Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

Protective effects of mitochondria-targeted antioxidant SkQ in aqueous and lipid membrane environments.

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

6-fluorodopamine selectively destroys neuroblastoma cells expressing the noradrenaline transporter.

BACKGROUND: 6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells. PROCEDURE: We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells. RESULTS: Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA. CONCLUSIONS: We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells.

Neuroblastoma cells expressing the noradrenaline transporter are destroyed more selectively by 6-fluorodopamine than by 6-hydroxydopamine.

6-Hydroxydopamine (6-OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6-OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6-OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6-fluorodopamine (6-FDA) hydrolyzes slowly to 6-OHDA at physiological pH. Oxygen consumption, H(2)O(2), and quinone production are found to be intermediate between those of 6-OHDA and dopamine (DA). Relative neurotoxicity of these compounds was assessed by cell viability and DNA damage in the human neuroblastoma lines SH-SY5Y and SK-N-LO, which express and lack the noradrenaline transporter, respectively. Specific uptake of DA and 6-FDA by SH-SY5Y cells was demonstrated by competitive m-[(131)I]iodobenzylguanidine uptake inhibition. The competition by 6-OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6-FDA toxicity was preferential for SH-SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6-FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6-OHDA cytotoxicity.

Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions.

One-electron reduction of quinones (Q) by ascorbate (AscH ); (1) AscH + Q --> Q*- + Asc*- + H+, followed by the oxidation of semiquinone (Q*-) by molecular oxygen; (2) Q*- + O2 --> Q + O2*-, results in the catalytic oxidation of ascorbate (with Q as a catalyst) and formation of active forms of oxygen. Along with enzymatic redox cycling of Q. this process may be related to Q cytotoxicity and underlie an antitumor activity of some Qs. In this work, the kinetics of oxygen consumption accompanied the interaction of ascorbate with 55 Qs including substituted 1,4- and 1,2-benzoquinones, naphthoquinones and other quinoid compounds were studied in 50 mM sodium phosphate buffer, pH 7.40, at 37 degrees C by using the Clark electrode technique. The capability of Q to catalyze ascorbate oxidation was characterized by the effective value of kEFF calculated from the initial rate of oxygen consumption (R(OX)) by the equation R(OX) = kEFF[Q][AscH-] as well as by a temporary change in R(OX). The correlation of kEFF with one-electron reduction potential, E(Q/Q*-), showed a sigma-like plot, the same for different kinds of Qs. Only the Qs which reduction potential E(Q/Q*-) ranged from nearly -250 to + 50 mV displayed a pronounced catalytic activity, kEFF increased with shifting E(Q/Q*-) to positive values. The following linear correlation between kEFF (in M (-1) s(-1)) and E(Q/Q*-) (in mV) might be suggested for these Qs: lg(kEFF)= 3.91 + 0.0143E(Q/Q*-). In contrast, Qs with E(Q/Q*-) < - 250 mV and E(Q/Q*-) > + 50 mV showed no measurable catalytic activity. The Qs studied displayed a wide variety in the kinetic regularities of oxygen consumption. When E(Q/Q*-) was more negative than - 100 mV, Q displayed a simple ('standard') kinetic behavior--R(OX) was proportional to [AscH-][Q] independently of concentration of individual reagents, [AscH-] and [Q]; R(OX) did not decrease with time if [AscH-] was held constant: Q recycling was almost reversible. Meanwhile, Qs with E(Q/Q*-) > - 100 mV demonstrated a dramatic deviation from the 'standard' behavior that was manifested by the fast decrease in R(OX) with time and non-linear dependence of even starting values of R(OX) on [Q] and [AscH-]. These deviations were caused basically by the participation of Q*- in side reactions different from (2). The above findings were confirmed by kinetic computer simulations. Some biological implications of Q-AscH- interaction were discussed.

Kinetics of redox interaction between substituted 1,4-benzoquinones and ascorbate under aerobic conditions: critical phenomena.

Redox cycling is believed to be the most general molecular mechanism of quinone (Q) cytotoxicity. Along with redox cycling induced by a reductase, a similar process is known to occur via electron transfer from ascorbate (AscH-) to Q with formation of a semiquinone radical (Q.-): (1) Q + AscH- (k1)--> Q.- + Asc.- + H+ (2) Q.- + O2 --> Q + O2.-. The net effect of reactions (1) and (2) provides for the catalytic oxidation of AscH-, with Q serving as a catalyst. In this work, the kinetics of oxygen consumption accompanying this process were studied with several substituted 1,4-benzoquinones (BQ) at 37 degrees C in phosphate buffer, pH 7.40, using the Clark electrode technique. The value of k1 determined from the initial rate of oxygen consumption was typically found to increase when the one-electron reduction potential E(Q/Q.-) shifted to more positive values. With Q, for which E(Q/Q.-) is less than -100 mV, the rate of oxygen uptake (R(OX)) was found to be directly correlated with the [Q][AscH-] value independent of the concentration of individual reagents, remaining constant for a long period. With mono- and dialkyl-substituted 1,4-BQs, for which E(Q/Q.-) is higher than -100 mV, significant deviations from the above simple kinetic regularities were observed. In particular, R(OX) decreased dramatically with time and critical phenomena (the existence of certain concentrations of Q and/or AscH- above or below which the catalytic oxidation of AscH- ceased completely after a non-stationary period of short duration) were observed. These abnormalities can be explained on the basis of the kinetic scheme which contains, in addition to reactions (1) and (2), several side reactions including that between Q.- and AscH-. Implications of critical phenomena discovered in this study for the problems of Q toxicity and vitamin C avitaminosis are discussed.

Fully reversible redox cycling of 2,6-dimethoxy-1,4-benzoquinone induced by ascorbate.

The kinetics of cyclic redox transformation of 2,6-dimethoxy-1, 4-benzoquinone (DMOBQ)--the well-known effective anticancer agent--induced by ascorbate (AscH-) were studied in phosphate buffer, pH 7.40, at 37 degreesC using the Clark electrode and ESR techniques. The process is due to the electron transfer from AscH- to quinone (Q): Q + AscH- --> Q*- + Asc.- + H+ (1), followed by semiquinone (Q.-) oxidation: Q.- + O2 --> Q + O2.- (2). DMOBQ, taken even at submicromolar concentrations, effectively catalyzed AscH- oxidation that manifested itself by intensive oxygen consumption and an increase in the steady-state concentration of the ascorbyl radical (Asc.-). The rate of oxygen consumption, ROX, was kept almost constant for a long time. ROX was found to be proportional to the [Q][AscH-] product and not dependent on the concentrations of the individual reagents. The rate constant for reaction (1) determined from ROX and [Asc.-] was as much as 380 +/- 40 and 280 +/- 30 M-1.sec-1, respectively. When DMOBQ was mixed with the corresponding hydroquinone, QH2, in oxygen-free buffer, the ESR signal of Q.- which formed due to the equilibrium Q + QH2 left and right arrow 2Q.- + 2H+ (3) was observed. The equilibrium constant K3 of (2.6 +/- 0.4).10-5 and the change in the reduction potential, DeltaE3 = E(Q/Q.-) - E(Q.-/QH2), of -280 mV were calculated from the steady-state concentration of Q.- at pH 7.4 and 37 degrees C. From combination of DeltaE3 determined in this study with E7(Q/Q.-) reported in the literature, a value of +190 mV was calculated for the standard second one-electron reduction potential E(Q*-/QH2). The latter is lower by 270-230 mV than that for all the studied 1, 4-hydroquinones. The very beneficial combination of E(Q/Q.-) and E(Q.-/QH2) was suggested to be the basic reason for the perfect work of DMOBQ as a redox cycling agent and its pronounced anticancer activity.

Ubiquinone-0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone) as effective catalyzer of ascorbate and epinephrine oxidation and damager of neuroblastoma cells.

The kinetics of ascorbate (AscH ) and epinephrine (EP) oxidation in the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ) were studied in 0.05 M phosphate buffer, pH 7.4, at 37 degrees C by using a Clark electrode and ESR techniques. UQ at nanomolar concentrations displayed a pronounced catalytic effect on AscH oxidation which exceeded that of all reported organic catalysts tested in this system. The process was accompanied by the intensive oxygen consumption and increase in the steady-state concentration of the ascorbyl radical Asc.-. The rate of oxygen consumption (R[OX]) was maximal at the moment of reagent mixing ((R[OX]0) and then reduced over a few minutes until a steady-state level ((R[OX])SS) was achieved. (R[OX])0 was found to be proportional to [UQ][AscH-] without regard to the concentrations of the individual reagents; (R[OX])SS was directly related to [UQ] at a given concentration of AscH-. The difference between (R[OX])0 and (R[OX])SS decreased as [AscH-] decreased. The presence of a lipid phase (sodium dodecylsulphate micelles) only moderately decreased UQ activity as a catalyst of AscH- oxidation. Adding micromolar concentrations of UQ induced the acceleration of EP autoxidation. The capability of UQ to catalyze the oxidation of EP exceeded by approximately 25 times that of adrenochrome, a quinoid product of EP oxidation. These catalytic properties of UQ allowed us to predict its pronounced cytotoxicity, especially in the presence of AscH- and to cells of the sympathetic nervous system which are rich in catecholamines. This possibility was confirmed by experiments with human neuroblastoma cells in culture. The capability of UQ to injure neuroblastoma cell line SK-N-SH exceeded that of well-known neurotoxic agents 6-hydroxydopamine and menadione.

Iron bound to ferritin catalyzes ascorbate oxidation: effects of chelating agents.

Ferritin is the main intracellular iron storage protein. Ferritin iron may be released by many reducing agents including ascorbate. In this work we report ferritin to catalyze the oxidation of ascorbate. The kinetics of this process were studied in detail in phosphate buffer (pH 7.40), at 37 degrees C by using the Clark electrode technique and ESR. The catalytic effect of ferritin manifested itself as the increase both in the rate of oxygen uptake and steady-state concentration of the ascorbate radical. The ferritin catalytic activity was found to be modified by iron chelators, EDTA. Desferal (DFO) as well as by ferrozine (FRZ) which is widely used in kinetic studies on ferritin iron release thanks to the formation of a coloured complex with Fe(II). While EDTA promotes the catalytic action of ferritin, DFO and FRZ diminished it. From the comparison of the kinetics of ascorbate oxidation obtained in the current work and data on the kinetics of ferritin iron release reported by Boyer and McCleary ((1987) Free Rad. Biol. Med. 3, 389-395), we conclude that iron bound to ferritin rather than the iron released is likely responsible for ferritin catalytic action. In addition, it has been concluded that the use of FRZ as an analytical reagent in kinetic studies of reductive ferritin iron release requires taking into account the competitive character of the formation of the Fe(II)-FRZ complex.

Reduction of ubiquinone-1 by ascorbic acid is a catalytic and reversible process controlled by the concentration of molecular oxygen.

To address whether reduction by vitamin C may contribute to the in vivo maintenance of coenzyme Q in the reduced form, we studied the reduction of ubiquinone-1 by ascorbate at pH 7.4. Addition of ascorbate to ubiquinone-1 resulted in rapid O2 consumption and an increase in the steady-state concentration of ascorbyl radical. The initial rate of O2 consumption was proportional to the product of [ubiquinone-1] and [ascorbate] whereas [ascorbyl radical] was proportional to the square root of this parameter; both dependencies were in quantitative agreement with each other. The extent of O2 consumption greatly exceeded the amounts of ubiquinone-1 initially present. Formation of ubiquinol-1 from ubiquinone-1 by ascorbate was reversible, moderate under aerobic conditions, but substantial in the absence or near absence of oxygen. At high O2 concentration, ascorbate promoted the oxidation of ubiquinol-1 to ubiquinone-1. Addition of sodium dodecyl sulphate dramatically decreased the rate of reaction between ubiquinone-1 and ascorbate, most likely as a result of phase separation of the reagents. A preliminary reaction scheme with putative rate constants for the relevant reactions is presented that quantitatively describes the kinetic behaviour of the process studied. The key reactions in the scheme are electron transfer from ascorbate to ubiquinone-1 with formation of the ascorbyl and ubisemiquinone radical. The reaction of the latter with O2 is postulated to be responsible for O2 consumption, with ubiquinone-1 acting as a catalyst. Together, the results demonstrate that the extent of reduction of ubiquinone-1 by ascorbate was controlled by the O2 concentration and the physical availability of the reactants. As the O2 concentration in human blood is relatively high and ubiquinone-10 is located exclusively within the lipid phase of lipoproteins where negatively charged ascorbate has little access, our results suggest that direct reduction by ascorbate is unlikely to be responsible for the high reduction percentage observed for plasma coenzyme Q.

Ascorbyl radical as natural indicator of oxidative stress: quantitative regularities.

The intensity of ESR spectrum associated with ascorbyl free radical (A.) was found to be sensitive to various pathologies and intoxications connected with oxidative stress. For this reason, A. has been suggested to be used as a natural noninvasive ESR indicator of oxidative stress. To specify factors controlling [A.] in biological tissues, the kinetic study of ascorbic acid (AH) oxidation in aqueous solutions catalyzed by Fe ions and methylene blue (MB) has been performed by using ESR and Clark electrode techniques. Concentration, temperature, and pH dependences of [A.] and rate of AH oxidation (R(ox)), effects of additives of biological importance (glutathione, uric acid, proteins, glucose, lipid) as well as that of Na dodecylsulfate (SDS) have been studied. The oxidation of 1 mol of AH is accompanied by the consumption of 1 mol of O2 and the generation of 1 mol of A.. [A.] has been found to be proportional to the square root of R(ox), no matter what reason is for R(ox) variation. A. has been shown to be inactive toward all substances tested and free radicals other than A.. It has been concluded that A. decays in biological tissues mostly by self-disproportionation, a value of [A.] gives the definitive quantitative information on the total rate of AH oxidative transformations (oxidation by O2 plus reactions with free radicals). SDS shows the retarding effect on AH oxidation induced by MB.

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides.

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.