Ascorbate 6-palmitate protects human erythrocytes from oxidative damage

It is well established that the process of thymocyte differentiation and maturation occurs in the thymus, where cell-to-cell communication is essential for providing the messages to T-cell precursors. At least two pathways are important for such communication: one via membrane surface molecules and the other via soluble mediators such as cytokines and some hormones. Recently, the presence of receptors for extracellular ATP has been demonstrated on thymocytes and microenvironment cells, and putative functions for this molecule have been proposed. Herein we focus on the recent evidence which supports the view of extracellular ATP and some related nucleotides as novel intrathymic signal molecules. In addition, we discuss the possible physiological implications of such purinergic receptors for the physiology of the thymus.     

Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells

To clarify the mechanisms of intracellular induction of oxidative DNA damage, we have investigated the concentrations of intracellular reactive oxygen species and the amounts of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic oxidative DNA damage, in human neutrophil-like cells, dimethylsulfoxide-differentiated HL60 (DMSO-HL60). We determined intracellular concentrations of hydrogen peroxide and superoxide by flow cytometry with dichlorofluorescein diacetate and hydroethidine, respectively. We determined the 8OHdG amounts with an electrochemical detector connected to HPLC after anaerobic sample processing. DMSO-HL60 releases superoxide upon stimulation with phorbol myristate acetate, and the released superoxide dismutates to hydrogen peroxide. Stimulation of DMSO-HL60 with 100 nM phorbol myristate acetate increased intracellular hydrogen peroxide, superoxide and 8OHdG (control). Addition of 1000 U/ml catalase decreased hydrogen peroxide (31.3% of control) and 8OHdG (20.3%). Addition of 100 U/ml SOD decreased superoxide (18.7%) and 8OHdG (41.6%). Addition of 1 mM deferoxamine decreased 8OHdG (30.4%), but increased hydrogen peroxide (129.6%). Addition of 200 microM 4-acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid decreased superoxide (59.9%) and 8OHdG (42.0%). Addition of 0.4% ethanol had no effect on superoxide concentration (102.2%), but tended to decrease hydrogen peroxide (83.5%) and 8OHdG (84.3%). Pretreatment of DMSO-HL60 with 0.1 mM FeSO4 increased 8OHdG (117.3%), but decreased hydrogen peroxide (75.8%). These findings indicate that the extracellularly released superoxide and hydrogen peroxide diffuse into the cell, but that such reactive oxygen species are not the direct molecules to induce 8OHdG. Our results suggest that 8OHdG is induced by the hydroxyl radical which is generated from intracellular hydrogen peroxide and superoxide-reduced Fe.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

DNA oxidative damage and life expectancy in houseflies

The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence.     

Serum carotenoids and oxidative DNA damage in human lymphocytes

Carotenoids are thought to act as antioxidants in vivo, decreasing oxidative damage to biomolecules and thus protecting against coronary heart disease and cancer. However, human intervention studies with beta-carotene have given equivocal results in terms of cancer incidence. In an alternative molecular epidemiological approach, we have employed the 'comet assay' (single cell alkaline gel electrophoresis) to measure strand breaks, oxidized pyrimidines and altered purines in the DNA of lymphocytes from volunteers supplemented with alpha/beta-carotene, lutein, lycopene or placebo. In addition, we measured concentrations of the main serum carotenoids, and vitamins E and C, by HPLC. We report a significant negative correlation between basal concentrations of total serum carotenoids and oxidized pyrimidines. A similar correlation was seen between individual carotenoids (notably lutein and beta-carotene) and oxidized pyrimidines. However, carotenoid supplementation did not have a significant effect on endogenous oxidative damage. This suggests that there are some factors in the basal diet, probably found in fruit and vegetables, that decrease oxidative damage to DNA. In this case, basal serum carotenoids may simply be markers of consumption of fruit and vegetables, they themselves having little or no protective value.     

Involvement of reactive oxygen species in kidney damage

There is considerable evidence suggesting that reactive oxygen species (ROS) are implicated in the pathogenesis of ischemic, toxic, and immunologically-mediated renal injury. In experimental renal ischemia, ROS sources include the electron transport chain, oxidant enzymes (xanthine oxidase), phagocytes, and auto-oxidation of epinephrine. ROS cause lipid peroxidation of cell and organelle membranes and, hence, disruption of the structural integrity and capacity for cell transport and energy production, especially in the proximal tubule segment. In experimental immune glomerulonephritis, ROS are generated by both infiltrating blood-borne cells (polymorphonuclear leukocytes and monocytes) and resident glomerular cells, mainly mesangial cells. Their formation results in morphologic lesions and in modifications of glomerular permeability to proteins through activation of proteases and reduction of proteoglycan synthesis. Additionally, they promote a reduction in glomerular blood flow and glomerular filtration rate through liberation of vasoconstrictory bioactive lipids (prostaglandins, thromboxane, and platelet activating factor) and, possibly, inactivation of relaxing nitric oxide. Further studies are needed to address the role of ROS in human glomerular diseases.     

Detection of active oxygen species in biological systems

1. Cypridina luciferin analogues, 2-methyl-6-(p-methoxyphenyl)-3,7- dihydroimidazo[1,2-a]pyrazin-3-one (MCLD) and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one(CLA ), react with O2- or 1O2 to emit light in visible region. Such chemiluminescences were used for the detection of O2- or 1O2 in activated leukocyte systems and myeloperoxidase (granulocyte-extract) + Br- + H2O2 systems in vitro. 2. The mechanisms of MCLA (CLA)-dependent luminescence is described in detail. Superoxide generated from sinusoidal cells in acute ethanol intoxication of rats was detected by MCLA-dependent luminescence from the surface of perfused rat liver (organ luminescence). 3. Furthermore, with alive animals, O2- generated in the lung of rats with necrotized pancreatitis and that in the stomach of rats after ischemia/reperfusion were detected by their organ luminescences.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC-->AT transitions and GC-->TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to assess the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Artificial background and induced levels of oxidative base damage in DNA from human cells

The pre-mutagenic oxidative DNA base damage of 8-hydroxy-guanine is present in DNA isolated from cells and the amount present increases with exposure of cells to oxidative stress. The oxidative DNA base damage may be present before isolation of DNA or it may be produced during isolation and processing of DNA. We have found that the amount of oxidative base damage measured in DNA can be reduced to a stable lower level by adding increasing concentrations of the antioxidants desferrioxamine, histidine and reduced glutathione immediately before cell lysis. Inclusion of these antioxidants after cell lysis did not affect the level of DNA damage. Oxidative DNA base damage produced by ultraviolet A irradiation of human cells was also reduced by adding antioxidants after irradiation and before cell lysis. Thus, unidentified oxidants induced by ultraviolet A irradiation may damage DNA significantly during extractions of DNA from cells subsequent to ultraviolet A irradiation.     

Comparison of oxidative base damage in mitochondrial and nuclear DNA

In 1994-1995, a child and five dogs from villages located between Jerusalem and Tel-Aviv, Israel were diagnosed with visceral leishmaniasis (VL). Based on these findings, the distribution of VL in domestic and wild canids in central Israel was examined. In the two villages where canine index cases were identified, a substantial proportion (11.5%, 14 of 122) of the dogs examined were seropositive. However, the rate of infection in five neighboring villages was only 1% (1 of 99). Parasites were cultured from 92% (12 of 13) of the seropositive dogs biopsied and the strains were characterized as Leishmania infantum by a clamped polymorphic-polymerase chain reaction, monoclonal antibodies, and/or excreted factor serology. The discovery of VL close to major urban centers is an important public health issue. The disease appears to have emerged recently in this area, and it is unclear whether the parasite was re-introduced or was continuously present at low levels in this region. The presence of seropositive wild canids, jackals (7.6%, 4 of 53) and red foxes (5%, 1 of 20), in central Israel, and the reappearance of the jackal population after near extinction suggests that wild canids may play a role in spreading this disease.     

Action of phenolic antioxidants on various active oxygen species

The action of phenolic antioxidants, such as probucol, on various active oxygen species was investigated using luminol chemiluminescence and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The various active oxygen species, including hydroxyl radicals (Fenton reaction), superoxide anions, singlet oxygen and hypochlorite ions were examined with phenolic antioxidants under aqueous and nonaqueous conditions. Probucol showed a quenching effect on both superoxide anions and hypochlorite ions in nonaqueous solution. However, it had no effect on hydroxyl radicals. alpha-Tocopherol, a natural phenolic antioxidant, showed a stronger quenching effect on superoxide anions and hypochlorite ions than probucol, and quenched hydroxyl radicals in nonaqueous solution. Furthermore, Trolox showed a quenching effect on all active oxygen species in both aqueous and nonaqueous solution. The antioxidants were studied under comparable conditions in a series of test systems and the reactivity profiles depicted as 'radar charts' which are helpful for characterizing antioxidant action.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.     

The amyloid beta protein induces oxidative damage of mitochondrial DNA

A 55-year-old man was admitted to our hospital with of hemoptysis, progression of anemia and renal failure in February, 1996. Idiopathic interstitial pneumonia had been diagnosed and he had been followed at a regional hospital since 1988. On the third day after admission, he suffered from sudden and massive hemoptysis. Goodpasture's syndrome was diagnosed because anti-GBM antibody was detected in serum. A high titer of MPO-ANCA was also recognized simultaneously. Steroid pulse therapy, immunosuppressive therapy, and plasmapheresis were begun, but he died on the 28th hospital day because of severe hypoxemia and multi-organ failure. Histological examination after autopsy revealed crescentic glomerulonephritis with linear deposition of IgG in the glomerular capillary wall, and interstitial pneumonia accompanied by massive alveolar hemorrhage. It was suggested that in this patient, not only anti-GBM antibody but also circulating MPO-ANCA might have participated in the progression of the crescentic glomerulonephritis and alveolar hemorrhage observed in Goodpasture's syndrome.     

Effects of smoking and aging on oxidative DNA damage of human lymphocytes

The effects of H2O2-induced oxidative DNA damage in 80 healthy individuals with relation to age (20-25 and 55-60 years old) and smoking has been investigated with the comet assay technique. Both factors have shown a significant effect upon basal DNA damage with smoking appearing to have the most impact. A differentiation of the four groups response to induced oxidative damage was also observed. A distinctly separate behavior of the younger non-smokers group, when compared with the rest of the categories, was found. This is attributed to the lower degree of initial basal damage that occurs in their lymphocytes.     

Protective activity of propofol, Diprivan and intralipid against active oxygen species

The purpose of this study was to assess the importance of stereolithographic models (SLMs) for preoperative diagnosis and planning in craniofacial surgery and to examine whether these models offer valuable additional information as compared to normal CT scans and 3D CT images. Craniofacial SLMs of 20 patients with craniomaxillofacial pathology were made. A helical volume CT scan of the anatomic area involved delivered the necessary data for their construction. These were built with an SLA 250 stereolithography apparatus (3D-Systems, Valencia, CA, USA), steered by FORM-IT/DCS software (University of Zurich, Switzerland). The stereolithography models were classified according to pathology, type of surgery and their relevance for surgical planning. Though not objectively measurable, it was beyond doubt that relevant additional information for the surgeon was obtained in cases of hypertelorism, severe asymmetries of the neuro- and viscerocranium, complex cranial synostoses and large skull defects. The value of these models as realistic "duplicates" of complex or rare dysmorphic craniofacial pathology for the purpose of creating a didactic collection should also be emphasized. The models proved to be less useful in cases of consolidated fractures of the periorbital and naso-ethmoidal complex, except where there was major dislocation.     

Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells

In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.