Peroxidation of proteins and lipids in suspensions of liposomes, in blood serum, and in mouse myeloma cells

There is growing evidence that proteins are early targets of reactive oxygen species, and that the altered proteins can in turn damage other biomolecules. In this study, we measured the effects of proteins on the oxidation of liposome phospholipid membranes, and the formation of protein hydroperoxides in serum and in cultured cells exposed to radiation-generated hydroxyl free radicals. Lysozyme, which did not affect liposome stability, gave 50% protection when present at 0.3 mg/ml, and virtually completely prevented lipid oxidation at 10 mg/ml. When human blood serum was irradiated, lipids were oxidized only after the destruction of ascorbate. In contrast, peroxidation of proteins proceeded immediately. Protein hydroperoxides were also generated without a lag period in hybrid mouse myeloma cells, while at the same time no lipid peroxides formed. These results are consistent with the theory that, under physiological conditions, lipid membranes are likely to be effectively protected from randomly-generated hydroxyl radicals by proteins, and that protein peroxyl radicals and hydroperoxides may constitute an important hazard to biological systems under oxidative stress.     

Protein and thiol oxidation in cells exposed to peroxyl radicals is inhibited by the macrophage synthesised pterin 7,8-dihydroneopterin

Monocyte cells are exposed to a range of reactive oxygen species (ROS) when they are recruited to a site of inflammation. In this study, we have examined the damage caused to the monocyte-like cell line U937 by peroxyl radicals and characterised the protective effect of the macrophage synthesised compound 7,8-dihydroneopterin.Exposure of U937 cells to peroxyl radicals, generated by the thermolytic breakdown of 2,2'-azobis(amidinopropane) dihydrochloride (AAPH), resulted in the loss of cell viability as measured by thiazolyl blue (MTT) reduction, and lactate dehydrogenase (LDH) leakage. The major form of cellular damage observed was cellular thiol loss and the formation of reactive protein hydroperoxides. Peroxyl radical oxidation of the cells only caused a small increase in cellular lipid oxidation measured. Supplementation of the media with increasing concentrations of 7,8-dihydroneopterin significantly reduced the cellular thiol loss and inhibited the formation of the protein hydroperoxides. High performance liquid chromatography (HPLC) analysis showed 7,8-dihydroneopterin was oxidised by both peroxyl radicals and preformed protein hydroperoxides to predominately 7,8-dihydroxanthopterin.The possibility that 7,8-dihydroneopterin is a cellular antioxidant protecting macrophage proteins during inflammation is discussed.     

Protein damage, induced by small amounts of photodynamically generated singlet oxygen or hydroxyl radicals

The influence of limited oxidation of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and myoglobin by singlet oxygen and by hydroxyl radicals was investigated. The intrinsic fluorescence of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase decreased rapidly during oxidation, indicating a conformational change of the protein molecules. The free energy of isothermal unfolding in urea solutions was increased by singlet oxygen, but decreased by hydroxyl radical attack. The velocity of refolding of the denatured protein after dilution of the denaturant was increased by exposure to either singlet oxygen or hydroxyl radicals, with one exception: the velocity of refolding of myoglobin, oxidized by singlet oxygen, was strongly decreased. Hydroxyl radicals produced covalently crosslinked protein aggregates and some fragmentation, whereas singlet oxygen produced only crosslinked aggregates with glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase. All oxidized proteins were more susceptible to proteolysis by elastase and proteinase K, as compared to the undamaged proteins. Singlet oxygen-induced crosslinked aggregates were degraded very rapidly by elastase. Hydroxyl radical-induced aggregates of glyceraldehyde-3-phosphate dehydrogenase were also degraded very rapidly by this enzyme, but hydroxyl radical-induced aggregates of alcohol dehydrogenase were resistent to enzymatic degradation. The results indicate that limited protein oxidation may have a pronounced influence on several properties of the protein. The effects vary, however, with varying proteins and with the oxidizing species.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

DNA degradation and protein peroxidation in cells exposed to hydroxyl free radicals

The kinetics of DNA and protein damage in two lines of cultured cells exposed to radiation-generated hydroxyl free radicals were measured. The results show that DNA damage is a relatively late event, preceded by the formation of protein hydroperoxides which may play a role in the degradation of the DNA.     

Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies

In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated.     

Antioxidant activity of extract from Polygonum cuspidatum

Numerous diseases are induced by free radicals via lipid peroxidation, protein peroxidation and DNA damage. It has been known that a variety of plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum cuspidatum Sieb. et Zuce has antioxidant activity is unknown. In this study, dried roots of Polygonum cuspidatum were extracted by ethanol and the extract was lyophilized. Free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays were employed to study antioxidant activities. The results indicate that the IC50 value oí Polygonum cuspidatum extract is 110 microg/ml in free radical scavenging assays, 3.2 microg/ml in superoxide radical scavenging assays, and 8 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum cuspidatum extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 641.1 +/- 42.6 mg/g and 62.3 +/- 6.0 mg/g. The results indicate that Polygonum cuspidatum extract clearly has antioxidant effects.     

Oxalomalate,a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase,enhances lipid peroxidation-mediated oxidative damage in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against lipid peroxidation-mediated oxidative damage in U937 cells was investigated in control cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the viability was lower and the protein oxidation, lipid peroxidation, and oxidative DNA damage, reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin, as well as the significant decrease in the intracellular GSH level in oxalomalate-treated U937 cells upon exposure to AAPH. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against lipid peroxidation-mediated oxidative damage through the removal of reactive oxygen species.     

Histochemical visualization of oxidant stress

Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Mechanisms of free radical-induced damage to DNA

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Membrane proteins are critical targets in free radical mediated cytolysis

The hypothesis that proteins are critical targets in free radical mediated cytolysis was tested using U937 mononuclear phagocytes as targets and iron together with hydrogen peroxide to generate radicals. Those conditions which, after a lag of approx. 30 min, led to drastic lysis were also associated with very rapid membrane depolarisation. Conversely, when the early membrane depolarisation was prevented (by the addition of chelator and catalase), so was lysis. A similar correlation between early membrane depolarisation and subsequent lysis was also observed when the cells were exposed to a toxin from Actinobacillus actinomycetemcomitans. Those conditions of radical attack which led to lysis normally caused substantial lipid peroxidation. However, depolarisation and subsequent lysis were not prevented even when lipid peroxidation was completely suppressed by exogenous antioxidant. ATP levels were not grossly affected within the critical first 30 min period. These data exclude lipids and ATP as the target for lytic damage. We argue therefore that proteins are probably amongst the primary targets in cytolysis by radicals.     

Antioxidant activity of extract from Polygonum aviculare L

Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation, and DNA damage. It has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum aviculare L. (Polygonaceae) has antioxidant activity is unknown. In this study, dried Polygonum aviculare L. was extracted by ethanol, and the extract was lyophilized. The antioxidant activities of extract powder were examined by free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays. The results show that the IC50 value of Polygonum aviculare L. extract is 50 microg/ml in free radical scavenging assays, 0.8 microg/ml in superoxide radical scavenging assays, and 15 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum aviculare L. extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 677.4 +/- 62.7 microg/g and 112.7 +/- 13 microg/g. The results indicate that Polygonum aviculare L. extract clearly has antioxidant effects.     

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation

TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.     

The effect of alpha-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. alpha-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP+ +NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

Chemical nature of in vivo DNA base damage in hydrogen peroxide-treated mammalian cells.

Hydrogen peroxide is generated in mammalian cells by normal metabolism or by treatment with external agents. Treatment of mammalian cells with this oxidizing agent results in DNA damage. Little is known about the chemical nature of hydrogen peroxide-mediated DNA damage in mammalian cells. Here we report on the chemical characterization of in vivo base damage to nuclear DNA in mammalian cells caused by exposure to H2O2. Chromatin was isolated from cells and analyzed by gas chromatography/mass spectrometry with selected-ion monitoring. Ten DNA base products were identified and quantitated. Modified bases identified were typical hydroxyl radical-induced products of DNA bases. Results indicate involvement of hydroxyl radicals in the mechanism of nuclear DNA damage in mammalian cells caused by H2O2.     

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.     

Mechanisms of free radical-induced damage to DNA

Abstract

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Isofraxidin,a potent reactive oxygen species (ROS) scavenger,protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner

Ionizing radiation (IR) leads to oxidizing events such as excessive reactive oxygen species (ROS) in the exposed cells, resulting in further oxidative damage to lipids, proteins and DNA. To screen the potential radio-protective drug, the intracellular ROS was measured in irradiated U937 cells pretreated with 80 candidate traditional herbal medicine, respectively. Isofraxidin (IF) was one possible radio-protector in these 80 drugs. This study investigated the radio-protective role of IF, a Coumarin compound, in human leukemia cell lines, for the first time. Results indicate that IF protects against IR-induced apoptosis in U937 cells in the time- and concentration- dependent manner. IF decreases IR-induced intracellular ROS generation, especially hydroxyl radicals formation, inhibits IR-induced mitochondrial membrane potential loss and reduces IR-induced high intracellular Ca²⁺ levels regardless of ER stress. IF down-regulates the expression of caspase-3, phospho-JNK, phospho-p38 and activates Bax in mitochondria. IF inhibits cytochrome c release from mitochondria to cytosol. IF also moderates IR-induced Fas externalization and caspase-8 activation. IF also exhibits significant protection against IR-induced cell death in other leukemia cell lines such as Molt-4 cells and HL60 cells regardless of p53. Taken together, the data demonstrate that IF protects leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in a p53-independent manner.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.