Michal Wozniak

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines

The functions of N‐acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp‐214 in HSA or Trp‐212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp‐134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue. Proteins 2000;40:39–48.

Influence of structure on the antioxidant activity of indolinic nitroxide radicals

An in vitro study was carried out to verify whether the chain length of a substituent on an indolinic nitroxide could influence its antioxidant activity in different biological environments subjected to oxidative stress. Three distinct indolinic nitroxides were synthesized and compared with vitamin E and Trolox (a hydrophilic analogue of vitamin E), where the only difference between the nitroxides was the length of the hydrocarbon chain in the 2-position of indole, namely 2 (C2), 10 (C10), and 18 (C18) carbons. All the nitroxides were effective in preventing oxidation of bovine serum albumin, but to different extents, with the longer chain derivatives being more efficient. However, the C2 compound was the most efficient in preventing lipid peroxidation in microsomal membranes. The C2 and C18 compounds, Trolox, and vitamin E protected microsomal protein oxidation to the same extent at the highest concentration used (13 microM). The nitroxide with a C10 chain was less effective in this system. The influence of these compounds on the enzymatic activity of two mitochondrial proteins subjected to oxidative stress was also studied by means of oxygraph measurements. Mitochondrial rotenone-sensitive NADH oxidase and succinate oxidase responded differently to BuOOH-induced radical chemistry, and the compounds under study also protected the activity of the two enzymes but to different extents. The results clearly demonstrate that indolinic nitroxides are very efficient antioxidants, protecting both lipids and proteins from peroxidation. The indole structure influences the antioxidant efficacy in biological systems.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Effects of indolinic and quinolinic aminoxyls on protein and lipid peroxidation of rat liver microsomes

A study on peroxyl radical induced oxidation of rat liver microsomal membranes in the presence of different indolinic and quinolinic aminoxyls (Scheme 1) was carried out in order to test their efficiency as antioxidants in lipid and protein peroxidation. The extent of lipid peroxidation was quantified by the amount of malondialdehyde (MDA) produced, and the measurement of carbonyl residues was used as an index of microsomal protein oxidation. The results obtained suggest that lipid soluble indolinic and quinolinic aminoxyls are efficient in protecting lipids and proteins of biological membranes against oxidation. The efficacy of these aminoxyls as protectors of lipids and proteins was much higher than the water soluble TEMPOL. Moreover, the hydrophobic aminoxyls were more effective in preventing protein than lipid oxidation at low concentrations (1-20 microM). However, at high concentration (100 microM), lipid as opposed to protein oxidation was almost completely inhibited. The data supports the hypothesis that proteins probably have a different oxidation pattern from lipids.

A NEW FLUORESCENCE METHOD TO DETECT SINGLET OXYGEN INSIDE PHOSPHOLIPID MODEL MEMBRANES

A fluorescence method for detecting singlet oxygen (1O2) in model membranes is proposed. 1O2 was generated by hydrogen peroxide/sodium hypochlorite system. 1,3-Diphenylisobenzofuran (DPBF), a specific 1O2 trap, dissolved in organic solvents gives a strong fluorescence spectrum when excited at 410 nm. A similar spectrum, with a maximum at 455 nm, is obtained when DPBF is incorporated in unilamellar dipalmitoylphosphatidylcholine liposomes. The intensity of fluorescence spectrum decreases when DPBF-labeled liposomes are exposed to singlet oxygen. This decrease is sensitive to 1O2 traps and quenchers like tryptophan and sodium azide, to lipid membrane fluidity and to the concentration of sodium hypochlorite and hydrogen peroxide.

Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics

There is growing evidence of mitochondrial membrane raft‐like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl‐beta‐cyclodextrin (MβCD), being a well‐known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MβCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride‐induced swelling. These data suggest that disruption of mitochondrial raft‐like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.

Fisetin prevents fluoride- and dexamethasone-induced oxidative damage in osteoblast and hippocampal cells.

Fluoride intoxication and dexamethasone treatment produce deleterious effects in bone and brain. The aim of this study was to evaluate the effect of fluoride (F) and dexamethasone (Dex) co-exposure on oxidative stress and apoptosis in osteoblast-like MC3T3-E1 and hippocampal HT22 cell lines. Co-exposure to F and Dex resulted in a concentration-dependent decrease in cell viability, induction of apoptosis and increased generation of reactive oxygen species (ROS) and nitric oxide (NO) following 72 h of incubation. Fluoride-induced apoptosis in MC3T3-E1 and HT22 cells was attenuated by catalase and L-NNMA, indicating a role for H2O2 and NO as mediators of cytotoxicity. Dexamethasone-induced apoptosis was associated with H2O2 generation in both cell lines and it was attenuated during co-incubation with catalase. These data indicate that co-exposure to F and Dex amplifies their respective cytotoxicity in H2O2- and NO-dependent manner. As flavonoid fisetin prevented F- and Dex-induced cytotoxicity the potential role of this product in pharmacology and diet may be considered.

N-acylethanolamines as membrane topological stress compromising agents.

The effect of different N-acylethanolamines on the phase behaviour of fully hydrated egg phosphatidylethanolamines is reported. In particular, in the presence of N-acylethanolamines, the transition from the liquid-crystalline lamellar (L alpha) to the inverse hexagonal (HII) phase is observed at higher temperature with respect to the temperature transition of pure phosphatidylethanolamine. Moreover, in correspondence of this transition, an intermediate Q224 (space group Pn3m) cubic phase has been detected. Since the structure of this cubic phase presents unique topological analogies with the lipid bilayer organization, these data suggest the possible role of N-acylethanolamines in stabilizing the biological membranes by avoiding a sudden change to a non-bilayer phase in those tissues which undergo stress conditions.