8-OHdG In DNA Research Articles

Reactive oxygen species, DNA damage and Solar-Earth communications

Reactive oxygen species play a significant role in regulating the main functions of the cell both in the normal conditions and when exposing the cell to exogenous and endogenous factors under electromagnetic and radiation emission at various levels of solar activity. The research studied the impact of low-intensity alternating magnetic field; the intensity level is comparable to the geomagnetic field, in the frequency range from 3 to 60 Hz and γ-radiation on the generation of the most stable ROS which are hydrogen peroxide, in vitro. A degree of oxidative DNA damage in the blood serum of health donors, after being exposed to the alternating magnetic field, was investigated by determining the content of 8- hydroxy-2-deoxyguanosine (8-OHdG) in the blood serum and DNA single breaks in the lymphocytes. An increase of the content of 8-OHdG in DNA by a factor of 1.5 – 2 in comparison with non-emitted samples, and non-linear change in the level of lipid peroxide in the blood serum with maxima at MF frequencies of 8 and 50 Hz. In lymphocytes are observed. When exposed to gamma radiation, the number of single-strand breaks (SSB) of DNA increased with increasing exposure time from 44.3 ± 0.4% (irradiation for 30 minutes) to 68.3 ± 0.8% (irradiation for 90 minutes) compared to the control samples. It is concluded that the obtained effects can be associated with the generation of reactive oxygen species in the aquatic environment with variations in solar activity and affect the adaptive capabilities of the human body as a whole.

ROS-induced Oxidative Injury involved in Pathogenesis of Fungal Keratitis via p38 MAPK Activation

This study was to explore the mechanism by which reactive oxygen species (ROS)-induced oxidative stress involved in the pathogenesis of fungal keratitis using an in vivo experimental keratitis mouse model and an in vitro culture model of human corneal epithelial cells (HCECs). Compared to normal control mice and HCECs, ROS production was markedly increased in fungal corneas and HCECs exposed to Candida albicans, accompanied by p38 mitogen-activated protein kinases (MAPK) activation. Increased products of oxidative markers, malondialdehyde (MDA), 4–hydroxynonenal (HNE), mitochondria DNA 8-OHdG and aconitase-2 were observed in fungal infected corneas and HCECs. Fungal infection also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), with suppressed levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPx1) and peroxiredoxin-4 (PRDX4). Interestingly, the levels of ROS, oxidative markers and oxygenases were significantly reduced by co-cultured p38 inhibitor SB203580. Furthermore, SB203580 restored the levels of antioxidant enzymes suppressed by fungus. Our findings demonstrated for the first time that ROS-induced oxidative injury is involved in pathogenesis of fungal keratitis via p38 MAPK pathway, suggesting the novel therapeutic targets for the potential treatment of fungal keratitis.

What causes human cancer? Approaches from the chemistry of DNA damage.

To prevent human cancers, environmental mutagens must be identified. A common mechanism of carcinogenesis is DNA damage, and thus it is quite possible that environmental mutagens can be trapped as adducts by DNA components. It is also important to identify new types of DNA damaging reactions and clarify their mechanisms. In this paper, I will provide typical examples of our efforts to identify DNA damage by environmental agents, from chemistry-based studies. 1) Oxidative DNA damage: 8-Hydroxydeoxyguanosine (8-OHdG, 8-oxodG) was discovered during a structural study of DNA modifications caused in vitro by heating glucose, which was used as a model of cooked foods. We found that various oxygen radical-forming agents induced the formation of 8-OHdG in DNA, in vitro and in vivo. Analyses of the urinary 8-OHdG levels are useful to assess the extent of oxidative DNA damage in a human population. 2) Lipid peroxide-derived DNA adducts: We searched for mutagens that react with deoxynucleosides, in model systems of lipid peroxidation. The reaction mixtures were analyzed by high performance liquid chromatography (HPLC), and we discovered various lipid peroxide-derived mutagens, including new mutagens. Some of these adducts were detected in human DNA. These mutagens may be involved in lipid peroxide-related cancers. 3) Methylation of cytosine by free radicals: Methylation of the cytosine C-5 position is an important mechanism of carcinogenesis, in addition to gene mutations. However, the actual mechanisms of de novo methylation in relation to environmental agents are not clear. We found that cytosine C-5 methylation occurred by a free radical mechanism. The possible role of this radical-induced DNA methylation in carcinogenesis will be discussed, in relation to the presently accepted concept of cancer epigenetics. In these studies, chemical analyses of the adducts formed in model reactions led to the discoveries of new mutagens and important types of DNA modifications, which seem to be involved in human carcinogenesis.

Number of manic episodes is associated with elevated DNA oxidation in bipolar I disorder

Bipolar disorder (BD) is a major public health problem characterized by progressive functional impairment. A number of clinical variables have been associated with progression of the disease, most notably number of affective episodes and presence of psychotic symptoms, both of which correlate with greater cognitive impairment, lower response rates for lithium, and possibly lower levels of neurotrophic factors. Oxidative damage to cytosine and guanosine (8-OHdG) has been described as a modulator of DNA methylation, but the extent of DNA oxidative damage involvement in BD remains unclear. The aim of this study was to evaluate the extent of DNA oxidative damage to 8-OHdG and 5-methylcytosine (5-HMec), as well as global methylation (5-Mec), in BD patients and healthy controls. Potential association with clinical variables was also investigated. DNA levels of 8-OHdG, 5-HMec and 5-Mec were measured in 50 BD type I patients and 50 healthy controls. DNA 8-OHdG levels were higher in BD patients compared to healthy controls and found to be positively influenced by number of previous manic episodes. BD subjects had lower levels of 5-HMec compared to controls, whereas this measure was not influenced by the clinical features of BD. Number of manic episodes was correlated with higher levels of 8-OHdG, but not of 5-Mec or 5-HMec. Lower demethylation activity (5-HMec) but no difference in global 5-Mec levels was observed in BD. This finding suggests that oxidative damage to 8-OHdG might be a potential marker of disease progression, although further prospective cross-sectional studies to confirm neuroprogression in BD are warranted.

DNA Damage in Human Pleural Mesothelial Cells Induced by Exposure to Carbon Nanotubes

Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

Association of Base Excision Repair Gene Polymorphisms with ESRD Risk in a Chinese Population

The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/106 dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process.

Bio-effects of Nano-TiO 2 on DNA and cellular ultrastructure with different polymorph and size

In this paper, we investigated the bio-effect of three kinds of nano-TiO 2 on the aspects of DNA strand breaking activity, generation of 8-hydroxy-2-deoxyguanosine (8-OHdG), cytotoxicity and cell morphology. Different extents of strand scission and the formation of 8-OHdG in DNA were obtained after treated with different types of nano-TiO 2 (10 – 20 nm anatase, 50 – 60 nm anatase and 50 – 60 nm rutile). Furthermore, MTT assay and TEM detection showed the toxic effects on CHO cells with a toxic order as 10 – 20 nm anatase > 50 – 60 nm anatase > 50 – 60 nm rutile, which was consistent with that of 8-OHdG generation and DNA strand breaking activities. It was interesting that three kind of nano TiO 2 all had much higher toxic effect on tumor cells than on normal cells, and nano-TiO 2 was observed in the cytoplasm of CHO cells. These results indicated that the toxic effect of nano-TiO 2 relates to the size and type of particles, in addition, their potential application for cancer treatment was discussed.

Assessment of Biomarkers in asbestos-exposed workers as indicators of cancer risk

Epidemiological studies have shown that mortality from malignant mesothelioma (MM) and lung cancer have increased with increasing cumulative exposure to asbestos. To investigate whether tumour-related biomarkers can contribute towards the evaluation of the carcinogenic risk in populations exposed to asbestos, the DNA adduct 8-hydroxy-2′-deoxyguanosine (8OHdG), interleukine-6 (IL-6), platelet-derived growth factor (PDGF-BB), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGFβ) and soluble mesothelin-related peptides (SMRPs) were analysed in a cohort of workers differently exposed to asbestos fibres at the workplace. To document biomarker levels in an unexposed population, 54 age-matched subjects were enrolled. A total of 119 subjects with a history of occupational exposure to asbestos underwent clinical examination and were interviewed by trained personnel, responding to a detailed questionnaire related to duration of asbestos exposure, smoking, and occupational task. According to the occupational tasks, asbestos-exposed subjects were analysed for their asbestos cumulative dose and the association with the biomarkers was evaluated. Among the occupational groups, maintenance workers, pipe fitters and electricians were exposed to a higher cumulative dose of asbestos fibres. Exposure to asbestos significantly increased the steady-state content of 8OHdG in DNA. Elevated levels of 8OHdG and IL-6 best reflected a high level of SMRPs, which is related to cell transformation. Subjects heavily exposed to asbestos [>60 (ff/cm 3) × years] showed also a higher level of angiogenic factors. A combination of angiogenic biomarkers with a specific mesothelioma-biomarker such as SMRPs could be used for close surveillance of workers with a history of asbestos exposure.

Extremely low frequency magnetic fields cause oxidative DNA damage in rats

Purpose: To detect the genotoxic effects of extremely low frequency (ELF) -magnetic fields (MF) on oxidative DNA base modifications [8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde)] in rat leucocytes, measured following exposure to ELF-MF.Materials and methods: After exposure to ELF-MF (50 Hz, 100 and 500 μT, for 2 hours/day during 10 months), DNA was extracted, and measurement of DNA lesions was achieved by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS).Results: Levels of FapyAde, FapyGua and 8OHdG in DNA were increased by both 100 μT and 500 μT ELF-MF as compared to a cage-control and a sham group; however, statistical significance was observed only in the group exposed to 100 μT.Conclusion: This is the first study to report that ELF-MF exposure generates oxidatively induced DNA base modifications which are mutagenic in mammalian cells, such as FapyGua, FapyAde and 8-OH-Gua, in vivo. This may explain previous studies showing DNA damage and genomic instability. These findings support the hypothesis that chronic exposure to 50-Hz MF may be potentially genotoxic. However, the intensity of ELF-MF has an important influence on the extent of DNA damage.

Evaluation of oxidative stress in a group of adolescents exposed to a high level of aflatoxin B1 a multi-center and multi-biomarker study

The association between aflatoxin B1 (AFB1) exposure and oxidative stress was extensively examined in 84 adolescents from an area at high risk for hepatocellular carcinoma in China. Plasma level of aflatoxin B1-albumin adducts (AAAs) was associated with AFB1 excretion in urine (r = 0.394, P < 0.001). Urinary AFB1 was also associated with both the urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG) (r > or = 0.479, P < 0.001) and 8-OHdG and hOGG1 levels in peripheral leukocytes (r > or = 0.308, P < or = 0.005). Similarly, AAA was significantly associated with both the urinary excretion of 8-OHdG (r > or = 0.259, P < or = 0.018) and the 8-OHdG and hOGG1 levels in peripheral leukocytes (r > or = 0.313, P < or = 0.004). In addition, urinary 8-OHdG was correlated with both the level of DNA 8-OHdG (r > or = 0.24, P < or = 0.05) and the expression of hOGG1 in peripheral leukocytes (r > or = 0.429, P < 0.001). Protein carbonyl content (PCC) level was significantly associated with not only the level of DNA 8-OHdG (r > or = 0.366, P < 0.001) and the urinary 8-OHdG (r > or = 0.258, P < or = 0.018) but also the expression of hOGG1 in peripheral leukocytes (r = 0.485, P < 0.001). A significant but weak association was found between high-performance liquid chromatograph-electrochemical detection (HPLC-ECD) and enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG (r = 0.334, P = 0.002) and between HPLC-ECD and flow cytometry assays for 8-OHdG in leucocytes (r = 0.395, P < 0.001). Significant associations were observed between AAA and PCC and liver function indices (alanine aminotransferase and aspartate aminotransferase). These findings suggest significant contribution from AFB1 exposure to oxidative stress and subsequent repair among adolescents that may impose substantial risk for hepatocarcinogenesis in adulthood in this region.

Increased Levels of 8-Hydroxy-2′-Deoxyguanosine in Drosophila Larval DNA after Irradiation with 364-nm Laser Light but not with X-rays

Exposure to UVA light causes damage to cellular components such as DNA and membrane lipids. We showed previously that UVA irradiation can induce mutations in Drosophila larvae and that the major lesions responsible for mutations were not thymidine dimers when wavelengths tested became longer. The use of a longer wavelength with UVA laser apparatus (364 nm) has made it possible to test the effects of this powerful light in biological organisms. In the present study, we irradiated third instar larvae of the urate-null Drosophila mutant strain y v ma-l, which is sensitive to oxidative stress, and compared the effects of 364 nm light irradiation with the effects of X-rays. To assay viability, some of the larvae were kept at 25 degrees C until they eclosed in order to obtain a measure of viability. The remaining larvae were used to measure the amount of 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative DNA damage. The amount of 8-OHdG increased and viability decreased in response to increased UV dose in both the y v ma-l and wild-type strains. With irradiation of 600 kJ m(-2), 8-OHdG/10(6)dG was 7.2 +/- 3.2 and 6.2 +/- 2.0 in y v ma-l and wild-type strains, respectively, whereas the respective levels were 2.2 +/- 0.6 and 2.3 +/- 0.8 without irradiation. Our results indicated that irradiation with a 364-nm laser light caused significant oxidative damage in Drosophila larval DNA; however, induction of the damage was not prohibited by urate. To the best of our knowledge, this is the first report of a study in whole animals that shows increased levels of 8-OHdG in response to 364-nm UVA. X-ray ionizing radiation is also thought to generate reactive oxygen species in irradiated cells. We found that the amount of 8-OHdG in DNA following X-ray radiation remained unchanged in both strains, though survival rates were affected. X-ray-generated oxidative damage in Drosophila cells was followed by cell death but not DNA base oxidation, and the damage was suppressed by urate. The overall results suggest significant differences in the major in vivo oxidative damage caused by 364-nm light and X-rays.

Contrasting Genome-Wide Distribution of 8-Hydroxyguanine and Acrolein-Modified Adenine during Oxidative Stress-Induced Renal Carcinogenesis

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

4-Nitroquinoline 1-Oxide Forms 8-Hydroxydeoxyguanosine in Human Fibroblasts through Reactive Oxygen Species

4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which forms 8-hydroxydeoxyguanosine (8OHdG), oxidative damage. To determine whether reactive oxygen species (ROS) are involved in the generation of 8OHdG by 4NQO, we used high-performance liquid chromatography and immunohistochemistry to measure the levels of 8OHdG in normal human fibroblasts treated with 4NQO. The extent of ROS induced by 4NQO was determined by using fluorescent probes to detect ROS, electron paramagnetic resonance spectrometry using a cell-free system, and measurement of intracellular glutathione (GSH) levels. In fibroblasts, 4NQO dose dependently increased 8OHdG levels. Hydrogen peroxide (H2O2) and superoxide were detected in cells treated with 4NQO by using dichlorofluorescin diacetate and hydroethidine, respectively. The addition of catalase to culture medium reduced 8OHdG levels and the intensity of dichlorofluorescin fluorescence, while 4NQO generated hydroxyl radicals in the cell-free system. These findings suggest that 4NQO treatment leads to formation of superoxide, H2O2, and hydroxyl radicals, resulting in the production of a substantial amount of 8OHdG in DNA. Neither the level of 8OHdG nor that of GSH had returned to the basal level 24 h after removal of 4NQO even at a concentration as low as 1 microM. Our results suggest that generation of ROS and depletion of GSH in cells are also important factors for the generation of 8OHdG by 4NQO. This paper describes practical and sensitive ways to detect ROS and 8OHdG and discusses a new functional pathway to elicit genotoxicity.

Evaluation of Oxidative Damage Induced by Natural Sunlight in Drosophila

The sunlight that reaches the surface of the earth is composed of polychromatic light with wavelengths ranging from 290 nm to 2500 nm. Ultraviolet (UV) light is a component of sunlight that is harmful to organisms. Although it is known that sunlight induces photoproducts and harmful effects such as major DNA damage caused by UV light in the tissues of many species including human skin, it is not clear whether sunlight induces oxidative damage in organisms. In this study, we investigated whether sunlight causes oxidative damage in exposed organisms using Drosophila. Third instar larvae were exposed to sunlight for an evaluation of viability and 8-hydroxydeoxyguanosine (8-OHdG) content. Urate-null Drosophila mutant larvae (ry506), which are sensitive to active oxygen-producing agents, were more sensitive to sunlight lethality than the wild-type strain under limited conditions. This sunlight-induced toxicity in ry506 larvae was partially prevented by pretreatment of larvae with 400 mM uric acid. The level of 8-OHdG in DNA showed no significant increase in both strains. In contrast, sunlight was significantly mutagenic for all seasons as reported previously. These results suggest that sunlight is partly responsible for oxidative damage in Drosophila and that 8-OHdG-formation plays little or no role in sunlight-induced mutation and toxicity.

Increased oxidative DNA damage, 8-hydroxydeoxy- guanosine, in human pterygium

Chronic exposure to ultraviolet (UV) light is a widely accepted aetiological factor in the development of pterygium. UV radiation may induce production of reactive oxygen species via photosensitized oxidation, thus causing oxidative damage. This study was conducted to test the hypothesis that oxidative damage to DNA is increased in pterygium. Immunohistochemical analysis employing a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), a ubiquitous maker of oxidative stress, was performed in three patients with primary pterygium. The levels of 8-OHdG in DNA isolated from the other 29 pterygium specimens and their adjacent normal conjunctival tissues were determined using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry of 8-OHdG showed a distinct pattern of more extensive and intense staining in the nuclei of pterygium tissue compared with that in their adjacent normal conjunctiva. ELISA also revealed that the average level of 8-OHdG in the pterygium tissues was 4.7-fold higher than that of the corresponding normal conjunctiva (P<0.001). The increased levels of 8-OHdG in the pterygium tissues indicate that oxidative stress could play a role in the development of pterygium. These findings provide new information to better understand the pathogenesis of pterygium and are useful in the prevention and treatment of this disease.

Generation of 8‐hydroxydeoxyguanosine from DNA using rat liver homogenates

In relation to carcinogenesis, aging and other pathologic conditions, urinary 8-hydroxydeoxyguanosine (8OHdG) is widely used as a marker for evaluating the effect of oxidative stress on DNA. Because no reports have described how 8OHdG is generated from DNA in vivo or by biological materials, and how it is excreted into urine, the authors investigated the generation of 8OHdG from DNA, using rat liver homogenate. Oxidatively damaged DNA samples containing different levels of 8OHdG were prepared using ultraviolet irradiation with three different concentrations of riboflavin. Following incubation of damaged DNA samples with rat liver homogenates, the generation of 8OHdG from the DNA was determined using high-performance liquid chromatography with electrochemical detection after ultrafiltration of the incubation mixtures. The generation of 8OHdG was also tested with an anti-8OHdG antibody. The quantity of 8OHdG generated from the DNA by rat liver homogenates was dependent on the 8OHdG levels in the DNA: almost all 8OHdG in the DNA was released as 8OHdG by rat liver homogenates. Generation of 8OHdG correlated with the degradation of DNA. Interestingly, the generated 8OHdG was stable in the presence of rat liver homogenates, whereas deoxyguanosine (dG) rapidly disappeared in the same conditions. Less than 1/10,000 of dG was converted to 8OHdG when dG was incubated with rat liver homogenate. Incubation of 8-hydroxyguanine with rat liver homogenates did not generate 8OHdG. These findings suggest that most of the 8OHdG in DNA is released as 8OHdG during DNA degradation and that, because of its stability, 8OHdG is excreted into urine, thus providing a convenient measure of oxidative damage to DNA.

Ascorbate as a “redox sensor” and protector against irradiation-induced oxidative stress in 32D CL 3 hematopoietic cells and subclones overexpressing human manganese superoxide dismutase

PurposeTo determine whether increased expression of manganese superoxide dismutase (MnSOD) protects cells from irradiation by preventing the production of reactive oxygen species (ROS), a new approach to detecting free radical intermediates using ascorbate as an endogenous spin trap was used. Materials and methodsCells from the 32D cl 3 hematopoietic cell line or a subclone overexpressing MnSOD (2C6) were incubated with dehydroascorbate for 30 min and irradiated to doses from 0 to 50 Gy. Radical intermediates reacting with spin traps or ascorbate were measured by electron spin resonance spectroscopy. Results were compared to irradiation-induced changes in thiol levels, irradiation survival curves, and accumulation of 8-OHdG as a measurement of DNA oxidative damage. ResultsManganese superoxide dismutase-overexpressing 2C6 cells maintained higher levels of ascorbate (5.4 ± 0.5 and 2.6 ± 0.5 nmol/106 cells, respectively) and thiols (14.0 ± 0.1 and 11.1 ± 0.2 nmol/106 cells) compared to 32D cl 3 parent cells. Cells overexpressing MnSOD produced lower levels of ROS than did the parental 32D cl 3 cells, as evidenced by lower expenditure of ascorbate and GSH after irradiation. Increased ascorbate levels protected both 32D cl 3 and 2C6 cells from irradiation killing, as demonstrated by an increased shoulder on survival curves and decreased DNA 8-OHdG accumulation. ConclusionsManganese superoxide dismutase overexpression protects 2C6 cells from irradiation damage by scavenging ROS that readily interact with major endogenous antioxidants—ascorbate and GSH—in nontransfected hematopoietic 32D cl 3 cells.

Increased Formation of 8-Hydroxy-2’-Deoxyguanosine in Peripheral Blood Leukocytes in Bladder Cancer

Introduction: Reactive oxygen species-induced damage to DNA plays a major role in carcinogenesis. Methods: In order to estimate the level of oxidative damage in bladder cancer, 8-hydroxy-2′-deoxyguanosine (8-OHdG) was determined in DNA isolated from peripheral blood leukocytes of healthy adults and patients with superficial transitional cell carcinoma. Patients with transitional cell carcinoma of the bladder and control individuals were similar in age. In this study, the level of 8-OHdG in DNA in male subjects was measured by the high-performance liquid chromatography-electrochemical detector method. Results: The 8-OHdG levels in DNA from leukocytes of bladder cancer patients were significantly higher than those in controls. Conclusion: Reduction of oxidative stress is thought to be a very important measure for primary prevention of bladder cancer.

Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease.

A large body of experimental evidence suggests that in Alzheimer disease (AD) pathogenesis an important role is played by oxidative stress, but there is still a lack of data on in vivo markers of free radical-induced damage. To evaluate levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative damage to DNA, in peripheral lymphocytes; to measure plasma concentrations of several nonenzymatic antioxidants; and to assess the relationships between any observed changes in lymphocyte DNA 8-OHdG content and plasma antioxidant levels in patients with AD and healthy aged control subjects. Forty elderly outpatients with AD and 39 healthy age- and sex-matched controls were studied. The level of 8-OHdG was determined in DNA extracted from lymphocytes and plasma levels of vitamin C, vitamin A, vitamin E, and carotenoids (zeaxanthin, beta-cryptoxanthin, lycopene, lutein, and alpha- and beta-carotene) were measured by high-performance liquid chromatography. Lymphocyte DNA 8-OHdG content was significantly higher and plasma levels of antioxidants (with the exception of lutein) were significantly lower in patients with AD compared with controls. In patients with AD, a significant inverse relationship between lymphocyte DNA 8-OHdG content and plasma levels of lycopene, lutein, alpha-carotene, and beta-carotene, respectively, was observed. Markers of oxidative damage are increased in AD and correlate with decreased levels of plasma antioxidants. These findings suggest that lymphocyte DNA 8-OHdG content in patients with AD reflects a condition of increased oxidative stress related to a poor antioxidant status.

8-Hydroxydeoxyguanosine in leukocyte DNA and urine of quartz-exposed workers and patients with silicosis.

To examine radical-induced DNA damage and its elimination in workers exposed to quartz and in patients with silicosis, and to assess the relationship of these effects to lung function. Blood and spontaneous urine samples were obtained from active, quartz-exposed workers without silicosis (n = 63), and from retired workers with silicosis (n = 42). Levels of 8-hydroxydeoxyguanosine (8-OHdG) were determined in peripheral blood leukocyte DNA and urine, by the use of high-performance liquid chromatography coupled with ultra violet- (UV) and electrochemical detection. No significant differences in the mean levels of 8-OHdG in leukocyte DNA and of urinary excretion of 8-OHdG were found between silicosis patients and quartz-exposed healthy workers. However, in the group of silicosis patients with increased oxidative DNA damage the urinary excretion of 8-OHdG was lower than in the corresponding group of active workers without silicosis. In the case of silicosis, urinary 8-OHdG correlated positively, and 8-OHdG in DNA correlated negatively, with forced expiratory volume in one second (FEV1) and forced vital capacity (FVC). Healthy workers with a personally estimated high dust exposure in the workplace showed higher levels of 8-OHdG in DNA than did workers with moderate dust exposure. No association of 8-OHdG formation and/or elimination with duration of employment, field of activity, smoking or age was found. Our findings suggest that a less effective repair of 8-OHdG is associated with a higher degree of pulmonary airway obstruction in patients with silicosis.