Ray-induced DNA Damage Research Articles

Photoprotective, antioxidant potential and DNA damage protection assay of leaf methanolic extract of Holoptelea integrifolia (Roxb) Planch and determination of some bioactive phenolic compounds by RP-HPLC

The methanolic leaf extract (MLE) of medicinally important plant Holoptelea integrifolia (Roxb.) Planch, a tree member of Ulmaceae family, exhibits prominent photoprotective and antioxidant activity mainly due to presence of phenols, flavonoids and tannins in notable amounts. Photoprotective efficacy (Sun Protection Factor value) of MLE was found to be 16.265 ± 0.0077, which can effectively block around 93% UV-B radiation. The transmission erythema (%Te) and transmission pigmentation (%Tp) values are lowest (2.4102 ± 0.620 and 3.050984 ± 0.183 respectively) at highest concentration of MLE (500 μg/ml). The antioxidant potential of MLE was evaluated by Reducing Power Assay (IC50 value: 88.52 ± 0.583 μg/ml), DPPH (IC50 value: 61.96 ± 0.721 μg/ml) and ABTS scavenging (IC50 value: 210.15 ± 0.329 μg/ml) activities. Ultraviolet (UV) ray induced DNA damage assay also revealed that MLE provide ample protection. Among the separated TLC fractions of MLE, four fractions (F2, F3, F4 & F7) were selected for further analysis. Six phenolic acids namely-tannic acid, gallic acid, hydroxybenzoic acid, p-coumaric acid, trans-cinnamic acid, caffeic acid and quercetin were identified from the RP-HPLC chromatograms. Subsequent quantification of these identified phenolics revealed tannic acid as a dominant compound in F2 (45.06 ± 0.241 μg/mg) and F7 (47.46 ± 0.189 μg/mg), along with p-coumaric acid (104.12 ± 0.226 μg/mg) and trans-cinnamic acid (108.19 ± 0.316 μg/mg) in F3 and F4 respectively. So above findings indicate that MLE is endowed with rich source of medicinally potential phytometabolites with prominent UV-blocking and antioxidant property, which offer immense pharmaceutical prospects.

Effect of Olaparib on proliferation activity and radiosensitization of human non-small cell lung cancer cells

Objective To investigate the effects of Olaparib on cell proliferation and radiosensitization of human non-small cell lung cancer cells. Methods Non-small cell lung H460 and H1299 cell lines were cultured in vitro and the cells in logarithmic growth phase were selected for experiments. MTT and colony formation assays were used to determine cell proliferation and radiosensitization, respectively. Single cell gel electrophoresis assay (comet assay) was used to detect irradiation-induced DNA damage. Results The results of MTT assay showed that Olaparib inhibited the proliferation of H460 and H1299 cells in a dose-dependent pattern (all P<0.05). H1299 cell line was more sensitive to Olaparib than H460 cells. The results of colony formation assay showed that Olaparib enhanced the radiosensitizition of H460 and H1299 cells (all P<0.05). The results of comet assay showed that Olaparib increased γ ray-induced DNA damage. Conclusions Olapani can enhance the radiosensitization of human non-small cell lung cancer cells, and the radiosensitization effect of Olaparib may be associated with the inhibition of cell proliferation and induction of irradiation-induced DNA damage. Key words: Olaparib; Non-small cell lung cancer cells; Radiosensitization; Proliferation; DNA damage

Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a 60Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a 60Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney. Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.

In vivo γ-rays Induced Initial DNA Damage and the Effect of Famotidine in Mouse Leukocytes as Assayed by the Alkaline Comet Assay

Ionizing radiation induces a variety of lesions in DNA, each of which can be used as a bio-indicator for biological dosimetry or the study of the radioprotective effects of substances. To assess gamma ray-induced DNA damage in vivo in mouse leukocytes at various doses and the effect of famotidine, blood was collected from Balb/c male mice after irradiation with 4 Gy gamma-rays at different time intervals post-irradiation. To assess the response, mice were irradiated with doses of gamma-rays at 1 to 4 Grays. Famotidine was injected intra-peritoneally (i.p) at a dose of 5 mg/kg at various time intervals before irradiation. Four slides were prepared from each sample and alkaline comet assay was performed using standard protocols. Results obtained show that radiation significantly increases DNA damage in leukocytes in a dose dependent manner (p < 0.01) when using appropriate sampling time after irradiation, because increasing sampling time after irradiation resulted in a time dependent disappearance of DNA damage. Treatment with only 5 mg/kg famotidine before 4 Gy irradiation led to almost 50% reduction in DNA damage when compared with those animals which received radiation alone. The radioprotective capability of famotidine might be attributed to radical scavenging properties and an anti-oxidation mechanism.

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668 Gy/min between 0 and 4°C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM ( P<0.001) and TL ( P<0.001) was obtained at all the radiation doses (2–8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model ( r TM=0.999 and r TL=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.

Stress-Inducible DNA Repair in Saccharomyces Cerevisiae

Saccharomyces cerevisiae shows altered radiation response under various stress conditions, such as nutrition depletion, nitrogen starvation, osmotic shock, heat shock, and mild chemical treatments. In general, the cells show higher levels of UV or gamma radiation resistance under the stress. However, not all the stress conditions affect the repair system in the same manner. For example, depletion of nitrogen supply in the growth medium has been shown to enhance the repair of gamma ray-induced DNA damage without significantly affecting the UV response of the cells. On the other hand, a mild treatment with alkali or hydrogen peroxide improves the response to UV light but not to gamma radiation. It has further been shown that the effect of these stresses are not additive, e.g., the alkali and hydrogen peroxide treatments given simultaneously show the same effect as either of them alone. Low levels of gamma and UV radiation exposures are also treated as stress in the present context. Studies show that irradiation of low-dose gamma rays results in enhanced excision repair of UV-induced pyrimidine dimers. However, in all the wild-type strains tested, none showed any effect on gamma rays response. The exposure to low doses of UV light did not show any effect on either the gamma rays or the UV response. It is suggested that the stress-induced enhancement of DNA repair can be of two types: 1) A general response to stress, which prepares the organism to survive in adverse circumstances (some of the proteins produced during this response also take part in the DNA repair), and 2) a particular response involving DNA damage, such as that caused by gamma irradiation. In this case, the DNA damage may act as a signal for enhancement of the DNA repair.

Measurement of 60Co-λ ray-induced DNA damage by capillary electrophoresis

Capillary electrophoresis was employed in this study to monitor 60Co-gamma ray-induced damage to a 1 kb DNA ladder which consists of restriction fragments ranging from 75 to 12,000 bp. DNA samples (0.5 mg/ml) were exposed to 0-60 Gy of gamma-radiation in the presence and absence of 110 mumol/l ethidium bromide (EB). The analysis showed peak broadening without significant changes in the size distribution of irradiated fragments. Radiation-induced conformational changes may account for this peak broadening. EB addition caused small increases in the retention times of DNA fragments without affecting the overall DNA damage. This indicates that the presence of intercalated EB during radiation will not stabilize the DNA against 60Co-gamma ray-induced damage.

Effect of DNA polymerase inhibitors on repair of γ ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-β- d-arabinofuranosylcytosine

The mammalian DNA polymerase inhibitors aphidicolin and 1-β- d-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by γ rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that seen with each inhibitor alone. Thus either modulates the rate of intracellular accumulation of araC 5′-triphosphate (araCTP), the presumed rate-limiting step in genotoxic action of araC, or aphidicolin and araC inhibit repair by different mechanisms. To explore these possibilities, we compared the effects of aphidicolin, araC, araCTP, and 2′,3′-dideoxythymidine triphosphate (ddTTP) on repair of DNA damage induced by 60Co γ radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in permeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 μg/ml; 10 min) and incubated for 2 h with either chemical. The extent of repair inhibition by each drug was comparable in intact and permeable cells, amounting to ∼ 1.1 sites/10 8 daltons/2 h upon exposure to upon Gy. The active metabolite of araC, araCTP, did not inhibit repair in intact cells, but did so in permeable cells to an extent within the range of that seen with araC or aphidicolin alone. The incidence of DNA strand breaks accumulating in γ-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was ∼ 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrations of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populations, we have reason to be confident that within the short post-irradiation period considered here, the observed drug-accumulated breaks truly represent functional repair inhibition adn not merely abortive pathological responses. We thus conclude that (1) the accumulation of araCTP in intact cells is not limiting the ability of araC to inhibit DNA repair; and (2) the mode of the inhibitory action of araC/araCTP on γ ray repair is different from that of aphidicolin. In constrast to the observations with these chemicals, ddTTP (20 μM), a potent inhibitor of DNA polymerase β, did not produce any measurable effect on DNA repair in γ-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair. These results strongly suggest that DNA polymerase β plays no significant role in the repair of γ radioproducts in human fibroblasts. The permeabilized cell system adopted here does not adversely influence the repair capability of human fibroblasts and may thus prove useful in studying the enzymology of DNA repair in proliferating cultures following exposure to ionizing radiation and other genotoxic agents.

Enhancement of X ray induced DNA damage by pre-treatment with halogenated pyrimidine analogs

The use of the halogenated pyrimidine analogs, including bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd), as potential clinical radiosensitizers is an area of renewed clinical research. Cellular radiation effects of these analogs using clinically achievable steady state plasma levels (10 −7−10 −5 M) were measured in vitro using exponentially growing Chinese hamster V79 cells. Radiation response was determined by clonogenic survival and by the production of DNA single strand (SSB) and double strand breaks (DSB) using filter elution techniques. Replacement of thymidine in DNA by BrdUrd or IdUrd was also determined. Drug exposure of BrdUrd or IdUrd for two population doublins (17 hr) prior to irradiation resulted in a progressive reduction of n̄ and D 0 compared to untreated controls over the drug dose range of 10 −7-10 −5 M. The percent thymidine replacement increased from 1% at 10 −7 M to 5% at 10 −6 M to 23% at 10−5 M. Using a 17 hr exposure at 10 −5 of BrdUrd of IdUrd, the production of SSB and DSB was increased by ≥2× and ≥1.5× respectively. No differences in the kinetics of repair of SSB and DSB were found following 3 hr of post-irradiation repair. We conclude that in this in vitro model, there appears to be a direct relationship between percent thymidine replacement, reduction of radiation survival parameters (n, D 0), and the production of DNA strand breaks in the clinically achievable dose range of these halogenated pyrimidine analogs. These filter elution assays may be adapted to in vivo studies and possibly may allow for monitoring of radiation-induced DNA damage and repair in clinical tumor specimens treated with these radiosensitizers.

Radiosensitization by diamide analogs and arsenicals

Several analogs of the glutathione (GSH) oxidizing reagent diamide (diazenedicarboxylic acid bis[(N,N′-diethylamide)] were tested as radiosensitizers of aerobic cells. In general, radiosensitization correlates with the rate of reaction with cellular reducing agents and occurs only when the reductive capacity of the cell is exceeded. SR-4077, [diazenedicarboxylic acid bis(N,N-piperidide], is particularly suitable for mechanistic studies, because it is less cytotoxic than diamide, but is equally reactive toward cellular GSH. SR-4077 sensitizes CHO cells to X rays under aerobic conditions, even when the drug is added after irradiation. Radiosensitization is expressed both as a change in the exponential slope of the radiation cell survival curve and as a decrease in the shoulder of the survival curve. Phenylarsine oxide, a dithiol-binding reagent, sensitizes aerobic CHO cells to X rays by modification of the shoulder of the survival curve. The results are consistent with the hypothesis that the shoulder-modifying effect of GSH oxidants is caused by the loss of protein thiols, which might be involved in the repair of X ray-induced DNA damage.