Spatio-temporal analysis of DNA damage repair using the X-ray microbeam

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

Investigations of DNA damage induction and repair resulting from cellular exposure to high dose-rate pulsed proton beams

Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/µm on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately following irradiation in a dose-dependent manner. The analysis of repair capability showed that the cells irradiated with 1 and 2 Gy almost completely recovered from the damage, but not, however, 3 Gy treated cells in which DNA damage was not recovered. In addition, the results indicate the importance of the use of an appropriate control in radiobiological in vitro analysis.

Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults

Background: Cruciferous vegetable (CV) consumption is associated with a reduced risk of several cancers in epidemiologic studies. Objective: The aim of this study was to determine the effects of watercress (a CV) supplementation on biomarkers related to cancer risk in healthy adults. Design: A single-blind, randomized, crossover study was conducted in 30 men and 30 women (30 smokers and 30 nonsmokers) with a mean age of 33 y (range: 19-55 y). The subjects were fed 85 g raw watercress daily for 8 wk in addition to their habitual diet. The effect of supplementation was measured on a range of endpoints, including DNA damage in lymphocytes (with the comet assay), activity of detoxifying enzymes (glutathione peroxidase and superoxide dismutase) in erythrocytes, plasma antioxidants (retinol, ascorbic acid, a-tocopherol, lutein, and beta-carotene), plasma total antioxidant status with the use of the ferric reducing ability of plasma assay, and plasma lipid profile. Results: Watercress supplementation (active compared with control phase) was associated with reductions in basal DNA damage (by 17%; P = 0.03), in basal plus oxidative purine DNA damage (by 23.9%; P = 0.002), and in basal DNA damage in response to ex vivo hydrogen peroxide challenge (by 9.4%; P = 0.07). Beneficial changes seen after watercress intervention were greater and more significant in smokers than in nonsmokers. Plasma lutein and P-carotene increased significantly by 100% and 33% (P < 0.001), respectively, after watercress supplementation. Conclusion: The results support the theory that consumption of watercress can be linked to a reduced risk of cancer via decreased damage to DNA and possible modulation of antioxidant status by increasing carotenoid concentrations.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p < 0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-L-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p < 0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells. (C) 2011 Elsevier B.V. All rights reserved.

Tomato oleoresin inhibits DNA damage but not diethylnitrosamine-induced rat hepatocarcinogenesis

Various studies have shown that lycopene, a non-provitamin A carotenoid, exerts antioxidant, antimutagenic and anticarcinogenic activities in different in vitro and in vivo systems. However, the results concerning its chemopreventive potential on rat hepatocarcinogenesis are ambiguous. The aim of the present study was to investigate the antigenotoxic and anticarcinogenic effects of dietary tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300ppm (administered 2 weeks before and during or 8 weeks after carcinogen exposure) on liver of male Wistar rats treated with a single intraperitoneal dose of 20 or 100 mg/kg of diethylnitrosamine (DEN), respectively. The level of DNA damage in liver cells and the development of putative preneoplastic single hepatocytes, minifoci and foci of altered hepatocytes (FHA) positive for glutathione S-transferase (GST-P) were used as endpoints. Significant reduction of DNA damage was detected when the highest lycopene concentration was administered before and during the DEN exposure (20 mg/kg). However, the results also showed that lycopene consumption did not reduce cell proliferation in normal hepatocytes or the growth of initiated hepatocytes into minifoci positive for GST-P during early regenerative response after 70% partial hepatectomy, or the number and area of GST-P positive FHA induced by DEN (100 mg/ kg) at the end of week 10. Taken together, the data suggest a chemopreventive effect of tomato oleoresin against DNA damage induced by DEN but no clear effectiveness in initiating or promoting phases of rat hepatocarcinogenesis. (c) 2008 Elsevier GmbH. All rights reserved.

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.

Assessment of DNA damage by extracts and fractions of Strychnos pseudoquina, a Brazilian medicinal plant with antiulcerogenic activity

Strychnos pseudoquina St. Hil. is a native plant of the Brazilian Savannah, used in popular medicine to treat a number of conditions. Since it contains large quantities of alkaloids with proven antiulcer activity, we tested the genotoxic potential of crude extracts and fractions containing alkaloids and flavonoids from the leaves of this plant, on Salmonella typhimurium and performed the micronucleus test on peripheral blood cells of mice treated in vivo. The results showed that the methanol extract of the leaves of S. pseudoquina is mutagenic to the TA98 (-S9) and TA100 (+S9, -S9) strains of Salmonella. The dichloromethane extract was not mutagenic to any of the tested strains. Fractions enriched with alkaloids or flavonoids were not mutagenic. In vivo tests were done on the crude methanol extract in albino Swiss mice, which were treated, by gavage, with three different doses of the extract. The highest dose tested (1800 mg/kg b.w.) induced micronuclei after acute treatment, confirming the mutagenic potential of the methanol extract of the leaves of S. pseudoquina. In high doses, constituents of S. pseudoquina compounds act on DNA, causing breaks and giving rise to micronuclei in the blood cells of treated animals. © 2006 Elsevier Ltd. All rights reserved.

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p < 0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells. © 2006 Elsevier Inc. All rights reserved.

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

Omega-3 improves glucose tolerance but increases lipid peroxidation and DNA damage in hepatocytes of fructose-fed rats

The high consumption of fructose is linked to the increase in various characteristics of the metabolic syndrome. Fish oil is beneficial for the treatment of these comorbidities, such as insulin resistance, dyslipidemia, and hepatic steatosis. The objective of this study was to evaluate the consequences of the administration of fish oil concomitant to fructose ingestion during the experiment (45 days) and during the final 15 days in high-fructose-fed rats. Male Wistar rats were divided into 5 groups: control; those receiving 10% fish oil (FO); those receiving 60% fructose (Fr); those receiving 60% fructose and 10% fish oil for 45 days (FrFO); and those receiving fructose plus soybean oil for 30 days and fish oil for the final 15 days of the study (FrFO15). There was an increase in triacylglycerol, serum total cholesterol, and hepatic volume in the Fr group. The FO and FrFO groups experienced an increase in lipid peroxidation and a decrease in serum reduced glutathione. The FrFO group suffered greater hepatic injury, with increased alanine aminotransferase levels and DNA damage. Marked n-3 incorporation occurred in the groups receiving fish oil, favoring a better response to the oral glucose tolerance test. Fructose induced comorbidities of the metabolic syndrome, and the use of fish oil promoted a better glucose tolerance, although it was accompanied by more hepatocyte damage.

Carbohydrate supplementation delays DNA damage in elite runners during intensive microcycle training

The aim of this study was to evaluate the effect of carbohydrate supplementation on free plasma DNA and conventional markers of training and tissue damage in long-distance runners undergoing an overload training program. Twenty-four male runners were randomly assigned to two groups (CHO group and control group). The participants were submitted to an overload training program (days 1-8), followed by a high-intensity intermittent running protocol (10 x 800 m) on day 9. The runners received maltodextrin solution (CHO group) or zero energy placebo solution as the control equivalent before, during, and after this protocol. After 8 days of intensive training, baseline LDH levels remained constant in the CHO group (before: 449.1 +/- 18.2, after: 474.3 +/- 22.8 U/L) and increased in the control group (from 413.5 +/- 23.0 to 501.8 +/- 24.1 U/L, p &lt; 0.05). On day 9, LDH concentrations were lower in the CHO group (509.2 +/- 23.1 U/L) than in the control group (643.3 +/- 32.9 U/L, p &lt; 0.01) post-intermittent running. Carbohydrate ingestion attenuated the increase of free plasma DNA post-intermittent running (48,240.3 +/- 5,431.8 alleles/mL) when compared to the control group (73,751.8 +/- 11,546.6 alleles/mL, p &lt; 0.01). Leukocyte counts were lower in the CHO group than in the control group post-intermittent running (9.1 +/- 0.1 vs. 12.2 +/- 0.7 cells/mu L; p &lt; 0.01) and at 80 min of recovery (10.6 +/- 0.1 vs. 13.9 +/- 1.1 cells/mu L; p &lt; 0.01). Cortisol levels were positively correlated with free plasma DNA, leukocytes, and LDH (all r &gt; 0.4 and p &lt; 0.001). The results showed that ingestion of a carbohydrate beverage resulted in less DNA damage and attenuated the acute post-exercise inflammation response, providing better recovery during intense training.