Number Of DNA Breaks Research Articles

Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

Biological effect of microwave electromagnetic radiation: methods of registration and protection

The development of modern electronics, communications, high-tech production and other technologies lead to a significant increase in the intensity of microwave electromagnetic radiation. This article discusses the problem of registration (including biological detection) and protection from microwave electromagnetic radiation. To consider the method of integral biological and instrumental recording of the intensity of microwave electromagnetic radiation and to evaluate the method of protection using a passive multifrequency re-emitter generator with a radio holographic antenna (trade mark EQVILUM). A series of experiments were carried out to determine the number of double-strand breaks in DNA (DS DNA) by the foci of γH2AX repair proteins, to assess proliferative activity and clonogenic ability after 3 hours of exposure to microwave electromagnetic radiation with a frequency radiation of 1200 MHz both with shielding and without it on a culture of human lung fibroblasts (MRC5 line). It is shown that no changes in the level of DS DNA were revealed in experimental cells, however, a decrease in the clonogenic ability of irradiated cells without shielding was observed against the background of their high proliferative activity. The mechanisms of the observed cellular effects formation are unclear and we suppose that they can be associated with both a change in the metabolism of irradiated cells and with the triggering of certain intracellular mechanisms initiated by electromagnetic radiation (EMR). This study made it possible to identify effective ways of integral assessment of the biological effects of microwave electromagnetic radiation as well as ways of protection against it. A film microcircuit – an autonomous module of a passive multi-frequency re-emitter generator with a radio holographic antenna (EQVILUM trademark) has shown its high efficiency in protection against man-made microwave electromagnetic radiation and can be recommended as an additional means of effective protection.

Calculation of direct and indirect damages of Auger electron-emitting radionuclides based on the atomic geometric model: A simulation study using Geant4-DNA toolkit

This study represents the detailed calculation of damage by Auger electrons of radionuclides 99mTc,111In,125I and 123I to DNA molecules at cellular scales, using Geant4-DNA toolkit. The average number of breaks in DNA are shown as a function of distance from the center of the DNA axis to position of Auger electron decay and compared with results obtained for different models of DNA used by others. The highest damage occurs for electrons with energies below 1 keV, and in proximity of DNA molecule. The calculation of DSB yield per Gy per Dalton is carried out for above radionuclides. The most considerable damage occurs when the distance between an Auger electron-emitting atom and DNA is about 2.5 nm, and 50% decreases happen when the distance is about 3.5 to 4 nm. DSB yield are comparable with other experimental and simulation data, and show a good agreement between our results and other works.

DNA Damage and p53 Restrict Proliferation of Müller Cells in the Mouse Retina in Response to the Influence of N-Methyl-N-Nitrosourea

Systemic administration of N-methyl-N-nitrosourea in rats resulted in the death of retinal photoreceptors followed by differentiation of retinal Muller glial cells into photoreceptor-like cells [27]. However, mammalian Muller glial cells exhibit an extremely limited proliferative capacity, which correlates with the expression of histone γH2AX and p21 protein. These proteins are known to be components of the cellular response to DNA damage [26]. The restriction of proliferation of human Muller glial cells prevents retinal replacement therapy by cell transplantation. On the other hand, the mechanism that limits the proliferation of Muller glia in the mammalian retina remains to be elucidated. We examined the Muller glial proliferative response and the DNA damage response in Muller glia in the postreplicative stage, as well as the expression of the p53 protein in response to the influence of retinotoxic N-methyl-N-nitrosourea. It was shown that N‑methyl-N-nitrosourea induced retinal degeneration in mice via apoptosis of photoreceptors, whereas the other retinal layers retained intact morphology. Nevertheless, the formation of DNA breaks and alkali-labile sites was observed in all retinal cells 5 h after the N-methyl-N-nitrosourea injection; these formations completely disappeared 15 h after N-methyl-N-nitrosourea injection. By 72 h, a significant increase in the number of DNA breaks in Muller glial cells was observed. The absence of bromodeoxyuridine incorporation into the retinal cells later testifies to the absence of proliferation of Muller glial cells and DNA repair synthesis. At the same time, an increased expression of the p53 protein, a universal marker of DNA damage, was observed in the retina. Thus, our findings support the concept of the “DNA damage response” with respect to Muller glial cells, according to which the DNA damage in Muller glial cell is related to the restricted proliferation of these cells in mice. Postreplicative repair is considered as a probable mechanism of the formation of DNA breaks in postreplicative Muller glial cells.

Chemopreventive effect and angiogenic activity of punicalagin isolated from leaves of Lafoensia pacari A. St.-Hil.

Punicalagin is the major ellagitannin constituent from leaves of Lafoensia pacari, a Brazilian medicinal plant widely used for the treatment of peptic ulcer and wound healing. Genotoxic, cytotoxic, antigenotoxic, and anticytotoxic effects of punicalagin were assessed using micronucleus (MN) test and comet assay in mice. Due to the extensive use of L. pacari in the wound healing process, we also assessed the angiogenic activity of punicalagin using the chick chorioallantoic membrane (CAM) angiogenic assay. The highest dose of punicalagin (50mg/kg) showed significant cytotoxic effect by MN test and in the co-treatment with cyclophosphamide (CPA), this cytotoxicity was enhanced. Co-treatment, pre-treatment and post-treatment of punicalagin with CPA led to a significant reduction in the number of DNA breaks and in the frequency of CPA-induced MN, indicating antigenotoxic effect. Using the CAM model, punicalagin exhibited angiogenic activity in all doses mainly at the lowest concentration (12.5μg/μL). Therefore, these findings indicate an effective chemopreventive role of punicalagin and a high capacity to induce DNA repair. Also, the angiogenic activity presented by punicalagin in this study could contribute for the processes of tissue repairing and wound healing.

Genotoxic effects of the antimalarial drug lumefantrine in human lymphocytes in vitro and computational prediction of the mechanism associated with its interaction with DNA.

Lumefantrine (LF) is an aryl-amino alcohol antimalarial drug used in artemisinin-based combination therapies against malaria worldwide. In this study, we investigated the genotoxic effects of LF in human lymphocytes in vitro, and the potential noncovalent interaction of LF with DNA using a 3D DNA-docking model. The number of DNA breaks and the frequency of nuclear buds (NBUDS) was significantly increased (P < 0.01 and P < 0. 05, respectively) at LF concentrations of 60, 80, and 100 µg/mL (LF60, LF80, and LF100, respectively). Frequency (‰) of micronuclei (MN) formation also increased after LF treatments. However, this was only significant for LF100 (P = 0.01) and LF80 (P = 0.001). LF did not affect the frequency of nucleoplasmic bridges (NPBs) (P = 0.12) or the nuclear division index (NDI) (P = 0.32). Computational analysis suggests that LF may interact noncovalently with DNA via the DNA minor groove surface with a predicted binding affinity energy of -7.2 kcal/mol and showing a favorable shape complementary to this groove. Our results suggest that LF has clastogenic effects in human lymphocytes in vitro due to noncovalent interaction with the minor groove of DNA.

Assessment of yeast chromosome XII instability: Single chromosome comet assay

The tools and techniques used in single-cell analysis of DNA damage in yeast Saccharomyces cerevisiae are limited. In this study, we modified the single cell gel electrophoresis assay, namely, the single chromosome comet assay based on DNA break analysis, at the chromosomal level. We studied the largest yeast chromosome XII, which contains the rDNA locus, and we investigated its instability using cell cycle checkpoint-, DNA damage- and antioxidative defence-deficient, and lifespan-deregulated yeast mutant strains. Moreover, we compared chromosome XII instability with the variability of nucleolar rDNA fluorescence signals. Three single-gene-deletion strains, cells lacking single-stranded DNA endonuclease, Rad1p; NAD+-dependent histone deacetylase, Sir2p; and gamma glutamylcysteine synthetase, Gsh1p, were more prone to chromosome XII instability compared to corresponding wildtype strains, indicating that DNA damage repair machinery, chromatin silencing and redox homeostasis may contribute to genome stability. Elevation in the number of DNA breaks was correlated with a high variability in the levels of nucleolar rDNA in the Δrad1 background, while unaffected chromosome XII and low variability in nucleolar rDNA fluorescence signals were observed in the Δtor1 longevity mutant. Taken together, the single chromosome comet assay may be successfully used to study DNA damage at the chromosomal level, which might be overlooked using whole population analysis on DNA breaks with PFGE separation.

Elevated APEX1 Endonuclease Is Associated with Increased DNA Breaks and Instability in Myeloma

Abstract 1805Exposure to DNA-damaging agents of both endogenous and exogenous origin, including ultraviolet (UV) radiation, give rise to apurinic/apyrimidinic (AP) or abasic sites in genome, probably the most common mutagenic DNA lesions. APEX1, the key protein involved in the repair of AP sites through base excision repair, plays an important role in the maintenance of genomic integrity. We have observed that APEX1 is overexpressed in multiple myeloma (MM) cell lines and primary cells. To evaluate its functional role MM cells were transduced with control (CS) or APEX1-specific lentivirus based shRNAs, targeting two different regions of the gene. Both shRNAs mediated >70% suppression of APEX1 expression. Control and APEX1-Knock out (KO) cells were treated with UV (20 J/m2), cultured for 24 hrs and evaluated for both the cell viability and phosphorylated-H2AX (p-H2AX), a marker for DNA breaks. Although cell viability did not change, the amount of p-H2AX was reduced in APEX1-KO cells, indicating that elevated APEX1 is associated with induction of DNA breaks in MM cells. To directly evaluate DNA breaks, transduced myeloma cells were exposed to UV (20 J/m2), cultured for a short period, and evaluated by comet assay, a sensitive gel-based technique for detection and assessment of DNA breaks in individual cells. The fraction of cells with DNA breaks and the intensity and size distribution of comets indicating the number of DNA breaks, were evaluated. In cells transduced with control shRNA, very large, large, medium and small comets were observed in 24%, 18%, 23% and 23% of cells, respectively. However in APEX1-KO MM cells, very large and large comets were not observed at all, comets of medium size were observed in 13% and small comets observed in 40% of the cells. A large fraction (47%) of APEX1-KO cells did not have any comets, indicating minimal or no DNA breaks even after UV exposure, under the experimental conditions used. These data show that the suppression of APEX1 significantly reduces the acquisition DNA breaks in MM cells following exposure to UV. We also evaluated the impact of reduced APEX1 levels on DNA integrity in MM cells. Control (non targeting shRNA transduced) and APEX1-KO cells were transfected with a plasmid, encoding secretory Gaussia luciferase (GLuc). Cells were plated at equal density in triplicate dishes, and beginning at 8 hrs, GLuc activity was measured in the supernatants at 12-hr intervals. In preliminary experiments we have found that plasmid DNA is more stable in APE-KO, compared to control cells, indicating that suppression of APEX1 in myeloma cells may stabilize DNA. We have also observed that chemical inhibition of endonuclease suppresses acquistionof new genomic change sin MM. We are currently evaluating the impact of APEX1 KO on acquisition of genomewide changes in myeloma cells over time. In summary, our data shows that elevated APEX1 endonuclease makes myeloma cells vulnerable to acquire DNA breaks following exposure to intrinsic or extrinsic DNA damaging agents. APEX-1 thus may be an important target to further understandgenomic instability in MM. Disclosures:No relevant conflicts of interest to declare.

Antimutagenic activity of wheat β-purothionin Tk-AMP-BP

Antimutagenic activity on human RD cells was studied for beta-purothionin Tk-AMP-BP isolated from seeds of wheat Triticum kiharae, which has a higher stress resistance. Cadmium chloride at 5 x 10(-6) M was used as a mutagen. The numbers of DNA breaks in mutagen-treated and intact cells were inferred from the single-stranded to double-stranded DNA ratio and expressed as protection coefficients. The protective effect was simultaneously assayed for aqueous plant extracts known to possess antioxidant properties. Wheat thionin was the most active among all of the antimutagens examined; its protection coefficient reached 85-88% at micromolar peptide concentrations (8-32 microg/ml). Thus, wheat beta-purothionin was for the first time demonstrated to be highly efficient in protecting human cell DNA from the damaging effect of cadmium chloride.

Comparison of DNA Breaks at Entrance Channel and Bragg Peak Induced by Fast C6+ Ions

When energetic carbon ion beam (GeV range) goes through the matter, inelastic processes such as electronic ionization, molecular and nuclear fragmentation occur. For carbontherapy (hadrontherapy) purpose, it is of interest to compare the number of DNA breaks -single SSB or double DSB- for a given dose at the entrance channel and at the Bragg peak to look for a possible differential effect in the number of DNA breaks induced at these two locations. Samples of free plasmids DNA and complexes of plasmids DNA added with molecules containing platinum have been placed at different locations of an experimental setup simulating penetration depths of the ion beam in water and irradiated by carbon ions 290 MeV/amu. The DNA breaks have been quantified by subsequent electrophoresis on agarose gels. To disentangle the respective role of the direct and indirect effect, a free radical scavenger of hydroxyl radicals HO degree-dimethylsulfoxide DMSO- has been added in some of the experiments. In the range of Linear Energy Transfer-LET 13 - 110 keV/microm-, the number of the DSB was found to be constant versus the LET for a given dose. Contrary, the number of the SSB decreases at the Bragg peak compared to the entrance channel. In the presence of platinum, the number of single and double breaks was considerably enhanced, and follows a similar behaviour than in the free-DNA experiments. Quantitative results on DNA damages do not show significant enhancement due to the nuclear or to the molecular fragmentation in the present experiments.

Study of the genotoxic effects of a proline-rich polypeptide using the comet assay

The genotoxicity of a proline-rich polypeptide was investigated in vitro in the human myeloid leukemia KCL-22 cell line. A comet assay, based on single-cell gel electrophoresis, was used for the analysis of the DNA damage and reparation rate. After electrophoresis, images resembling comets were formed; the intensity of the “tail” relative to the “head” reflected the number of DNA breaks. We demonstrated that the level of DNA damage depended on the polypeptide concentration and incubation time. Estimation of DNA damage levels by the comet assay is recommended for the evaluation of the genotoxicity of new pharmaceuticals.

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C6+ and Fe26+

Purpose: In order to study the role of the Linear Energy Transfer (LET) of fast atomic ions in platinum-DNA complexes inducing breaks, DNA Plasmids were irradiated by C6+ and Fe26+ ions.Material and methods: DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C6+ ions and Fe26+ ions. The LET values ranged between 13.4 keV/μm and 550 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added.Results: In all experiments, a significant increase in DNA strand breaks was observed when platinum was present. The yield of breaks induced per Gray decreased when the LET increased. The yield of single and double strand breaks per plasmid per track increased with the LET, indicating that the number of DNA breaks per Gray was related to the number of tracks through the medium.Conclusions: These findings show that more DNA breaks are induced by atomic ions when platinum is present. This effect increases for low LET heavy atoms. As DSB induction may induce cell death, these results could open new perspectives with the association of hadrontherapy and chemotherapy. Thus the therapeutic index might be improved by loading the tumour with platinum salts.

Measurement of DNA adducts and strand breaks in dab ( Limanda limanda) collected in the field: effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA damage

In the Eastern English Channel, the potential application of the comet assay and post-labelling technique in dab was evaluated for genotoxicity monitoring of the marine environment. The effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA lesions were also studied. Female and male dab of two class of size (juvenile and adult) were collected by trawling in different sites in Seine Bay and Somme Bay during September 2001. Single-strand breaks and adducts were, respectively, measured in erythrocytes and the liver. Results obtained for the adult female were compared with those collected during a first cruise in March 2001 [Akcha et al., Mutat Res. 534 (1–2) (2003) 21]. Significant effects of sex and age were demonstrated on the level of strand breaks. Moreover, a significant interaction between age and sex was shown that might indicate the complex influence of other factors on the extent of DNA damage (i.e. reproduction status). In the adult dab, the level of breaks is higher in the male than in the female, whereas the opposite trend was observed for the juvenile. Whatever the sex, the number of DNA breaks is higher in the adult than in the juvenile. For the female dab, significant differences were observed with the comet assay between the Seine Bay and the Somme Bay in March but not in September. This may be due to seasonal variations in the formation of DNA lesions related to variations in lipid content and levels of biotransformation activities and/or to spawning cycles. The presence of genotoxic substances in the study areas was also confirmed by the detection of DNA adducts in each sample analysed. Whereas no effect was shown on the total level of adducts for the tested biotic and abiotic factors, qualitative differences in adduct profiles were observed for each of these factors. For the female dab, comparison of adduct profiles obtained in March and September with one generated by hepatic microsomal activation in dab of a PAH mixture indicated a PAH contamination of the study areas in autumn. These results show the importance of studying the effects of biotic and abiotic factors on the genotoxic endpoints considered to correctly assess the contribution of chemical contamination to the measured biological responses.

Development of new dosimetry using extended DNA fibers

We applied fluorescent microscopy to monitor the damage of DNA upon exposure to γ radiation. Our developed dosimetry demonstrated that the number of breaks in DNA is proportional to the dose of the irradiation but is not dependent on dose rate of the irradiation and the GC content of DNA.

Development of New Dosimetry Using Extended DNA Fibers

We applied fluorescent microscopy to monitor the damage of DNA upon exposure to γ radiation. Our developed dosimetry demonstrated that the number of breaks in DNA is proportional to the dose of the irradiation but is not dependent on dose rate of the irradiation and the GC content of DNA.

The Repair of DNA Damage Induced in Human Peripheral Lymphocytes with Styrene Oxide

Isolated human peripheral lymphocytes were treated in vitro with styrene-7, 8-oxide (SO) and the kinetics of the repair of induced DNA damage was assessed by comet assay during further incubation of lymphocytes. Using a modified assay we measured simultaneously the number of single strand breaks in DNA (SSBs) and the sites sensitive to endonuclease III (endo III) that most probably represent abasic sites in DNA molecules. SO induced DNA damage in a dose-dependent manner and both SSBs and endo III sites were removed from the DNA by a repair process with a half time about 2-4 hours. The damage was repaired completely within 12 hours after the treatment.

The estimation of molecular and cytogenetic effects in mice exposed to chronic low dose gamma-radiation

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate gamma-radiation (62 cGy/year) for 40, 80, 120, 210, and 365 days. After 40 days of exposure (6.7 cGy), spleen lymphocyte susceptibility to hydrogen peroxide was shown to increase. However, beginning from the day 120 of the treatment (20.4 cGy), the opposite effect was observed. An increase in number of the DNA-protein crosslinks was recorded in spleen lymphocytes only on day 40 of the experiment. The number of DNA breaks increased significantly beginning from day 120 of the experiment, as shown by the DNA-comet method. On the day 210 of irradiation, the frequency of abnormal sperm heads in the mice significantly increased. The number of normochromatic micronucleated erythrocytes of the peripheral blood remained unchanged.

The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway.

Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB. © 2001 Cancer Research Campaign http://www.bjcancer.com

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes.

Phorbol ester treatment of granulocytes triggers release of superoxide (O2.-) and a concomitant burst of DNA strand breaks. The relationship between the amount of O2.- and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O2.- generated over a 40-min period by 1 x 10(6) granulocytes/mL, a discontinuous "10-min pulse" method employing cytochrome c was used; 140 nmol O2.- per 1 x 10(6) cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O2.- released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1-10 mM) and staurosporine (2-10 nM) both inhibited O2.- production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O2.-. Zinc chloride (50-200 microM) inhibited both O2.- and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O2.- production more effectively than it inhibited DNA breaks. O2.- dismutes to H2O2, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H2O2 had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O2.- or H2O2 in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway.

Elevated frequency of p53-independent apoptosis after irradiation increases levels of DNA breaks in ataxia telangiectasia lymphoblasts

. Ataxia telangiectasia is a recessive genetic disease featuring cerebellar degeneration, developmental abnormalities, high cancer risk, immunodeficiency, and radiosensitivity. Increased levels of unrepaired DNA breaks have been observed in irradiated ataxia telangiectasia cells compared to normal cells but no specific DNA break rejoining rate deficiency has been defined. Alterations in radiation-induced p53-dependent apoptosis have been reported for ataxia telangiectasia cells. This study investigated the radiation response of ataxia telangiectasia lymphoblastoid cells using the comet assay and uncovered a new feature of this technique, namely its capacity to preferentially detect living cells. It is shown here that early after exposure to gamma -rays, ataxia telangiectasia lymphoblasts exhibit an elevated frequency of cells committed to die via apoptosis. The observed apoptosis, which is likely to be independent of p53, leads to a higher number of DNA breaks during the first 3 h post irradiation in ataxia telangiectasia cells, relative to controls. Apart from cells undergoing apoptosis, ataxia telangiectasia lymphoblasts have an identical capacity to rejoin radiation-induced DNA breaks as controls. Results suggest that p53-independent apoptosis may contribute to the radiosensitivity and the immune defects of ataxia telangiectasia patients.