DNA damage by peroxynitrite characterized with DNA repair enzymes.

The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relatively low. This DNA damage profile caused by peroxynitrite is significantly different from that obtained with hydroxyl radicals or with singlet molecular oxygen. The effects of various radical scavengers and other additives (t-butanol, selenomethionine, selenocystine, desferrioxamine) were the same for single-strand breaks and Fpg-sensitive modifications and indicate that a single reactive intermediate but not peroxynitrite itself is responsible for the damage.     

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.     

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.     

Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.

Ultraviolet A (UVA; 320-400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1, 4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.     

Protection against peroxynitrite

Peroxynitrite formed in vivo from superoxide and nitric oxide can mediate oxidation, nitration, or nitrosation reactions, leading to impaired function, toxicity, and alterations in signaling pathways. Protection against peroxynitrite is important for defense of normal tissue, especially during inflammation. Biological protection against peroxynitrite is organized in three categories: prevention, interception, and repair. Prevention is the control of the formation of peroxynitrite precursors, nitric oxide and superoxide. Interception is by direct reaction with peroxynitrite, leading to non-toxic products. In this regard, organoselenium compounds, metalloporphyrin derivatives, and peroxidases (e.g. glutathione peroxidase and myeloperoxidase) exhibit high second-order rate constants with peroxynitrite. Ebselen, like glutathione peroxidase, protects in a catalytic fashion utilizing glutathione as reductant in the peroxynitrite reductase reaction. Protection by metalloporphyrins can be maintained through glutathione or ascorbate. Repair processes remove damaged products and restitute intact biomolecules.     

A half-marathon and a marathon run induce oxidative DNA damage, reduce antioxidant capacity to protect DNA against damage and modify immune function in hobby runners

This study investigated whether a 21.1 km (half-marathon) or a 42.195 km (marathon) run modulates DNA damage, antioxidant capacity in lymphocytes and plasma, and the immune system in healthy hobby runners. Ten and 12 volunteers who completed the Baden-Marathon race in Karlsruhe with a running distance of 21.1 km and 41.195 km, respectively, were assessed 10 days before and immediately after the finish. There was no increase in the levels of endogenous DNA strand breaks immediately after half-marathon or marathon races. A statistically significant increase in the levels of oxidative DNA damage in lymphocytes was found using endonuclease III but not formamidopyrimidine glycolase (Fpg). The resistance of DNA to oxidative damage induced by hydrogen peroxide in isolated lymphocytes was significantly decreased after both races. The levels of plasma antioxidants such as alpha-tocopherol, beta-carotene and lycopene were close to, or higher than, those considered optimal for reducing the risk of cardiovascular diseases and there were no significant changes after the races in antioxidant capacity of LDL (lag-time test) or plasma in ORAC, TEAC or paraoxonase assays. The number and percentage of granulocytes and monocytes able to generate oxidative burst were significantly increased after both races, but the lytic activity of NK cells was significantly increased at the end of the half-marathon; no effect was observed in the marathon runners. Thus, oxidative DNA damage in lymphocytes, decreased the antioxidant capacity to protect lymphocytes against DNA strand breaks and increased the formation of reactive species by phagocytes in well-nourished hobby runners indicating moderate oxidative damage during such high-intensity exercise.     

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.     

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera)

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.     

Interaction of peroxynitrite with carotenoids in human low density lipoproteins

Interaction of peroxynitrite, the product of the reaction between nitric oxide and superoxide, with carotenes (lycopene, alpha-carotene, and beta-carotene) and oxocarotenoids (beta-cryptoxanthin, zeaxanthin, and lutein) was studied both in homogeneous solution and in human low-density lipoproteins (LDL). All carotenoids prevented the formation of rhodamine 123 from dihydrorhodamine 123 caused by peroxynitrite, suggesting that the carotenoids react with peroxynitrite. Oxocarotenoids were as effective as biothiols, known scavengers of peroxynitrite, whereas lycopene, alpha-carotene, and beta-carotene exhibited a considerably more pronounced effect. Moreover, peroxynitrite caused a loss of carotenoids in LDL as was revealed by HPLC. The concentration of peroxynitrite causing half-maximal loss of carotenoids in LDL ranged from 13 +/- 3 to 68 +/- 3 microM for lycopene and lutein, respectively. Again, oxocarotenoids were less reactive in this system. A correlation between efficiency of carotenoids in the competitive assay with dihydrorhodamine 123 and the concentration of peroxynitrite causing half-maximal loss of carotenoids in LDL was observed (r(2) = 0.91). These findings suggest that carotenoids can efficiently react with peroxynitrite and perform the role of scavengers of peroxynitrite in vivo.