Ruuge Ek

An attempt to prevent senescence: A mitochondrial approach

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

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, C1/2 values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1·10−11 and 8·10−13 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 Δψ 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 1.3·108 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.

Fatty acids as natural uncouplers preventing generation of O⋅−2 and H2O2 by mitochondria in the resting state

Both natural (laurate) and artificial (m‐chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O⋅− 2 and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate, an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the releasing effect of carboxyatractylate. GDP is much less effective than carboxyatractylate in releasing the laurate inhibition of reactive oxygen species (ROS) formation. Micromolar laurate concentrations arresting the ROS formation cause strong inhibition of reverse electron transfer from succinate to NAD+, whereas State 4 respiration and the transmembrane electric potential difference (ΔΨ) level are affected only slightly. It is suggested that (i) free fatty acids operate as natural ‘mild uncouplers’ preventing the transmembrane electrochemical H+ potential difference ( ) from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of the respiratory chain, and (ii) it is the ATP/ADP‐antiporter, rather than uncoupling protein 2, that is mainly involved in this antioxidant mechanism of heart muscle mitochondria.

Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain.

Antimycin, 2‐nonyl‐4‐hydroxyquinoline N‐oxide and funiculosin induce O·− 2 generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3‐dimercaptopropanol inhibit O·− 2 generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto‐oxidation of unstable ubisemiquinone formed in centre o of the Q‐cycle generates most of the O·− 2 radicals in the cytochrome bc 1‐site of the mitochondrial respiratory chain.

Magnetic fluids as drug carriers : targeted transport of drugs by a magnetic field

Abstract Some important biomedical applications of various types of magnetically controlled drug carrier systems are presented. Model investigations of the possibility of using ferrofluids for drug localization in blood vessels and in hollow organs are described. The main factors that affect magnetic holding of a drop of ferrofluid on the tube wall in the stream of nonmagnetic fluid medium are investigated.

Superoxide generation by the respiratory chain of tumor mitochondria.

O2-. generation by the succinate oxidase segment of the respiratory chain of mitochondria and submitochondrial particles from hepatoma 22a and hepatoma Zajdela has been studied with the use of the Tiron method. In the presence of succinate, superoxide generation is induced by antimycin, 2-n-4-hydroxyquinoline N-oxide or funiculosin, and is inhibited by mucidin, myxothiazol or cyanide. The rate of O2-. generation in the antimycin-inhibited state is maximal at the [succinate]/[fumarate] ratio of 1:10 and diminishes at more positive and more negative redox potentials. These characteristics of O2-.-generation are the same as observed earlier in submitochondrial particles from normal tissues. Accordingly, the mechanism of superoxide production is suggested to be the same in tumor and normal mitochondria, namely, autoxidation of the unstable ubisemiquinone in the ubiquinol-oxidizing centre o of cytochrome bc1 complex. With respect to the rate of O2-. generation, the hepatoma mitochondrial membranes are approximately twice as active as bovine heart submitochondrial particles and exceed those from rat liver by more than one order of magnitude.

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies.

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.

Electron transport control in chloroplasts. Effects of photosynthetic control monitored by the intrathylakoid pH

Abstract (1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pHin). A decrease in pHin due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg2+-ADP with Pi); Mg2+-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700+ reduction. In chloroplasts, without adding Mg2+-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 ( Δ pH ⋍ 0.5–1.0 ). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700+ reduction in the presence of Mg2+-ADP (with Pi) and without adding Mg2+-ADP is dependent on external pH variations, showing a maximum value of K ⋍ 3.5 at pHout 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg2+-ADP and Pi.

Semiquinone Q in the respiratory chain of electron transport particles: electron spin resonance studies.

The specific role of coenzyme Q semiquinone (QH.) in the respiratory chain has been discussed extensively [l-7] , but few experimental data are available on this point. Though evidence for the QH. contribution to the ESR signal at g 2.00 in mitochondria [8] , ETP [9] and bacteria [lo] had been previously obtained, (for references, see [g-12]) significant interference from flavosemiquinones obscured the results of those early observations. We have recently chosen ETP poised with the succinate/fumarate redox couple as a convenient experimental system for the QH’ ESR studies [ 11 ,121. The rationale for this choice was that at low succinate/ fumarate ratios (Eh +50 mV) reduction of the SDH flavin (Em = -40 mV [13] or -90 mV [14]) and, the more so, of the NADH-dehydrogenase flavin should be thermodynamically unfavourable while the yield of QH* should be about maximal (I!?, QH2/Q = +40 mV at pH 7.4 [15]). Some data

Magnetic fluid as a possible drug carrier for thrombosis treatment

Abstract The possibility of using a composition of ferrofluid with fibrinolytic enzyme for thrombosis treatment has been demonstrated. The main factors that affect the magnetic capture in the occluded branch of the blood vessel of such a composition and the factors that affect magnetically holding the ferrofluid drop on the tube wall in the stream of a medium were obtained.