Inhibition Of Oxidative DNA Damage Research Articles

Aconitine induces autophagy via activating oxidative DNA damage-mediated AMPK/ULK1 signaling pathway in H9c2 cells

Ethnopharmacological relevanceAconitum species, with a medicinal history of 2000 years, was traditionally used in the treatment of rheumatism, arthritis, bruises, and pains. However, many studies have reported that Aconitum species can cause arrhythmia in experimental animals, resulting in myocardial fibrosis and cardiomyocyte damage. Cardiotoxicity is the main toxic effect of aconitine, but the detailed mechanism remains unclear. Aim of the studyThis study aimed to explore the effects and underlying mechanism of autophagy in H9c2 cardiomyocytes induced by aconitine. Materials and methodsH9c2 cells were incubated with different concentrations of aconitine for 24 h, and the intervention sections were pretreated with various inhibitors for 1 h. The effects of aconitine on the oxidative DNA damage, autophagy and viability of H9c2 cells were evaluated by flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and Western blot. ResultsIn H9c2 cells, the cell viability declined, LDH release rate, the number of autophagosomes, protein expression levels of LC3 and Beclin-1 increased significantly after 24 h of aconitine incubation. The pretreatment of autophagy inhibitor 3-MA decreased markedly autophagosomes and protein expression levels of LC3 and Beclin-1, which suggested that aconitine could induce cell autophagy. The significant increase of ROS and 8-OHdG showed that aconitine could cause oxidative DNA damage through ROS accumulation. Meanwhile, treatment of aconitine dramatically increased AMPKThr172 and ULK1Ser317 phosphorylation, and Compound C inhibited AMPKThr172 and ULK1Ser317 phosphorylation, which proved that aconitine induced autophagy via AMPK activation mediated ULK1 phosphorylation. Antioxidant NAC significantly reduced LDH, ROS and 8-OHdG, inhibited the phosphorylation of AMPKThr172 and ULK1Ser317, and down-regulated autophagosomes and proteins expression levels of LC3 and Beclin-1. Consequently, the inhibition of oxidative DNA damage and AMPK/ULK1 signaling pathway alleviated the aconitine-induced autophagic death of H9c2 cells. ConclusionsThese results showed that aconitine induces autophagy of H9c2 cardiomyocytes by activating AMPK/ULK1 signaling pathway mediated by oxidative DNA damage. The autophagy induced by aconitine in cardiomyocytes is dependent on the activation of the AMPK pathway, which may provide novel insights into the prevention of aconitine-related toxicity.

Inhibition of Oxidative DNA Damage in the Presence of Spinach Extract

Oxidative damage can induce DNA protein cross‐linking. The present study aimed to evaluate whether aqueous extracts of spinach can inhibit oxidative cross‐linking between DNA and protein. In the flash‐quench method, the intercalator Ru(phen)2dppz2+ (phen = phenanthroline, dppz = dipyridophenazine) is excited with blue light, releasing an electron to the quencher, Co(NH3)5Cl2+. The oxidized intercalator then extracts an electron from guanine and the guanine radical reacts to produce DNA‐protein cross‐links. Samples containing Co(NH3)5Cl2+, histone, Ru(phen)2dppz2+, and DNA were subjected to flash quench treatment in the absence or presence of a spinach extract. Cross‐linking was then detected in two ways—chloroform extraction assay and gel shift assays. With calf thymus DNA, the chloroform extraction assay was used, and this UV spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to crosslinking visible in a spectra and a plot of % crosslinked vs time. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross‐linked material visible within the wells. In the presence of an aqueous extract of spinach, cross‐linking is strongly inhibited and the DNA runs as a single band. This inhibition is maintained out to significant dilutions of the green extract. More recently, we have examined kaempferol, one antioxidant found in spinach, and have observed protection of DNA via a nicking assay. A single component of spinach would not be a universal protector against oxidative stress‐induced disease. Lipophilic components are also abundant, which is why two of them, vitamin K and β‐Carotene, are currently being examined.

Inhibition of Oxidative DNA‐protein Crosslinking Via an Aqueous Extract of Kale

Oxidative damage is involved in the formation of free radicals, and if deregulated, can ultimately facilitate a variety of diseases ranging from Alzheimer’s disease to cancer. DNA‐protein crosslinking is a recognized form of oxidative stress that involves proteins interacting with radical‐induced lesions in the DNA molecule. In DNA, this damage is observed primarily at guanine (G) since it is the most easily oxidized base. Studies on oxidative stress show that substances – the most familiar being kale and spinach – high on antioxidants aid in protection from the excessive oxidizing effects of free radical species. The exact mechanism of inhibition has been hypothesized as a redox mechanism between the antioxidant and the radical. Therefore, the primary counteraction used to observe this mechanism and to reverse oxidative DNA‐protein crosslinking in this experiment was kale.The flash‐quench technique is a method used for guanine oxidation and can induce DNA‐protein crosslinking. The intercalator, Ru(phen)2dppz2+[phen = phenanthroline, dppz = dipyridophenazine], is excited with a laser and gives an electron to the quencher, Co(NH3)5Cl2+. The intercalator then takes an electron from guanine, creating the guanine radical, which then reacts with protein. Samples containing Ru(phen)2dppz2+, Co(NH3)5Cl2+, histone protein, PUC19 DNA and kale were irradiated with blue laser light for 0 to 140 seconds to induce guanine damage. The extent of crosslinking was determined by the gel shift assay where SDS was added to the samples to disrupt the noncovalent interactions between the DNA and protein. Furthermore, the samples were compared with the control which substituted water for the kale extract. The inhibition of oxidative DNA damage by kale was studied further by focusing on kaempferol, a component of kale. Likewise, the gel shift assay was performed on samples containing water and two different concentrations – 50 μM and 200 μM – of the antioxidant. The interaction between kaempferol and the guanine radical was then displayed through transient absorption spectroscopy. Since kaempferol holds known antioxidative properties, the inhibition of DNA oxidation at the guanine site was also expected. Indeed, upon comparison, minimal DNA protein crosslinking was observed when kale or kaempferol were present in the samples. Specifically, as the irradiation time increased, the absorption of free DNA was low, assuming that a redox mechanism between the antioxidant and the guanine radical occurred.Future directions include determining the lowest concentration at which kaempferol is active, assessing the effects of additional antioxidants from kale, and examining a pure sample of kaempferol via high performance liquid chromatography (HPLC).Support or Funding InformationSupported and Funded by:Loring‐Denault Endowed Chair in Chemistry, Dr. Eleanor Siebert, and Professor Emeritus

Total flavonoids of Epimedium reduce ageing-related oxidative DNA damage in testis of rats via p53-dependent pathway.

Increased DNA damage in testis is considered as a major factor for ageing-related dysfunction. Total flavonoids of Epimedium (TFE), the main active compositions of Epimedium, have been used to treat sexual dysfunction and delay ageing. However, whether TFE could improve ageing-related testicular dysfunction remains unknown. Therefore, we investigated the protection effects of TFE and its mechanisms of action in a naturally ageing rat model. Eighteen-month-old SD rats were randomised to receive either vehicle or TFE (10 and 20mg/kg). Nine-month-old SD rats were used as adult controls. Morphology, protein expression and immunohistochemistry were determined. Compared with adult control group, intragastric administration of TFE for 6months significantly improved testicular morphology, increased the activities of SOD and decreased the levels of MDA of testis. In addition, TFE decreased γH2AX expression levels and γH2AX focal formation in spermatogonia and primary spermatocyte with concomitant downregulation of 8-OHdG levels. Furthermore, TFE inhibited p-P53/p21 and chk1/chk2 expression levels. Collectively, TFE effectively reduce oxidative DNA damage in testis of ageing rats via a p53-dependent pathway. Thus, inhibition of oxidative DNA damage is likely to represent a promising strategy for restoration of ageing-related testicular dysfunction.

Antioxidative Activities and Inhibition Effects on Oxidative DNA Damage of Valeriana fauriei

Background: Valeriana fauriei (Valerianaceae) has been used to as a traditional medicine to treat a variety of symptoms, including headache, insomnia, hypertension, and menstrual irregularity. However, the present study investigates the species' antioxidant activity and its inhibition of oxidative DNA damage, which have yet to be studied. Methods and Results: The antioxidant activity was assessed using radical scavenging assays with 1,1-diphenyl-2-picryl hydrazyl (DPPH) and, 2, 2'-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid) diammonium salt (ABTS) and a reducing power assay. The total phenol content was also analyzed, and phenolic compounds were detected using HPLC/UV, whereas the inhibitory effect of Valeriana fauriei on oxidative DNA damage was measured using φ-174 RF I plasmid DNA cleavage assay. The DPPH and ABTS radical scavenging activity were 75.17 ± 3.55% and 95.83 ± 0.63%, repectively, and the reducing power was 93.14 ± 1.74 at 200 ㎍/㎖. The total phenol content was 10.24 ± 0.04㎎/g, whereas chlorogenic acid, catechin, caffeic acid and epicatechin were identified using HPLC/UV, and the φ-174 RF I plasmid DNA cleavage assay indicated that V. fauriei provided protection against oxidative damage. Conclusions: The results of the present study suggest that V. fauriei has powerful antioxidant activity that can provide protective effects against the oxidative DNA damage caused by free radicals. The species, therefore, provides a valuable resource for the development of natural pharmaceutical to treat aging, cancer, and degenerative diseases.

Inhibition of radical-induced oxidative DNA damage by antioxidants loaded in electrospun polylactide nanofibers

Polylactide (PLA) nanofibers loaded with antioxidants, i.e. vitamin B6 or pyridoxine (PN) and its analogue pyridoxal (PL), and with hydroxycinnamic acids, i.e. p-coumaric acid (CUM) and caffeic acid (CAF), were prepared by the electrospinning technique. The control consisted of nanofibers loaded with trolox (TRX). Experimentally, these new materials were tested on the inhibition of oxidative DNA damage caused by free radicals initiated by 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH). This damage was assessed in vitro and in vivo by measuring the conversion of supercoiled pUC19 plasmid DNA to open circular and linear forms. It was found that these antioxidants, in solution, could significantly inhibit oxidative DNA damage, and that the antioxidants loaded in the PLA nanofibers maintained in vitro and in vivo their protective role against oxidative DNA damage. Thus, electrospun mats derived from PLA nanofibers loaded with the considered compounds have the capacity to protect DNA against oxidative damage, and appear interesting, for their use in the purification of plasmidic or genomic DNA.

Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer

BackgroundEstrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.MethodsFemale ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2.ResultsThe expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage.ConclusionsOur studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.

Combined effect of cadmium, lead, and UV rays on Bacillus cereus using comet assay and oxidative stress parameters

Exposure to environmental chemicals and oxidative stress particularly at low dose levels may produce additive or synergistic interactions not seen in single component exposure. Exposure to cadmium, lead, and ultraviolet rays occurs in many occupational settings, such as pigment and battery production, galvanization, and recycling of electric tools. However, little is known about interactions between heavy metals and ultraviolet rays. This study aimed to evaluate the interactions of ultraviolet rays of 254 nm (UV-B) with cadmium or lead on Bacillus cereus. B. cereus was treated with different concentrations of cadmium or lead followed by exposure to UV-B radiation as combined effect. Photoirradiation of B. cereus with UV-B with exposure to cadmium or lead results in DNA damage, cytotoxicity, depletion of glutathione, and formation of lipid peroxidation. UV-B rays alone enhanced glutathione production which was depleted with lead and high doses of cadmium. Lead alone does not increase DNA breaking. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or lead. Lipid peroxidation was increased with exposure to UV-B and cadmium or lead. DNA, glutathione, and lipid peroxidation can be used as biomarkers to identify possible environmental contamination in bacteria. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium or lead and UV rays.

Synthesis of novel carbazole chalcones as radical scavenger, antimicrobial and cancer chemopreventive agents

A series of novel carbazole chalcones has been synthesised and evaluated for radical scavenging activity, cytotoxicity and antimicrobial activities. Compounds 12m, 12o and 12c exhibited good 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, compounds 12e, 12m and 12d were excellent hydroxyl radical scavengers and compounds 12a, 12e, 12g, 12n and 12m have shown inhibition of oxidative DNA damage induced by 2,2′-azobis (2-amidinopropane hydrochloride). Compounds 12j, 12i, 12n, 12c, 12m and 12e were most active against the selected cancer cell lines. Compounds 12a, 12e and 12m showed good antibacterial activity and compounds 12h and 12m have shown good antifungal activity. All the compounds were subjected for absorption, distribution, metabolism and excretion (ADME) predictions by computational method and found that these molecules could be considered as potential candidates for oral drug development.

Chemoprevention of rat mammary carcinogenesis by Azadirachta indica leaf fractions: Modulation of hormone status, xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation and apoptosis

We evaluated the chemopreventive potential of the ethyl acetate fraction (EAF) and methanolic fraction (MF) of Azadirachta indica (neem) leaf on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Estradiol and estrogen receptor status, xenobiotic-metabolizing enzyme activities, redox status, DNA and protein modifications, and the expression of cell proliferation, and apoptosis related proteins in the mammary gland and liver were used as biomarkers of chemoprevention. Administration of both EAF and MF at a dose of 10 mg/kg bw effectively suppressed tumour incidence. Chemoprevention by neem leaf fractions was associated with modulation of hormone and receptor status, xenobiotic-metabolising enzymes, and lipid and protein oxidation, with upregulation of antioxidants, inhibition of oxidative DNA damage, protein modification, and cell proliferation, and induction of apoptosis. However EAF rich in constituent phytochemicals was more effective than MF in modulating multiple molecular targets. These results provide evidence for the chemopreventive efficacy of neem leaf fractions in the rat mammary tumour model.

Antioxidant prenylflavonoids from Artocarpus communis and Artocarpus elasticus

Two new prenylflavonoids, cyclogeracommunin (1) and artoflavone A (2), were isolated from the cortex of roots of Artocarpus communis. Their structures were determined by spectroscopic methods. Compounds 1, 2 and known compounds, artomunoisoxanthone (3), artocommunol CC (4), artochamin D (5), artochamin B (6), and dihydroartomunoxanthone (7), isolated from A. communis, and known compounds, cycloartelastoxanthone (8), artelastoheterol (9), cycloartobiloxanthone (10) and artonol A (11), isolated from Artocarpus elasticus, all showed inhibition of oxidative DNA damage. Compounds 2 and 8-10 significantly showed 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity with IC50 values of 24.2 ± 0.8, 18.7 ± 2.2, 42.2 ± 2.8 and 26.8 ± 1.2 μM, respectively, while compounds 1 and 11 significantly displayed inhibitory effects on xanthine oxidase (XO) activity with IC50 values of 73.3 ± 19.1 and 43.3 ± 8.1 μM, respectively. These findings indicate that 1–11 are promising antioxidants.

Phloroglucinols with Antioxidant Activity and Xanthonolignoids from the Heartwood of Hypericum geminiflorum

A new phloroglucinol, hyperielliptone HA (1/1a), a new spirophloroglucinol possessing an unprecedented skeleton, hyperielliptone HB (2/2a), and two new xanthonolignoids, hyperielliptones HC (3) and HD (4), were isolated from the heartwood of Hypericum geminiflorum. Compounds 1/1a and 2/2a were obtained as tautomeric pairs. The structures and relative configurations of these compounds were elucidated by spectroscopic methods. In biological testing, compound 2/ 2a revealed significant inhibition of oxidative DNA damage and an inhibitory effect on xanthine oxidase.

Pretreatment With Black Tea Polyphenols Modulates Xenobiotic-Metabolizing Enzymes in an Experimental Oral Carcinogenesis Model

The objective of this study was to evaluate the chemopreventive potential of the black tea polyphenols Polyphenon-B and BTF-35 during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Hamsters were divided into six groups. Animals in groups 2 and 3 received diet containing Polyphenon-B and BTF-35, respectively, 4 weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, animals in groups 1, 2, and 3 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 4 and 5 were given Polyphenon-B and BTF-35 alone, respectively, as in groups 2 and 3. Animals in group 6 served as control. All the animals were sacrificed after an experimental period of 18 weeks. Phase I and phase II xenobiotic-metabolizing enzymes and 8-hydroxy-deoxyguanosine (8-OH-dG) in the buccal pouch and liver were used as biomarkers of chemoprevention. Hamsters painted with DMBA showed increased expression of 8-OH-dG and enhanced activities of phase I (CYP450; total as well as CYP1A1, 1A2, and 2B isoforms and cytochrome b5) and phase II (GST and quinone reductase) xenobiotic-metabolizing enzymes with increased immunohistochemical expression of CYP1A1, and CYP1B1 isoforms in the buccal pouch. This was accompanied by increased phase I and decreased phase II enzyme activities in the liver. Administration of Polyphenon-B and BTF-35 significantly decreased tumor incidence, oxidative DNA damage, phase I enzyme activities as well as expression of CYP1A1 and CYP1B1 isoforms, while enhancing phase II enzyme activities in the buccal pouch and liver. Our results provide a mechanistic basis for the chemopreventive potential of black tea polyphenols. Furthermore, the greater efficacy of BTF-35 in chemoprevention of HBP carcinomas via inhibition of oxidative DNA damage and modulation of xenobiotic-metabolizing enzymes may have a major impact in human oral cancer prevention.

Berry phytochemicals, genomic stability and cancer: Evidence for chemoprotection at several stages in the carcinogenic process

Consumption of a diet high in plant-based foods is associated with a decreased risk of epithelial cell cancers at several sites. Cytoprotectants in fruits and vegetables include vitamins, minerals and numerous micronutrients. While there is little evidence uniquely linking berry consumption with lower cancer risk, berries contain high levels of compounds believed to reduce malignant transformation, including the polyphenol flavonoids and anthocyanins. There is strong and convincing evidence that berry extracts and berry phytochemicals modulate biomarkers of DNA damage and indicators of malignant transformation in vitro and in vivo. Data from numerous cell culture and animal models indicate that berry components such as the anthocyanins are potent anticarcinogenic agents and are protective against genomic instability at several sites in the carcinogenic pathway. Anticarcinogenic mechanisms include modulation of carcinogen activation and detoxification, decreased DNA binding of the carcinogen, inhibition of oxidative DNA damage, alteration in cell signalling and malignant transformation and inhibition of cell invasiveness and metastasis. Exactly which berry constituents are cytoprotective remains uncertain and in the majority of in vitro and in vivo studies the concentration of extract or phytochemical employed is non-nutritional. Evidence for an anticarcinogenic effect in human studies is weak.

Inhibition of oxidative DNA damage by plant antioxidants

Reactive oxygen species (ROS), from both endogenous and exogenous sources, may be involved in aging and different human diseases such as atherosclerosis, cancer, diabetes, neurodegenerative diseases and acquired immunodeficiency syndrome (AIDS) (Olinski et a!., 2002). Natural antioxidants contained in medicinal and aromatic plants, fruits and vegetables may be useful in preventing the deleterious consequences of oxidative damage caused by ROS and therefore they are considered as possible chemopreventive agents. They can possess a variety of biological activities e.g. anti-mutagenic, anti-carcinogenic, anti-proliferative, scavenging of free radicals or activated mutagens/carcinogens, they can modulate DNA repair and other enzyme activities or even regulate gene expression (Brigelius-Flohe and Traber, 1999; Craig, 1999; Heo et a l., 2001; Mi t i c et a!., 2001; Lazarova and Slamenova, 2004; Niko li c et a!., 2004). In our previous work, we have shown that terpenoids from sage (Salvia officinalis L.) and basil (Ocimum basilicum L.) possess antimutagenic potential in prokaryotic and lower eukaryotic test systems (Vukovi6Gac i c et a!., 1993; Simi c et a!., 1998,2000; Mi ti c et a!', 2001). For estimation of their mechanism of action, it is very important to determine the potential to prevent oxidative DNA damage.

Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, cobalt and lead as more dangerous than hitherto expected.

Co-exposure to cadmium, cobalt, lead and other heavy metals occurs in many occupational settings, such as pigment and batteries production, galvanization and recycling of electric tools. However, little is known about interactions between several heavy metals. In the present study we determined DNA single strand break (DNA-SSB) induction and repair capacity for 8-oxoguanine in mononuclear blood cells of 78 individuals co-exposed to cadmium (range of concentrations in air: 0.05-138.00 micro g/m(3)), cobalt (range: 0-10 micro g/m(3)) and lead (range: 0-125 micro g/m(3)). Exposure to heavy metals was determined in air, blood and urine. Non-parametric correlation analysis showed a correlation between cadmium concentrations in air with DNA-SSB (P = 0.001, R = 0.371). Surprisingly, cobalt air concentrations correlated even better (P < 0.001, R = 0.401), whereas lead did not correlate with DNA-SSB. Logistic regression analysis including 11 possible parameters of influence resulted in a model showing that cobalt in air, cadmium in air, cadmium in blood and lead in blood influence the level of DNA-SSB. The positive result with cobalt was surprising, since exposure levels were much lower compared with the TRK-value of 100 micro g/m(3). To examine, whether the positive result with cobalt is stable, we applied several logistic regression models with two blocks, where all factors except cobalt were considered preferentially. All strategies resulted in the model described above. Logistic regression analysis considering also all possible interactions between the relevant parameters of influence finally resulted in the following model: Odds ratio = 1.286(Co in air) x 1.040(Cd in air) x 3.111(Cd in blood) x 0.861(Pb in air) x 1.023(Co in air x Pb in air). This model correctly predicts an increased level of DNA-SSB in 91% of the subjects in our study. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium, cobalt and lead. For instance increasing lead air concentrations from 1.6 to 50 micro g/m(3) in the presence of constant exposures to cobalt and cadmium (8 micro g/m(3) and 3.8 micro g/m(3)) leads to an almost 5-fold increase in the odds ratio, although lead alone does not increase DNA-SSB. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or cobalt. Indeed, repair of 8-oxoguanine decreased with increasing exposures and inversely correlated with the level of DNA-SSB (P = 0.001, R = -0.427). Protein expression patterns of individuals exposed to cobalt concentrations of approximately 10 micro g/m(3) were compared with those of unexposed individuals using two-dimensional gel electrophoresis. Qualitative and apparent quantitative alterations in protein expression were selective and certainly occurred in <0.1% of all proteins. In conclusion, the hazard due to cobalt exposure - that has been classified only as IIB by the IARC - seems to be underestimated, especially when individuals are co-exposed to cadmium or lead. Co-exposure may cause genotoxic effects, even if the concentrations of individual heavy metals do not exceed TRK-values.

The neuroprotectant ebselen inhibits oxidative DNA damage induced by dopamine in the presence of copper ions

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound with glutathione peroxidase-like activity, has been shown to be protective against brain ischemic injury and Parkinson's disease. This study was undertaken to investigate the protective effects of ebselen on oxidative DNA damage induced by dopamine in the presence of copper ions. Incubation of φX-174 plasmid DNA with micromolar dopamine in the presence of Cu(II) resulted in a concentration-dependent induction of DNA strand breaks. Both a Cu(II)/Cu(I) redox cycle and H 2O 2 formation were critically involved in the induction of DNA strand breaks by the dopamine/Cu(II) system. The presence of ebselen at micromolar concentrations led to a marked concentration-dependent inhibition of DNA strand breaks induced by the dopamine/Cu(II) system. Further studies showed that ebselen did not affect either the Cu(II)-mediated oxidation of dopamine to dopamine quinone or the reduction of Cu(II) to Cu(I) by dopamine. Instead, the presence of ebselen resulted in a marked decrease in the levels of H 2O 2 derived from the Cu(II)-mediated oxidation of dopamine. Taken together, our results demonstrate for the first time that ebselen is able to inhibit the dopamine/Cu(II)-induced oxidative DNA damage, which appears to be attributable to the ability of ebselen to decrease the levels of H 2O 2 derived from the dopamine/Cu(II) system. Since oxidative DNA damage has been implicated in the pathogenesis of various neurodegenerative diseases, the inhibition of oxidative DNA damage by ebselen may be responsible, at least partially, for its neuroprotective activities observed in both humans and experimental animals.

Inhibition of ultraviolet light-induced oxidative events in the skin and internal organs of hairless mice by isoflavone genistein

We have previously demonstrated that soybean isoflavone genistein inhibits ultraviolet-B (UVB)-induced skin tumorigenesis in hairless mice. In the present study, we further investigated the possible mechanism(s) of action whereby genistein inhibits photocarcinogenesis with focuses on UVB-induced oxidative events, including hydrogen peroxide (H 2O 2) production, lipid peroxidation (as represented by malondialdehyde, MDA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in vivo. We demonstrated that subacute exposure to UVB substantially increased the level of H 2O 2, lipid peroxides, and 8-OHdG in skin of hairless mice. In addition, chronic exposure to low-dose UVB (0.9–1.2 kJ/m 2 for 20 weeks) substantially increased the levels of 8-OHdG not only in the epidermis, but also in the internal organs such as liver, brain, and spleen of mice with exception of kidney. However, genistein did not affect the level of UVB-induced pyrimidine dimmers in the same UVB exposed mouse skin, indicating selective inhibition of oxidative DNA damage by genistein. Induction of H 2O 2 was independent of UVB fluences whereas the levels of MDA and 8-OHdG were induced in an UVB fluence-dependent manner. The results suggest that H 2O 2 be generated as an acute cutaneous response to UVB irradiation, while MDA and 8-OHdG are accumulated with increasing UVB exposure and more closely related to chronic effects of UVB radiation. Pre-treatment of animals with 10 μmol of genistein 1 h prior to UVB exposure significantly inhibited UVB-induced H 2O 2 and MDA in skin and 8-OHdG in epidermis as well as internal organs. Suppression of 8-OHdG formation by genistein has been corroborated in purified DNA irradiated with UVA and B. In summary, our results suggest that UVB irradiation elicit a series of oxidative events, which can be substantially inhibited by isoflavonoid genistein through either direct quenching of reactive oxygen species or indirect antiinflammatory effects. Thus, the antioxidative properties of genistein may explain for the mechanisms of anti-photocarcinogenic action of genistein.

Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention

Epidemiological studies have suggested that the use of aspirin is associated with a decreased incidence of human malignancies, particularly colorectal cancer. Since reactive oxygen species (ROS) are critically involved in multistage carcinogenesis, this study was undertaken to examine the ability of aspirin to inhibit ROS-mediated DNA damage. Hydrogen peroxide (H 2O 2)+Cu(II) and hydroquinone (HQ) + Cu(II) were used to cause oxidative DNA strand breaks in φX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.5–2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a marked inhibition of oxidative DNA damage induced by either H 2O 2/Cu(II) or HQ/Cu(II). The inhibition of oxidative DNA damage by aspirin was exhibited in a concentration-dependent manner. Moreover, aspirin was found to be much more potent than the hydroxyl radical scavengers, mannitol and dimethyl sulfoxide, in protecting against the H 2O 2/Cu(II)-mediated DNA strand breaks. Since the reduction of Cu(II) to Cu(I) is crucially involved in both H 2O 2/Cu(II)- and HQ/Cu(II)-mediated formation of hydroxyl radical or its equivalent, and the subsequent oxidative DNA damage, we examined whether aspirin could inhibit this Cu(II)/Cu(I) redox cycle. It was observed that aspirin at concentrations that showed the inhibitory effect on oxidative DNA damage did not alter the Cu(II)/Cu(I) redox cycle in either H 2O 2/Cu(II) or HQ/Cu(II) system. In addition, aspirin was not found to significantly scavenge H 2O 2. This study demonstrates for the first time that aspirin potently inhibits both H 2O 2/Cu(II)- and HQ/Cu(II)-mediated oxidative DNA strand breaks most likely through scavenging the hydroxyl radical or its equivalent derived from these two systems. The potent inhibition of oxidative DNA damage by aspirin may thus partially contribute to its anticancer activities observed in humans.

Inhibition of oxidative DNA damage in vitro by extracts of Brussels sprouts

Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2′-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light.The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC50 values were 6–20 μg/ml corresponding to the extract of 5–20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C.Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.