondrial swelling was deaccelerated in mitochondria. Melatonin prevented mPTP and impaired the release of cytochrome c and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) from mitochondria of both young and aged rats. Our data suggest that melatonin retains СNPase inside mitochondria, thereby providing the protection of the protein against deleterious effects of 2′,3′-cAMP in aging. [ABSTRACT FROM AUTHOR]

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10-3 × 10 М) inhibit K outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10-1 × 10 M), manganese activates K outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis. [ABSTRACT FROM AUTHOR]

[Display omitted] • (Carboxymethyl)triphenylphosphonium alkyl esters CMTPP-C n (n = 8–14) were studied. • Due to acidic C H bond between P and ester group these TPP salts are ylide precursors. • CMTPP-C 10 induced proton-selective current through planar bilayer lipid membrane. • CMTPP-C n uncoupled rat liver mitochondria collapsing their membrane potential. • CMTPP-C 12 caused dissipation of pH gradient on chloroplast membranes. Triphenylphosphonium ylides are commonly used as key intermediates in the Wittig reaction. Based on the known acidities of stabilized ylide precursors, we proposed that a methylene group adjacent to phosphorus in these compounds can ensure proton shuttling across lipid membranes. Here, we synthesized (decyloxycarbonylmethyl)triphenylphosphonium bromide (CMTPP-C 10) by reaction of triphenylphosphine with decyl bromoacetate. This phosphonium salt precursor of the ester-stabilized phosphorus ylide along with its octyl (CMTPP-C 8) and dodecyl (CMTPP-C 12) analogues was found to be a carrier of protons in mitochondrial, chloroplast and artificial lipid membranes, suggesting that it can reversibly release hydrogen ions and diffuse through the membranes in both zwitterionic (ylide) and cationic forms. The CMTPP-C 10 -mediated electrical current across planar bilayer lipid membranes exhibited pronounced proton selectivity. Similar to conventional protonophores, known to uncouple electron transport and ATP synthesis, CMTPP-C n (n = 8, 10, 12) stimulated mitochondrial respiration, while decreasing membrane potential, at micromolar concentrations, thereby showing the classical uncoupling activity in mitochondria. CMTPP-C 12 also caused dissipation of transmembrane pH gradient on chloroplast membranes. Importantly, CMTPP-C 10 exhibited substantially lower toxicity in cell culture, than C 12 TPP. Thus, we report the finding of a new class of ylide-type protonophores, which is of substantial interest due to promising therapeutic properties of uncouplers. [ABSTRACT FROM AUTHOR]

The effect of mitochondrial ATP-dependent K+-channel (K+АТР-channel) opener diazoxide (DZ) on the oxygen consumption, functional state and the opening of cyclosporine-sensitive pore in the rat liver mitochondria has been studied. It has been established that K+АТР-channel activation results in the increase of the oxygen consumption rate (V4S) and the uncoupling due to the acceleration of K+-cycling, the decrease in state 3 respiration rate (V3) and the respiratory control ratio (RCR). Under K+АТР-channel activation an inhibition of oxidative phosphorylation takes place which reduces the rate of ATP synthesis and hydrolysis as well as ATP production and consequently results in the seeming increase of P/O ratio. It has been shown that the increase in ATP-dependent K+-uptake accompanied by the opening of mitochondrial permeability transition pore (MPTP) leads to dramatic uncoupling of the respiratory chain due to simultaneous activation of K+– and Ca2+-cycling supported by MPTP and Ca2+-uniporter as well as K+-channels and K+/H+-exchange. K+АТР-channel activation leads to the partial inhibition of MPTP, but insufficient for the restoration of mitochondrial functions. Elimination of Ca2+-cycling after MPTP opening is necessary to return mitochondrial functions back to the control level which shows that MPTP could serve as the mechanism of reversible modulation of bioenergetic effects of K+АТР-channel activation.

Oxidative stress is one of the major factors underlying mitochondrial dysfunctions. One of the most promising approaches for alleviating or preventing oxidative stress is the use of cationic uncouplers that accumulate in mitochondria in accordance to the level of the membrane potential, producing 'mild' uncoupling. Based on this theoretical background, cationic rhodamine 19 butyl ester (CR1) was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these tests were presented (Khailova et al. (2014) Biochim. Biophys. Acta, 1837, 1739-1747), but one publication could not accommodate all data on interactions of CR1 with isolated mitochondria. In addition to previously presented data, we found that the effect of CR1 on the rate of oxygen uptake is subject to temporal variations, which probably reflects variable rates of CR1 entry into the mitochondria. Consequently, transient stimulation of respiration can be followed by inhibition. CR1 was found not to shunt electron flow from complex I of the respiratory chain; it largely acted as an inhibitor of complex I in the respiratory chain and showed antioxidant activity. CR1 taken at low, non-uncoupling concentrations enhanced the uncoupling activity of fatty acids (e.g. palmitate). Relatively low CR1 concentrations stimulated opening of a nonspecific Ca/P-dependent pore. ATP synthesis and hydrolysis were substantially inhibited by CR1 at low concentrations that had no appreciable effects on respiration in states 4 and 3 and only slightly decreased the membrane potential. Besides, conditions were revealed allowing correct evaluation of the membrane potential generated at the inner mitochondrial membrane with safranin O upon oxidation of both succinate and NAD-dependent substrates in the presence of CR1. [ABSTRACT FROM AUTHOR]

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC 50 : 6.48 ± 2.74 μM) > celecoxib (IC 50 : 14.92 ± 6.40 μM) > valdecoxib (IC 50 : 161.4 ± 28.6 μM) > rofecoxib (IC 50 : 238.4 ± 79.2 μM) > etoricoxib (IC 50 : 405.1 ± 116.3 μM). Mechanism based inhibition of ATP synthesis (K inact 0.078 min − 1 and K I 21.46 μM and K inact /K I ratio 0.0036 min − 1 μM − 1 ) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r 2 = 0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs and these metabolites did not alter the inhibition profile of ATP synthesis of the parent compound. The results suggest that coxibs themselves could be involved in the hepatotoxic action through inhibition of ATP synthesis. [ABSTRACT FROM AUTHOR]

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in ‘c’ conformation by phenylarsine oxide (PAO), tert -butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO 3 and KNO 3 stimulated the Tl + -induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨ mito ), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in ‘m’ conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl + -induced MPTP in the inner membrane of Ca 2+ -loaded rat liver mitochondria. Using the Tl + -induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds. [ABSTRACT FROM AUTHOR]

Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca2+), phosphate (Pi), and triiodothyronine (T3) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM Pi, 50 μM Ca2+ and 25 μM T3 when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca2+ and Pi are used as activating factors, the addition of T3 doubled the rate of swelling. T3 has also an activating effect on mitochondrial membrane potential. The T3 activating effect was also found after in vivo application of T3. Our data thus demonstrate that T3 has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions. [ABSTRACT FROM AUTHOR]

Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4′-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4′-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2–5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (K inact ) of 2.64 ± 0.15 min − 1 and half maximal rate of inactivation (K I ) of 7.69 ± 2.48 μM with K inact /K I ratio of 0.343 min − 1 μM − 1 . Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis. [ABSTRACT FROM AUTHOR]

Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen- N -acetyl cysteine-thioester (F-NAC) and fenbufen- S -glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen- O- carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen- N -acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ATP synthesis with K inact of 4.4 min − 1 and K I of 0.88 μM and K inact /K I ratio of 5.01 min − 1 μM − 1 . Data show that fenbufen did not act through opening MPT pore, nor did incubation of mitochondria with reduced GSH and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria. The results from the present experiments provide evidence that fenbufen and its metabolites could be involved in mitochondrial toxicity through inhibition of ATP synthesis. [ABSTRACT FROM AUTHOR]

Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca 2 + overload and matrix glutathione loss. Fixing the ‘m’ conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP. Oxidants (diamide or tert -butylhydroperoxide (tBHP)) fix the ANT in ‘c’ conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl + -induced MPTP opening, which was accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl + -induced MPTP in the inner membrane of Ca 2+ -loaded rat liver mitochondria. [ABSTRACT FROM AUTHOR]

It is known that Ca 2+ and heavy metals more actively induce MPTP opening in mitochondria, energized by the I complex substrates. Thus, a rise in a Tl + -induced MPTP was proposed in experiments on isolated rat liver mitochondria energized by the complex I substrate (glutamate and malate). Expose of the mitochondria to Ca 2+ into a medium containing TlNO 3 , glutamate, and malate as well as sucrose or KNO 3 resulted in a decrease in state 3, state 4, or DNP-stimulated respiration as well as an increase of both mitochondrial swelling and Δ Ψ mito dissipation. The MPTP inhibitors, CsA and ADP, almost completely eliminated the effect of Ca 2+ , which was more pronounced in the presence of the complex I substrates than the complex II substrate (succinate) and rotenone (Korotkov and Saris, 2011). The present study concludes that Tl + -induced MPTP opening is more appreciable in mitochondria energized by glutamate and malate but not succinate in the presence of rotenone. We assume that the Tl + -induced MPTP opening along with followed swelling and possible structural deformations of the complex I in Ca 2+ -loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl + to Tl 3+ by mitochondrial oxygen reactive species is proposed for the mechanism. [ABSTRACT FROM AUTHOR]

The antioxidant effect of succinate and 3-hydroxybutyrate oxidation on the kinetics of lipid peroxidation induced by ATP-Fe complex in isolated rat liver mitochondria of old (1-1.5 years) and young (3 months) male rats was investigated. Rate of induced lipid peroxidation V in rat liver mitochondria was recorded polarographically by oxygen consumption and the half-time of maximum oxygen consumption Δ t, which includes a lag period and the lipid peroxidation initiation phase, was determined. Without exogenous oxidative substrates V is slightly higher in mitochondria of old animals, but the onset of lipid peroxidation cascade is significantly earlier than in mitochondria of young animals. Incubation of intact mitochondria with 5 mM succinate for 1 min inhibited V by 15 and 35% in young and old animals respectively, however only in mitochondria of old animals Δ t increased by 19%. Oxidation of 3-hydroxybutyrate in mitochondria of young animals did not significantly change the rate of lipid peroxidation, while in mitochondria of old animals it was reduced by 19% with a slight increase of Δ t. To simulate age-dependent dysfunction we damaged isolated mitochondria by a session of freeze-thaw cycles, which caused a significant increase of V in mitochondria of both age groups. Succinate oxidation inhibited V in damaged mitochondria in all cases by 56%, as compared to V without oxidative substrates, and extended Δ t twofold in mitochondria of young animals. Oxidation of 3-hydroxybutyrate had no effect on V in damaged mitochondria regardless of animal age and extended Δ t by 48% in mitochondria of young animals. Thus, the antioxidant effect of succinate oxidation can prevent lipid peroxidation damage and may exhibit geroprotective action on aging mitochondria. Addition of either substrate had no effect on lipid peroxidation parameters in phospholipid emulsion. Therefore, the antioxidant effect is a result of the process of substrate oxidation in the respiratory chain and not an interaction of their structures with membrane lipids per se. [ABSTRACT FROM AUTHOR]

It is known that a closure of ATP sensitive (mitoK) or BK-type Ca activated (mitoK) potassium channels triggers opening of the mitochondrial permeability transition pore (MPTP) in cells and isolated mitochondria. We found earlier that the Tl-induced MPTP opening in Ca-loaded rat liver mitochondria was accompanied by a decrease of 2,4-dinitrophenol-uncoupled respiration and increase of mitochondrial swelling and ΔΨ dissipation in the medium containing TlNO and KNO. On the other hand, our study showed that the mitoK inhibitor, 5-hydroxydecanoate favored the Tl-induced MPTP opening in the inner membrane of Ca-loaded rat heart mitochondria (Korotkov et al. ). Here we showed that 5-hydroxydecanoate increased the Tl-induced MPTP opening in the membrane of rat liver mitochondria regardless of the presence of mitoK modulators (diazoxide and pinacidil). This manifested in more pronounced decrease in the uncoupled respiration and acceleration of both the swelling and the ΔΨ dissipation in isolated rat liver mitochondria, incubated in the medium containing TlNO, KNO, and Ca. A slight delay in Ca-induced swelling of the mitochondria exposed to diazoxide could be result of an inhibition of succinate oxidation by the mitoK modulator. Mitochondrial calcium retention capacity (CRC) was markedly decreased in the presence of the mitoK inhibitor (5-hydroxydecanoate) or the mitoK inhibitor (paxilline). We suggest that the closure of mitoK or mitoK in calcium loaded mitochondria favors opening of the Tl-induced MPTP in the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]

We showed earlier that diminution of 2,4-dinitrophenol (DNP)-stimulated respiration and increase of both mitochondrial swelling and electrochemical potential (ΔΨ mito ) dissipation in medium containing TlNO 3 and KNO 3 were caused by opening of Tl + -induced mitochondrial permeability transition pore (MPTP) in the inner membrane of Ca 2 + -loaded rat liver mitochondria. The MPTP opening was studied in the presence of bivalent metal ions (Sr 2 + , Ba 2 + , Mn 2 + , Co 2 + and Ni 2 + ), trivalent metal ions (Y 3 + and La 3 + ), and ruthenium red. We found that these metal ions (except Ba 2 + and Co 2 + ) as well as ruthenium red inhibited to the MPTP opening that manifested in preventing both diminution of the DNP-stimulated respiration and increase of the swelling and of the ΔΨ mito dissipation in medium containing TlNO 3 , KNO 3 , and Ca 2 + . Inhibition of the MPTP opening by Sr 2 + and Mn 2 + is suggested because of their interaction with high affinity Ca 2 + sites, facing the matrix side and participating in the MPTP opening. The inhibitory effects of metal ions (Y 3 + , La 3 + , and Ni 2 + ), and ruthenium red are accordingly discussed in regard to competitive and noncompetitive inhibition of the mitochondrial Ca 2 + -uniporter. High concentrations (50 μM) of Y 3 + and La 3 + favored of MPTP opening in the inner membrane of rat liver mitochondria in Ca 2 + free medium containing TlNO 3 . The latter MPTP opening was markedly eliminated by MPTP inhibitors (cyclosporine A and ADP). [ABSTRACT FROM AUTHOR]

Consequences of gestational protein restriction (GPR) on liver metabolism in rat offspring were investigated. Pregnant dams were divided into groups: normal (NP, 17 % casein) or low-protein diet (LP, 6 % casein). Livers were collected from 30-day-old offspring (d30) for analysis or isolation of mitochondria. At d30, hepatic and muscle glycogen was increased in LP group. Mitochondrial swelling and oxygen uptake (recorded with a Clark-type electrode) were significantly reduced in NP female and LP pups. Thiobarbituric acid reactive substances production was lower in females (NP or LP), suggesting significant inhibition of lipid peroxidation. Measurement of mitochondrial respiration (states 3 and 4 stimulated by succinate) showed a higher ADP/O ratio in LP pups, particularly females, suggesting higher phosphorylation efficiency. In the 1st month of life, under our experimental conditions, GPR protects liver mitochondria against oxidative stress and females seem to be more resistant or more suitable for survival. [ABSTRACT FROM AUTHOR]

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce the ΔΨ value and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop in ΔΨ able to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Commonly used organic solvents inhibited oxidative phosphorylation (ATP synthesis) in rat liver mitochondria. [•] Acetonitrile and acetone were strong inhibitors of ATP synthesis compared to ethanol, methanol and DMSO. [•] All the organic solvents showed potentiation of the inhibition of 2,4-dinitrophenol. [•] Selection of solvent and concentration of solvent used for oxidative phosphorylation were critical. [Copyright &y& Elsevier]

Abstract: Fish oil (FO) and tetradecylthioacetic acid (TTA) — a synthetic modified fatty acid have beneficial effects in regulating lipid metabolism. In order to dissect the mechanisms underlying the molecular action of those two fatty acids we have investigated the changes in mitochondrial protein expression in a long-term study (50weeks) in male Wistar rats fed 5 different diets. The diets were as follows: low fat diet; high fat diet; and three diets that combined high fat diet with fish oil, TTA or combination of those two as food supplements. We used two different proteomics techniques: a protein centric based on 2D gel electrophoresis and mass spectrometry, and LC-MSE based peptide centric approach. As a result we provide evidence that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate that fatty acid metabolism; lipid oxidation, amino acid metabolism and oxidative phosphorylation pathways are involved in fish oil and TTA action. Evidence for the involvement of PPAR mediated signalling is provided. Additionally we postulate that down regulation of components of complexes I and II contributes to the strong antioxidant properties of TTA. Biological significance: This study for the first time explores the effect of fish oil and TTA — tetradecyl-thioacetic acid and the combination of those two as diet supplements on mitochondria metabolism in a comprehensive and systematic manner. We show that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate in a large scale that fatty acid metabolism and lipid oxidation are affected by fish oil and TTA, a phenomenon already known from more directed molecular biology studies. Our approach, however, shows additionally that amino acid metabolism and oxidative phosphorylation pathways are also strongly affected by TTA and also to some extent by fish oil administration. Strong evidence for the involvement of PPAR mediated signalling is provided linking the different metabolic effects. The global and systematic viewpoint of this study compiles many of the known phenomena related to the effects of fish oil and fatty acids giving a solid foundation for further exploratory and more directed studies of the mechanisms behind the beneficial and detrimental effects of fish oil and TTA diet supplementation. This work is already a second article in a series of studies conducted using this model of dietary intervention. In the previous study (Vigerust et al., [21]) the effects of fish oil and TTA on the plasma lipids and cholesterol levels as well as key metabolic enzymes in the liver have been studied. In an ongoing study more work is being done to explore in detail for example the link between the down regulation of the components of the respiratory chain (observed in this study) and the strong antioxidant effects of TTA. The reference diet in this study has been designed to mimic an unhealthy – high fat diet that is thought to contribute to the development of metabolic syndrome – a condition that is strongly associated with diabetes, obesity and heart failure. Fish oil and TTA are known to have beneficial effects for the fatty acid metabolism and have been shown to alleviate some of the symptoms of the metabolic syndrome. To date very little is known about the molecular mechanisms behind these beneficial effects and the potential pitfalls of the consumption of those two compounds. Only studies of each compound separately and using only small scale molecular biology approaches have been carried out. The results of this work provide an excellent starting point for further studies that will help to understand the metabolic effects of fish oil and TTA and will hopefully help to design dietary programs directed towards reduction of the prevalence of metabolic syndrome and associated diseases. [Copyright &y& Elsevier]

Abstract: Toxic agents, derived from bee or hornet venoms and from fungi – melittin, mastoparan, and alamethicin are able to permeabilize biological membranes. We studied the initial steps of pore formation by these peptides in rat liver mitochondria preparations (RLM) generating transmembrane potential (ΔΨ). RLM has been used as a potassium transmembrane current (PTC) sensor. The PTC induced in RLM depends linearly on the degree of steady-state activation of RLM respiration. The concentration order of such activation by melittin in a “potassium” incubation medium containing 6mM Mg2+ was 2.01±0.15. In the case of mastoparan, the reaction order was 1.83±0.23. The first steady-state phase of activation of RLM respiration by alamethicin was not detected in “Tris” incubation medium; it appeared only after addition of KCl. The order of the reaction limiting such activation was 1.92±0.07. It is suggested that PTC in this phase is determined by the channels with the lowest degree of oligomerization formed by “dimers”. The ratio of equally active membrane concentrations of peptides obviously reflects the ratio of average lifetimes (ALT) for corresponding “dimers” (alamethicin and melittin, 38.5; mastoparan and melittin, 0.32). It is concluded that the results of this investigation may be useful for comparative testing of perspective pharmaceuticals. [Copyright &y& Elsevier]

Abstract: The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO2) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7m2 g−1 is achieved by TiO2 aerogel, much higher than those obtained by commercial TiO2 nanoparticles and by the latest reported mesoporous TiO2 spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO2 microparticles (173 vs 28μmolg−1). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO2 aerogel, a detection limit as low as 30amol for analyzing phosphopeptides in β-casein digest was achieved. TiO2 aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO2 aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study. [Copyright &y& Elsevier]

Antioxidant activity of gentisic acid has been studied using fast chemical kinetics and two in vitro models, namely the isolated rat liver mitochondria (RLM) and the human erythrocytes. The presence of gentisic acid (GA) during irradiation significantly reduced the levels of gamma radiation induced damages to lipids and proteins in RLM. Further, GA imparted protection to the human erythrocytes against exposure to gamma radiation. Molecular mechanism of free radical scavenging reactions has been evaluated with the help of rate constants and transients obtained from gentisic acid using pulse radiolysis technique. GA efficiently scavenged hydroxyl radical (k == 1.1 × 1010 dm3mol−1s−1) to produce reducing adduct radical (∼76%) and oxidizing phenoxyl radical (∼24%). GA has also scavenged organohaloperoxyl radical (k == 9.3 × 107 dm3 mol−1s−1). Ascorbate has been found to repair phenoxyl radical of GA (k == 1.0 × 107 dm3mol−1s−1). Redox potential value of GA•/GA couple (0.774 V vs NHE) obtained by cyclic voltammetry is less than those of physiologically important oxidants, which supports the observed antioxidant capacity of GA. We, therefore, propose that the antioxidant and radioprotective properties of GA are exerted by its phenoxyl group. [ABSTRACT FROM AUTHOR]

We present a novel method of assessing damage to the liver using fluorescence analysis of hepatic mitochondria following ischaemia/reperfusion of the small intestine in a rat model. This work is of substantial importance in understanding the syndrome of multiorgan failure after ischemia/transplantation of the small intestine. Mitochondria were isolated from six sample groups that had undergone three different experimental treatments: a control group; a treatment with ischaemia followed by reperfusion of the small intestine (IRx); and, a one hour ischaemia followed by reperfusion after transplant of the small intestine (TRx). The IR treatment was further subdivided into three groups: 1, 24 h and 30 days reperfusion - IR1, IR24, IR720, respectively. Concomitantly, the TR treatment was further subdivided: one group underwent a 1 h reperfusion and another group a 6 h reperfusion following ischaemia and transplant - groups TR1 and TR6, respectively. Once treatment had been undergone, mitochondria were isolated and all five experimental groups - IR1, IR24, IR720, Tr1, Tr6 - and their emission matrices were analysed compared with that of the control group (C). Comparing fluorescence values in zone A of all experimental groups with those of the control group indicated a reduction in aromatic amino acids in the mitochondria of all experimental groups. Comparison of fluorescent zone B of experimental groups with the control group identified a lack of oxygen in samples IR1, IR24, which was indicated though an increase in the fluorescence of the reduced pyridine nucleotide NADH+H+. [ABSTRACT FROM AUTHOR]

Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, MnTMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of MnTMPyP. SOD and cytochrome c antioxidant activities were also investigated. MnTMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, MnTMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for MnTMPyP and do not exclude cytochrome c-like activity. However, considering that para MnTMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the MnTMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of MnTMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of MnTMPyP, whose efficiency is dependent on the NADPH/GSH content in cells. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200μmolL−1 flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200μmolL−1. Flavone at certain doses (75–200μmolL−1) reduced mitochondrial swelling in the presence of valinomycin and KNO3, suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones. [ABSTRACT FROM AUTHOR]

The mechanism of cyanide's inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ) and the enzyme affinity to oxygen ( p value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60-80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects. [ABSTRACT FROM AUTHOR]

The aim of the present work was to investigate the mechanisms of oxidative damage of rat liver mitochondria in vitro, under hypochlorous acid (HOCl)-induced oxidative stress, and in vivo, under acute carbon tetrachloride-induced intoxication in rats. Hypochlorous acid (50–300 μM), the main inflammatory agent, inhibited liver mitochondria respiratory activity and caused uncoupling in the respiratory and phos-porylation processes. The toxic damage of rat liver after 24 h of acute carbon tetrachloride-induced intoxication (4 g/kg, intragastrically) was accompanied by a significant reduction in succinate- and glutamate-dependent respiration rate in state 3 (by 65%, p < 0.001, and by 50%, p < 0.01, respectively). The respiration control ratio approached 1, reflecting the loss of respiration control. The phosphorylation coefficient significantly decreased due to uncoupling of the oxidation and phosphorylation processes. The mitochondrial alterations were associated with oxidation of intramitochondrial GSH by 25% ( p < 0.05), the marked inhibition of succinate dehydrogenase (complex II) by 35% ( p < 0.05), and the rise of blood plasma nitric oxide level by 45% ( p < 0.05). The impairment of mitochondrial respiratory function may result from the inhibition of enzymatic activities in the respiratory chain and the damage of mitochondrial membrane during intoxication and plays a key role in the development of the CCl4-induced hepatotoxicity. Melatonin administration under CCl4-induced intoxication (three times at a dose of 10 mg/kg) increased the rate of succinate oxidation in state 3 by 30% ( p < 0.05) and reversed the increase in glutathione peroxidase activity. Melatonin prevented an elevation of nitric oxide level in the blood plasma of intoxicated animals but did not protect mitochondrial functions under acute intoxication. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca2+. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle. [Copyright &y& Elsevier]

Abstract: Mitochondria consume nitric oxide (NO) mainly through reaction with superoxide anion (). Here, we analyzed the sources for NO degradation by isolated rat liver mitochondria. Electron leakage from complex III and reverse electron transport to complex I accounted for -dependent NO degradation by mitochondria in the presence of a protonmotive force. Mitochondria incubated with NAD(P)H also presented intense generation and NO degradation rates that were insensitive to respiratory inhibitors and abolished after proteinase treatment. These results suggest that an outer membrane-localized NAD(P)H oxidase activity, in addition to the electron leakage from the respiratory chain, promotes -dependent NO degradation in rat liver mitochondria. [Copyright &y& Elsevier]