Gamma Radiation-induced DNA Damage Research Articles

Evaluation of gamma radiation–induced genotoxicity in the workers of Italian honey bees, Apis mellifera ligustica, using single cell gel electrophoresis (comet) assay

The environment exposed to radionuclide faces detrimental effects of ionizing radiations and these radiations can damage DNA by inducing genotoxicity in living organisms. The gamma radiation induced genotoxicity was evaluated by employing Single cell gel electrophoresis (comet) assay in the foragers of Apis mellifera ligustica by exposing to different doses (1 to 60 Gy) of gamma radiation. DNA damage was measured as the percentage of comet tail DNA. The results showed a dose dependent increase and post irradiation time (1-24 h) dependent decrease in % tail DNA level in bee foragers. A significant increase in DNA damage was observed in all the samples and the extent of damage was proportional to the dose of gamma radiation received by the bees except in those treated with the lower doses (1, 5 and 10 Gy) of gamma radiation. The highest amount of DNA damage was recorded at 1 h post-irradiation which decreased gradually with time, that is, at 3 h and 6 h post-irradiation. However, there were no significant differences in the % tail DNA of bees of 12 h and 24 h post irradiation. The present study shows gamma radiation induced DNA damage in foragers of A. m. ligustica and also the comet assay is a suitable technique for detection of gamma radiation induced DNA damage.

The sequence preference of gamma radiation-induced DNA damage as determined by a polymerase stop assay

Purpose: The aim of this paper was to investigate the sequence preference of ionizing radiation (IR)-induced DNA damage as assessed by a linear amplification/polymerase stop (LA/PS) assay. The LA/PS assay is able to detect a wide range of IR-induced DNA lesions and this technique was utilized to quantitatively determine the preferential sites of gamma irradiation-induced DNA lesions in three different DNA sequences.Materials and methods: This analysis was performed on an automated DNA sequencer with capillary electrophoresis and laser-induced fluorescence detection.Results: The main outcome of this study was that G nucleotides were preferentially found at IR-induced polymerase stop sites. The individual nucleotides at the IR-induced DNA damage sites were analyzed and a consensus sequence of 5′-GG* (where * indicates the damaged nucleotide) was observed. In a separate method of analysis, the dinucleotides and trinucleotides at the IR-induced DNA damage sites were examined and 5′-GG* and 5′-G*G dinucleotides and 5′-GG*G trinucleotides were found to be the most prevalent. The use of the LA/PS assay permits a large number of IR-induced DNA lesions to be detected in the one procedure including: double- and single-strand breaks, apurinic/apyrimidinic sites and base damage.Conclusions: It was concluded that 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G) and the degradation products of 8-oxoG were possibly the main lesions detected. To our knowledge, this is the first occasion that the DNA sequence preference of IR-induced DNA damage as detected by a LA/PS assay has been reported.

Evaluation of gamma radiation-induced DNA damage in Aedes aegypti using the comet assay.

The study was undertaken to evaluate gamma radiation-induced DNA damage in Aedes aegypti. The comet assay was employed to demonstrate the extent of DNA damage produced in adult male A. aegypti exposed to seven different doses of gamma radiation, ranging from 1 Gy to 50 Gy. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in all samples; the extent of damage being proportional to the dose of gamma radiation the organism received, except in those treated with 1 Gy. The highest amount of DNA damage was noticed at 1 h postirradiation, which decreased gradually with time, that is, at 3, 6 and 12 h postirradiation. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the postirradiation time increased. The comet assay serves as a sensitive and rapid technique to detect gamma radiation-induced DNA damage in A. aegypti. This could be used as a potential biomarker for environmental risk assessment.

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways

BackgroundRadiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown. MethodsTumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed. ResultsThe combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation. ConclusionThe loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair. General significanceThe radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.

Paf15 expression correlates with rectal cancer prognosis, cell proliferation and radiation response.

Paf15, which participates in DNA repair, is overexpressed in numerous solid tumors. Blocking of Paf15 inhibits the growth of many types of cancer cells; while simultaneously enhancing cellular sensitivity to UV radiation. However, its expression and function in rectal cancer (RC) remain unknown. The current study was undertaken to assess the association of Paf15 expression with RC prognosis, as well as to explore the participation of Paf15 in the response of RC cells to irradiation. Increased Paf15 expression was observed in RC tissues and associated with pTNM stage and poor survival. In vitro, Paf15 induced increased RC cell proliferation while accelerating cell cycle progression, inhibiting cell death, and protecting against gamma radiation-induced DNA damage in RC cells. In conclusion, increased Paf15 expression is associated with increased RC proliferation, decreased patient survival, and a worse radiotherapeutic response.

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2–10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution.

Extracellularly synthesized ZnO nanoparticles interact with DNA and augment gamma radiation induced DNA damage through reactive oxygen species

ZnONPs were synthesized using ferulic acid as the reductant and the intricate twofold role as DNA binders and radio sensitizers was revealed, which can pave the way for anticancer therapy.

Emodin, an anthraquinone derivative, protects against gamma radiation-induced toxicity by inhibiting DNA damage and oxidative stress

Purpose: In the present study, we explored the modulatory effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone, C15H10O5) against gamma radiation-induced DNA damage and oxidative stress in acellular and cellular systems, respectively.Materials and methods: For cellular systems, concanavalin A (ConA)-stimulated murine splenocytes were used. Cytotoxic effect of emodin (0–400 μM), radiation (3–12 Gy) and emodin + radiation was measured by MTT [3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. Gamma radiation (3–12 Gy)-induced production of reactive oxygen species (ROS), an increase in nitric oxide (NO) level and its inhibition by emodin were estimated by DCFDA (2ʹ,7ʹ-dichlorofluorescein diacetate) and Griess regent, respectively. Analysis of radiation-induced apoptosis was performed using flow cytometery and acridine orange/ethidium bromide staining. DNA damage was evaluated in acellular system using pBR322 plasmid relaxation assay.Results: Emodin was able to effectively scavenge radiation- induced free radicals (ROS and NO) in murine splenocytes. Radiation-induced apoptosis and cell death was also inhibited by emodin pre-treatment. It could significantly prevent radiation-induced DNA damage.Conclusions: Protection against gamma radiation-induced cell death and DNA damage by emodin could be attributed to its free radical scavenging activity. The present study is the first report of the radioprotective role of emodin in mammalian cells.

Effects of a Mangifera indica L. stem bark extract and mangiferin on radiation‐induced DNA damage in human lymphocytes and lymphoblastoid cells

Mangifera indica L. (mango) stem bark aqueous extract (MSBE) that has antioxidant, anti-inflammatory and immunomodulatory properties, can be obtained in Cuba. It is rich in polyphenols, where mangiferin is the main component. In this study, we have tested DNA damage and protection effects of MSBE and mangiferin on primary human lymphocytes and lymphoblastoid cells. Cell suspensions were incubated with the products (50-1000 μg/ml) for experiments on damage induction, and evaluation of any potential protective effects (5-100 μg/ml) for 60 min at 37 °C. Irradiation was performed using a γ-ray source, absorbed dose 5 Gy. At the end of exposure, DNA damage, protection and repair processes were evaluated using the comet assay. MSBE (100-1000 μg/ml) induced DNA damage in a concentration dependent manner in both cell types tested, primary cells being more sensitive. Mangiferin (200 μg/ml) only induced light DNA damage at higher concentrations. DNA repair capacity was not affected after MSBE or mangiferin exposure. On the other hand, MSBE (25 and 50 μg/ml) and mangiferin (5-25 ug/ml) protected against gamma radiation-induced DNA damage. These results show MSBE has protector or harmful effects on DNA in vitro depending on the experimental conditions, which suggest that the extract could be acting as an antioxidant or pro-oxidant product. Mangiferin was involved in protective effects of the extract.

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae

To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4Gy of radiation, and biochemical and molecular damage became substantial from 8Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae.

Age- and time interval-specific gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky, assessed using comet assays

The gamma radiation-induced DNA damage in adult maize weevils, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), was assessed using single-cell electrophoresis (comet assay). Analysis of DNA damage following 0.5 and 1.0 kGy of gamma radiation was performed using cells from 1- and 15-day-old adults. Gamma-irradiated adults from both age groups showed typical DNA fragmentation, whereas cells from non-irradiated adults showed more intact DNA than young S. zeamais. Investigations using the comet assay showed that tail length, % tail DNA and % DNA damage all increased in adults of both age groups when compared to the control insects. A maximum comet length of 227.33 μm was recorded for 15-day-old adults at 24 h after irradiation with 1.0 kGy and a minimum of 50.12 μm for 1-day-old adults at 0 h after irradiation with 0.5 kGy. The percentage of DNA damage increased up to 57.31% and 68.15% for 1- and 15-day-old adults, respectively, at 24 h after irradiation with 1.0 kGy, whereas only 8.58% and 12.22% DNA damage were observed in the control batches. The results also showed that percentage of DNA damage increased at 24 h after irradiation compared to that at 0 h. However, further studies are needed to confirm these results.

Quercetin ameliorates gamma radiation-induced DNA damage and biochemical changes in human peripheral blood lymphocytes

We investigated the radioprotective efficacy of quercetin (QN), a naturally occurring flavonoid against gamma radiation-induced damage in human peripheral blood lymphocytes and plasmid DNA. In plasmid study, QN at different concentrations (3, 6, 12, 24 and 48 μM) were pre-incubated with plasmid DNA for 1 h followed by exposure of 6 Gy radiation. Among all concentrations of QN used, 24 μM showed optimum radioprotective potential. To establish the most effective protective concentration of QN in lymphocytes, the cells were pre-incubated with 3, 6, 12, 24 and 48 μM of QN for 30 min and then exposed to 4 Gy γ-radiation. The concentration-dependent effects of QN were evaluated by scoring micronuclei (MN) frequencies. The results showed that QN decreased the MN frequencies dose dependently, but the effect was more pronounced at 24 μM. Thus, 24 μM of QN was selected as the optimum concentration and was further used to evaluate its radioprotective effect in lymphocytes. For that a separate experiment was carried out, in which lymphocytes were incubated with QN (24 μM) for 30 min and exposed to different doses of radiation (1, 2, 3 and 4 Gy). Genetic damage (MN, dicentric aberration and comet attributes) and biochemical changes were measured to evaluate the effect of QN on γ-radiations (1–4 Gy). Radiation exposed showed significant increases in the genetic damage and thiobarbituric acid reactive substances (TBARS) accompanied by a significant decrease in the antioxidant status. QN pretreatment significantly decreased the genetic damage and TBARS and improved antioxidant status through its antioxidant potential. Altogether, our findings encourage further mechanistic and in vivo studies to investigate radioprotective efficacy of QN.

Molecular mechanisms of response of human peripheral blood mononuclear cells to ionizing radiation

Introduction: The aim of this study was to compare reaction of quiescent and proliferating PHA (mitogenic lectin phytohemagglutinin)-stimulated human peripheral blood mononuclear cells (PBMCs) to γ-radiation (IR) and analyze changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53 and its phosphorylations on serine 15 and 392, and p21. Materials and methods: PBMC were acquired from venous blood of normal donors by centrifugation over Histopague. The lymphocytes were cultured in IMDM medium suplemented with 20% fetal calf serum and antibiotics. For mitogenic stimulation of lymphocytes was added 10 μg/ml PHA. The viability of the PBMC has been identified by flow cytometry. Western blotting has been used for p53 and its phosphorylation form, p21 and H2AX detection. T-lymphocytes and PBMC were irradiated and lysed. To enhance detection sensitivity the Total p53 and PathScan p53ser15 Sandwich ELISA Kits were used. Results: PBMCs were stimulated to enter the cell cycle by treatment with the PHA. After 72 h long stimulation by PHA 96 % of CD3+cells were CD25+ and 12 % entered cell cycle (S+ and G2 phase). PHA-stimulation itself causes increase in γH2A.X, p53, and p21, but not phosphorylation of p53. After the irradiation of these stimulated PBMCs we detected increase in p53 and its phosphorylations on serine 15 and 392, and further increase in p21 from 4 h after the irradiation. Also level of γH2A.X increased significantly. Increase of p53 phosphorylation on serine 15 is dose-dependent 4 h after the irradiation in the whole studied dose range (0.5-7.5 Gy) in stimulated PBMCs. We compared p53, p53ser15 and ser392, p21 and H2AX between grouply of phytohemaglutinin-stimulated and non-stimulated PBMC 4 hours after 0,5-7,5Gy as well as 1-72 hours after 4Gy irradiation.. We were unable to detect p53 accumulation or phosphorylation in response unstimulated PBMC to gamma radiation induced DNA damage. We induced cell cycling by phytohemaglutinin in the T-cell fraction of PMBC preparations. Althougly total p53 protein expression was unchanged after 4 Gy irradiation, we found p53ser392 time-dependent expression (1-72 hours, 4 Gy) and p53ser15 dose-dependent expression in range of 0,5-7,5 Gy 4 hours after irradiation in phytohemaglutinin- stimulated PBMC. Neither total p53 protein nor p53ser392 was not altered by irradiation in non-stimulated PBMC. Conclusions: We suggested that p53 protein accumulation is a common mechanism for induction apoptosis of irradiated cells. We suggest that this pathway is activated in proliferating lymphocytes, unlike quiescent lymphocytes.

Moderate acute intake of de-alcoholised red wine, but not alcohol, is protective against radiation-induced DNA damage ex vivo—Results of a comparative in vivo intervention study in younger men

Moderate intake of wine is associated with reduced risk of cardiovascular disease and possibly cancer however it remains unclear whether the potential health benefits of wine intake are due to alcohol or the non-alcoholic fraction of wine. We therefore tested the hypothesis that the non-alcoholic fraction of wine protects against genome damage induced by oxidative stress in a crossover intervention study involving six young adult males aged 21–26 years. The participants adhered to a low plant phenolic compound diet for 48 h prior to consuming 300 mL of complete red wine, dealcoholised red wine or ethanol on separate occasions 1 week apart. Blood samples were collected 0.5, 1.0 and 2.0 h after beverage consumption. Baseline and radiation-induced genome damage was measured using the cytokinesis-block micronucleus assay and total plasma catechin concentration was measured. Consumption of dealcoholised red wine significantly decreased the gamma radiation-induced DNA damage at 1 and 2 h post-consumption by 20%. In contrast alcohol tended to increase radiation-induced genome damage and complete wine protected against radiation-induced genome damage relative to alcohol. The observed effects were only weakly correlated with the concentration of total plasma catechin ( R = −0.23). These preliminary data suggest that only the non-alcoholic fraction of red wine protects DNA from oxidative damage but this effect cannot be explained solely by plasma catechin.

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.

Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae.

Sanazole/DNA repair/Hypoxic radiosensitization/DNA polymerases/Saccharomyces cerevisiae Yeast Saccharomyces cerevisiae can exist in two physiological states, namely anaerobic and aerobic. They differ in their response to gamma- radiation and radiomodification. We report hereon our results concerning radiosensitization by Sanazole (AK-2123), a well-known hypoxic radio sensitizer, whose mechanism of action has been studied extensively. The results have revealed that Sanazole (1 mM) when present during irradiation could specifically sensitize wild-type anaerobic yeast cells with a DMF of 2.4. In a radiation-sensitive mutant which lacks a DNA repair pathway specific for the recovery from gamma-radiation induced DNA damage, the extent of sensitization was considerably lower and the DMF was only 1.3. Studies on the liquid holding recovery of cells of both wild- type and rad52 yeast cells exposed to radiation in presence of Sanazole revealed that sensitization by Sanazole is due to a preferential increase in the DNA damage, and not by impairing DNA repair. This system thus holds promise for screening potential hypoxic chemical radiosensitizers.