Induction Of Oxidative DNA Damage Research Articles

Neutrophil-derived ROS contribute to oxidative DNA damage induction by quartz particles

The carcinogenicity of respirable quartz is considered to be driven by reactive oxygen species (ROS) generation in association with chronic inflammation. The contribution of phagocyte-derived ROS to inflammation, oxidative stress, and DNA damage responses was investigated in the lungs of C57BL/6 J wild-type and p47 phox−/− mice, 24 h after pharyngeal aspiration of DQ12 quartz (100 mg/kg bw). Bone-marrow-derived neutrophils from wild-type and p47 phox−/− mice were used for parallel in vitro investigations in coculture with A549 human alveolar epithelial cells. Quartz induced a marked neutrophil influx in both wild-type and p47 phox−/− mouse lungs. Significant increases in mRNA expression of the oxidative stress markers HO-1 and γ-GCS were observed only in quartz-treated wild-type animals. Oxidative DNA damage in lung tissue was not affected by quartz exposure and did not differ between p47 phox−/− and WT mice. Differences in mRNA expression of the DNA repair genes OGG1, APE-1, DNA Polβ, and XRCC1 were also absent. Quartz treatment of cocultures containing wild-type neutrophils, but not p47 phox−/− neutrophils, caused increased oxidative DNA damage in epithelial cells. Our study demonstrates that neutrophil-derived ROS significantly contribute to pulmonary oxidative stress responses after acute quartz exposure, yet their role in the associated induction of oxidative DNA damage could be shown only in vitro.

Genoprotective effects of green tea (Camellia sinensis) in human subjects: results of a controlled supplementation trial

Green tea is rich in polyphenolic antioxidants and has widely reported but largely unsubstantiated health benefits. In the present study, genoprotective effects of two types of green tea were studied both in an in vitro and in a human supplementation trial. For the in vitro study, human lymphocytes were pre-incubated in tea (0·005-0·1 %, w/v), washed and subjected to oxidant challenge induced by H2O2. In a placebo-controlled, cross-over supplementation study, eighteen healthy volunteers took 2 x 150 ml/d of 1% (w/v) green tea ('Longjing' green tea or 'screw-shaped' green tea) or water (control) for 4 weeks (n 6). Subjects took all the three treatments in a random order, with 6 weeks' washout between each treatment. Fasting blood and urine were collected before and after each treatment. The comet assay was used to measure the resistance of lymphocytic DNA to H2O2-induced challenge. Basal oxidation-induced DNA damage was measured using the formamidopyrimidine glycosylase (Fpg) enzyme-assisted comet assay. Urine 7,8-dihydro-2-deoxyguanosine (8-oxodG, mol/mmol creatinine), a biomarker of whole-body oxidative stress, was measured by liquid chromatography with tandem MS. In vitro testing results of tea-treated cells showed increased (P < 0·05) resistance of DNA to the challenge. In the supplementation trial, a significant (P < 0·05) increase in resistance was also observed. Furthermore, the FPg comet data showed .20% decrease in DNA damage with tea supplementation: mean and standard deviation changes in %DNA in comet tail in the Fpg-assisted comet assay were: -5·96 (SD 3·83) % after Longjing tea; -6·22 (SD 3·34) % after screw-shaped tea; +0·91 (SD 5·79) % after water (P < 0·05). No significant changes in urine 8-oxodG were seen. The results indicate that green tea has significant genoprotective effects and provide evidence for green tea as a 'functional food'.

Reactions of the OOH radical with guanine: Mechanisms of formation of 8-oxoguanine and other products

The mutagenic product 8-oxoguanine (8-oxoGua) is formed due to intermediacy of peroxyl (OOR) radicals in lipid peroxidation and protein oxidation-induced DNA damage. The mechanisms of these reactions are not yet understood properly. Therefore, in the present study, the mechanisms of formation of 8-oxoGua and other related products due to the reaction of the guanine base of DNA with the hydroperoxyl radical (OOH) were investigated theoretically employing the B3LYP and BHandHLYP hybrid functionals of density functional theory and the polarizable continuum model for solvation. It is found that the reaction of the OOH radical with guanine can occur following seven different mechanisms leading to the formation of various products including 8-oxoGua, its radicals, 5-hydroxy-8-oxoguanine and CO 2. The mechanism that yields 8-oxoGua as an intermediate and 5-hydroxy-8-oxoGua as the final product was found to be energetically most favorable.

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts

Melanomas occur mainly in sunlight-exposed skin. Xeroderma pigmentosum (XP) patients have 1,000-fold higher incidence of melanoma, suggesting that sunlight-induced "bulky" photoproducts are responsible for melanomagenesis. Sunlight induces a high level of reactive oxygen species in melanocytes (MCs); oxidative DNA damage (ODD) may thus also contribute to melanomagenesis, and XP gene products may participate in the repair of ODD. We examined the effects of melanin on UVA (320-400 nm) irradiation-induced ODD and UV photoproducts and the repair capacity in MC and XP cells for ODD and UV-induced photoproducts. Our findings indicate that UVA irradiation induces a significantly higher amount of formamidopyrimidine glycosylase-sensitive ODD in MCs than in normal human skin fibroblasts (NHSFs). In contrast, UVA irradiation induces an insignificant amount of UvrABC-sensitive sites in either of these two types of cells. We also found that, compared to NHSFs, MCs have a reduced repair capacity for ODD and photoproducts; H(2)O(2) modified- and UVC-irradiated DNAs induce a higher mutation frequency in MCs than in NHSFs; and, XP complementation group A (XPA), XP complementation group C, and XP complementation group G cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSFs. These results suggest that: (i) melanin sensitizes UVA in the induction of ODD but not bulky UV photoproducts; (ii) the high susceptibility to UVA-induced ODD and the reduced DNA repair capacity in MCs contribute to carcinogenesis; and (iii) the reduced repair capacity for ODD contributes to the high melanoma incidence in XP patients.

Abstract 2986: Melanocytes are deficient in repair of oxidative DNA damage and UVC induced photoproducts

Abstract Melanomas occur mainly in sunlight-exposed skin, xeroderma pigmentosum complementation (XP) patients, defective in nucleotide excision repair function, have 1000-fold higher incidence of melanoma. These findings indicate that sunlight induced “bulky” photoproducts are responsible for melanomagenesis. On the other hand, sunlight also induces a high level of reactive oxygen species (ROS) in melanoctes (MC), oxidative DNA damage (ODD) therefore may contribute to melanomagenesis and that the XP protein may participate in ODD repair. To address these questions we determined: 1) the effect of melanin on UVA (320-400 nm) irradiation induced ODD and UV photoproducts using formamidopyrimidine glycosylase (Fpg) and UvrABC incision assays, 2) the repair capacity for ODD and UV-induced photoproducts in MC and XPA cells using host cell reactivation and in vitro DNA repair synthesis assays. We found: 1) UVA irradiation-inducing a significantly higher amount of Fpg sensitive ODD in MC than in normal human skin fibroblasts (NHSF). In contrast, UVA irradiation induces an insignificant amount of UvrABC sensitive sites in either of these two types of cells. 2) Compared to NHSF, MC have a reduced repair capacity for ODD and photoproducts. 3) H2O2 modified DNA induces a higher mutation frequency in MC than in NHSF. 4) XPA cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSF. Based on these results, we propose: 1) UVA is a major etiological agent for melanomagenesis and the mechanism is through melanin sensitizes UVA in induction of ODD and reduced repair capacity in MC; 2) the reduced repair capacity for ODD contributes to the high melanoma incidence in XPA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2986.

Recommendations for Standardized Description of and Nomenclature Concerning Oxidatively Damaged Nucleobases in DNA

The field of oxidative stress, and the study of oxidatively damaged DNA, in particular, is a subject of intense, and growing interest. This has, in part, benefited from the availability of kits from commercial suppliers which are advertised as reporting on markers of oxidative stress. Such widespread use has inevitably led to an increase in the number of concerns, amongst experts in the field, editors and referees, over appropriateness of terminology and methodology. Thus, the widely used term “oxidative DNA damage” is misleading as it implies that the damage, i.e. the lesion per se, is oxidative and thus capable of oxidising other substrates. We would encourage the use of such terms as ‘oxidatively damaged DNA’, ‘oxidatively generated DNA damage’, ‘oxidatively-derived damage to DNA’ or ‘oxidation-induced DNA damage’ to describe the consequence of the interaction of reactive oxygen species with DNA. One of the most studied nucleic acid-derived biomarkers of oxidative stress is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Yet, in the literature, this compound has been referred to using a number of different terms, sometimes leading to confusion over the designation of the modified nucleobase or (2′-deoxy)ribonucleoside. Standardisation of nomenclature would not only simplify literature searches, but also clarify the lesion in question. Herein, we provide justification for our preferred nomenclature, and suggest a number of steps by which we may work towards standardisation of calibration, and with it improved inter-laboratory agreement, for assays of 8-oxodG, in order to achieve accurate measurements.

Elevation of cell proliferation via generation of reactive oxygen species by piperonyl butoxide contributes to its liver tumor-promoting effects in mice

Piperonyl butoxide (PBO) is a pesticide synergist used with pyrethroids as a domestic insecticide, and it acts as a non-genotoxic hepatocarcinogen in rats and mice. To clarify whether oxidative stress is involved in the liver tumor-promoting effect of PBO in mice, male mice were subjected to two-thirds partial hepatectomy, followed by N-diethylnitrosamine (DEN) treatment, and given a diet containing 0.6% PBO for 25 weeks. The incidences of cytokeratin (CK) 8/18-positive foci, adenomas, and carcinomas significantly increased in the DEN + PBO group compared with the DEN-alone group. The PCNA-positive ratio significantly increased in non-tumor hepatocytes, CK8/18-positive foci and adenomas in the DEN + PBO group compared with the DEN-alone group. PBO increased reactive oxygen species (ROS) production in microsomes but did not change oxidative DNA damage as assessed by 8-hydroxydeoxyguanosine (8-OHdG). In real-time RT-PCR, PBO upregulated the expression of genes related to metabolism, such as Cytochrome P450 1a1, 2a5, and 2b10, and metabolic stress, such as Por and Nqo1, but downregulated Egfr and Ogg1. PBO also increased early response genes downstream of mitogen-activated protein kinase (MAPK), such as c-Myc that is induced by excessive ROS production, and G1/S transition-related genes, such as E2f1 and Ccnd1. Thus, PBO can generate ROS via the metabolic pathway without any induction of oxidative DNA damage, activate cell growth, increase c-Myc- and E2F1-related pathways, and act as a liver tumor promoter of DEN-induced hepatocarcinogenesis in mice.

Genotoxicity of Soluble and Particulate Cadmium Compounds: Impact on Oxidative DNA Damage and Nucleotide Excision Repair

Water-soluble and particulate cadmium compounds are carcinogenic to humans. While direct interactions with DNA are unlikely to account for carcinogenicity, induction of oxidative DNA damage and interference with DNA repair processes might be more relevant underlying modes of action (recently summarized, for example, in Joseph , P. (2009) Tox. Appl. Pharmacol. 238 , 271 - 279). The present study aimed to compare genotoxic effects of particulate CdO and soluble CdCl(2) in cultured human cells (A549, VH10hTert). Both cadmium compounds increased the baseline level of oxidative DNA damage. Even more pronounced, both cadmium compounds inhibited the nucleotide excision repair (NER) of BPDE-induced bulky DNA adducts and UVC-induced photolesions in a dose-dependent manner at noncytotoxic concentrations. Thereby, the uptake of cadmium in the nuclei strongly correlated with the repair inhibition of bulky DNA adducts, indicating that independent of the cadmium compound applied Cd(2+) is the common species responsible for the observed repair inhibition. Regarding the underlying molecular mechanisms in human cells, CdCl(2) (as shown before by Meplan, C., Mann, K. and Hainaut, P. (1999) J. Biol. Chem. 274 , 31663 - 31670 ) and CdO altered the conformation of the zinc binding domain of the tumor suppressor protein p53. In further studies applying only CdCl(2), cadmium decreased the total nuclear protein level of XPC, which is believed to be the principle initiator of global genome NER. This led to diminished association of XPC to sites of local UVC damage, resulting in decreased recruitment of further NER proteins. Additionally, CdCl(2) strongly disturbed the disassembly of XPC and XPA. In summary, our data indicate a general nucleotide excision repair inhibition by cadmium compounds, which is most likely caused by a diminished assembly and disassembly of the NER machinery. These data reveal new insights into the mechanisms involved in cadmium carcinogenesis and provide further evidence that DNA repair inhibition may be one predominant mechanism in cadmium induced carcinogenicity.

Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions

How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

Roles of polysaccharide from Branchiostoma belcheri in anti-DNA oxidation and anti-tumor activity in S180 mice

In this study, we isolated a polysaccharide from Branchiostoma belcheri (PBB) by enzymatic protein hydrolysis and alcohol precipitation. We investigated the effects of PBB supplementation on DNA oxidation and growth of the transplanted tumor cells Sarcoma (S180) in mice. Sixty healthy Kunming mice weighing between 18 and 25 g were randomly assigned to 6 groups, each consisting of 10 animals. All the mice, except for the blank control group, were inoculated with S180 sarcoma cells into the axilla of the left foreleg. PBB was given to mice by gavage at doses of 0 (model control), 25, 50, or 100 mg/kg b.w. in 0.2 ml saline for 30 days. The fifth group of S180-mice was given cytoxan (50 mg/kg) by peritoneal injection as a positive control group. The animals had free access to food and water. The mice were sacrificed after the final treatment and blood was quickly collected. Spontaneous and oxidized DNA damage of peripheral lymphocytes induced by H2O2 were analyzed by SCGE. O6-methyl-guanine (O6-MeG) was measured by high-performance capillary zone electrophoresis. The average tumor weights (0.856–1.118 g) of the three PBB groups were significantly lower than that of the model control group (1.836 g) (p<0.05). The tumor inhibition ratios of the PBB groups were 39.1%–53.4% and similar to the cytoxan positive group (57.5%). There were no significant differences in spontaneous DNA damage in peripheral lymphocytes among the groups. The oxidative DNA damage induced by 10 µmol/L H2O2 in the 50 and 100 mg/kg b.w. groups were 246.1 AU and 221.7 AU, respectively, both of which were significantly lower than that in the model group (289.0 AU; p<0.05). The plasma concentrations of O6-MeG in the 25, 50, and 100 mg/kg supplemented groups were 2.09 µmol/L, 1.86 µmol/L, and 1.63 µmol/L, respectively, all of which were significantly lower than that of the model group (2.67 µmol/L; p<0.05). These results indicated that PBB may have antioxidative activity and thus reduce oxidation-induced DNA damage.

Potassium Bromate Enhances N-Ethyl-N-Hydroxyethylnitrosamine–Induced Kidney Carcinogenesis Only at High Doses in Wistar Rats: Indication of the Existence of an Enhancement Threshold

As susceptibility to carcinogens varies considerably among different strains of experimental animals, evaluation of dose-response relationships for genotoxic carcinogen in different strains is indispensable for risk assessment. Potassium bromate (KBrO(3)) is a genotoxic carcinogen inducing kidney cancers at high doses in male F344 rats, but little is known about its carcinogenic effects in other strains of rats. The purpose of the present study was to determine dose-response relationships for carcinogenic effects of KBrO(3) on N-ethyl-N-hydroxyethylnitrosamine (EHEN)-induced kidney carcinogenesis in male Wistar rats. We found that KBrO(3) showed significant enhancement effects on EHEN-induced kidney carcinogenesis at above 250 ppm but not at doses of 125 ppm and below when evaluated in terms of induction of either preneoplastic lesions or tumors in male Wistar rats. Furthermore, KBrO(3) significantly increased the formation of oxidative DNA damage at doses of 125 and above but not at doses of 30 ppm and below in kidneys. These results demonstrated that low doses of KBrO(3) exert no effects on development of EHEN-initiated kidney lesions and induction of oxidative DNA damage. Taking account of previous similar findings in male F344 rats, it is strongly suggested that a threshold dose exists for enhancement effects of KBrO(3) on kidney carcinogenesis in rats.

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV-signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose-dependent generation of hydrogen peroxide in UV-irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with alpha-melanocortin (alpha-MSH) reduced the UV-induced generation of 7,8-dihydro-8-oxyguanine (8-oxodG), a major form of oxidative DNA damage. Pretreatment with alpha-MSH also increased the protein levels of catalase and ferritin. The effect of alpha-MSH on 8-oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss-of-function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP-YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by alpha-MSH. Our data unravel some mechanisms by which alpha-MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss-of-function MC1R with melanoma.

GSTT2, a phase II gene induced by apple polyphenols, protects colon epithelial cells against genotoxic damage

The potential protective effect of a polyphenol-rich diet for colon carcinogenesis is of great scientific and medical interest. Apples are a main source of polyphenols, and apple juice has been shown to attenuate chemically induced colon carcinogenesis in animal models. In addition to an antioxidant and antiproliferative activity, apple polyphenols have been shown to elevate expression of the phase II gene glutathione S-transferase T2 (GSTT2) in colon epithelial cells. We hypothesized that apple polyphenols may thereby provide protection against oxidant-induced DNA damage. Using GSTT2 promoter constructs and luciferase reporter assays, we found that polyphenolic apple extracts (AE) can directly enhance GSTT2 promoter activity. Comet assays demonstrated that the genotoxicity of the GSTT2 substrate cumene hydroperoxide (CumOOH) was significantly reduced when HT29 colon epithelial cells were pretreated with AE. Overexpression of GSTT2 in HT29 cells significantly reduced CumOOH induced DNA damage, whereas shRNA mediated knockdown of GSTT2 gene expression resulted in higher damage. Our results causally link GSTT2 levels with protection from genotoxic stress, and provide evidence that the antigenotoxic effects of apple polyphenols in vitro are at least in part due to an induction of GSTT2 expression. Induction of phase II genes may contribute to primary chemoprevention of colon cancer by apple polyphenols.

Repair of Nitric Oxide-damaged DNA in β-Cells Requires JNK-dependent GADD45α Expression

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately beta-cell death. Not only does nitric oxide cause beta-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged beta-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in beta-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45alpha, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45alpha expression attenuates the repair of nitric oxide-induced beta-cell DNA damage. These findings show that beta-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45alpha mediate the p53-independent repair of this DNA damage.

Contribution of glutathione status to oxidant-induced mitochondrial DNA damage in colonic epithelial cells

Although oxidative stress induces mitochondrial DNA (mtDNA) damage, a role for redox in modulating mtDNA oxidation and repair is relatively unexplored. This study examines the contribution of cellular glutathione (GSH) redox status to menadione (MQ)-induced mtDNA damage and postoxidant mtDNA recovery in a nontransformed NCM460 colonic cell line. We show that MQ caused dose-dependent increases in mtDNA damage that were blunted by N-acetylcysteine, a thiol antioxidant. Damage to mtDNA paralleled mitochondrial protein disulfide formation and glutathione disulfide increases in the cytosol and mitochondria and was exacerbated by inhibition of GSH synthesis in accordance with decreased cytosolic and mitochondrial GSH. Blockade of mitochondrial GSH (mtGSH) transport potentiated mtDNA damage, which was prevented by overexpression of the oxoglutarate mtGSH carrier, underscoring a link between mtGSH and mtDNA responsiveness to oxidative stress. The removal of MQ posttreatment elicited mtDNA recovery to basal levels by 4 h, indicating complete repair. Notably, mtDNA recovery was preceded by restored cytosolic and mtGSH levels at 2 h, suggesting a connection between the maintenance of cell GSH and effective mtDNA repair. The MQ-induced dose-dependent increase in mtDNA damage was attenuated by overexpressing mitochondrial 8-oxoguanine DNA glycosylase (Ogg1), consistent with 7,8-dihydro-8-oxoguanine being a major oxidative mtDNA lesion. Collectively, the results show that oxidative mtDNA damage in colonic cells is highly responsive to the mtGSH status and that postoxidant mtDNA recovery may also be GSH sensitive.

Electrochemical detection of natural DNA damage induced by ferritin/ascobic acid/H 2O 2 system and amplification of DNA damage by endonuclease Fpg

Oppositely charged natural DNA and chitosan (CS) were assembled into (CS/DNA) n layer-by-layer films on electrode surface, and Ru(bpy) 3 2+ (bpy = bipyridyl) in solution was used as electroactive catalyst to detect damage of DNA in the films after incubation of the films in ferritin/AA/H 2O 2 solutions (AA = ascorbic acid). The mechanism of DNA damage caused by the ferritin/AA/H 2O 2 system was similar to that of Fenton reaction, where the reaction of ferritin with AA would release some Fe(II) ions from ferritin and the following reaction between Fe(II) ions and H 2O 2 would produce hydroxyl radical, which could induce DNA oxidative damage. This system provided an in vitro model to imitate the DNA damage indirectly induced by ferritin in real bio-systems. In addition, formamidopyrimidine DNA glycosylase (Fpg), a key endonuclease enzyme in repair of oxidatively damaged DNA, was used to amplify the DNA damage caused by ferritin/AA/H 2O 2 system through conversion of oxidative purine bases into single-strand breaks. The high sensitivity of electrocatalytic method with Ru(bpy) 3 2+ as the catalyst in detection of DNA damage and the magnification function of Fpg may provide a novel idea to detect natural DNA lesion sensitively.

Genista sessilifolia DC. and Genista tinctoria L. inhibit UV light and nitric oxide-induced DNA damage and human melanoma cell growth

Many Genista species (Leguminosae), containing isoflavones as biologically active substances, show interesting biological properties such as hypoglycemic, antiinflammatory, antiulcer, spasmolytic, antioxidant, estrogenic and cytotoxic activity against different human cancer cell lines. In this work, we describe the chemical composition of the methanolic extracts from aerial parts of Genista sessilifolia DC. and Genista tinctoria L., and their biological activity testing the effect on pBR322 DNA cleavage induced by hydroxyl radicals ( OH), generated from UV-photolysis of hydrogen peroxide (H 2O 2) and by nitric oxide (NO). In addition, we investigated the growth inhibitory activity of these natural products against human melanoma cell line (M14). The extracts of G. sessilifolia and G. tinctoria, for their isoflavone components, showed a protective effect on UV light and nitric oxide-mediated plasmid DNA damage, and inhibited the growth of melanoma cells. The data of the present study also suggest that these natural products could trigger apoptotic death in M14 cells. In fact, a high DNA fragmentation (COMET assay) and a significant increase of caspase-3 activity, not correlated to LDH release, a marker of membrane breakdown, occurred in melanoma cells exposed to these extracts. The significant production of reactive oxygen species (ROS) evidenced in these experimental conditions could contribute to trigger the apoptosis cascades.

Obesity, insulin resistance and oxidative stress: implication for breast carcinogenesis.

Abstract Abstract #6025 Background: Obesity is associated with a modest increase in the risk of postmenopausal breast cancer (RR = 1.5 – 2.0); but, because nearly a third of the U.S. population is obese, the population attributable risk is estimated at 15%. Obesity can cause insulin resistance culminating in Type II diabetes. Notably, breast cancer incidence is significantly increased in the years preceding a diagnosis of type II diabetes. Because IGFBP-1 expression is tightly regulated by insulin, it is an excellent marker of insulin resistance in healthy individuals.&amp;#x2028; Methods: These data are based on well-annotated prospectively acquired baseline blood and breast tissue samples from 72 high risk women between the ages of 37 and 86 years who participated in a chemoprevention trial. None of the women had been diagnosed with Type II diabetes. Women with plasma IGFBP1 levels in the lowest tertile (mean 2.1 ng/ml) were classified as insulin-resistant.&amp;#x2028; Results: Plasma IGFBP1 was strongly inversely correlated with BMI (R2 = 0.247, P &amp;lt; 0.0001). Insulin-resistant women had marginally higher mean plasma free estradiol levels than women not classified as insulin-resistant (2.13 x 10-12M versus 1.53 x 10-12M, P = 0.072). There was no difference in plasma IGF1, IGF2, or IGFBP3 levels. Illumina whole genome expression microarray data was available for breast tissue from 55 women. Women classified as insulin-resistant showed evidence of an adaptive response to oxidative stress based on significant upregulation of NQO1, GSTK1, CYP4ZP2, and SRXN1 (P &amp;lt; 0.001).&amp;#x2028; Conclusions: Marginally increased circulating estradiol may contribute to the increased breast cancer risk observed in insulin-resistant women. However, insulin resistance increases oxidative stress in breast tissue and may promote carcinogenesis through induction of oxidative DNA damage. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6025.

Chemopreventive action of dexamethasone and α-tocopherol in oxidative stressed cells

Introduction: Recent research indicates a close connection of inflammation and cancer as presumed by Virchow in 1893. The growing understanding of cellular signalling and regulatory pathways reveals multiple links between inflammation and cancer. This study was designed to evaluate the influence of the anti-inflammatory drug dexamethasone and the antioxidant α-tocopherol on oxidative induced DNA damage, a major factor in the development of malignancies. Material and methods: Miniorgan cultures (MOC) of fresh biopsied human nasal mucosa were used to keep cells in their microenvironment and thus to mimic in vivo conditions. MOC were pretreated with dexamethasone and α-tocopherol in different concentrations on 1 or on 5 days before oxidative DNA damage was introduced by hydrogen peroxide. The effect of these substances on DNA damage was evaluated using the alkaline single cell microgel electrophoresis (Comet Assay). Results: Dexamethasone induced slight, but considerable DNA fragmentation by itself. It effectively protected cells from hydrogen peroxide induced DNA damage, leading to a maximum decrease of about 45% when preincubated on 5 days at 20 μM. α-Tocopherol most effectively reduced oxidative DNA fragmentation by about 38% when MOC were pretreated 5 days at 20 μM. Discussion: Our experimental data clearly shows the DNA protective action of dexamethasone and α-tocopherol with regard to oxidatively induced DNA damage, a major pathogenetic factor that inflammation and cancer have in common.