Oxidative stress and aging in Caenorhabditis elegans

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. A mev-1 (kn1) mutant of Caenorhabditis elegans, isolated on the basis of its methyl viologen (paraquat) hypersensitivity, is also hypersensitive to elevated oxygen levels. Unlike the wild type, its life span decreases dramatically as oxygen concentrations are increased from 1% to 60%. Strains, which bear this mutation, accumulate fluorescent materials and protein carbonyl groups, markers of aging, at faster rates than the wild type. We have cloned mev-1 gene by transformation rescue and found that it is, in fact, the previously sequenced gene (cyt-1) that encodes succinate dehydrogenase cytochrome b. A missense mutation abolishes complex II activity in the mitochondrial membrane but not succinate dehydrogenase enzyme activity per se. These data suggest that CYT-1 directly participates in electron transport from FADH₂ to coenzyme Q. Moreover, mutational inactivation of this process renders animals susceptible to oxidative stress and, as a result, leads to premature aging.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 μM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 μM. H1004, a reagent used as control for H-7, did not affect (at 10 μM) or increased little (at 100 μM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 μM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.     

D(+) galactosamine induced oxidative and nitrosative stress-mediated renal damage in rats via NF-κB and inducible nitric oxide synthase (iNOS) pathways is ameliorated by a polyphenol xanthone,mangiferin

The present study investigated the possible protective effect of mangiferin against D(+) galactosamine (DGal)-induced nephrotoxicity. DGal intoxication increased reactive oxygen species (ROS), reactive nitrogen species and tumor necrosis factor-α (TNF-α) production and disturbed the antioxidant machineries in the kidney tissue. Mangiferin treatment post to DGal exposure reduced all these DGal-induced adverse effects. Signal transduction studies showed that DGal significantly increased the protein expression of Bax, cytochrome c, caspase 3/9 and inducible nitric oxide synthase (iNOS) in the cytosol and NF-κB in nuclear fraction. The same exposure, on the other hand, reduced the protein expression of Bcl-2 in the cytosol. Mangiferin treatment could, however, reduce the DGal-induced up-regulation of cytochrome c, NF-κB, iNOS, caspase 3/9 and alter the reciprocal regulation of Bcl-2 family proteins. Histological studies also revealed the nephroprotective effect of mangiferin against DGal induced nephrotoxicity. Combining, results suggest that mangiferin protects rat's kidney in DGal-induced oxidative/nitrosative stress and acute nephrotoxicity via its antioxidant activities.     

Generation of superoxide and singlet oxygen from α-tocopherolquinone and analogues

Three potential routes to generation of reactive oxygen species (ROS) from α-tocopherolquinone (α-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 10⁸ dm³/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10^{? 6} s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 10⁹ dm³/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid–soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A₂ superfamily (PLA₂) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.