Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic. Insights into the interaction of ebselen with mitochondria

Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.     

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.     

Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary

Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.     

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2(+/-) mice

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.     

Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development

Two distinct thioredoxin/thioredoxin reductase systems are present in the cytosol and the mitochondria of mammalian cells. Thioredoxins (Txn), the main substrates of thioredoxin reductases (Txnrd), are involved in numerous physiological processes, including cell-cell communication, redox metabolism, proliferation, and apoptosis. To investigate the individual contribution of mitochondrial (Txnrd2) and cytoplasmic (Txnrd1) thioredoxin reductases in vivo, we generated a mouse strain with a conditionally targeted deletion of Txnrd1. We show here that the ubiquitous Cre-mediated inactivation of Txnrd1 leads to early embryonic lethality. Homozygous mutant embryos display severe growth retardation and fail to turn. In accordance with the observed growth impairment in vivo, Txnrd1-deficient embryonic fibroblasts do not proliferate in vitro. In contrast, ex vivo-cultured embryonic Txnrd1-deficient cardiomyocytes are not affected, and mice with a heart-specific inactivation of Txnrd1 develop normally and appear healthy. Our results indicate that Txnrd1 plays an essential role during embryogenesis in most developing tissues except the heart.     

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.     

Association of mitochondrial SOD deficiency with salt-sensitive hypertension and accelerated renal senescence.

Mitochondria are the major source of superoxide (O(2)(-)) in the aerobic organisms. O(2)(-) produced by the mitochondria is converted to hydrogen peroxide by mitochondrial superoxide dismutase (SOD2). Mice with complete SOD2 deficiency (SOD2(-/-)) exhibit dilated cardiomyopathy and fatty liver leading to neonatal mortality, whereas mice with partial SOD2 deficiency (SOD2(+/-)) show evidence of O(2)(-)-induced mitochondrial damage resembling cell senescence. Since earlier studies have provided compelling evidence for the role of oxidative stress and tubulointerstitial inflammation in the pathogenesis of hypertension, we tested the hypothesis that partial SOD2 deficiency may result in hypertension. Wild-type (SOD2(+/+)) and partial SOD2-deficient (SOD2(+/-)) mice had similar blood pressures at 6-7 mo of age, but at 2 yr SOD2(+/-) mice had higher blood pressure. Oxidative stress, renal interstitial T-cell and macrophage infiltration, tubular damage, and glomerular sclerosis were all significantly increased in 2-yr-old SOD2(+/-) mice. High-salt diet induced hypertension in 6-mo-old SOD2-deficient mice but not in wild-type mice. In conclusion, partial SOD2 deficiency results in oxidative stress and renal interstitial inflammation, changes compatible with accelerated renal senescence and salt-sensitive hypertension. These findings are consistent with the pattern described in numerous other models of salt-sensitive hypertension and resemble that commonly seen in elderly humans.     

The mitochondria-targeted antioxidant MitoQ decreases features of the metabolic syndrome in ATM+/-/ApoE-/- mice

A number of recent studies suggest that mitochondrial oxidative damage may be associated with atherosclerosis and the metabolic syndrome. However, much of the evidence linking mitochondrial oxidative damage and excess reactive oxygen species (ROS) with these pathologies is circumstantial. Consequently the importance of mitochondrial ROS in the etiology of these disorders is unclear. Furthermore, the potential of decreasing mitochondrial ROS as a therapy for these indications is not known. We assessed the impact of decreasing mitochondrial oxidative damage and ROS with the mitochondria-targeted antioxidant MitoQ in models of atherosclerosis and the metabolic syndrome (fat-fed ApoE(-/-) mice and ATM(+/-)/ApoE(-/-) mice, which are also haploinsufficient for the protein kinase, ataxia telangiectasia mutated (ATM). MitoQ administered orally for 14weeks prevented the increased adiposity, hypercholesterolemia, and hypertriglyceridemia associated with the metabolic syndrome. MitoQ also corrected hyperglycemia and hepatic steatosis, induced changes in multiple metabolically relevant lipid species, and decreased DNA oxidative damage (8-oxo-G) in multiple organs. Although MitoQ did not affect overall atherosclerotic plaque area in fat-fed ATM(+/+)/ApoE(-/-) and ATM(+/-)/ApoE(-/-) mice, MitoQ reduced the macrophage content and cell proliferation within plaques and 8-oxo-G. MitoQ also significantly reduced mtDNA oxidative damage in the liver. Our data suggest that MitoQ inhibits the development of multiple features of the metabolic syndrome in these mice by affecting redox signaling pathways that depend on mitochondrial ROS such as hydrogen peroxide. These findings strengthen the growing view that elevated mitochondrial ROS contributes to the etiology of the metabolic syndrome and suggest a potential therapeutic role for mitochondria-targeted antioxidants.     

Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.     

IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice

Cardiac hypertrophy, a risk factor for heart failure, is associated with enhanced oxidative stress in the mitochondria, resulting from high levels of reactive oxygen species (ROS). The balance between ROS generation and ROS detoxification dictates ROS levels. As such, disruption of these processes results in either increased or decreased levels of ROS. In previous publications, we have demonstrated that one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is to control the mitochondrial redox balance, and thereby mediate the cellular defense against oxidative damage, via the production of NADPH. To explore the association between IDH2 expression and cardiac function, we measured myocardial hypertrophy, apoptosis, and contractile dysfunction in IDH2 knockout (idh2^−/−) and wild-type (idh2^+/+) mice. As expected, mitochondria from the hearts of knockout mice lacked IDH2 activity and the hearts of IDH2-deficient mice developed accelerated heart failure, increased levels of apoptosis and hypertrophy, and exhibited mitochondrial dysfunction, which was associated with a loss of redox homeostasis. Our results suggest that IDH2 plays an important role in maintaining both baseline mitochondrial function and cardiac contractile function following pressure-overload hypertrophy, by preventing oxidative stress.     

Overexpressed human mitochondrial thioredoxin confers resistance to oxidant-induced apoptosis in human osteosarcoma cells

Oxidative damage to mitochondria is a central mechanism of apoptosis induced by many toxic chemicals. Thioredoxin family proteins share a conserved Cys-X-X-Cys motif at their active center and play important roles in control of cellular redox state and protection against oxidative damage. In addition to the well studied cytosolic and extracellular form (Trx1), rat and avian mitochondrial forms of thioredoxin (mtTrx) have been reported. In this study, we cloned the full-length human mtTrx cDNA and performed localization and functional studies in 143B human osteosarcoma cells. The coding sequence of human mtTrx consists of a region with homology to Trx1 as well as a putative mitochondrial localization signal (MLS) at its N terminus. In stably transfected cell lines, mtTrx had a mitochondrial localization as measured by subcellular fractionation studies and by confocal fluorescence microscopy. Deletion of the MLS rendered mtTrx to be solely expressed in the cytosolic fraction. On SDS-PAGE, transfected mtTrx had the same apparent molecular weight as the MLS truncated form, indicating that the leader sequence is cleaved during or after mitochondrial import. Treatment with the oxidant tert-butylhydroperoxide induced apoptosis in 143B cells. This oxidant-induced apoptosis was inhibited by overexpressing the full-length mtTrx in 143B cells. Thus, human mtTrx is a member of the thioredoxin family of proteins localized to mitochondria and may play important roles in protection against oxidant-induced apoptosis.     

Vasomotor responses in MnSOD-deficient mice

MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD(-/-) mice die soon after birth, and MnSOD(+/-) mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD(+/-) mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2alpha (PGF2alpha), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD(+/-) mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2alpha and impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD(+/-) mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD(+/-) mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD(+/-) mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2alpha similarly in aortas of WT and MnSOD(+/-) mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD(+/-) mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Efficiency of propolis extract against mitochondrial stress induced by antineoplasic agents (doxorubicin and vinblastin) in rats

To assess the oxidative stress and mitochondrial dysfunction associated with disease, toxic process and aging, in vivo and in vitro preventive effect of propolis extract against mitochondrial oxidative stress induced by two anticancer drugs (doxorubicin and vinblastin) have been investigated in female wistar rat using liver and heart mitochondria. The results show that doxorubicin and vinblastin altered mitochondrial functions as observed by a decrease in respiratory control value, an activation of swelling and overproduction of superoxide anion. Myocardial tissue from doxorubicin treated rats showed a marked increase in malondialdehyde production, a depletion of reduced glutathione contents and an inhibition of catalase and superoxide dismutase activities. Similar results were also observed in liver tissue. Pretreatment of rats with propolis extract (100 mg/kg/day po) (10(-4) M ip) administered 4 days prior to doxorubicin (20 mg/kg) and/or vinblastin (2 mg/kg) injection, substantially reduced the peroxidative damage in myocardium and hepatic tissues and markedly restored the tissues catalase and SOD activities. The results strongly suggest that propolis extract protects heart and liver tissues from oxidative stress by protecting the mitochondria.     

Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis

Heart mitochondria from heterozygous (Sod2(-/+)) knockout mice have a 50% reduction in manganese superoxide dismutase (MnSOD) activity. The decrease in MnSOD activity was associated with increased mitochondrial oxidative damage as demonstrated by a decrease in the activities of iron sulfhydryl proteins sensitive to oxygen stress (aconitase and reduced nicotinamide adenine dinucleotide-oxidoreductase). Mitochondrial function was altered in the Sod2(-/+) mice, as shown by decreased respiration by complex I and an increase in the sensitivity of the permeability transition to induction by calcium and t-butylhydroperoxide. The increased induction of the permeability transition in heart mitochondria from Sod2(-/+.)mice was associated with increased release of cytochrome c and an increase in DNA fragmentation. Cardiomyocytes isolated from neonatal Sod2(-/+) and Sod2(-/-) mice were more sensitive to cell death than cardiomyocytes from Sod2(+/+) mice after t-butylhydroperoxide treatment, and this increased sensitivity was prevented by inhibiting the permeability transition with cyclosporin A. These experiments demonstrate that MnSOD may play an important role in the induction of the mitochondrial pathway of apoptosis in the heart, and this appears to occur primarily through the permeability transition.