Free Radical Lipid Peroxidation Induced by Reactive Halogen Species.

The review is devoted to the mechanisms of free radical lipid peroxidation (LPO) initiated by reactive halogen species (RHS) produced in mammals, including humans, by heme peroxidase enzymes, primarily myeloperoxidase (MPO). It has been shown that RHS can participate in LPO both in the initiation and branching steps of the LPO chain reactions. The initiation step of RHS-induced LPO mainly involves formation of free radicals in the reactions of RHS with nitrite and/or with amino groups of phosphatidylethanolamine or Lys. The branching step of the oxidative chain is the reaction of RHS with lipid hydroperoxides, in which peroxyl and alkoxyl radicals are formed. The role of RHS-induced LPO in the development of human inflammatory diseases (cardiovascular and neurodegenerative diseases, cancer, diabetes, rheumatoid arthritis) is discussed in detail.

The Cytotoxic Action of Cytochrome C/Cardiolipin Nanocomplex (Cyt-CL) on Cancer Cells in Culture.

PURPOSE: The effect of existing anti-cancer therapies is based mainly on the stimulation of apoptosis in cancer cells. Here, we have demonstrated the ability of a catalytically-reactive nanoparticle-based complex of cytochrome c with cardiolipin (Cyt-CL) to induce the apoptosis and killing of cancer cells in a monolayer cell culture. METHODS: Cyt-CL nanoparticles were prepared by complexing CytC with different molar excesses of CL. Following characterization, cytotoxicity and apoptosis inducing effects of nanoparticles were investigated. In an attempt to identify the anticancer activity mechanism of Cyt-CL, pseudo-lipoxygenase and lipoperoxidase reaction kinetics were measured by chemiluminescence. RESULTS: Using chemiluminescence, we have demonstrated that the Cyt-CL complex produces lipoperoxide radicals in two reactions: by decomposition of lipid hydroperoxides, and by lipid peroxidation under the action of H2O2. Antioxidants inhibited the formation of lipid radicals. Cyt-CL nanoparticles, but not the CytC alone, dramatically enhanced the level of apoptosis and cell death in two cell lines: drug-sensitive (A2780) and doxorubicin-resistant (A2780-Adr). The proposed mechanism of the cytotoxic action of Cyt-CL involves either penetration through the cytoplasm and outer mitochondrial membrane and catalysis of lipid peroxidation reactions at the inner mitochondrial membrane, or/and activation of lipid peroxidation within the cytoplasmic membrane. CONCLUSIONS: Here we propose a new type of anticancer nano-formulation, with an action based on the catalytic action of Cyt-CL nanoparticles on the cell membrane and and/or mitochondrial membranes that results in lipid peroxidation reactions, which give rise to activation of apoptosis in cancer cells, including multidrug resistant cells.

Cytochrome c Complexes with Cardiolipin Monolayer Formed under Different Surface Pressure.

The formation of the complex of cytochrome c (Cytc) with a phospholipid cardiolipin (CL) in mitochondria is a crucial event in apoptosis development. There are two viewpoints on the structure of the complex. (1) Cytc is bound on the surface of the lipid bilayer. (2) The complex is a hydrophobic nanoparticle Cytc-CL formed by Cytc molten globule, covered by CL monolayer.1 In the present work, we attempted to bridge the gap between these two structures. We investigated the interaction between Cytc and Langmuir monolayers of CL. The surface pressure increase during incorporation of Cytc into CL monolayer obeys the equation: π = π0 + Δπ∞[1 - exp(-ßt)], where ß is pseudo-first-order rate constant of Cytc binding, directly proportional to the initial Cytc concentration c0. Parameters Δπ∞ and the rate ß measured in different conditions were virtually equal for natural bovine CL and peroxidation-resistant tetraoleoyl CL in all experiments. Surface area-surface pressure isotherms of Cytc alone and in combination with a CL monolayer were similar in shape. Apparently, the protein exposes hydrophilic groups to the water phase and hydrophobic to the air or to the hydrocarbon chains of CL. The 30% ethanol dramatically accelerated the adsorption of Cytc on the water surface. The protein-lipid surface films showed, in compression-expansion cycles, that hysteresis loops were observed always when Cytc present, reproducible in repeating cycles. Taken together, our data show that when incorporated in a lipid monolayer or after adsorption on the water-air interface, Cytc undergoes conformational transition. In that, one part of the globule sphere becomes predominantly hydrophobic and the other, hydrophilic and charged ("stratified" Cytc). We hypothesize that in CL-containing bilayer membranes, Cytc incorporation into the lipid monolayer would result in membrane folding with subsequent formation of either catalytically reactive "bubbles" inside the bilayer, formed by Cytc-CL, or the appearance of hydrophilic pores. The role of lipid peroxidation catalyzed by Cytc-CL in the appearance of pores and apoptosis is also discussed.

Are the mitochondrial respiratory complexes blocked by NO the targets for the laser and LED therapy?

Effects of laser (442 and 532 nm) and light-emitting diode (LED) (650 nm) radiation on mitochondrial respiration and mitochondrial electron transport rate (complexes II-III and IV) in the presence of nitric oxide (NO) were investigated. It was found that nitric oxide (300 nM-10 µM) suppresses mitochondrial respiration. Laser irradiation of mitochondria (442 nm, 3 J cm(-2)) partly restored mitochondrial respiration (approximately by 70 %). Irradiation with green laser (532 nm) or red LED (650 nm) in the same dose had no reliable effect. Evaluation of mitochondrial electron transport rate in complexes II-III and IV and effects of nitric oxide demonstrated almost similar sensitivity of complex II-III and IV to NO, with approximately 50 % inhibition at NO concentration of 3 µM. Subsequent laser or LED irradiation (3 J cm(-2)) showed partial recovery of electron transport only in complex IV and only under irradiation with blue light (442 nm). Our results support the hypothesis of the crucial role of cytochrome c oxidase (complex IV) in photoreactivation of mitochondrial respiration suppressed by NO.

Effects of low-level laser therapy on mitochondrial respiration and nitrosyl complex content.

Among the photochemical reactions responsible for therapeutic effects of low-power laser radiation, the photolysis of nitrosyl iron complexes of iron-containing proteins is of primary importance. The purpose of the present study was to compare the effects of blue laser radiation on the respiration rate and photolysis of nitrosyl complexes of iron-sulfur clusters (NO-FeS) in mitochondria, subjected to NO as well as the possibility of NO transfer from NO-FeS to hemoglobin. It was shown that mitochondrial respiration in State 3 (V3) and State 4 (V4), according to Chance, dramatically decreased in the presence of 3 mM NO, but laser radiation (λ = 442 nm, 30 J/cm(2)) restored the respiration rates virtually to the initial level. At the same time, electron paramagnetic resonance (EPR) spectra showed that laser irradiation decomposed nitrosyl complexes produced by the addition of NO to mitochondria. EPR signal of nitrosyl complexes of FeS-clusters, formed in the presence of 3 mM NO, was maximal in hypoxic mitochondria, and disappeared in a dose-dependent manner, almost completely at the irradiation dose 120 J/cm(2). EPR measurements showed that the addition of lysed erythrocytes to mitochondria decreased the amount of nitrosyl complexes in iron-sulfur clusters and produced the accumulation of NO-hemoglobin. On the other hand, the addition of lysed erythrocytes to mitochondria, preincubated with nitric oxide, restored mitochondrial respiration rates V3 and V4 to initial levels. We may conclude that there are two possible ways to destroy FeS nitrosyl complexes in mitochondria and recover mitochondrial respiration inhibited by NO: laser irradiation and ample supply of the compounds with high affinity to nitric oxide, including hemoglobin.

Effects of Laser Radiation on Mitochondria and Mitochondrial Proteins Subjected to Nitric Oxide.

The biological roles of heme and nonheme nitrosyl complexes in physiological and pathophysiological conditions as metabolic key players are considered in this study. Two main physiological functions of protein nitrosyl complexes are discussed-(1) a depot and potential source of free nitric oxide (NO) and (2) a controller of crucial metabolic processes. The first function is realized through the photolysis of nitrosyl complexes (of hemoglobin, cytochrome c, or mitochondrial iron-sulfur proteins). This reaction produces free NO and subsequent events are due to the NO physiological functions. The second function is implemented by the possibility of NO to bind heme and nonheme proteins and produce corresponding nitrosyl complexes. Enzyme nitrosyl complex formation usually results in the inhibition (or enhancement in the case of guanylate cyclase) of its enzymatic activity. Photolysis of protein nitrosyl complexes, in this case, will restore the original enzymatic activity. Thus, cytochrome c acquires peroxidase activity in the presence of anionic phospholipids, and this phenomenon can be assumed as a key step in the programmed cell death. Addition of NO induces the formation of cytochrome c nitrosyl complexes, inhibits its peroxidase activity, and hinders apoptotic reactions. In this case, photolysis of cytochrome c nitrosyl complexes will reactivate cytochrome c peroxidase activity and speed up apoptosis. Control of mitochondrial respiration by NO by formation or photolytic decay of iron-sulfur protein nitrosyl complexes is an effective instrument to modulate mitochondrial metabolism. These questions are under discussion in this study.

A mitochondrial pathway for biosynthesis of lipid mediators.

The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes.

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.