Analysis of metal-induced oxidative DNA damage in cultured mammalian cells

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.     

ISOLATION OF V79 FIBROBLAST CELL LINES CONTAINING ELEVATED METALLOTHIONEIN LEVELS THAT HAVE INCREASED RESISTANCE TO THE CYTOTOXIC EFFECTS OF ULTRAVIOLET-A RADIATION

Abstract Isolated clones of V79 Chinese hamster lung fibroblasts, selected for resistance against cadmium toxicity, were exposed to monochromatic 365 nm ultraviolet-A (UVA, 320 nm to visible light) radiation and examined for cell survival. All three of the Cd-resistant V79 clones (V79Cd) tested exhibited significant increases in survival after irradiation compared with control cultures similar to the increased survival observed in Zn acetate-induced V79 cells. Dose-modifying factors calculated for these survival experiments were all approximately 1.5. When characterized for steady-state levels of metallothionein (MT) mRNA and associated Cd-binding activity, all of the Cd-resistant V79Cd clones demonstrated elevated constitutive levels of both, implicating MT as the mechanism responsible for the observed cellular resistance to Cd and also to 365 nm UVA radiation. However, whereas levels of intracellular MT protein correlated with differences in survival against Cd, MT intracellular levels did not correlate well with protection against 365 nm UVA. Increased cell survival after exposure to 365 nm UVA radiation mediated by MT appeared to reach a threshold level and MT only provided a limited degree of protection. Since UVA radiation is known to cause cell death mediated through the intracellular generation of reactive oxygen species (ROS), these results suggest that the role of MT in ameliorating cellular photooxidative damage produced by UVA is by reducing intracellular ROS.     

Acetoaminophen-induced accumulation of 8-oxodeoxyguanosine through reduction of Ogg1 DNA repair enzyme in C6 glioma cells

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome p450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.     

Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation

Ageing process in cells is associated with oxidative stress. Ultraviolet A produces reactive oxygen species responsible for accumulation of DNA and cellular damage. After the evaluation of antioxidant enzyme activities and oxidative stress markers at the basal state, we have studied the responses to UVA stress of coetaneous fibroblasts, isolated from different male donors (2-88 years, n=23) in terms of cytotoxicity, genotoxicity and DNA repair capacities. For this purpose, we have determined level of DNA damage using the comet assay (single strand breaks and alkali-labile sites) and the cell cycle distribution after a 5 J/cm2 irradiation. No differences with age were observed for antioxidant enzyme activities and oxidative stress markers. DNA strand breaks after UVA irradiation (5-20 J/cm2), was found to be age-dependent. DNA repair was slow and also significantly affected by ageing. The cell cycle distribution analysis showed that high repair correlated with high proliferative capacities at basal level. Twenty-four hours after the stress, fraction of young fibroblasts blocked in G1 phase was significantly increased whereas significant modifications concerned the G2-M phase for adult and older fibroblasts. These results indicate an age-dependent decline in the DNA repair capacities correlated with modifications of the cell cycle parameters.     

PHOTOREACTIVATION OF LETHAL DAMAGE AND DAMAGE LEADING TO CHROMATID DELETIONS INDUCED IN G1 PHASE HAMSTER x Xenopus HYBRID CELLS BY UV

A86 Xenopus cells, cloned from a Xenopus line that exhibited a high level of photoreactivation of UV-induced lethal damage, and V79M1 hamster cells, cloned from a hamster line that did not exhibit efficient photoreactivation of such damage, were fused to produce the V79M1 x A86 cell line--a hybrid line in which approximately 84% of the cells contained the entire V79M1 and A86 genomes. Ultraviolet and UV plus photoreactivation fluence-survival relations were then determined and compared for hybrid and parental G1 phase cells in a first attempt to elucidate interactions of the parental genetic potentials for photoreactivation in the hybrid. Specifically, it was anticipated that the combined V79M1 and A86 genomes in the hybrid would produce photoreactivating enzymes sufficient to efficiently photoreactivate UV-induced lethal damage in both A86 and V79M1 DNA and little difference would be observed in the levels of photoreactivation exhibited by V79M1 x A86 and A86 G1 phase cells. To the contrary, the level of photoreactivation observed for the hybrid did not closely approach that observed for the A86 line. To assist in the interpretation of this somewhat unexpected observation, three additional studies were performed: (1) comparison of 'optimal' schemes for photoreactivation of UV-induced lethal damage in the hybrid and parental G1 phase cells, (2) comparison of the effects of some different types of growth medium on photoreactivation of UV-induced lethal damage in hybrid and parental G1 phase cells, and (3) comparison of the levels of photoreactivation of UV-induced chromatid deletions in the V79M1 and A86 chromosomes of G1 phase hybrid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ evaluation of chromium–DNA damage using a DNA-electrochemical biosensor

The in situ evaluation of the direct interaction of chromium species with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine bases. The results obtained revealed the interaction with dsDNA of the Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation by O₂ dissolved in the solution bound to dsDNA. This interaction leads to different modifications and causes oxidative damage in the B-DNA structure. Using polyhomonucleotides of guanine and adenine, it was shown that the interaction between reactive intermediates Cr(IV) and Cr(V)–DNA causes oxidative damage and preferentially takes place at guanine-rich segments, leading to the formation of 8-oxoguanine, the oxidation product of guanine residues and a biomarker of DNA oxidative damage. The interaction of Cr(VI) with dsDNA causes breaking of hydrogen bonds, conformational changes, and unfolding of the double helix, which enables easier access of other oxidative agents to interact with DNA, and the occurrence of oxidative damage to DNA.     

Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.     

Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser-induced fluorescence detection in cultured cell lines.

A capillary zone electrophoretic (CZE) method coupled with laser-induced fluorescence (LIF) was developed for the simultaneous determination of two important intracellular parameters related to oxidative stress (i.e. reactive oxygen species, ROS, and reduced glutathione, GSH). This rapid and sensitive method was applied to the study of oxidative stress in cultured V79 fibroblast cells. The fluorogenic reagents selected were: (i) dihydrorhodamine-123 (DHR-123) which is converted intracellularly by ROS to the fluorescent rhodamine-123 dye (Rh-123), and (ii) naphthalene-2,3-dicarboxaldehyde (NDA), which reacts quickly with GSH in cell extracts to produce a fluorescent adduct. Separation of Rh-123, GSH-NDA and gamma-glutamylcysteine-NDA adducts was performed using an uncoated fused-silica capillary and a 100 mM borate buffer, pH 9.2, at 20 degrees C and at an applied voltage of 25 kV; LIF detection was operated using an argon laser. The cell line was also tested for its ability to alleviate oxidative stress induced by tert-butylhydroperoxide (t-BuOOH). Exposure to t-BuOOH (up to 3 mm for 2 h) did not affect the intracellular ROS and GSH concentrations. At higher (4-10 mM) t-BuOOH concentrations, an inverse relationship between the concentrations of ROS and GSH was obtained, showing that the present method can readily evaluate the gradual consumption of the primary cellular scavenger of ROS which occurs simultaneously with the increase of oxidative insult.     

Damage to rat retinal DNA induced in vivo by visible light

Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.     

Antiproliferation and Induction of Apoptosis in Ca9-22 Oral Cancer Cells by Ethanolic Extract of <em>Gracilaria tenuistipitata</em>

The water extract of Gracilaria tenuistipitata have been found to be protective against oxidative stress-induced cellular DNA damage, but the biological function of the ethanolic extracts of G. tenuistipitata (EEGT) is still unknown. In this study, the effect of EEGT on oral squamous cell cancer (OSCC) Ca9-22 cell line was examined in terms of the cell proliferation and oxidative stress responses. The cell viability of EEGT-treated OSCC cells was significantly reduced in a dose-response manner (p < 0.0001). The annexin V intensity and pan-caspase activity of EEGT-treated OSCC cells were significantly increased in a dose-response manner (p < 0.05 to 0.0001). EEGT significantly increased the reactive oxygen species (ROS) level (p < 0.0001) and decreased the glutathione (GSH) level (p < 0.01) in a dose-response manner. The mitochondrial membrane potential (MMP) of EEGT-treated OSCC cells was significantly decreased in a dose-response manner (p < 0.005). In conclusion, we have demonstrated that EEGT induced the growth inhibition and apoptosis of OSCC cells, which was accompanied by ROS increase, GSH depletion, caspase activation, and mitochondrial depolarization. Therefore, EEGT may have potent antitumor effect against oral cancer cells.     

Increased level of oxidative stress in genomically unstable cell clones

Recently, we reported that ultraviolet radiation induces delayed mutations in mammalian cells. At the same level of cell death the oxidative component of sunlight (ultraviolet A radiation) was as potent in inducing this kind of genomic instability as ultraviolet B radiation. Ultraviolet B radiation predominantly harms cells by direct damage to DNA and thus is much more mutagenic than ultraviolet A radiation. From that study, clones with a significantly increased mutation rate in the hypoxanthine phosphoribosyl transferase gene were obtained. These genomically unstable clones were also found to have a higher variance in the number of chromosomes than the unirradiated control cells, indicating chromosomal instability. The mechanisms for induction and maintenance of radiation induced genomic instability are not known, but some studies suggest that reactive oxygen species might be involved. In the present study, we have measured the level of potentially mutagenic peroxides in the genomically unstable clones. The levels of intracellular peroxides and lipid peroxides were measured using the probes dihydrorhodamine 123 and diphenyl-1-pyrenyl-phosphine, respectively. The unstable clones had elevated levels of oxidants, supporting the hypothesis that intermediate reactive oxygen species might have a role in the maintenance of genomic instability induced by ultraviolet radiation.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells

The crucial role of DNA polymerase eta in protecting against sunlight‐induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP‐V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XP‐V cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP‐V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP‐V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and γH2AX signaling. Corroborating ATR participation in UVA‐DDR, a significant increase in Chk1 protein phosphorylation, as well as S‐phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N‐acetylcysteine (NAC), which significantly protects XP‐V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA‐induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells.     

Differential response induced by exposure to low-dose ionizing radiation in SHSY-5Y and normal human fibroblast cells

Radiation therapy has been used in the treatment of a wide variety of cancers for nearly a century and is one of the most effective ways to treat cancer. Low-dose ionizing radiation (IR) can interfere with cell division of cancer and normal cells by introducing oxidative stress and injury to DNA. The differences in the response to IR-induced DNA damage and increased reactive oxygen species between normal human fibroblasts (NHFs) and cancerous SHSY-5Y cells were considered. H2AX staining and comet assays revealed that NHF cells responded by initiating a DNA repair sequence whereas SHSY-5Y cells did not. In addition, NHF cells appeared to quench the oxidative stress induced by IR, and after 24 h no DNA damage was present. SHSY-5Y cells, however, did not repair their DNA, did not quench the oxidative stress, and showed characteristic signs that they were beginning to undergo apoptosis. These results indicate that there is a differential response between this cancerous and normal cell line in their ability to respond to low-dose IR, and these differences need to be exploited in order to treat cancer effectively. Further study is needed in order to elucidate the mechanism by which SHSY-5Y cells undergo apoptosis following radiation and why these normal cells are better equipped to deal with IR-induced double-strand breaks and oxidative stress.     

Oxidative Stress,Genomic Integrity,and Liver Diseases

Excess reactive oxygen species production and free radical formation can lead to oxidative stress that can damage cells, tissues, and organs. Cellular oxidative stress is defined as the imbalance between ROS production and antioxidants. This imbalance can lead to malfunction or structure modification of major cellular molecules such as lipids, proteins, and DNAs. During oxidative stress conditions, DNA and protein structure modifications can lead to various diseases. Various antioxidant-specific gene expression and signal transduction pathways are activated during oxidative stress to maintain homeostasis and to protect organs from oxidative injury and damage. The liver is more vulnerable to oxidative conditions than other organs. Antioxidants, antioxidant-specific enzymes, and the regulation of the antioxidant responsive element (ARE) genes can act against chronic oxidative stress in the liver. ARE-mediated genes can act as the target site for averting/preventing liver diseases caused by oxidative stress. Identification of these ARE genes as markers will enable the early detection of liver diseases caused by oxidative conditions and help develop new therapeutic interventions. This literature review is focused on antioxidant-specific gene expression upon oxidative stress, the factors responsible for hepatic oxidative stress, liver response to redox signaling, oxidative stress and redox signaling in various liver diseases, and future aspects.     

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H₂O₂) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H₂O) and oxygen (O₂) in order to reduce the harm caused by H₂O₂. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H₂O₂ accumulates excessively, it produces radical-(HO-) through Fe^2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H₂O₂ by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.     

Differential activation of ERK 1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions

The mechanisms by which mitogen-activated protein kinases (MAPK) respond to the input of UV-induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC-irradiated cells. The intracellular oxidative level of normal human fibroblast-like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N-acetyl-L-cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV-irradiation was significantly reduced. Among MAPK, extracellular-signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c-Jun N-terminal kinase (JNK) activation was inhibited in NAC-treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV-induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.     

Mechanism of DNA damage photosensitized by trisbipyrazyl ruthenium complex. Unusual role of Cu/Zn superoxide dismutase

Trisbipyrazyl ruthenium(II) (Ru[bpz]3(2+)) was examined as DNA photosensitizer. Damage resulting from the photolysis of synthetic oligonucleotides has been monitored by polyacrylamide gel electrophoresis. Photoadduct formation is found on both single- and double-stranded oligonucleotides. On oligonucleotide duplex, oxidative damage occurs selectively at the 5'G of the 5'GG3' site and to a lesser extent at the 5'G of a GA sequence. These findings suggest the involvement of electron transfer and show that this mechanism is the main DNA damaging process involved in Ru(bpz)3(2+) photosensitization. In addition, photoadducts and oxidative damage are both highly affected by an increase of salt concentration in the reaction medium, stressing the importance of direct interactions between nucleic acid bases and the excited ruthenium complex for efficient electron transfer. On single-stranded oligonucleotides, all the guanines are oxidized to the same extent. In this case, oxidative damage, which is not affected by an increase of salt in the solution, has been attributed, in part, to singlet oxygen. More importantly, Cu/Zn superoxide dismutase (SOD) strongly enhances the yield of all damage, correlated to an increase of both electron transfer and singlet oxygen production. This original activity of SOD is the first example of bioactivation of a polyazaaromatic ruthenium complex.     

Oxidation of bovine serum albumin: identification of oxidation products and structural modifications

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long‐lived proteins. The metal‐catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off‐line nano‐liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time‐points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time‐course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step‐ladder mechanism. Copyright © 2009 John Wiley & Sons, Ltd.