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Sample records for decreased dna damage

  1. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

  2. A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence.

    Science.gov (United States)

    Kikuchi, Ikue; Nakayama, Yuji; Morinaga, Takao; Fukumoto, Yasunori; Yamaguchi, Naoto

    2010-05-04

    Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration-dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin-induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated beta-galactosidase activity. In DNA damage-induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage-induced senescence.

  3. Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

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    Wojewodzka, M., E-mail: marylaw@ichtj.waw.pl [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Gradzka, I.; Buraczewska, I.; Brzoska, K.; Sochanowicz, B. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Goncharova, R.; Kuzhir, T. [Institute of Genetics and Cytology, Belarussian National Academy of Sciences, Minsk (Belarus); Szumiel, I. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland)

    2009-12-01

    Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

  4. Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

    Science.gov (United States)

    Pedro, Dalila F N; Ramos, Alice A; Lima, Cristovao F; Baltazar, Fatima; Pereira-Wilson, Cristina

    2016-02-01

    Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption. Copyright © 2015 John Wiley & Sons, Ltd.

  5. Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Kalpana Mujoo

    2017-11-01

    Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

  6. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation

    Energy Technology Data Exchange (ETDEWEB)

    Korzeneva, Inna B., E-mail: inna.korzeneva@molgen.vniief.ru [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Kostuyk, Svetlana V.; Ershova, Liza S. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation); Osipov, Andrian N. [Federal Medial and Biological Center named after Burnazyan of the Federal Medical and Biological Agency (FMBTz named after Burnazyan of FMBA), Moscow (Russian Federation); State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Zhivopisnaya, 46, Moscow, 123098 (Russian Federation); Zhuravleva, Veronika F.; Pankratova, Galina V. [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Porokhovnik, Lev N.; Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation)

    2015-09-15

    Highlights: • The chronic exposure to low-dose IR induces DSBs in human lymphocytes (TM index). • Exposure to IR decreases the level of human circulating DNA (cfDNA index). • IR induces an increase of DNase1 activity (DNase1 index) in plasma. • IR induces an increase of the level of antibodies to DNA (Ab DNA index) in plasma. • The ratio cfDNA/(DNase 1 × Ab DNA × TM) is a potential marker of human exposure to IR. - Abstract: The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism’s cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1 × Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab

  7. Keratin23 (KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Karin Birkenkamp-Demtröder

    Full Text Available Keratin 23 (KRT23 is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.

  8. Oral and intraperitoneal administration of quercetin decreased lymphocyte DNA damage and plasma lipid peroxidation induced by TSA in vivo.

    Science.gov (United States)

    Chan, Shu-Ting; Lin, Yi-Chin; Chuang, Cheng-Hung; Shiau, Rong-Jen; Liao, Jiunn-Wang; Yeh, Shu-Lan

    2014-01-01

    Our previous study showed that quercetin enhances the anticancer effect of trichostatin A (TSA) in xenograft mice given quercetin intraperitoneally (10 mg/kg, 3 times/week). Herein, we investigate whether quercetin administered orally exerts such an effect and prevents the cytotoxic side effects of TSA. We found that quercetin given orally (20 and 100 mg/kg, 3 times/week) failed to enhance the antitumor effect of TSA although it increased the total quercetin concentration more than quercetin administered intraperitoneally in the plasma. The compound quercetin-3-glucuronide (Q3G) increased the most. However, quercetin administered intraperitoneally increased the total quercetin level in tumor tissues more than oral quercetin. Oral and intraperitoneal administration of quercetin similarly decreased lymphocyte DNA damage and plasma lipid peroxidation level induced by TSA. Furthermore, we found that the enhancing effect of Q3G on the antitumor effect of TSA and the incorporation of Q3G was less than that of quercetin in A549 cells. However, we found that A549 cells possessed the ability to convert Q3G to quercetin. In conclusion, different from quercetin administered intraperitoneally, quercetin administered orally failed to enhance the antitumor effect of TSA because of its metabolic conversion. However, it prevented TSA-induced DNA damage and lipid peroxidation.

  9. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  10. The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

    Science.gov (United States)

    Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

    2017-09-01

    Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

  11. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation.

    Science.gov (United States)

    Korzeneva, Inna B; Kostuyk, Svetlana V; Ershova, Liza S; Osipov, Andrian N; Zhuravleva, Veronika F; Pankratova, Galina V; Porokhovnik, Lev N; Veiko, Natalia N

    2015-09-01

    The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly - at survival of the cells, including the cells with the damaged DNA. Copyright © 2015. Published by Elsevier B.V.

  12. Immunoassay of DNA damage

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Santella, R.M.

    1988-01-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

  13. Immunoassay of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Gasparro, F P; Santella, R M

    1988-09-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

  14. DNA damage and polyploidization.

    Science.gov (United States)

    Chow, Jeremy; Poon, Randy Y C

    2010-01-01

    A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

  15. Soybean and fish oil mixture increases IL-10, protects against DNA damage and decreases colonic inflammation in rats with dextran sulfate sodium (DSS colitis

    Directory of Open Access Journals (Sweden)

    Carvalho Patrícia O

    2010-07-01

    Full Text Available Abstract It was investigated whether dietary polyunsaturated fatty acids (PUFA could influence colonic injury, tissue DNA damage, cytokines and myeloperoxidase activity (MPO and plasma corticosterone in DSS-induced colitis rats. Male weaning Wistar rats were fed for 47 days with an AIN-93 diet with control (C, fish (F or a mixture of fish and soybean oil (SF. The colitis was induced from day 36 until day 42 by 3% DSS in drinking water. On day 48, blood samples were collected for corticosterone determination. The distal colon was excised for histological analysis and to quantify the cytokine (IL-4, IL-10 and INF-γ, MPO and DNA damage. The disease activity index (DAI was recorded daily during colitis induction. The DAI, MPO, histological analyses showed decreases only in the SF group compared with the C group. IL-10 was increased and DNA damage was reduced in the groups F and SF, and an inverse correlation between these variables was found. There were no differences in corticosterone, IFN-γ and IL-4 levels. Soybean and fish oil mixture may be effective in improving colonic injury and DNA damage, and it could be an important complementary therapy in UC to reduce the use of anti-inflammatory drugs and prevent colorectal cancer.

  16. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.

    1978-01-01

    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

  17. DNA Damage and Pulmonary Hypertension

    Science.gov (United States)

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  18. Keratin23 (KRT23) knockdown decreases proliferation and affects the DNA damage response of colon cancer cells

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Hahn, Stephan; Mansilla, Francisco

    2013-01-01

    correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed...... response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced......Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation...

  19. Decreased paraoxonase1 activity and increased malondialdehyde and oxidative DNA damage levels in primary open angle glaucoma

    Science.gov (United States)

    Mumcu, Ugur Yilmaz; Kocer, Ibrahim; Ates, Orhan; Alp, H. Hakan

    2016-01-01

    To investigate the malondialdehyde (MDA) levels, paraoxonase1 (PON1) activity and 8-hydroxy 2-deoxyguanosine (8-OHdG) levels in the primary open angle glaucoma (POAG) patient. Blood samples from 52 healthy individuals and 53 patients with POAG were analyzed for MDA and 8-OHdG by HPLC (high-performance liquid chromatography) and PON1 by spectrophotometry. The data obtained were analyzed statistically. MDA levels were 10.46±8.4 and 4.70±1.79 µmol; PON1 levels were 121±39.55 and 161.62±60.22 U/mL; and 8-OHdG values were 1.32±0.53/106 dG and 0.47±0.27/106 dG in the POAG patients and the control group, respectively. The difference was significant in MDA levels, 8-OHdG levels and PON1 activity in POAG patients in comparison with controls (P<0.001). We concluded that the observed increase in MDA and 8-OHdG levels may be correlated with decreased PON1 activity. Oxidative stress plays an important role in glaucoma development. PMID:27803873

  20. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

    Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

    2015-06-15

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  1. DNA damage in neurodegenerative diseases

    International Nuclear Information System (INIS)

    Coppedè, Fabio; Migliore, Lucia

    2015-01-01

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  2. DNA Damage, Mutagenesis and Cancer

    Directory of Open Access Journals (Sweden)

    Ashis K. Basu

    2018-03-01

    Full Text Available A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

  3. DNA damage by Auger emitters

    International Nuclear Information System (INIS)

    Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.

    1988-01-01

    125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

  4. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    Stelow, R.B.

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

  5. Vitamin C for DNA damage prevention

    International Nuclear Information System (INIS)

    Sram, Radim J.; Binkova, Blanka; Rossner, Pavel

    2012-01-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  6. Vitamin C for DNA damage prevention

    Energy Technology Data Exchange (ETDEWEB)

    Sram, Radim J., E-mail: sram@biomed.cas.cz [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic); Binkova, Blanka; Rossner, Pavel [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic)

    2012-05-01

    The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2 Prime -deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 {mu}mol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with {gamma}-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 {mu}mol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 {mu}mol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

  7. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  8. Radiation damage to DNA constituents

    International Nuclear Information System (INIS)

    Bergene, R.

    1977-01-01

    The molecular changes of the DNA molecule, in various systems exposed to inoizing radiation, have been the subject of a great number of studies. In the present work electron spin resonance spectroscopy (ESR) has been applied to irradiated crystalline systems, in particular single crystals of DNA subunits and their derivatives. The main conclusions about the molecular damage are based on this technique in combination with molecular orbital calculations. It should be emphasized that the ESR technique is restricted to damage containing unpaired electrons. These unstable intermediates called free radicals seem, however, to be involved in all molecular models describing the action of radiation on DNA. One of the premises for a detailed theory of the radiation induced reactions at the physico-chemical level seems to involve exact knowledge of the induced free radicals as well as the modes of their formation and fate. For DNA, as such, it is hardly possible to arrive at such a level of knowledge since the molecular complexity prevents selective studies of the many different radiation induced products. One possible approach is to study the free radicals formed in the constituents of DNA. In the present work three lines of approach should be mentioned. The first is based on the observation that radical formation in general causes only minor structural alterations to the molecule in question. The use of isotopes with different spin and magnetic moment (in particular deuterium) may also serve a source of information. Deuteration leads to a number of protons, mainly NH - and OH, becoming substituted, and if any of these are involved in interactions with unpaired protons the resonance pattern is influeneed. The third source of information is molecular orbital calculation. The electron spin density distribution is a function in the three dimensional space based on the system's electronic wave functions. This constitutes the basis for the idea that ESR data can be correlated with

  9. The DNA damage response during mitosis

    NARCIS (Netherlands)

    Heijink, Anne Margriet; Krajewska, Malgorzata; van Vugt, Marcel A. T. M.

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

  10. DNA Damage, Repair, and Cancer Metabolism

    Science.gov (United States)

    Turgeon, Marc-Olivier; Perry, Nicholas J. S.; Poulogiannis, George

    2018-01-01

    Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors. PMID:29459886

  11. DNA damage in plant herbarium tissue.

    NARCIS (Netherlands)

    Staats, M.; Cuenca, A.; Richardson, J.E.; Ginkel, R.V.; Petersen, G.; Seberg, O.; Bakker, F.T.

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of

  12. DNA damage and repair in plants

    International Nuclear Information System (INIS)

    Britt, A.B.

    1996-01-01

    The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

  13. DNA damage repair and radiosensitivity

    International Nuclear Information System (INIS)

    Suzuki, Norio

    2003-01-01

    Tailored treatment is not new in radiotherapy; it has been the major subject for the last 20-30 years. Radiation responses and RBE (relative biological effectiveness) depend on assay systems, endpoints, type of tissues and tumors, radiation quality, dose rate, dose fractionation, physiological and environmental factors etc, Latent times to develop damages also differ among tissues and endpoints depending on doses and radiation quality. Recent progress in clarification of radiation induced cell death, especially of apoptotic cell death, is quite important for understanding radiosensitivity of tumor cure process as well as of tumorigenesis. Apoptotic cell death as well as dormant cells had been unaccounted and missed into a part of reproductive cell death. Another area of major progress has been made in clarifying repair mechanisms of radiation damage, i.e., non-homologous end joining (NHEJ) and homologous recombinational repair (HRR). New approaches and developments such as cDNA or protein micro arrays and so called informatics in addition to basic molecular biological analysis are expected to aid identifying molecules and their roles in signal transduction pathways, which are multi-factorial and interactive each other being involved in radiation responses. (authors)

  14. Oxidative DNA damage & repair: An introduction.

    Science.gov (United States)

    Cadet, Jean; Davies, Kelvin J A

    2017-06-01

    This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. DNA damages induced by Ar F laser

    Energy Technology Data Exchange (ETDEWEB)

    Chapel, C.; Rose, S.; Chevrier, L.; Cordier, E.; Courant, D. [CEA Fontenay-aux-Roses, 92 (France). Dept. de Radiobiologie et de Radiopathologie

    2006-07-01

    The photo ablation process used in corneal refractive surgery by the Argon Fluoride (Ar F) laser emitting in ultraviolet C at 193 nm, exposes viable cells round the irradiated zone to sub ablative doses (< 400 joules.m -2). Despite that DNA absorption is higher at 193 nm than 254 nm, cytotoxicity of 193 nm laser radiation is lower than radiation emitted by 254 nm UV-C lamps. In situ, DNA could be protected of laser radiation by cellular components. Consequently, some authors consider that this radiation does not induce genotoxic effect whereas others suspect it to be mutagenic. These lasers are used for fifteen years but many questions remain concerning the long term effects on adjacent cells to irradiated area. The purpose of this study is to describe the effect of 193 nm laser radiation on DNA of stromal keratocytes which are responsible of the corneal structure. The 193 nm laser irradiation induces directly DNA breakage in keratocytes as it has been shown by the comet assay under alkaline conditions. Two hours post irradiation, damages caused by the highest exposure (150 J.m-2) are not repaired as it has been measured with the Olive Tail Moment (product of tail length and tail DNA content). They give partly evidence of induction of an apoptotic process in cells where DNA could be too damaged. In order to characterize specifically double strand breaks, a comparative analysis by immunofluorescence of the H2 Ax histone phosphorylation (H2 Ax) has been performed on irradiated keratocytes and unirradiated keratocytes. Results show a dose dependent increase of the number of H2 Ax positive cells. Consequences of unrepaired DNA lesions could be observed by the generation of micronuclei in cells. Results show again an increase of micronuclei in laser irradiated cells. Chromosomal aberrations have been pointed out by cytogenetic methods 30 mn after irradiation. These aberrations are dose dependent (from 10 to 150 J.m-2). The number of breakage decreases in the long run

  16. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    Science.gov (United States)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  17. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1985-07-01

    Radiation damage to DNA results from the direct interaction of radiation with DNA where positive ions, electrons and excited states are formed in the DNA, and the indirect effect where radical species formed in the surrounding medium by the radiation attack the DNA. The primary mechanism proposed for radiation damage, by the direct effect, is that positive and negative ions formed within the DNA strand migrate through the stacked DNA bases. The ions can then recombine, react with the DNA bases most likely to react by protonation of the anion and deprotonation or hydroxylation of the cation or transfer out of the DNA chain to the surrounding histone protein. This work as aimed at understanding the possible reactions of the DNA base ion radicals, as well as their initial distribution in the DNA strand. 31 refs

  18. Cellular responses to environmental DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  19. uv photobiology: DNA damage and repair

    International Nuclear Information System (INIS)

    Sutherland, B.M.

    1978-01-01

    The following topics are discussed: targets that determine the fate of the cell when uv light interacts with a cell; comparison of action spectrum for a given biological effect with the absorption spectrum of different biological macromolecules; biological effects of damage to DNA; measurement of mutations; chemical damage to DNA; photoreactivation; role of pyrimidine dimers in induction of skin cancer by uv

  20. DNA damage in plant herbarium tissue.

    Science.gov (United States)

    Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

    2011-01-01

    Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

  1. DNA Damage Signals and Space Radiation Risk

    Science.gov (United States)

    Cucinotta, Francis A.

    2011-01-01

    Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

  2. Decreased stability of DNA in cells treated with alkylating agents

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Cedars Medical Center, Miami, FL (United States))

    1990-12-01

    A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

  3. SIRT participates at DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

    2009-05-15

    Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

  4. Repair of DNA damage in Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Evans, D.M.

    1984-01-01

    The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

  5. The DNA damage response in mammalian oocytes

    Directory of Open Access Journals (Sweden)

    John eCarroll

    2013-06-01

    Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

  6. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed

  7. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

    2013-10-15

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  8. The DNA damage response during mitosis.

    Science.gov (United States)

    Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Processing of free radical damaged DNA bases

    International Nuclear Information System (INIS)

    Wallace, S.

    2003-01-01

    Free radicals produced during the radiolysis of water gives rise to a plethora of DNA damages including single strand breaks, sites of base loss and a wide variety of purine and pyrimidine base lesions. All these damages are processed in cells by base excision repair. The oxidative DNA glycosylases which catalyze the first step in the removal of a base damage during base excision repair evolved primarily to protect the cells from the deleterious mutagenic effects of single free radical-induced DNA lesions arising during oxidative metabolism. This is evidenced by the high spontaneous mutation rate in bacterial mutants lacking the oxidative DNA glycosylases. However, when a low LET photon transverses the DNA molecule, a burst of free radicals is produced during the radiolysis of water that leads to the formation of clustered damages in the DNA molecule, that are recognized by the oxidative DNA glycosylases. When substrates containing two closely opposed sugar damages or base and sugar damages are incubated with the oxidative DNA glycosylases in vitro, one strand is readily incised by the lyase activity of the DNA glycosylase. Whether or not the second strand is incised depends on the distance between the strand break resulting from the incised first strand and the remaining DNA lesion on the other strand. If the lesions are more than two or three base pairs apart, the second strand is readily cleaved by the DNA glycosylase, giving rise to a double strand break. Even if the entire base excision repair system is reconstituted in vitro, whether or not a double strand break ensues depends solely upon the ability of the DNA glycosylase to cleave the second strand. These data predicted that cells deficient in the oxidative DNA glycosylases would be radioresistant while those that overproduce an oxidative DNA glycosylase would be radiosensitive. This prediction was indeed borne in Escherichia coli that is, mutants lacking the oxidative DNA glycosylases are radioresistant

  10. Molecular mechanisms in radiation damage to DNA

    International Nuclear Information System (INIS)

    Osman, R.

    1991-01-01

    The objectives of this work are to elucidate the molecular mechanisms that are responsible for radiation-induced DNA damage. The overall goal is to understand the relationship between the chemical and structural changes produced by ionizing radiation in DNA and the resulting impairment of biological function expressed as carcinogenesis or cell death. The studies are based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA. These mechanistic explorations should lead to the formulation of testable hypothesis regarding the processes of impairment of regulation of gene expression, alternation in DNA repair, and damage to DNA structure involved in cell death or cancer

  11. Biologically important radiation damage in DNA

    International Nuclear Information System (INIS)

    Ward, J.F.

    1994-01-01

    Most DNA damage by the hydroxyl radical is confined to the bases, and this base damage represents an important component of locally multiply demanded sites (LMOS). The yields of the major damaged bases have been determined by gas chromatography mass spectrometry. For our propose, it was necessary to convert a known fraction of these damaged bases to strand breaks and then assay these labile sites as the increase in strand break yield over the normally observed level. Three potential agents by which this strategy of conversion of base damage to strand break could be implemented were identified in the original application: 1, Sl nuclease; 2, piperidine; and 3, base damage specific enzymes

  12. DNA damage induced by radionuclide internal irradiation

    International Nuclear Information System (INIS)

    Cui Fengmei; Zhao Jingyong; Hong Chengjiao; Lao Qinhua; Wang Liuyi; Yang Shuqin

    2004-01-01

    Objective: To study the DNA damage of peripheral blood mononuclear cell (PBMC) in rats exposed to radionuclide internal irradiation. Methods: The radionuclides were injected into the rats and single cell get electrophoresis (SCGE) was performed to detect the length of DNA migration in the rat PBMC. Results: DNA migration in the rat PBMC increased with accumulative dose or dose-rate. It showed good relationship of dose vs. response and of dose-rate vs. response, both relationship could be described as linear models. Conclusion: Radionuclide internal irradiation could cause DNA damage in rat PBMC. (authors)

  13. Clustered DNA damage induced by proton and heavy ion irradiation

    International Nuclear Information System (INIS)

    Davidkova, M.; Pachnerova Brabcova, K; Stepan, V.; Vysin, L.; Sihver, L.; Incerti, S.

    2014-01-01

    Ionizing radiation induces in DNA strand breaks, damaged bases and modified sugars, which accumulate with increasing density of ionizations in charged particle tracks. Compared to isolated DNA damage sites, the biological toxicity of damage clusters can be for living cells more severe. We investigated the clustered DNA damage induced by protons (30 MeV) and high LET radiation (C 290 MeV/u and Fe 500 MeV/u) in pBR322 plasmid DNA. To distinguish between direct and indirect pathways of radiation damage, the plasmid was irradiated in pure water or in aqueous solution of one of the three scavengers (coumarin-3-carboxylic acid, dimethylsulfoxide, and glycylglycine). The goal of the contribution is the analysis of determined types of DNA damage in dependence on radiation quality and related contribution of direct and indirect radiation effects. The yield of double strand breaks (DSB) induced in the DNA plasmid-scavenger system by heavy ion radiation was found to decrease with increasing scavenging capacity due to reaction with hydroxyl radical, linearly with high correlation coefficients. The yield of non-DSB clusters was found to occur twice as much as the DSB. Their decrease with increasing scavenging capacity had lower linear correlation coefficients. This indicates that the yield of non-DSB clusters depends on more factors, which are likely connected to the chemical properties of individual scavengers. (authors)

  14. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1993-12-01

    In this project the author has proposed several mechanisms for radiation damage to DNA and its constituents, and has detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work he has completed several experiments on the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges and their reactions on DNA, investigated protonation reactions in DNA base anions, and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics evolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this years work to a consideration of ionization energies of various components of the DNA deoxyribose backbone and resulting neutral sugar radicals. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, four are in press, one is submitted and several more are in preparation. Four papers have been presented at scientific meetings. This years effort will include another review article on the open-quotes Electron Spin Resonance of Radiation Damage to DNAclose quotes

  15. DNA damage in the oocytes SACs

    Czech Academy of Sciences Publication Activity Database

    Macůrek, Libor

    2016-01-01

    Roč. 15, č. 4 (2016), s. 491-492 ISSN 1538-4101 Institutional support: RVO:68378050 Keywords : DNA damage response * oocyte * meiosis * checkpoint Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

  16. Early models of DNA damage formation

    International Nuclear Information System (INIS)

    Śmiałek, Małgorzata A

    2012-01-01

    Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

  17. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  18. Profiling DNA damage response following mitotic perturbations

    DEFF Research Database (Denmark)

    Pedersen, Ronni Sølvhøi; Karemore, Gopal; Gudjonsson, Thorkell

    2016-01-01

    that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells. Unexpectedly, we find that only a subset of these correlations are functionally linked. We identify the genuine mitosis-born DNA damage events and sub-classify them according to penetrance of the observed...

  19. (UVB)-induced DNA damage

    African Journals Online (AJOL)

    Jane

    2011-08-17

    dependent cytogenetic lesions were assessed by the micronucleus test (MNT). It was found that POE effectively reduced the extent of DNA breakages and cytogenetic lesions upon exposure to UVB (erythemal ultraviolet (EUV);.

  20. DNA damage induction of ribonucleotide reductase.

    OpenAIRE

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

  1. Carcinogen-induced damage to DNA

    International Nuclear Information System (INIS)

    Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

    1979-01-01

    Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

  2. Parvovirus infection-induced DNA damage response

    Science.gov (United States)

    Luo, Yong; Qiu, Jianming

    2014-01-01

    Parvoviruses are a group of small DNA viruses with ssDNA genomes flanked by two inverted terminal structures. Due to a limited genetic resource they require host cellular factors and sometimes a helper virus for efficient viral replication. Recent studies have shown that parvoviruses interact with the DNA damage machinery, which has a significant impact on the life cycle of the virus as well as the fate of infected cells. In addition, due to special DNA structures of the viral genomes, parvoviruses are useful tools for the study of the molecular mechanisms underlying viral infection-induced DNA damage response (DDR). This review aims to summarize recent advances in parvovirus-induced DDR, with a focus on the diverse DDR pathways triggered by different parvoviruses and the consequences of DDR on the viral life cycle as well as the fate of infected cells. PMID:25429305

  3. Radiation damage of DNA. Model for direct ionization of DNA

    International Nuclear Information System (INIS)

    Kobayashi, Kazuo; Tagawa, Seiichi

    2004-01-01

    Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

  4. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  5. Molecular models for DNA damaged by photoreaction

    International Nuclear Information System (INIS)

    Pearlman, D.A.; Holbrook, S.R.; Pirkle, D.H.; Kim, S.H.

    1985-01-01

    Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells

  6. Delayed chromosomal instability induced by DNA damage

    International Nuclear Information System (INIS)

    Morgan, W.F.; Marder, B.A.; Day, J.P.

    1994-01-01

    Cellular exposure to DNA damaging agents rapidly results in a dose dependent increase in chromosomal breakage and gross structural chromosomal rearrangements. Over recent years, evidence has been accumulating indicating genomic instability can manifest multiple generations after cellular exposure to physical and chemical DNA damaging agents. Genomic instability manifests in the progeny of surviving cells, and has been implicated in mutation, gene application, cellular transformation, and cell killing. To investigate chromosome instability following DNA damage, we have used fluorescence in situ hybridization to detect chromosomal rearrangements in a human/hamster somatic hybrid cell line following exposure to ionizing radiation. Delayed chromosomal instability was detected when multiple populations of uniquely arranged metaphases were observed in clonal isolates raised from single cells surviving X-irradiation many generations after exposure. At higher radiation doses, chromosomal instability was observed in a relatively high frequency of surviving clones and, in general, those clones showed delayed chromosome instability also showed reduced survival as measured by colony forming ability

  7. DNA damage response during mouse oocyte maturation

    Czech Academy of Sciences Publication Activity Database

    Mayer, Alexandra; Baran, Vladimír; Sakakibara, Y.; Brzáková, Adéla; Ferencová, Ivana; Motlík, Jan; Kitajima, T.; Schultz, R. M.; Šolc, Petr

    2016-01-01

    Roč. 15, č. 4 (2016), s. 546-558 ISSN 1538-4101 R&D Projects: GA MŠk LH12057; GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : double strand DNA breaks * DNA damage * MRE11 * meiotic maturation * mouse oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

  8. DNA damage and repair in age-related macular degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Szaflik, Jacek P. [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Zaras, Magdalena [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Wozniak, Katarzyna [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland); Szaflik, Jerzy [Department of Ophthalmology, Medical University of Warsaw and Samodzielny Publiczny Szpital Okulistyczny, Sierakowskiego 13, 03-710 Warsaw (Poland); Blasiak, Janusz, E-mail: januszb@biol.uni.lodz.pl [Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz (Poland)

    2009-10-02

    Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

  9. DNA damage and repair in age-related macular degeneration

    International Nuclear Information System (INIS)

    Szaflik, Jacek P.; Janik-Papis, Katarzyna; Synowiec, Ewelina; Ksiazek, Dominika; Zaras, Magdalena; Wozniak, Katarzyna; Szaflik, Jerzy; Blasiak, Janusz

    2009-01-01

    Age-related macular degeneration (AMD) is a retinal degenerative disease that is the main cause of vision loss in individuals over the age of 55 in the Western world. Clinically relevant AMD results from damage to the retinal pigment epithelial (RPE) cells thought to be mainly caused by oxidative stress. The stress also affects the DNA of RPE cells, which promotes genome instability in these cells. These effects may coincide with the decrease in the efficacy of DNA repair with age. Therefore individuals with DNA repair impaired more than average for a given age may be more susceptible to AMD if oxidative stress affects their RPE cells. This may be helpful in AMD risk assessment. In the present work we determined the level of basal (measured in the alkaline comet assay) endogenous and endogenous oxidative DNA damage, the susceptibility to exogenous mutagens and the efficacy of DNA repair in lymphocytes of 100 AMD patients and 110 age-matched individuals without visual disturbances. The cells taken from AMD patients displayed a higher extent of basal endogenous DNA damage without differences between patients of dry and wet forms of the disease. DNA double-strand breaks did not contribute to the observed DNA damage as checked by the neutral comet assay and pulsed field gel electrophoresis. The extent of oxidative modification to DNA bases was grater in AMD patients than in the controls, as probed by DNA repair enzymes NTH1 and Fpg. Lymphocytes from AMD patients displayed a higher sensitivity to hydrogen peroxide and UV radiation and repaired lesions induced by these factors less effectively than the cells from the control individuals. We postulate that the impaired efficacy of DNA repair may combine with enhanced sensitivity of RPE cells to blue and UV lights, contributing to the pathogenesis of AMD.

  10. Mechanisms for radiation damage in DNA

    International Nuclear Information System (INIS)

    Sevilla, M.D.

    1987-01-01

    Several mechanisms are proposed for radiation damage to DNA and its constituents, and a series of experiments utilizing electron spin resonance spectrometry have been used to test the proposed mechanisms. In the past we have concentrated chiefly on investigating irradiated systems of DNA constituents. In this year's effort we have concentrated on radiation effects on DNA itself. In addition studies of radiation effects on lipids and model compounds have been performed which shed light on the only other proposed site for cell kill, the membrane

  11. Damage and repair of ancient DNA

    DEFF Research Database (Denmark)

    Mitchell, David; Willerslev, Eske; Hansen, Anders

    2005-01-01

    degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes...... such as extinct horses, cave bears, the marsupial wolf, the moa, and Neanderthal. In the past few years, this technology has been extended to the study of infectious disease in ancient Egyptian and South American mummies, the dietary habits of ancient animals, and agricultural practices and population dynamics......, and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived...

  12. FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

    Science.gov (United States)

    This paper describes a fiber optic biosensor for the rapid and sensitive detection of radiation-induced or chemically-induced oxidative DNA damage. The assay is based on the hybridization and temperature-induced dissociation (melting curves) of synthetic oligonucleotides. The...

  13. Experimental study of oxidative DNA damage

    DEFF Research Database (Denmark)

    Loft, Steffen; Deng, Xin-Sheng; Tuo, Jingsheng

    1998-01-01

    Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical ...

  14. The Intertwined Roles of DNA Damage and Transcription

    OpenAIRE

    Di Palo, Giacomo

    2016-01-01

    DNA damage and transcription are two interconnected events. Transcription can induce damage and scheduled DNA damage can be required for transcription. Here, we analyzed genome-wide distribution of 8oxodG-marked oxidative DNA damage obtained by OxiDIP-Seq, and we found a correlation with transcription of protein coding genes.

  15. Repair of DNA damage in light sensitive human skin diseases

    Energy Technology Data Exchange (ETDEWEB)

    Horkay, I.; Varga, L.; Tam' asi P., Gundy, S.

    1978-12-01

    Repair of uv-light induced DNA damage and changes in the semiconservative DNA synthesis were studied by in vitro autoradiography in the skin of patients with lightdermatoses (polymorphous light eruption, porphyria cutanea tarda, erythropoietic protoporphyria) and xeroderma pigmentosum as well as in that of healthy controls. In polymorphous light eruption the semiconservative DNA replication rate was more intensive in the area of the skin lesions and in the repeated phototest site, the excision repair synthesis appeared to be unaltered. In cutaneous prophyrias a decreased rate of the repair incorporation could be detected. Xeroderma pigmentosum was characterized by a strongly reduced repair synthesis.

  16. Decrease DNA contamination in the laboratories

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Børsting, Claus; Morling, Niels

    2017-01-01

    In this study, we tested various approaches to remove DNA from hard laboratory surfaces. We contaminated clean surfaces with four different concentrations of massively parallel sequencing libraries. The DNA was dried and left for 45 min and for 24 h, respectively, before any treatment. The surfaces.......9–1.8% hypochlorite in order to eliminate laboratory contamination and simultaneously minimise any poisonous gases....

  17. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900 MHz radiofrequency fields

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yulong; Zong, Lin; Gao, Zhen [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Zhu, Shunxing [Laboratory Animal Center, Nantong University, Nantong, Jiangsu Province (China); Tong, Jian [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Cao, Yi, E-mail: yicao@suda.edu.cn [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China)

    2017-03-15

    Highlights: • Increased reactive oxygen species. • Decreased mitochondrial transcription Factor A and polymerase gamma. • Decreased mitochondrial transcripts (ND1 and 16S) and mtDNA copy number. • Increased 8-hydroxy-2′deoxyguanosine. • Decreased adenosine triphosphate. - Abstract: HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900 MHz radiofrequency fields (RF) at 120 μW/cm{sup 2} power intensity for 4 h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2′-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.

  18. Evaluation of DNA damage using microwave dielectric absorption spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu [Research Institute of Nuclear Engineering, University of Fukui, Fukui (Japan); Sunagawa, Takeyoshi [Fukui University of Technology, Fukui (Japan)

    2016-12-15

    Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy.

  19. Evaluation of DNA damage using microwave dielectric absorption spectroscopy

    International Nuclear Information System (INIS)

    Hirayama, Makoto; Matuo, Youichrou; Izumi, Yoshinobu; Sunagawa, Takeyoshi

    2016-01-01

    Evaluation of deoxyribonucleic acid (DNA)-strand break is important to elucidate the biological effect of ionizing radiations. The conventional methods for DNA-strand break evaluation have been achieved by Agarose gel electrophoresis and others using an electrical property of DNAs. Such kinds of DNA-strand break evaluation systems can estimate DNA-strand break, according to a molecular weight of DNAs. However, the conventional method needs pre-treatment of the sample and a relatively long period for analysis. They do not have enough sensitivity to detect the strand break products in the low-dose region. The sample is water, methanol and plasmid DNA solution. The plasmid DNA pUC118 was multiplied by using Escherichia coli JM109 competent cells. The resonance frequency and Q-value were measured by means of microwave dielectric absorption spectroscopy. When a sample is located at a center of the electric field, resonance curve of the frequency that existed as a standing wave is disturbed. As a result, the perturbation effect to perform a resonance with different frequency is adopted. The resonance frequency shifted to higher frequency with an increase in a concentration of methanol as the model of the biological material, and the Q-value decreased. The absorption peak in microwave power spectrum of the double-strand break plasmid DNA shifted from the non-damaged plasmid DNA. Moreover, the sharpness of absorption peak changed resulting in change in Q-value. We confirmed that a resonance frequency shifted to higher frequency with an increase in concentration of the plasmid DNA. We developed a new technique for an evaluation of DNA damage. In this paper, we report the evaluation method of DNA damage using microwave dielectric absorption spectroscopy

  20. DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice

    International Nuclear Information System (INIS)

    Wang, Degui; Yu, Tianyu; Liu, Yongqiang; Yan, Jun; Guo, Yingli; Jing, Yuhong; Yang, Xuguang; Song, Yanfeng; Tian, Yingxia

    2016-01-01

    Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. - Highlights: • This study explore contribution of DNA damage to neurodegeneration in Parkinson's disease mice. • A53T-α-Syn MEF cells show a prolonged DNA damage repair process and senescense phenotype. • DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice. • DNA damage decrease the number of nigrostriatal dopaminergic neurons. • Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages.

  1. Progress on clustered DNA damage in radiation research

    International Nuclear Information System (INIS)

    Yang Li'na; Zhang Hong; Di Cuixia; Zhang Qiuning; Wang Xiaohu

    2012-01-01

    Clustered DNA damage which caused by high LET heavy ion radiation can lead to mutation, tumorigenesis and apoptosis. Promoting apoptosis of cancer cells is always the basis of cancer treatment. Clustered DNA damage has been the hot topic in radiobiology. The detect method is diversity, but there is not a detail and complete protocol to analyze clustered DNA damage. In order to provide reference for clustered DNA damage in the radiotherapy study, the clustered DNA damage characteristics, the latest progresses on clustered DNA damage and the detecting methods are reviewed and discussed in detail in this paper. (authors)

  2. DNA damage, homology-directed repair, and DNA methylation.

    Directory of Open Access Journals (Sweden)

    Concetta Cuozzo

    2007-07-01

    Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

  3. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    Science.gov (United States)

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Immunochemical detection of oxidatively damaged DNA

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Šrám, Radim

    2012-01-01

    Roč. 46, č. 4 (2012), s. 492-522 ISSN 1071-5762 R&D Projects: GA MŽP(CZ) SP/1B3/50/07; GA MŠk 2B08005; GA ČR GAP503/11/0084 Institutional research plan: CEZ:AV0Z50390703 Institutional support: RVO:68378041 Keywords : oxidative DNA damage * ELISA * immunohistochemistry Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.279, year: 2012

  5. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  6. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  7. Chromatin remodeling in the UV-induced DNA damage response

    NARCIS (Netherlands)

    Ö.Z. Aydin (Özge)

    2014-01-01

    markdownabstract__Abstract__ DNA damage interferes with transcription and replication, causing cell death, chromosomal aberrations or mutations, eventually leading to aging and tumorigenesis (Hoeijmakers, 2009). The integrity of DNA is protected by a network of DNA repair and associated

  8. High LET radiation and mechanism of DNA damage repair

    International Nuclear Information System (INIS)

    Furusawa, Yoshiya

    2004-01-01

    Clarifying the mechanism of repair from radiation damage gives most important information on radiation effects on cells. Approximately 10% of biological experiments groups in Heavy Ion Medical Accelerator in Chiba (HIMAC) cooperative research group has performed the subject. They gave a lot of new findings on the mechanism, and solved some open questions. The reason to show the peak of relative biological effectiveness RBE at around 100-200 keV/μm causes miss-repair of DNA damage. Sub-lethal damage generated by high linear energy transfer (LET) radiation can be repaired fully. Potentially lethal damages by high-LET radiation also repaired, but the efficiency decreased with the LET, and so on. (author)

  9. Inflammation, oxidative DNA damage, and carcinogenesis

    International Nuclear Information System (INIS)

    Lewis, J.G.; Adams, D.O.

    1987-01-01

    Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is though that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. The authors and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H 2 O 2 and oxidized lipid products. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin that C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H 2 O 2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice. These data support the hypothesis that inflammation and the release of genotoxic oxidants may be one mechanism whereby initiated cells receive further genetic insults. They also further complicate risk assessment by suggesting that some environmental agents may work indirectly by subverting host systems to induce damage rather than maintaining homeostasis

  10. Radiation damage to DNA in DNA-protein complexes.

    Science.gov (United States)

    Spotheim-Maurizot, M; Davídková, M

    2011-06-03

    The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

  11. Solar radiation and mitochondrial DNA damage

    International Nuclear Information System (INIS)

    Hill, H.Z.; Locitzer, J.; Nassrin, E.; Ogbonnaya, A.; Hubbard, K.

    2003-01-01

    The 16.6 kB human mitochondrial DNA contains two homologous 13 base pair direct repeats separated by about 5 kB. During asynchronous mitochondrial DNA replication, the distant repeat sequences are thought to anneal, resulting in the looping out of a portion of the non-template strand which is subsequently deleted as a result of interaction with reactive oxygen species (ROS). A normal daughter and a deleted daughter mitochondrion result from such insults. This deletion has been termed the common deletion as it is the most frequent of the known mitochondrial DNA deletions. The common deletion is present in high frequency in several mitochondrial disorders, accumulates with age in slow turnover tissues and is increased in sun-exposed skin. Berneburg, et al. (Photochem. Photobiol. 66: 271, 1997) induced the common deletion in normal human fibroblasts after repeated exposures to UVA. In this study, the common deletion has been shown to be induced by repeated non-lethal exposures to FS20 sunlamp irradiation. Increases in the common deletion were demonstrated using nested PCR which produced a 303 bp product that was compared to a 324 bp product that required the presence of the undeleted 5 kB region. The cells were exposed to 10 repeated doses ranging from 0.5 (UVB) - 0.24 (UVA) J/sq m to 14.4 (UVB) - 5.8 J/sq m (UVA) measured using a UVX digital radiometer and UVB and UVA detectors respectively. Comparison with the earlier study by Berneberg, et al. suggests that this type of simulated solar damage is considerably more effective in fewer exposures than UVA radiation alone. The common deletion provides a cytoplasmic end-point for ROS damage produced by low dose chronic irradiations and other low level toxic exposures and should prove useful in evaluating cytoplasmic damage produced by ionizing radiation as well

  12. DNA methylation in human fibroblasts following DNA damage and repair

    International Nuclear Information System (INIS)

    Kastan, M.B.

    1984-01-01

    Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

  13. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents.

  14. Protection of DNA damage by radiation exposure

    International Nuclear Information System (INIS)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents

  15. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents.

  16. DNA damage caused by ionizing radiation

    International Nuclear Information System (INIS)

    Sachs, R.K.; Peili Chen; Hahnfeldt, P.J.; Klatky, L.R.

    1992-01-01

    A survey is given of continuous-time Markov chain models for ionizing radiation damage to the genome of mammalian cells. In such models, immediate damage induced by the radiation is regarded as a batch-Poisson arrival process of DNA double-strand breaks (DSBs). Enzymatic modification of the immediate damage is modeled as a Markov process similar to those described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete-exchange model. The model postulates that, after being induced by radiation, DSBs subsequently either undergo enzymatically mediated restitution (repair) or participate pairwise in chromosome exchanges. Some of the exchanges make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. Methods for analyzing the Markov chains include using an approximate model for expected values, the discrete-time Markov chain embedded at transitions, partial differential equations for generating functions, normal perturbation theory, singular perturbation theory with scaling, numerical computations, and certain matrix methods that combine Perron-Frobenius theory with variational estimates. Applications to experimental results on expected values, variances, and statistical distributions of DNA lesions are briefly outlined. Continuous-time Markov chains are the most systematic of those radiation damage models that treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g., ignore diffusion limitations). They contain virtually all other relevant homogeneous models and semiempirical summaries as special cases, limiting cases, or approximations. However, the Markov models do not seem to be well suited for studying spatial dependence of DSB interactions. 51 refs., 5 figs

  17. Repair of Clustered Damage and DNA Polymerase Iota.

    Science.gov (United States)

    Belousova, E A; Lavrik, O I

    2015-08-01

    Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

  18. DNA Damage Response and Immune Defence: Links and Mechanisms

    Directory of Open Access Journals (Sweden)

    Björn Schumacher

    2016-08-01

    Full Text Available DNA damage plays a causal role in numerous human pathologies including cancer, premature aging and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signalling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signalling. We highlight evidence gained into (i which molecular and cellular pathways of DDR activate immune signalling, (ii how DNA damage drives chronic inflammation, and (iii how chronic inflammation causes DNA damage and pathology in humans.

  19. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    International Nuclear Information System (INIS)

    Huselton, C.A.; Hill, H.Z.

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals

  20. MicroRNAs, the DNA damage response and cancer

    International Nuclear Information System (INIS)

    Wouters, Maikel D.; Gent, Dik C. van; Hoeijmakers, Jan H.J.; Pothof, Joris

    2011-01-01

    Many carcinogenic agents such as ultra-violet light from the sun and various natural and man-made chemicals act by damaging the DNA. To deal with these potentially detrimental effects of DNA damage, cells induce a complex DNA damage response (DDR) that includes DNA repair, cell cycle checkpoints, damage tolerance systems and apoptosis. This DDR is a potent barrier against carcinogenesis and defects within this response are observed in many, if not all, human tumors. DDR defects fuel the evolution of precancerous cells to malignant tumors, but can also induce sensitivity to DNA damaging agents in cancer cells, which can be therapeutically exploited by the use of DNA damaging treatment modalities. Regulation of and coordination between sub-pathways within the DDR is important for maintaining genome stability. Although regulation of the DDR has been extensively studied at the transcriptional and post-translational level, less is known about post-transcriptional gene regulation by microRNAs, the topic of this review. More specifically, we highlight current knowledge about DNA damage responsive microRNAs and microRNAs that regulate DNA damage response genes. We end by discussing the role of DNA damage response microRNAs in cancer etiology and sensitivity to ionizing radiation and other DNA damaging therapeutic agents.

  1. DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice.

    Science.gov (United States)

    Wang, Degui; Yu, Tianyu; Liu, Yongqiang; Yan, Jun; Guo, Yingli; Jing, Yuhong; Yang, Xuguang; Song, Yanfeng; Tian, Yingxia

    2016-12-02

    Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The effect of higher order chromatin structure on DNA damage and repair

    International Nuclear Information System (INIS)

    Yasui, L.S.; Warters, R.L.; Higashikubo, R.

    1985-01-01

    Alterations in chromatin structure are thought to play an important role in various radiobiological end points, i.e., DNA damage, DNA damage repair and cell survival. The authors use here the isoleucine deprivation technique to decondense higher order chromatin structure and asses X-ray induced DNA damage, DNA damage repair and cell survival on cells with decondensed chromatin as compared to controls. This chromatin decondensation manifests itself as a 30 fold decrease in nuclear area occupied by heterochromatin, an increased rate of Micrococcal nuclease digestion, 15% increased ethidium bromide intercalation and an altered binding capacity of Hl histone. These chromatin/nuclear changes do not affect X-ray induced DNA damage as measured by the alkaline elution technique or cell survival but slows DNA damage repair by 2 fold. Therefore, even though the chromatin appears more accessible to DNA damage and repair processes, these particular nuclear changes do not affect the DNA damaging effects of X-rays and in addition, repair is not enhanced by the ''relaxed'' state of chromatin. It is proposed that the altered metabolic state of isoleucine deprived cells provides a less efficient system for the repair of X-ray induced DNA damage

  3. Effect of low energy electron irradiation on DNA damage by Cu{sup 2+} ion

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Hyung Ah; Cho, Hyuck [Dept. of Physics, Chungnam National University, Daejeon (Korea, Republic of); Park, Yeun Soo [Plasma Technology Research Center, National Fusion Research Institute, Gunsan (Korea, Republic of)

    2017-03-15

    The combined effect of the low energy electron (LEE) irradiation and Cu{sup 2+} ion on DNA damage was investigated. Lyophilized pBR322 plasmid DNA films with various concentrations (1–15 mM) of Cu{sup 2+} ion were independently irradiated by monochromatic LEEs with 5 eV. The types of DNA damage, single strand break (SSB) and double strand break (DSB), were separated and quantified by gel electrophoresis. Without electron irradiation, DNA damage was slightly increased with increasing Cu ion concentration via Fenton reaction. LEE-induced DNA damage, with no Cu ion, was only 6.6% via dissociative electron attachment (DEA) process. However, DNA damage was significantly increased through the combined effect of LEE-irradiation and Cu ion, except around 9 mM Cu ion. The possible pathways of DNA damage for each of these different cases were suggested. The combined effect of LEE-irradiation and Cu ion is likely to cause increasing dissociation after elevated transient negative ion state, resulting in the enhanced DNA damage. For the decrease of DNA damage at around 9-mM Cu ion, it is assumed to be related to the structural stabilization due to DNA inter- and intra-crosslinks via Cu ion.

  4. Molecular mechanisms in radiation damage to DNA: Final report

    International Nuclear Information System (INIS)

    Osman, R.

    1996-01-01

    The objectives of this work were to elucidate the molecular mechanisms that were responsible for radiation-induced DNA damage. The studies were based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA

  5. DNA damage-induced inflammation and nuclear architecture.

    Science.gov (United States)

    Stratigi, Kalliopi; Chatzidoukaki, Ourania; Garinis, George A

    2017-07-01

    Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Cellular responses of Saccharomyces cerevisiae to DNA damage

    International Nuclear Information System (INIS)

    Ciesla, Z.; Sledziewska-Gojska, E.; Nowicka, A.; Mieczkowski, P.; Fikus, M.U.; Koprowski, P.

    1998-01-01

    Full text. Several experimental strategies have been used to study responses of S. cerevisiae cells to DNA damage. One approach was based on the isolation of novel genes, the expression of which is induced by lesions in DNA. One of these genes, DIN7, was cloned and partially characterized previously. The product of DIN7 belongs to a large family of proteins involved in DNA repair and mutagenesis. This family includes Rad2, Rad27 and ExoI proteins of S. cerevisiae and their respective human homologues, all of which are endowed with DNA nuclease activity. To study cellular function of Din7 we constructed the pPK3 plasmid carrying DIN7 fused to the GAL1 promoter. Effects of DIN7 overproduction on the phenotypes of wild-type cells and of rad27 and exoI mutants were examined. Overproduction of Din7 does not seem to affect the proficiency of wild-type S. cerevisiae cells in recombination and mutagenesis. Also, overexpression of DIN7 does not suppress the deficiency of the EXOI gene product, the closest homologue of Din7, both in recombination and in controlling the fidelity of DNA replication. Unexpectedly, we found that elevated levels of Din7 result in a very high frequency of mitochondrial rho - mutants. A high frequency of production of rho - mutants wa s also observed in strains defective in the functioning of the Dun1 protein kinase involved in signal transmission in cells exposed to DNA damaging agents. Interestingly, deficiency of Dun1 results also in a significant derepression of the DIN7 gene. Experiments are under way to distinguish whether a high cellular level of Din7 specifically decreases stability of mitochondrial DNA or affects stability of chromosomal DNA as well. Analysis of previously constructed S. cerevisiae strains carrying random geno mic fusions with reporter lacZ gene, allowed us to identify the reading frame YBR173c, on chromosome II as a novel damage inducible gene - DIN8. We have shown that DIN8-lacZ fusion is induced in yeast cells treated

  7. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

    2013-06-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

  8. Quantitative measurement of ultraviolet-induced damage in cellular DNA by an enzyme immunodot assay

    International Nuclear Information System (INIS)

    Wakizaka, A.; Nishizawa, Y.; Aiba, N.; Okuhara, E.; Takahashi, S.

    1989-01-01

    A simple enzyme immunoassay procedure was developed for the quantitative determination of 254-nm uv-induced DNA damage in cells. With the use of specific antibodies to uv-irradiated DNA and horseradish peroxidase-conjugated antibody to rabbit IgG, the extent of damaged DNA in uv-irradiated rat spleen mononuclear cells was quantitatively measurable. Through the use of this method, the amount of damaged DNA present in 2 X 10(5) cells irradiated at a dose of 75 J/m2 was estimated to be 7 ng equivalents of the standard uv-irradiated DNA. In addition, when the cells, irradiated at 750 J/m2, were incubated for 1 h, the antigenic activity of DNA decreased by 40%, suggesting that a repair of the damaged sites in DNA had proceeded to some extent in the cells

  9. Melanogenesis: a photoprotective response to DNA damage?

    International Nuclear Information System (INIS)

    Agar, Nita; Young, Antony R.

    2005-01-01

    Exposure to ultra violet radiation (UVR) is associated with significant long-term deleterious effects such as skin cancer. A well-recognised short-term consequence of UVR is increased skin pigmentation. Pigmentation, whether constitutive or facultative, has widely been viewed as photoprotective, largely because darkly pigmented skin is at a lower risk of photocarcinogenesis than fair skin. Research is increasingly suggesting that the relationship between pigmentation and photoprotection may be far more complex than previously assumed. For example, photoprotection against erythema and DNA damage has been shown to be independent of level of induced pigmentation in human white skin types. Growing evidence now suggests that UVR induced DNA photodamage, and its repair is one of the signals that stimulates melanogenesis and studies suggest that repeated exposure in skin type IV results in faster DNA repair in comparison to skin type II. These findings suggest that tanning may be a measure of inducible DNA repair capacity, and it is this rather than pigment per se which results in the lower incidence skin cancer observed in darker skinned individuals. This evokes the notion that epidermal pigmentation may in fact be the mammalian equivalent of a bacterial SOS response. Skin colour is one of most conspicuous ways in which humans vary yet the function of melanin remains controversial. Greater understanding of the role of pigmentation in skin is vital if one is to be able to give accurate advice to the general public about both the population at risk of skin carcinogenesis and also public perceptions of a tan as being healthy

  10. Melanogenesis: a photoprotective response to DNA damage?

    Energy Technology Data Exchange (ETDEWEB)

    Agar, Nita [St. John' s Institute of Dermatology, Guy' s, Kings and St. Thomas' School of Medicine, Kings College London, London (United Kingdom); Young, Antony R. [St. John' s Institute of Dermatology, Guy' s, Kings and St. Thomas' School of Medicine, Kings College London, London (United Kingdom)]. E-mail: antony.r.young@kcl.ac.uk

    2005-04-01

    Exposure to ultra violet radiation (UVR) is associated with significant long-term deleterious effects such as skin cancer. A well-recognised short-term consequence of UVR is increased skin pigmentation. Pigmentation, whether constitutive or facultative, has widely been viewed as photoprotective, largely because darkly pigmented skin is at a lower risk of photocarcinogenesis than fair skin. Research is increasingly suggesting that the relationship between pigmentation and photoprotection may be far more complex than previously assumed. For example, photoprotection against erythema and DNA damage has been shown to be independent of level of induced pigmentation in human white skin types. Growing evidence now suggests that UVR induced DNA photodamage, and its repair is one of the signals that stimulates melanogenesis and studies suggest that repeated exposure in skin type IV results in faster DNA repair in comparison to skin type II. These findings suggest that tanning may be a measure of inducible DNA repair capacity, and it is this rather than pigment per se which results in the lower incidence skin cancer observed in darker skinned individuals. This evokes the notion that epidermal pigmentation may in fact be the mammalian equivalent of a bacterial SOS response. Skin colour is one of most conspicuous ways in which humans vary yet the function of melanin remains controversial. Greater understanding of the role of pigmentation in skin is vital if one is to be able to give accurate advice to the general public about both the population at risk of skin carcinogenesis and also public perceptions of a tan as being healthy.

  11. Quantification of complex DNA damage by ionising radiation. An experimental and theoretical approach

    International Nuclear Information System (INIS)

    Fulford, J.

    2000-05-01

    Ionising radiation potentially produces a broad spectrum of damage in DNA including single and double strand breaks (ssb and dsb) and base damages. It has been hypothesised that sites of complex damage within cellular DNA have particular biological significance due to an associated decreased efficiency in repair. The aim of this study is to gain further understanding of the formation of complex DNA damage. Irradiations of plasmid DNA illustrate that an increase in ionising density of the radiation results in a decrease in ssb yields/Gy but an increase in dsb per ssb, indicative of an increase in the number of complex damage sites per simple isolated damage site. As the mechanism for damage formation shifts from purely indirect at low scavenging capacities to a significant proportion of direct at higher scavenging capacities the proportion of complex damage increases. Comparisons with the yields of ssb and dsb simulated by Monte-Carlo calculations for Al K USX and α-particles also indicate this correspondence. The ionisation density of low energy, secondary electrons produced by photons was assessed experimentally from the dependence of the yield of OH radicals escaping intra-track recombination on photon energy. As energy decreases the OH radical yield initially decreases reflecting an increased ionisation density. However, with further decrease in photon energy the yield of OH radicals increases in line with theoretical calculations. Base damage yields were determined for low and high ionising density radiation over a range of scavenging capacities. As scavenging capacity increases the base damage: ssb ratios increases implying a contribution from electrons to base damage. It is proposed that base damage contributes to DNA damage complexity. Complex damage analysis reveals that at cell mimetic scavenging capacities, 23% and 72% of ssb have an additional spatially close damage site following γ-ray and α-particle irradiation respectively. (author)

  12. Slow elimination of DNA damaged bases in the liver of old gamma-irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A I; Malakhova, L V; Fomenko, L A [AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki

    1981-01-01

    Elimination of the DNA damaged bases in the liver of old and young mice after their gamma-irradiation is studied. It is established that the incision rate of DNA gamma-damaged bases in the liver of old mice is lower than in the liver of the young ones. It is supposed to be connected with the decrease of the activity of DNA reparation ferments or with the presence of limitations in chromatin for the access of these ferments to the damaged parts of DNA in the cells of old animals.

  13. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  14. DNA damage response pathway in radioadaptive response.

    Science.gov (United States)

    Sasaki, Masao S; Ejima, Yosuke; Tachibana, Akira; Yamada, Toshiko; Ishizaki, Kanji; Shimizu, Takashi; Nomura, Taisei

    2002-07-25

    Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to

  15. Short communication Sperm DNA damage in relation to lipid ...

    African Journals Online (AJOL)

    Leyland Fraser

    Short communication. Sperm DNA ... (Received 21 January 2017; Accepted 28 February2017; First published online 8 March 2017) ... This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage.

  16. Single Molecule Scanning of DNA Radiation Oxidative Damage, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal will develop an assay to map genomic DNA, at the single molecule level and in a nanodevice, for oxidative DNA damage arising from radiation exposure;...

  17. Aging and oxidatively damaged nuclear DNA in animal organs

    DEFF Research Database (Denmark)

    Møller, Peter; Løhr, Mille; Folkmann, Janne K

    2010-01-01

    Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs...... with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies...... evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation....

  18. Damages to DNA that result in neoplastic transformation

    International Nuclear Information System (INIS)

    Setlow, R.B.

    1975-01-01

    Some topics discussed are: correlation between carcinogens and mutagens; defective DNA repair in uv-damaged xeroderma pigmentosum cells; analysis of nucleotide damage to DNA following exposure to chemicals or radiations; photoreactivation in uv-irradiated Escherichia coli; tumor development in fish; excision repair as an aid in identifying damage; detection of excision repair; role of endonucleases in repair of uv damage; and alkylation products and tumors

  19. Oxidative DNA damage during night shift work.

    Science.gov (United States)

    Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

    2017-09-01

    We previously reported that compared with night sleep, day sleep among shift workers was associated with reduced urinary excretion of 8-hydroxydeoxyguanosine (8-OH-dG), potentially reflecting a reduced ability to repair 8-OH-dG lesions in DNA. We identified the absence of melatonin during day sleep as the likely causative factor. We now investigate whether night work is also associated with reduced urinary excretion of 8-OH-dG. For this cross-sectional study, 50 shift workers with the largest negative differences in night work versus night sleep circulating melatonin levels (measured as 6-sulfatoxymelatonin in urine) were selected from among the 223 shift workers included in our previous study. 8-OH-dG concentrations were measured in stored urine samples using high performance liquid chromatography with electrochemical detection. Mixed effects models were used to compare night work versus night sleep 8-OH-dG levels. Circulating melatonin levels during night work (mean=17.1 ng/mg creatinine/mg creatinine) were much lower than during night sleep (mean=51.7 ng/mg creatinine). In adjusted analyses, average urinary 8-OH-dG levels during the night work period were only 20% of those observed during the night sleep period (95% CI 10% to 30%; psleep, is associated with reduced repair of 8-OH-dG lesions in DNA and that the effect is likely driven by melatonin suppression occurring during night work relative to night sleep. If confirmed, future studies should evaluate melatonin supplementation as a means to restore oxidative DNA damage repair capacity among shift workers. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  20. Assessment of DNA damage in ceramic workers.

    Science.gov (United States)

    Anlar, Hatice Gul; Taner, Gokce; Bacanli, Merve; Iritas, Servet; Kurt, Turker; Tutkun, Engin; Yilmaz, Omer Hinc; Basaran, Nursen

    2018-02-24

    It is known that ceramic workers are potentially exposed to complex mixture of chemicals such as silica, inorganic lead, lime, beryllium and aluminum that can be associated with an increased risk of several diseases. All operations in the ceramic industries such as mixing, moulding, casting, shaking out and finishing jobs, have been associated with the higher exposure levels and in most of the silica-related industries, average overall exposure exceeded permissible exposure levels for respirable crystalline silica. The aim of this study was to evaluate the possible genotoxic damage in ceramic workers exposed to complex mixture of chemicals mainly crystalline silica. For this purpose, the blood and buccal epithelial cell samples were taken from the ceramic workers (n = 99) and their controls (n = 81). The genotoxicity was assessed by the alkaline comet assay in isolated lymphocytes and whole blood. Micronucleus (MN), binucleated (BN), pyknotic (PYC), condensed chromatin (CC), karyolytic (KYL), karyorrhectic (KHC) and nuclear bud (NBUD) frequencies in buccal epithelial cells and plasma 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels were also evaluated. In the study, 38 workers were diagnosed with silicosis, 9 workers were suspected to have silicosis, whereas 52 workers were found to be healthy. DNA damage in blood and lymphocytes; MN, CC + KHC, PYC frequencies in buccal epithelial cells and 8-oxodG levels in plasma were increased in workers compared to their controls. These results showed that occupational chemical mixture exposure in ceramic industry may cause genotoxic damage that can lead to important health problems in the workers.

  1. DNA Damage Induction and Repair Evaluated in Human Lymphocytes Irradiated with X-Rays an Neutrons

    International Nuclear Information System (INIS)

    Niedzwiedz, W.; Cebulska-Wasilewska, A.

    2000-12-01

    The objective of this study was to evaluate the kinetic of the DNA damage induction and their subsequent repair in human lymphocytes exposed to various types of radiation. PBLs cells were isolated from the whole blood of two young healthy male subjects and one skin cancer patient, and than exposed to various doses of low LET X-rays and high LET neutrons from 252 Cf source. To evaluate the DNA damage we have applied the single cell get electrophoresis technique (SCGE) also known as the comet assay. In order to estimate the repair efficiency, cells, which had been irradiated with a certain dose, were incubated at 37 o C for various periods of time (0 to 60 min). The kinetic of DNA damage recovery was investigated by an estimation of residual DNA damage persisted at cells after various times of post-irradiation incubation (5, 10, 15, 30 and 60 min). We observed an increase of the DNA damage (reported as a Tail DNA and Tail moment parameters) in linear and linear-quadratic manner, with increasing doses of X-rays and 252 Cf neutrons, respectively. Moreover, for skin cancer patient (Code 3) at whole studied dose ranges the higher level of the DNA damage was observed comparing to health subjects (Code 1 and 2), however statistically insignificant (for Tail DNA p=0.056; for Tail moment p=0.065). In case of the efficiency of the DNA damage repair it was observed that after 1 h of post-irradiation incubation the DNA damage induced with both, neutrons and X-rays had been significantly reduced (from 65% to 100 %). Furthermore, in case of skin cancer patient we observed lover repair efficiency of X-rays induced DNA damage. After irradiation with neutrons within first 30 min, the Tail DNA and Tail moment decreased of about 50%. One hour after irradiation, almost 70% of residual and new formed DNA damage was still observed. In this case, the level of unrepaired DNA damage may represent the fraction of the double strand breaks as well as more complex DNA damage (i.e.-DNA or DNA

  2. Repair of radiation-induced DNA damage in rat epidermis as a function of age

    International Nuclear Information System (INIS)

    Sargent, E.V.; Burns, F.J.

    1985-01-01

    The rate of repair of radiation-induced DNA damage in proliferating rat epidermal cells diminished progressively with increasing age of the animal. The dorsal skin was irradiated with 1200 rad of 0.8 MeV electrons at various ages, and the amount of DNA damage was determined as a function of time after irradiation by the method of alkaline unwinding followed by S 1 nuclease digestion. The amount of DNA damage immediately after irradiation was not age dependent, while the rate of damage removal from the DNA decreased with increasing age. By fitting an exponential function to the relative amount of undamaged DNA as a function of time after irradiation, DNA repair halftimes of 20, 27, 69, and 107 min were obtained for 28, 100-, 200-, and 400-day-old animals, respectively

  3. UV and ionizing radiations induced DNA damage, differences and similarities

    Science.gov (United States)

    Ravanat, Jean-Luc; Douki, Thierry

    2016-11-01

    Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

  4. Decreased Circulating mtDNA Levels in Professional Male Volleyball Players.

    Science.gov (United States)

    Nasi, Milena; Cristani, Alessandro; Pinti, Marcello; Lamberti, Igor; Gibellini, Lara; De Biasi, Sara; Guazzaloca, Alessandro; Trenti, Tommaso; Cossarizza, Andrea

    2016-01-01

    Exercise exerts various effects on the immune system, and evidence is emerging on its anti-inflammatory effects; the mechanisms on the basis of these modifications are poorly understood. Mitochondrial DNA (mtDNA) released from damaged cells acts as a molecule containing the so-called damage-associated molecular patterns and can trigger sterile inflammation. Indeed, high plasma levels of mtDNA are associated to several inflammatory conditions and physiological aging and longevity. The authors evaluated plasma mtDNA in professional male volleyball players during seasonal training and the possible correlation between mtDNA levels and clinical parameters, body composition, and physical performance. Plasma mtDNA was quantified by real-time PCR every 2 mo in 12 professional volleyball players (PVPs) during 2 consecutive seasons. As comparison, 20 healthy nonathlete male volunteers (NAs) were analyzed. The authors found lower levels of mtDNA in plasma of PVPs than in NAs. However, PVPs showed a decrease of circulating mtDNA only in the first season, while no appreciable variations were observed during the second season. No correlation was observed among mtDNA, hematochemical, and anthropometric parameters. Regular physical activity appeared associated with lower levels of circulating mtDNA, further confirming the protective, anti-inflammatory effect of exercise.

  5. Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

    International Nuclear Information System (INIS)

    Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

    1989-01-01

    Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

  6. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.

    2013-01-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time...

  7. Chromatin structure influence the sensitivity of DNA to ionizing radiation induced DNA damage

    International Nuclear Information System (INIS)

    Gupta, Sanjay

    2016-01-01

    Chromatin acts as a natural hindrance in DNA-damage recognition, repair and recovery. Histone and their variants undergo differential post-translational modification(s) and regulate chromatin structure to facilitate DNA damage response (DDR). During the presentation we will discuss the importance of chromatin organization and histone modification(s) during IR-induced DNA damage response in human liver cells. Our data shows G1-phase specific decrease of H3 serine10 phosphorylation in response to DNA damage is coupled with chromatin compaction in repair phase of DDR. The loss of H3Ser10P during DNA damage shows an inverse correlation with gain of γH2AX from a same mono-nucleosome in a dose-dependent manner. The loss of H3Ser10P is a universal phenomenon as it is independent of origin of cell lines and nature of genotoxic agents in G1 phase cells. The reversible reduction of H3Ser10P is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. The present study suggests distinct reversible histone marks are associated with G1-phase of cell cycle and plays a critical role in chromatin organization which may facilitate differential sensitivity against radiation. Thus, the study raises the possibility of combinatorial modulation of H3Ser10P and histone acetylation with specific inhibitors to target the radio-resistant cancer cells in G1-phase and thus may serve as promising targets for cancer therapy. (author)

  8. Cellular radiosensitivity and DNA damage in primary human fibroblasts

    International Nuclear Information System (INIS)

    Wurm, R.; Burnet, N.G.; Duggal, N.

    1994-01-01

    To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

  9. Mitochondrial and Nuclear DNA Damage and Repair in Age-Related Macular Degeneration

    Directory of Open Access Journals (Sweden)

    Janusz Blasiak

    2013-01-01

    Full Text Available Aging and oxidative stress seem to be the most important factors in the pathogenesis of age-related macular degeneration (AMD, a condition affecting many elderly people in the developed world. However, aging is associated with the accumulation of oxidative damage in many biomolecules, including DNA. Furthermore, mitochondria may be especially important in this process because the reactive oxygen species produced in their electron transport chain can damage cellular components. Therefore, the cellular response to DNA damage, expressed mainly through DNA repair, may play an important role in AMD etiology. In several studies the increase in mitochondrial DNA (mtDNA damage and mutations, and the decrease in the efficacy of DNA repair have been correlated with the occurrence and the stage of AMD. It has also been shown that mitochondrial DNA accumulates more DNA lesions than nuclear DNA in AMD. However, the DNA damage response in mitochondria is executed by nucleus-encoded proteins, and thus mutagenesis in nuclear DNA (nDNA may affect the ability to respond to mutagenesis in its mitochondrial counterpart. We reported that lymphocytes from AMD patients displayed a higher amount of total endogenous basal and oxidative DNA damage, exhibited a higher sensitivity to hydrogen peroxide and UV radiation, and repaired the lesions induced by these factors less effectively than did cells from control individuals. We postulate that poor efficacy of DNA repair (i.e., is impaired above average for a particular age when combined with the enhanced sensitivity of retinal pigment epithelium cells to environmental stress factors, contributes to the pathogenesis of AMD. Collectively, these data suggest that the cellular response to both mitochondrial and nuclear DNA damage may play an important role in AMD pathogenesis.

  10. DNA damage assessment by visualization and quantification of DNA damage response

    International Nuclear Information System (INIS)

    Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

    2017-01-01

    DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

  11. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  12. Chromatin modifications and the DNA damage response to ionizing radiation

    International Nuclear Information System (INIS)

    Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

  13. Ultraviolet induced DNA damage and hereditary skin cancer

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Francis, A.A.

    1984-01-01

    Clearly, cells from normal individuals possess the ability to repair a variety of damage to DNA. Numerous studies indicate that defects in DNA repair may increase an individual's susceptibility to cancer. It is hoped that continued studies of the exact structural changes produced in the DNA by environmental insults, and the correlation of specific DNA changes with particulr cellular events, such as DNA repair, will lead to a better understanding of cell-killing, mutagenesis and carbinogenesis. 1 figure, 2 tables

  14. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

  15. DNA damage protection and 5-lipoxygenase inhibiting activity of ...

    African Journals Online (AJOL)

    DNA damage caused by free radical is associated with mutation-based health impairment. The protective effect on DNA damage mediated by hydroxyl radical and peroxynitrite radical, and the inhibiting activity on 5-lipoxygenase of areca inflorescence extracts were studied in vitro. The results show that the boiling water ...

  16. Sperm DNA damage in relation to lipid peroxidation following ...

    African Journals Online (AJOL)

    This study investigated the relationships between lipid peroxidation (LPO) and sperm DNA damage following freezing-thawing of boar semen in different extenders. The comet assay was used to measure the extent of sperm DNA damage in a cryoprotectant-free extender or in cryoprotectant-based extenders after single ...

  17. Assessment of DNA damage by panmasala, gutkha chewing and ...

    African Journals Online (AJOL)

    In the present study the comet assay was performed in buccal epithelial cells to evaluate DNA damage among pan masala or gutkha chewers and smokers. The assay is a rapid, suitable and sensitive method for detecting various forms of DNA damage at individual cell level. The study comprises 300 individuals of which 50 ...

  18. Transcription and DNA Damage: Holding Hands or Crossing Swords?

    Science.gov (United States)

    D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

    2017-10-27

    Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. DNA Damage among Wood Workers Assessed with the Comet Assay

    Science.gov (United States)

    Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

    2016-01-01

    Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

  20. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  1. Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

    Science.gov (United States)

    Gredilla, R; Barja, G; López-Torres, M

    2001-10-01

    Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

  2. Damage of DNA by radiation and it's recovery, 3

    International Nuclear Information System (INIS)

    Narita, Noboru; Matsuura, Tomio; Sato, Hiroyuki.

    1974-01-01

    The damage and recovery of DNA was investigated by the incorporation of thymine derivatives (DHT, I trans, II trans, cis and glycol) into exponentially growing Tetrahymena cells. The strain employed was Tetrahymena pyriformis, Variety I, mating type IV. It is well known that these thymine derivatives are induced in vivo by radiation. The in vivo damage of DNA induced by radiation, and its recovery, were confirmed experimentally by means of gradient separation of sucrose density and by analytical ultra centrifugation (UVC). The recovery of DNA, its excision repair and its recombinational repair were compared with the recovery of Bacillus subtilis whose recovery kinetics were already known. 1) The damage of DNA was more sensitive to glycol than to II trans and cis. On the other hand, DHT is not sensitive for breaking DNA strand. 2) In its recovery damaged DNA was no more sensitive to glycol than to hhp as was true for Bacillus subtilis. (author)

  3. Evaluating In Vitro DNA Damage Using Comet Assay.

    Science.gov (United States)

    Lu, Yanxin; Liu, Yang; Yang, Chunzhang

    2017-10-11

    DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

  4. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

  5. Electrochemical behavior of antioxidants: Part 3. Electrochemical studies of caffeic Acid–DNA interaction and DNA/carbon nanotube biosensor for DNA damage and protection

    Directory of Open Access Journals (Sweden)

    Refat Abdel-Hamid

    2016-05-01

    Full Text Available Multi-walled carbon nanotubes-modified glassy carbon electrode biosensor was used for electrochemical studies of caffeic acid–dsDNA interaction in phosphate buffer solution at pH 2.12. Caffeic acid, CAF, shows a well-defined cyclic voltammetric wave. Its anodic peak current decreases and the peak potential shifts positively on the addition of dsDNA. This behavior was ascribed to an interaction of CAF with dsDNA giving CAF–dsDNA complex by intercalative binding mode. The apparent binding constant of CAF–dsDNA complex was determined using amperometric titrations. The oxidative damage caused to DNA was detected using the biosensor. The damage caused by the reactive oxygen species, hydroxyl radical (·−OH generated by the Fenton system on the DNA-biosensor was detected. It was found that CAF has the capability of scavenging the hydroxide radical and protecting the DNA immobilized on the GCE surface.

  6. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  7. Primary DNA Damage in Dry Cleaners with Perchlorethylene Exposure

    Directory of Open Access Journals (Sweden)

    Mohammad Azimi

    2017-10-01

    Full Text Available Background: Perchloroethylene is a halogenated solvent widely used in dry cleaning. International agency of research on cancer classified this chemical as a probable human carcinogen. Objective: To evaluate the extent of primary DNA damage in dry cleaner workers who were exposed to perchloroethylene as compared to non-exposed subjects. The effect of exposure modifying factors such as use of personal protective equipment, perceived risk, and reported safe behaviors on observed DNA damage were also studied. Methods: 59 exposed and non-exposed workers were selected from Yazd, Iran. All the 33 exposed workers had work history at least 3 months in the dry cleaning shops. Peripheral blood sampling was performed. Microscope examination was performed under fluorescent microscope (400×. Open comet software was used for image analysis. All biological analysis was performed in one laboratory. Results: Primary DNA damage to leukocytes in dry cleaners was relatively high. The median tail length, %DNA in tail, and tail moment in exposed group were significantly higher than those in non-exposed group. There was no significant difference between smokers and nonsmokers in terms of tail length, tail moment, and %DNA in tail. There was no significant correlation between duration of employment in dry cleaning and observed DNA damage in terms of tail length, tail moment and %DNA in tail. Stratified analysis based on exposed and nonexposed category showed no significant relationship between age and observed DNA damage. Conclusion: Occupationally exposure to perchloroethylene can cause early DNA damage in dry cleaners.

  8. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

    NARCIS (Netherlands)

    Buma, A.G.J.; Van Hannen, E.J.; Veldhuis, M.; Gieskes, W.W.C.

    1996-01-01

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  9. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp.

    NARCIS (Netherlands)

    Buma, A.G.J.; van Hannen, E.J; Veldhuis, M.J W; Gieskes, W.W C

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  10. Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

    Science.gov (United States)

    Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

    2017-08-31

    Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

  11. Chemical determination of free radical-induced damage to DNA.

    Science.gov (United States)

    Dizdaroglu, M

    1991-01-01

    Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

  12. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  13. Endogenous DNA Damage and Repair Enzymes

    Directory of Open Access Journals (Sweden)

    Arne Klungland

    2016-06-01

    Full Text Available Tomas Lindahl completed his medical studies at Karolinska Institute in 1970. Yet, his work has always been dedicated to unraveling fundamental mechanisms of DNA decay and DNA repair. His research is characterized with groundbreaking discoveries on the instability of our genome, the identification of novel DNA repair activities, the characterization of DNA repair pathways, and the association to diseases, throughout his 40 years of scientific career.

  14. Phosphorylation of human INO80 is involved in DNA damage tolerance

    International Nuclear Information System (INIS)

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

    2012-01-01

    Highlights: ► Depletion of hINO80 significantly reduced PCNA ubiquitination. ► Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. ► Western blot analyses showed phosphorylated hINO80 C-terminus. ► Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

  15. Molecular mechanisms in radiation damage to DNA. Progress report

    International Nuclear Information System (INIS)

    Osman, R.

    1994-01-01

    The objectives of this work are to elucidate the molecular mechanisms that are responsible for radiation-induced DNA damage. The overall goal is to understand the relationship between the chemical and structural changes produced by ionizing radiation in DNA and the resulting impairment of biological function expressed as carcinogenesis or cell death. The studies are based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA. These mechanistic explorations should lead to the formulation of testable hypotheses regarding the processes of impairment of regulation of gene expression, alteration in DNA repair, and damage to DNA structure involved in cell death or cancer

  16. Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

    Science.gov (United States)

    Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

    2017-11-20

    DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N 2 -2'-deoxyguanosine (N 2 -dG) and N 6 -2'-deoxyadenosine (N 6 -dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N 2 -dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

  17. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  18. Imaging the DNA damage response with PET and SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Knight, James C.; Koustoulidou, Sofia; Cornelissen, Bart [University of Oxford, CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom)

    2017-06-15

    DNA integrity is constantly challenged by endogenous and exogenous factors that can alter the DNA sequence, leading to mutagenesis, aberrant transcriptional activity, and cytotoxicity. Left unrepaired, damaged DNA can ultimately lead to the development of cancer. To overcome this threat, a series of complex mechanisms collectively known as the DNA damage response (DDR) are able to detect the various types of DNA damage that can occur and stimulate the appropriate repair process. Each DNA damage repair pathway leads to the recruitment, upregulation, or activation of specific proteins within the nucleus, which, in some cases, can represent attractive targets for molecular imaging. Given the well-established involvement of DDR during tumorigenesis and cancer therapy, the ability to monitor these repair processes non-invasively using nuclear imaging techniques may facilitate the earlier detection of cancer and may also assist in monitoring response to DNA damaging treatment. This review article aims to provide an overview of recent efforts to develop PET and SPECT radiotracers for imaging of DNA damage repair proteins. (orig.)

  19. Role of the Checkpoint Clamp in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2013-01-01

    Full Text Available DNA damage occurs during DNA replication, spontaneous chemical reactions, and assaults by external or metabolism-derived agents. Therefore, all living cells must constantly contend with DNA damage. Cells protect themselves from these genotoxic stresses by activating the DNA damage checkpoint and DNA repair pathways. Coordination of these pathways requires tight regulation in order to prevent genomic instability. The checkpoint clamp complex consists of Rad9, Rad1 and Hus1 proteins, and is often called the 9-1-1 complex. This PCNA (proliferating cell nuclear antigen-like donut-shaped protein complex is a checkpoint sensor protein that is recruited to DNA damage sites during the early stage of the response, and is required for checkpoint activation. As PCNA is required for multiple pathways of DNA metabolism, the checkpoint clamp has also been implicated in direct roles in DNA repair, as well as in coordination of the pathways. Here we discuss roles of the checkpoint clamp in DNA damage response (DDR.

  20. Visualization of complex DNA damage along accelerated ions tracks

    Science.gov (United States)

    Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

    2018-04-01

    The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

  1. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    Science.gov (United States)

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  3. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    International Nuclear Information System (INIS)

    Zheng, Juanjuan; Zhang, Yu; Xu, Wentao; Luo, YunBo; Hao, Junran; Shen, Xiao Li; Yang, Xuan; Li, Xiaohong; Huang, Kunlun

    2013-01-01

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ m ). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in

  4. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Juanjuan; Zhang, Yu [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Xu, Wentao, E-mail: xuwentaoboy@sina.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Luo, YunBo [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Hao, Junran [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Shen, Xiao Li [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Yang, Xuan [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Li, Xiaohong [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Huang, Kunlun, E-mail: hkl009@163.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China)

    2013-04-15

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by

  5. Epigenetic telomere protection by Drosophila DNA damage response pathways.

    Science.gov (United States)

    Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

    2006-05-01

    Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

  6. Global chromatin fibre compaction in response to DNA damage

    International Nuclear Information System (INIS)

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-01-01

    Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

  7. Radiation-induced DNA damage as a function of DNA hydration

    International Nuclear Information System (INIS)

    Swarts, S.G.; Miao, L.; Wheeler, K.T.; Sevilla, M.D.; Becker, D.

    1995-01-01

    Radiation-induced DNA damage is produced from the sum of the radicals generated by the direct ionization of the DNA (direct effect) and by the reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). The indirect effect has been believed to be the predominant contributor to radiation-induced intracellular DNA damage, mainly as the result of reactions of bulk water radicals (e.g., OH·) with DNA. However, recent evidence suggests that DNA damage, derived from the irradiation of water molecules that are tightly bound in the hydration layer, may occur as the result of the transfer of electron-loss centers (e.g. holes) and electrons from these water molecules to the DNA. Since this mechanism for damaging DNA more closely parallels that of the direct effect, the irradiation of these tightly bound water molecules may contribute to a quasi-direct effect. These water molecules comprise a large fraction of the water surrounding intracellular DNA and could account for a significant proportion of intracellular radiation-induced DNA damage. Consequently, the authors have attempted to characterize this quasi-direct effect to determine: (1) the extent of the DNA hydration layer that is involved with this effect, and (2) what influence this effect has on the types and quantities of radiation-induced DNA damage

  8. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  9. DNA damage caused by UV- and near UV-irradiation

    International Nuclear Information System (INIS)

    Ohnishi, Takeo

    1986-01-01

    Much work with mutants deficient in DNA repair has been performed concerning UV-induced DNA damage under the condition where there is no artificial stimulation. In an attempt to infer the effects of solar wavelengths, the outcome of the work is discussed in terms of cellular radiation sensitivity, unscheduled DNA synthesis, and mutation induction, leading to the conclusion that some DNA damage occurs even by irradiation of the shorter wavelength light (270 - 315 nm) and is repaired by excision repair. It has been thought to date that pyrimidine dimer (PD) plays the most important role in UV-induced DNA damage, followed by (6 - 4) photoproducts. As for DNA damage induced by near UV irradiation, the yield of DNA single-strand breaks and of DNA-protein crosslinking, other than PD, is considered. The DNA-protein crosslinking has proved to be induced by irradiation at any wavelength of UV ranging from 260 to 425 nm. Near UV irradiation causes the inhibition of cell proliferation to take place. (Namekawa, K.)

  10. DNA Damage, Fruits and Vegetables and Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Thompson, Henry

    2002-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

  11. DNA Damage, Fruits and Vegetables and Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Thompson, Henry

    2001-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

  12. DNA Damage, Fruits and Vegetables and Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Thompson, Henry

    2003-01-01

    The purpose of this project was to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

  13. DNA Damage, Fruits and Vegetables and Breast Cancer Prevention

    National Research Council Canada - National Science Library

    Thompson, Henry

    2000-01-01

    The purpose of this project is to evaluate the effect(s) of increasing fruit and vegetable intake on oxidative DNA damage and lipid peroxidation in a population of women at elevated risk for breast cancer...

  14. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-01-18

    Jan 18, 2010 ... (Fluitest Glu, Biocon Solutions Pte Ltd, Singapore). Cholesterol, ... migration in the comet tail was taken as an estimate of DNA damage and is ..... fever, and dietary energy intake on weight gain in rural Bangladeshi children.

  15. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  16. Assessment of DNA damage induced by terrestrial UV irradiation of dried bloodstains: forensic implications.

    Science.gov (United States)

    Hall, Ashley; Sims, Lynn M; Ballantyne, Jack

    2014-01-01

    Few publications have detailed the nature of DNA damage in contemporary (i.e. non-ancient) dried biological stains. The chief concern, from a forensic standpoint, is that the damage can inhibit polymerase-mediated primer extension, ultimately resulting in DNA typing failure. In the work described here, we analyzed the effects of UVA and UVB irradiation on cell-free solubilized DNA, cell-free dehydrated DNA and dehydrated cellular DNA (from bloodstains). After UV exposure ranging from 25 J cm(-2) to 1236 J cm(-2), we assayed for the presence of bipyrimidine photoproducts (BPPPs), oxidative lesions and strand breaks, correlating the damage with the inhibition of STR profiling. Subsequent to irradiation with either UVA and UVB, the incidence of BPPPs, oxidative products and strand breaks were observed in decreasing quantities as follows: cell-free solubilized DNA>cell-free dehydrated DNA>bloodstain DNA. UVA irradiation did not result in even the partial loss of a STR profile in any sample tested. Somewhat different results were observed after genetic analysis of UVB exposed samples, in that the ability to produce a complete STR profile was affected earliest in bloodstain DNA, next in cell-free solubilized DNA and not at all in cell-free dehydrated DNA. Therefore, it is likely that other types of damage contributed to allele-drop-out in these samples but remained undetected by our assays, whereby the endonucleases did not react with the lesions or the presence of the lesions was masked by strand breaks. Under the conditions of the study, strand breaks appeared to be the predominant types of damage that ultimately resulted in DNA typing failure from physiological stains, although some evidence suggested oxidative damage may have played a role as well. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Protection of vanillin derivative VND3207 on plasmid DNA damage induced by different LET ionizing radiation

    International Nuclear Information System (INIS)

    Xu Huihui; Wang Li; Sui Li; Guan Hua; Wang Yu; Liu Xiaodan; Zhang Shimeng; Xu Qinzhi; Wang Xiao; Zhou Pingkun

    2011-01-01

    Objective: To evaluate the radioprotective effect of vanillin derivative VND3207 on DNA damage induced by different LET ionizing radiation. Methods: The plasmid DNA in liquid was irradiated by 60 Co γ-rays, proton or 7 Li heavy ion with or without VND3207. The conformation changes of plasmid DNA were assessed by agarose gel electrophoresis and the quantification was done using gel imaging system. Results: The DNA damage induced by proton and 7 Li heavy ion was much more serious as compared with that by 60 Co γ-rays, and the vanillin derivative VND3207 could efficiently decrease the DNA damage induced by all three types of irradiation sources, which was expressed as a significantly reduced ratio of open circular form (OC) of plasmid DNA. The radioprotective effect of VND3207 increased with the increasing of drug concentration. The protective efficiencies of 200 μmol/L VND3207 were 85.3% (t =3.70, P=0.033), 73.3% (t=10.58, P=0.017) and 80.4% (t=8.57, P=0.008) on DNA damage induction by 50 Gy of γ-rays, proton and 7 Li heavy ion, respectively. It seemed that the radioprotection of VND3207 was more effective on DNA damage induced by high LET heavy ion than that by proton. Conclusions: VND3207 has a protective effect against the genotoxicity of different LET ionizing radiation, especially for γ-rays and 7 Li heavy ion. (authors)

  18. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    Science.gov (United States)

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  19. MDM2 Antagonists Counteract Drug-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Anna E. Vilgelm

    2017-10-01

    Full Text Available Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.

  20. Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage.

    Science.gov (United States)

    Vande Loock, Kim; Ciardelli, Roberta; Decordier, Ilse; Plas, Gina; Haumont, Dominique; Kirsch-Volders, Micheline

    2012-09-01

    Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H(2)O(2) induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

  1. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    International Nuclear Information System (INIS)

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-01-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA III ); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA III [URO-MSC(+)] and after subsequent removal of MMA III [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA III , URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA III . URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA III exposure, suggesting the presence of MMA III is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA III results in: the induction of DNA damage that remains elevated upon removal of MMA III ; increased levels of ROS that play a role in MMA III induced-DNA damage; and decreased PARP activity in the presence of MMA III .

  2. Radiation damage to DNA: the effect of LET

    Energy Technology Data Exchange (ETDEWEB)

    Ward, J F; Milligan, J R [California Univ., San Diego, La Jolla, CA (United States). School of Medicine

    1997-03-01

    Mechanisms whereby ionizing radiation induced damage are introduced into cellular DNA are discussed. The types of lesions induced are summarized and the rationale is presented which supports the statement that radiation induced singly damaged sites are biologically unimportant. The conclusion that multiply damaged sites are critical is discussed and the mechanisms whereby such lesions are formed are presented. Structures of multiply damaged sites are summarized and problems which they present to cellular repair systems are discussed. Lastly the effects of linear energy transfer on the complexity of multiply damaged sites are surveyed and the consequences of this increased complexity are considered in terms of cell survival and mutation. (author)

  3. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    International Nuclear Information System (INIS)

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

    1990-01-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

  4. The complexity of DNA damage: relevance to biological consequences

    International Nuclear Information System (INIS)

    Ward, J.F.

    1994-01-01

    Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics). The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. (Author)

  5. Damage severity estimation from the global stiffness decrease

    International Nuclear Information System (INIS)

    Nitescu, C; Gillich, G R; Manescu, T; Korka, Z I; Abdel Wahab, M

    2017-01-01

    In actual damage detection methods, localization and severity estimation can be treated separately. The severity is commonly estimated using fracture mechanics approach, with the main disadvantage of involving empirically deduced relations. In this paper, a damage severity estimator based on the global stiffness reduction is proposed. This feature is computed from the deflections of the intact and damaged beam, respectively. The damage is always located where the bending moment achieves maxima. If the damage is positioned elsewhere on the beam, its effect becomes lower, because the stress is produced by a diminished bending moment. It is shown that the global stiffness reduction produced by a crack is the same for all beams with a similar cross-section, regardless of the boundary conditions. One mathematical relation indicating the severity and another indicating the effect of removing damage from the beam. Measurements on damaged beams with different boundary conditions and cross-sections are carried out, and the location and severity are found using the proposed relations. These comparisons prove that the proposed approach can be used to accurately compute the severity estimator. (paper)

  6. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2004-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  7. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2005-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  8. Choreography of the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C

    2004-01-01

    DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication...... stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous...... recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting...

  9. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic Antoine Alexandre

    2013-01-01

    for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often DNA is recognized as nutrient source...... of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations...

  10. Vitamin C for DNA damage prevention

    Czech Academy of Sciences Publication Activity Database

    Šrám, Radim; Binková, B.; Rössner ml., Pavel

    2012-01-01

    Roč. 733, 1-2 (2012), s. 39-49 ISSN 0027-5107 R&D Projects: GA MŠk 2B08005; GA MŽP(CZ) SP/1B3/50/07 Institutional research plan: CEZ:AV0Z50390703 Keywords : Chromosomal aberrations * DNA adducts * DNA repair Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.902, year: 2012

  11. Role of oxidative DNA damage in genome instability and cancer

    International Nuclear Information System (INIS)

    Bignami, M.; Kunkel, T.

    2009-01-01

    Inactivation of mismatch repair (MMR) is associated with a dramatic genomic instability that is observed experimentally as a mutator phenotype and micro satellite instability (MSI). It has been implicit that the massive genetic instability in MMR defective cells simply reflects the accumulation of spontaneous DNA polymerase errors during DNA replication. We recently identified oxidation damage, a common threat to DNA integrity to which purines are very susceptible, as an important cofactor in this genetic instability

  12. Visualization of DNA clustered damage induced by heavy ion exposure

    International Nuclear Information System (INIS)

    Tomita, M.; Yatagai, F.

    2003-01-01

    Full text: DNA double-strand breaks (DSBs) are the most lethal damage induced by ionizing radiations. Accelerated heavy-ions have been shown to induce DNA clustered damage, which is two or more DNA lesions induced within a few helical turns. Higher biological effectiveness of heavy-ions could be provided predominantly by induction of complex DNA clustered damage, which leads to non-repairable DSBs. DNA-dependent protein kinase (DNA-PK) is composed of catalytic subunit (DNA-PKcs) and DNA-binding heterodimer (Ku70 and Ku86). DNA-PK acts as a sensor of DSB during non-homologous end-joining (NHEJ), since DNA-PK is activated to bind to the ends of double-stranded DNA. On the other hand, NBS1 and histone H2AX are essential for DSB repair by homologous recombination (HR) in higher vertebrate cells. Here we report that phosphorylated H2AX at Ser139 (named γ-H2AX) and NBS1 form large undissolvable foci after exposure to accelerated Fe ions, while DNA-PKcs does not recognize DNA clustered damage. NBS1 and γ-H2AX colocalized with forming discrete foci after exposure to X-rays. At 0.5 h after Fe ion irradiation, NBS1 and γ-H2AX also formed discrete foci. However, at 3-8 h after Fe ion irradiation, highly localized large foci turned up, while small discrete foci disappeared. Large NBS1 and γ-H2AX foci were remained even 16 h after irradiation. DNA-PKcs recognized Ku-binding DSB and formed foci shortly after exposure to X-rays. DNA-PKcs foci were observed 0.5 h after 5 Gy of Fe ion irradiation and were almost completely disappeared up to 8 h. These results suggest that NBS1 and γ-H2AX can be utilized as molecular marker of DNA clustered damage, while DNA-PK selectively recognizes repairable DSBs by NHEJ

  13. The intersection between DNA damage response and cell death pathways.

    Science.gov (United States)

    Nowsheen, S; Yang, E S

    2012-10-01

    Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

  14. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

    2009-01-01

    overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were...... attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. METHODS: Mother...

  15. Characterization of hepatic DNA damage induced in rats by the pyrrolizidine alkaloid monocrotaline

    Energy Technology Data Exchange (ETDEWEB)

    Petry, T.W.; Bowden, G.T.; Huxtable, R.J.; Sipes, I.G.

    1984-04-01

    Hepatic DNA damage induced by the pyrrolizidine alkaloid monocrotaline was evaluated following i.p. administration to adult male Sprague-Dawley rats. Animals were treated with various doses ranging upward from 5 mg/kg, and hepatic nuclei were isolated 4 hr later. Hepatic nuclei were used as the DNA source in all experiments. DNA damage was characterized by the alkaline elution technique. A mixture of DNA-DNA interstrand cross-links and DNA-protein cross-links was induced. Following an injection of monocrotaline, 30 mg/kg i.p., DNA-DNA interstrand cross-linking reached a maximum within 12 hr or less and thereafter decreased over a protracted period of time. By 96 hr postadministration, the calculated cross-linking factor was no longer statistically different from zero. No evidence for the induction of DNA single-strand breaks was observed, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. These DNA cross-links may be related to the hepatocarcinogenic, hepatotoxic, and/or antimitotic effects of monocrotaline.

  16. Mechanisms of free radical-induced damage to DNA.

    Science.gov (United States)

    Dizdaroglu, Miral; Jaruga, Pawel

    2012-04-01

    Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.

  17. Study on DNA damages induced by UV radiation

    International Nuclear Information System (INIS)

    Doan Hong Van; Dinh Ba Tuan; Tran Tuan Anh; Nguyen Thuy Ngan; Ta Bich Thuan; Vo Thi Thuong Lan; Tran Minh Quynh; Nguyen Thi Thom

    2015-01-01

    DNA damages in Escherichia coli (E. coli) exposed to UV radiation have been investigated. After 30 min of exposure to UV radiation of 5 mJ/cm"2, the growth of E. coli in LB broth medium was about only 10% in compared with non-irradiated one. This results suggested that the UV radiation caused the damages for E. coli genome resulted in reduction in its growth and survival, and those lesions can be somewhat recovered. For both solutions of plasmid DNAs and E. coli cells containing plasmid DNA, this dose also caused the breakage on single and double strands of DNA, shifted the morphology of DNA plasmid from supercoiled to circular and linear forms. The formation of pyrimidine dimers upon UV radiation significantly reduced when the DNA was irradiated in the presence of Ganoderma lucidum extract. Thus, studies on UV-induced DNA damage at molecular level are very essential to determine the UV radiation doses corresponding to the DNA damages, especially for creation and selection of useful radiation-induced mutants, as well as elucidation the protective effects of the specific compounds against UV light. (author)

  18. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...... shows that normal mitochondrial function is prerequisite for retaining stable dNTP pools upon DNA damage. Therefore it is likely that mitochondrial deficiency defects may cause an increase in DNA mutations by disrupting dNTP pool balance....

  19. Oxidative DNA damage during sleep periods among nightshift workers.

    Science.gov (United States)

    Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

    2016-08-01

    Oxidative DNA damage may be increased among nightshift workers because of suppression of melatonin, a cellular antioxidant, and/or inflammation related to sleep disruption. However, oxidative DNA damage has received limited attention in previous studies of nightshift work. From two previous cross-sectional studies, urine samples collected during a night sleep period for 217 dayshift workers and during day and night sleep (on their first day off) periods for 223 nightshift workers were assayed for 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, using high-performance liquid chromatography with electrochemical detection. Urinary measures of 6-sulfatoxymelatonin (aMT6s), a marker of circulating melatonin levels, and actigraphy-based sleep quality data were also available. Nightshift workers during their day sleep periods excreted 83% (p=0.2) and 77% (p=0.03) of the 8-OH-dG that dayshift workers and they themselves, respectively, excreted during their night sleep periods. Among nightshift workers, higher aMT6s levels were associated with higher urinary 8-OH-dG levels, and an inverse U-shaped trend was observed between 8-OH-dG levels and sleep efficiency and sleep duration. Reduced excretion of 8-OH-dG among nightshift workers during day sleep may reflect reduced functioning of DNA repair machinery, which could potentially lead to increased cellular levels of oxidative DNA damage. Melatonin disruption among nightshift workers may be responsible for the observed effect, as melatonin is known to enhance repair of oxidative DNA damage. Quality of sleep may similarly impact DNA repair. Cellular levels of DNA damage will need to be evaluated in future studies to help interpret these findings. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  20. Histone modifications in response to DNA damage

    International Nuclear Information System (INIS)

    Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques

    2007-01-01

    The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity

  1. Damaging the Integrated HIV Proviral DNA with TALENs.

    Directory of Open Access Journals (Sweden)

    Christy L Strong

    Full Text Available HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs to target a highly conserved sequence in the transactivation response element (TAR of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

  2. DNA damage, repair and tanning acceleration

    NARCIS (Netherlands)

    Vink, A.A.; Berg, P.T.M. van den; Roza, L.

    1999-01-01

    Exposure of the skin to solar ultraviolet radiation (UV) leads to various adverse effects, such as the induction of cellular damage and mutations, suppression of the skin's immune system, and the induction of skin cancer. These effects are the consequence of various molecular alterations in the skin

  3. Inhibition of DNA chain elongation in Chinese hamster cells by damage localized behind the replication fork

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Hur, E [Israel Atomic Energy Commission, Beersheba. Nuclear Research Center-Negev; Hagan, M P [Armed Forces Radiobiology Research Inst., Bethesda, MD (USA)

    1984-05-01

    Chinese hamster fibroblasts were pulse labelled with 5-bromodeoxyuridine and exposed at time intervals (Tsub(i)) to near-ultraviolet (U.V.A.) light in the presence of a bisbenzimidazole derivative (Hoechst 33342). The sensitivity of the cells in terms of colony forming ability fluctuated depending on Tsub(i). Inhibition of DNA synthesis also depended on Tsub(i) and was maximal when Tsub(i)=O. Using the alkaline elution technique it was shown that the effect of a large dose of light was to inhibit both initiation and elongation of DNA chains. These effects were most pronounced for Tsub(i)=O. It is concluded that DNA damage in an active replicon can inhibit initiation of new replicons and that damage localized behind the replication fork can retard elongation of nascent DNA chains. This effect on chain elongation decreases with increased distance of the damage from the replication fork.

  4. Fanconi anemia: a disorder defective in the DNA damage response.

    Science.gov (United States)

    Kitao, Hiroyuki; Takata, Minoru

    2011-04-01

    Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

  5. Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

    International Nuclear Information System (INIS)

    Park, Yoo Kyoung; Park, Eunju; Kim, Jung-Shin; Kang, Myung-Hee

    2003-01-01

    Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19-57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75±1.55 μm versus after supplementation: 70.25±1.31 μm; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels

  6. Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoo Kyoung; Park, Eunju; Kim, Jung-Shin; Kang, Myung-Hee

    2003-08-28

    Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19-57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75{+-}1.55 {mu}m versus after supplementation: 70.25{+-}1.31 {mu}m; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels.

  7. Repair of damaged DNA in vivo: Final technical report

    International Nuclear Information System (INIS)

    Hanawalt, P.C.

    1987-09-01

    This contract was initiated in 1962 with the US Atomic Energy Commission to carry out basic research on the effects of radiation on the process of DNA replication in bacteria. Within the first contract year we discovered repair replication at the same time that Setlow and Carrier discovered pyrimidine dimer excision. These discoveries led to the elucidation of the process of excision-repair, one of the most important mechanisms by which living systems, including humans, respond to structural damage in their genetic material. We improved methodology for distinguishing repair replication from semiconservative replication and instructed others in these techniques. Painter then was the first to demonstrate repair replication in ultraviolet irradiated human cells. He, in turn, instructed James Cleaver who discovered that skin fibroblasts from patients with xeroderma pigmentosum were defective in excision-repair. People with this genetic defect are extremely sensitive to sunlight and they develop carcinomas and melanomas of the skin with high frequency. The existence of this hereditary disease attests to the importance of DNA repair in man. We certainly could not survive in the normal ultraviolet flux from the sun if our DNA were not continuously monitored for damage and repaired. Other hereditary diseases such as ataxia telangiectasia, Cockayne's syndrome, Blooms syndrome and Fanconi's anemia also involve deficiencies in DNA damage processing. The field of DNA repair has developed rapidly as we have learned that most environmental chemical carcinogens as well as radiation produce repairable damage in DNA. 251 refs

  8. Repair of Alkylation Damage in Eukaryotic Chromatin Depends on Searching Ability of Alkyladenine DNA Glycosylase.

    Science.gov (United States)

    Zhang, Yaru; O'Brien, Patrick J

    2015-11-20

    Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair pathway by excising alkylated and deaminated purine lesions. In vitro biochemical experiments demonstrate that AAG uses facilitated diffusion to efficiently search DNA to find rare sites of damage and suggest that electrostatic interactions are critical to the searching process. However, it remains an open question whether DNA searching limits the rate of DNA repair in vivo. We constructed AAG mutants with altered searching ability and measured their ability to protect yeast from alkylation damage in order to address this question. Each of the conserved arginine and lysine residues that are near the DNA binding interface were mutated, and the functional impacts were evaluated using kinetic and thermodynamic analysis. These mutations do not perturb catalysis of N-glycosidic bond cleavage, but they decrease the ability to capture rare lesion sites. Nonspecific and specific DNA binding properties are closely correlated, suggesting that the electrostatic interactions observed in the specific recognition complex are similarly important for DNA searching complexes. The ability of the mutant proteins to complement repair-deficient yeast cells is positively correlated with the ability of the proteins to search DNA in vitro, suggesting that cellular resistance to DNA alkylation is governed by the ability to find and efficiently capture cytotoxic lesions. It appears that chromosomal access is not restricted and toxic sites of alkylation damage are readily accessible to a searching protein.

  9. Microfabricated electrochemical sensor for the detection of radiation-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D.H.; Cai, X.; Parrado, C. [New Mexico State Univ., Las Cruces, NM (United States)

    1997-04-01

    An electrochemical biosensor protocol for the detection of radiation-induced DNA damage is described. The procedure employs a dsDNA-coated screen-printed electrode and relies on changes in the guanine-DNA oxidation signal upon exposure to ultraviolet radiation. The decreased signal is ascribed primarily to conformational changes in the DNA and to the photoconversion of the guanine-DNA moiety to a nonelectroactive monomeric base product. Factors influencing the response of these microfabricated DNA sensors, such as irradiation time, wavelength, and distance, are explored, and future prospects are discussed. Similar results are given for the use of bare strip electrodes in connection with irradiated DNA solutions. 8 refs., 4 figs.

  10. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

    Energy Technology Data Exchange (ETDEWEB)

    Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

    2004-11-22

    Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

  11. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

    International Nuclear Information System (INIS)

    Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

    2004-01-01

    Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

  12. Mexamine used to decrease radiation damage to Wistar rat embryogenesis

    International Nuclear Information System (INIS)

    Palyga, G.F.; Zakoshchikov, K.F.

    1987-01-01

    In experiments with 330 Wistar rats experiencing their pregnancy and 1430 neonatal rats of the first generation a study was made on the toxicity and radioprotective efficiency of a single subcutaneous injection of 10 mg/kg mexamine on days 3, 11 and 19 pregnancy. The agent caused various abnormalities in pregnancy, delivery and postnatal development of the offspring of nonirradiated animals, and it was almost ineffective when used for the prevention of radiation damages during the anrnatal ontogenesis

  13. DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

    Science.gov (United States)

    Boiteux, Serge; Jinks-Robertson, Sue

    2013-01-01

    DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

  14. Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response.

    Science.gov (United States)

    Fudrini Olivencia, Begonia; Müller, Andreas U; Roschitzki, Bernd; Burger, Sibylle; Weber-Ban, Eilika; Imkamp, Frank

    2017-10-25

    Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.

  15. Xeroderma Pigmentosum Group C Deficiency Alters Cigarette Smoke DNA Damage Cell Fate and Accelerates Emphysema Development.

    Science.gov (United States)

    Sears, Catherine R; Zhou, Huaxin; Justice, Matthew J; Fisher, Amanda J; Saliba, Jacob; Lamb, Isaac; Wicker, Jessica; Schweitzer, Kelly S; Petrache, Irina

    2018-03-01

    Cigarette smoke (CS) exposure is a major risk factor for the development of emphysema, a common disease characterized by loss of cells comprising the lung parenchyma. The mechanisms of cell injury leading to emphysema are not completely understood but are thought to involve persistent cytotoxic or mutagenic DNA damage induced by CS. Using complementary cell culture and mouse models of CS exposure, we investigated the role of the DNA repair protein, xeroderma pigmentosum group C (XPC), on CS-induced DNA damage repair and emphysema. Expression of XPC was decreased in mouse lungs after chronic CS exposure and XPC knockdown in cultured human lung epithelial cells decreased their survival after CS exposure due to activation of the intrinsic apoptosis pathway. Similarly, cell autophagy and apoptosis were increased in XPC-deficient mouse lungs and were further increased by CS exposure. XPC deficiency was associated with structural and functional changes characteristic of emphysema, which were worsened by age, similar to levels observed with chronic CS exposure. Taken together, these findings suggest that repair of DNA damage by XPC plays an important and previously unrecognized role in the maintenance of alveolar structures. These findings support that loss of XPC, possibly due to chronic CS exposure, promotes emphysema development and further supports a link between DNA damage, impaired DNA repair, and development of emphysema.

  16. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  17. Radiation damage to DNA-protein complexes

    Czech Academy of Sciences Publication Activity Database

    Spotheim-Maurizot, M.; Davídková, Marie

    2011-01-01

    Roč. 261, zima (2011), s. 1-10 ISSN 1742-6588. [COST Chemistry CM0603-MELUSYN Joint Meeting Damages Induced in Biomolecules by Low and High Energy Radiations. Paříž, 09.03.2010-12.03.2010] R&D Projects: GA AV ČR IAA1048103; GA AV ČR KJB4048401; GA MŠk 1P05OC085; GA MŠk OC09012; GA AV ČR IAB1048901 Institutional research plan: CEZ:AV0Z10480505 Keywords : radiolysis * molecular-dynamics simulation * hydroxyl radical attack * induced strand breakage Subject RIV: BO - Biophysics

  18. DNA damage response in monozygotic twins discordant for smoking habits.

    Science.gov (United States)

    Marcon, Francesca; Carotti, Daniela; Andreoli, Cristina; Siniscalchi, Ester; Leopardi, Paola; Caiola, Stefania; Biffoni, Mauro; Zijno, Andrea; Medda, Emanuela; Nisticò, Lorenza; Rossi, Sabrina; Crebelli, Riccardo

    2013-03-01

    Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

  19. An extended sequence specificity for UV-induced DNA damage.

    Science.gov (United States)

    Chung, Long H; Murray, Vincent

    2018-01-01

    The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  20. Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

    Science.gov (United States)

    Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

    2005-06-01

    Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

  1. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    Science.gov (United States)

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  2. DNA damage response in nephrotoxic and ischemic kidney injury

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Mingjuan; Tang, Chengyuan [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Ma, Zhengwei [Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States); Huang, Shuang [Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL (United States); Dong, Zheng, E-mail: zdong@augusta.edu [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States)

    2016-12-15

    DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

  3. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    Energy Technology Data Exchange (ETDEWEB)

    Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

    2012-08-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  4. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    International Nuclear Information System (INIS)

    Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

    2012-01-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  5. DNA damage mediated transcription arrest: Step back to go forward.

    Science.gov (United States)

    Mullenders, Leon

    2015-12-01

    The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Spatiotemporal kinetics of γ-H2AX protein on charged particles induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Niu, H., E-mail: hniu@mx.nthu.edu.tw [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Chang, H.C. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China); Cho, I.C. [Institute for Radiological Research, Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, C.H. [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, C.S. [Cancer Center of Taipei Veterans General Hospital, Taipei, Taiwan (China); Chou, W.T. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China)

    2014-08-15

    Highlights: • Charged particles can induce more complex DNA damages, and these complex damages have higher ability to cause the cell death or cell carcinogenesis. • In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in particle irradiated HeLa cells. • The HeLa cells were irradiated by 400 keV alpha-particles in four different dosages. • The result shows that a good linear relationship can be observed between foci number and radiation dose. • The data shows that the dissolution rate of γ-H2AX foci agree with the two components DNA repairing model, and it was decreasing as the radiation dose increased. • These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair. - Abstract: In several researches, it has been demonstrated that charged particles can induce more complex DNA damages. These complex damages have higher ability to cause the cell death or cell carcinogenesis. For this reason, clarifying the DNA repair mechanism after charged particle irradiation plays an important role in the development of charged particle therapy and space exploration. Unfortunately, the detail spatiotemporal kinetic of DNA damage repair is still unclear. In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in alpha-particle irradiated HeLa cells. The result shows that the intensity of γ-H2AX foci increased gradually, and reached to its maximum at 30 min after irradiation. A good linear relationship can be observed between foci intensity and radiation dose. After 30 min, the γ-H2AX foci intensity was decreased with time passed, but remained a large portion (∼50%) at 48 h passed. The data show that the dissolution rate of γ-H2AX foci agreed with two components DNA repairing model. These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA

  7. Linking abnormal mitosis to the acquisition of DNA damage

    Science.gov (United States)

    Pellman, David

    2012-01-01

    Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis. PMID:23229895

  8. The current state of eukaryotic DNA base damage and repair.

    Science.gov (United States)

    Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2015-12-02

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. DNA damage and repair in human skin in situ

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

    1987-01-01

    Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs.

  10. DNA damage and repair in human skin in situ

    International Nuclear Information System (INIS)

    Sutherland, B.M.; Gange, R.W.; Freeman, S.E.; Sutherland, J.C.

    1987-01-01

    Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells - including human cells in culture - there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair, as well as qualitative differences, including the presence or absence of repair mechanisms. Quantitation of DNA damage and repair in human skin required the development of new approaches for measuring damage at low levels in nanogram quantities of non-radioactive DNA. The method allows for analysis of multiple samples and the resulting data should be related to behavior of the DNA molecules by analytic expressions. Furthermore, it should be possible to assay a variety of lesions using the same methodology. The development of new analysis methods, new technology, and new biochemical probes for the study of DNA damage and repair are described. 28 refs., 4 figs

  11. Diagnosis of Lung Cancer by Fractal Analysis of Damaged DNA

    Directory of Open Access Journals (Sweden)

    Hamidreza Namazi

    2015-01-01

    Full Text Available Cancer starts when cells in a part of the body start to grow out of control. In fact cells become cancer cells because of DNA damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to study the cancer genes, DNA walk plots of genomes of patients with lung cancer were generated using a program written in MATLAB language. The data so obtained was checked for fractal property by computing the fractal dimension using a program written in MATLAB. Also, the correlation of damaged DNA was studied using the Hurst exponent measure. We have found that the damaged DNA sequences are exhibiting higher degree of fractality and less correlation compared with normal DNA sequences. So we confirmed this method can be used for early detection of lung cancer. The method introduced in this research not only is useful for diagnosis of lung cancer but also can be applied for detection and growth analysis of different types of cancers.

  12. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

    2013-01-01

    knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

  13. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  14. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    International Nuclear Information System (INIS)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

    2014-01-01

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting

  15. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

  16. Natural transformation of bacteria by fragmented, damaged and ancient DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren

    with fullgenome comparisons that the process has general relevance in extant bacteria. Our findings reveal that the large environmental reservoir of short and damaged DNA retains capacity for natural transformation, even after thousands of years. This describes for the first time a process by which cells can...... transfer playing an important role early in the evolution of life. The published article explains the chemical structure behind an observed degradation difference between the two purine-nucleotides guanosine and adenosine in ancient DNA. We also point at new uses for high-through-put DNA sequencing...

  17. ATM signaling and genomic stability in response to DNA damage

    International Nuclear Information System (INIS)

    Lavin, Martin F.; Birrell, Geoff; Chen, Philip; Kozlov, Sergei; Scott, Shaun; Gueven, Nuri

    2005-01-01

    DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed

  18. DNA damage in synchronized hela cells irradiated with ultraviolet

    International Nuclear Information System (INIS)

    Downes, C.S.; Collins, A.R.S.; Johnson, R.T.

    1979-01-01

    The lethal effect of uv radiation on HeLa cells is least in mitosis and greatest in late G 1 -early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of uv absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage

  19. DNA damage by carbonyl stress in human skin cells

    International Nuclear Information System (INIS)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L.

    2003-01-01

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N ε -(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress

  20. DNA damage by carbonyl stress in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L

    2003-01-28

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the {alpha}-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N{sup {epsilon}}-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

  1. Activation of DNA damage repair pathways by murine polyomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Katie; Nicholas, Catherine; Garcea, Robert L., E-mail: Robert.Garcea@Colorado.edu

    2016-10-15

    Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.

  2. Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Marie-Jo Halaby

    2015-01-01

    Full Text Available Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES that is located at the 5′-untranslated region (UTR of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80 has increased binding to the p53 mRNA in vivo following DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress.

  3. Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants.

    Science.gov (United States)

    Gichner, Tomás; Znidar, Irena; Száková, Jirina

    2008-04-30

    Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.

  4. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dizdaroglu, Miral

    1999-05-12

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  5. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    International Nuclear Information System (INIS)

    Dizdaroglu, Miral

    1999-01-01

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee ampersand Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of ''naked DNA'' for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  6. Detecting DNA damage with a silver solid amalgam electrode

    Czech Academy of Sciences Publication Activity Database

    Kuchaříková, Kateřina; Novotný, Ladislav; Josypčuk, Bohdan; Fojta, Miroslav

    2004-01-01

    Roč. 16, č. 5 (2004), s. 410-414 ISSN 1040-0397 R&D Projects: GA AV ČR IAA4004108; GA AV ČR IBS5004355 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA damage * silver solid amalgam electrode * HMDE Subject RIV: BO - Biophysics Impact factor: 2.038, year: 2004

  7. Biomarkers of oxidative damage to DNA and repair

    DEFF Research Database (Denmark)

    Loft, Steffen; Høgh Danielsen, Pernille; Mikkelsen, Lone

    2008-01-01

    environmental factors, including particulate air pollution, cause oxidative damage to DNA, whereas diets rich in fruit and vegetables or antioxidant supplements may reduce the levels and enhance repair. Urinary excretion of 8-oxodG, genotype and expression of OGG1 have been associated with risk of cancer...

  8. Cancer risk and oxidative DNA damage in man

    DEFF Research Database (Denmark)

    Loft, S; Poulsen, H E

    1996-01-01

    with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion...

  9. DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas

    DEFF Research Database (Denmark)

    Herrtwich, Laura; Nanda, Indrajit; Evangelou, Konstantinos

    2016-01-01

    to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid...

  10. DNA damage and plasma homocysteine levels are associated with ...

    African Journals Online (AJOL)

    This study describes the association between levels of DNA damage and homocysteine (Hcy) in persistent diarrheic (PD) patients and correlates them with serum biochemical metabolites and mineral components. PD patients (n = 36) age 4 - 6 years from Faisalabad hospitals were examined for anthropometric factors, ...

  11. Function of ZFAND3 in the DNA Damage Response

    Science.gov (United States)

    2013-06-01

    Cantor SB, Naka- tani Y, Livingston DM. 2006. Multifactorial contribu- tions to an acute DNA damage response by BRCA1/ BARD1-containing complexes. Genes...Cutaneous T Cell Lymphoma. PLoS ONE 8(7): e68915. doi:10.1371/journal.pone.0068915 Editor: Sue Cotterill, St. Georges University of London, United

  12. SUMO boosts the DNA damage response barrier against cancer

    Czech Academy of Sciences Publication Activity Database

    Bartek, Jiří; Hodný, Zdeněk

    2010-01-01

    Roč. 17, č. 1 (2010), s. 9-11 ISSN 1535-6108 R&D Projects: GA ČR GA301/08/0353 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage response * ubiquitylation * sumoylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 26.925, year: 2010

  13. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... crosslinks can also affect the structure of DNA significantly. ... H2O2 by converting it into water, reaction of H2O2 with ..... Damaged nucleotide flipping by (a) AGT due to intercalation of an amino acid (Arg128) (pdb 1t38) and ...

  14. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    Science.gov (United States)

    A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

  15. The DNA-damage response in human biology and disease

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management....

  16. Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Lorenzo, Y.; Karlsen, A.; Carlsen, M. H.; Novosadová, Vendula; Blomhoff, R.; Vodička, Pavel; Collins, A. R.

    2014-01-01

    Roč. 16, APR 2014 (2014), s. 66-73 ISSN 1568-7864 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA damage * DNA repair capacity * diet Subject RIV: EB - Genetics ; Molecular Biology; EI - Biotechnology ; Bionics (BTO-N) Impact factor: 3.111, year: 2014

  17. Role of DNA repair in repair of cytogenetic damages. Contribution of repair of single-strand DNA breaks to cytogenetic damages repair

    International Nuclear Information System (INIS)

    Rozanova, O.M.; Zaichkina, S.I.; Aptikaev, G.F.; Ganassi, E.Eh.

    1989-01-01

    The comparison was made between the results of the effect of poly(ADP-ribosylation) ingibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase ingibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronuclear test, and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modyfying effect with regard to radiation cytogenetic damages and kynetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes

  18. Carnosine attenuates cyclophosphamide-induced bone marrow suppression by reducing oxidative DNA damage

    Directory of Open Access Journals (Sweden)

    Jie Deng

    2018-04-01

    Full Text Available Oxidative DNA damage in bone marrow cells is the main side effect of chemotherapy drugs including cyclophosphamide (CTX. However, not all antioxidants are effective in inhibiting oxidative DNA damage. In this study, we report the beneficial effect of carnosine (β-alanyl-l-histidine, a special antioxidant with acrolein-sequestering ability, on CTX-induced bone marrow cell suppression. Our results show that carnosine treatment (100 and 200 mg/kg, i.p. significantly inhibited the generation of reactive oxygen species (ROS and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, and decreased chromosomal abnormalities in the bone marrow cells of mice treated with CTX (20 mg/kg, i.v., 24 h. Furthermore, carnosine evidently mitigated CTX-induced G2/M arrest in murine bone marrow cells, accompanied by reduced ratios of p-Chk1/Chk1 and p-p53/p53 as well as decreased p21 expression. In addition, cell apoptosis caused by CTX was also suppressed by carnosine treatment, as assessed by decreased TUNEL-positive cell counts, down-regulated expressions of Bax and Cyt c, and reduced ratios of cleaved Caspase-3/Caspase-3. These results together suggest that carnosine can protect murine bone marrow cells from CTX-induced DNA damage via its antioxidant activity. Keywords: Carnosine, Cyclophosphamide, Oxidative DNA damage, Sister chromatid exchange, Apoptosis, Cell cycle arrest

  19. Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

    Science.gov (United States)

    Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

    2014-01-01

    DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

  20. Oxidatively generated DNA/RNA damage in psychological stress states

    DEFF Research Database (Denmark)

    Jørgensen, Anders

    2013-01-01

    age-related somatic disorders. The overall aim of the PhD project was to investigate the relation between psychopathology, psychological stress, stress hormone secretion and oxidatively generated DNA and RNA damage, as measured by the urinary excretion of markers of whole-body DNA/RNA oxidation (8...... between the 24 h urinary cortisol excretion and the excretion of 8-oxodG/8-oxoGuo, determined in the same samples. Collectively, the studies could not confirm an association between psychological stress and oxidative stress on nucleic acids. Systemic oxidatively generated DNA/RNA damage was increased......Both non-pathological psychological stress states and mental disorders are associated with molecular, cellular and epidemiological signs of accelerated aging. Oxidative stress on nucleic acids is a critical component of cellular and organismal aging, and a suggested pathogenic mechanism in several...

  1. Radiation damage to DNA: The importance of track structure

    CERN Document Server

    Hill, M A

    1999-01-01

    A wide variety of biological effects are induced by ionizing radiation, from cell death to mutations and carcinogenesis. The biological effectiveness is found to vary not only with the absorbed dose but also with the type of radiation and its energy, i.e., with the nature of radiation tracks. An overview is presented of some of the biological experiments using different qualities of radiation, which when compared with Monte Carlo track structure studies, have highlighted the importance of the localized spatial properties of stochastic energy deposition on the nanometer scale at or near DNA. The track structure leads to clustering of damage which may include DNA breaks, base damage etc., the complexity of the cluster and therefore its biological repairability varying with radiation type. The ability of individual tracks to produce clustered damage, and the subsequent biological response are important in the assessment of the risk associated with low-level human exposure. Recent experiments have also shown that...

  2. Targeting Ongoing DNA Damage in Multiple Myeloma: Effects of DNA Damage Response Inhibitors on Plasma Cell Survival

    Directory of Open Access Journals (Sweden)

    Ana Belén Herrero

    2017-05-01

    Full Text Available Human myeloma cell lines (HMCLs and a subset of myeloma patients with poor prognosis exhibit high levels of replication stress (RS, leading to DNA damage. In this study, we confirmed the presence of DNA double-strand breaks (DSBs in several HMCLs by measuring γH2AX and RAD51 foci and analyzed the effect of various inhibitors of the DNA damage response on MM cell survival. Inhibition of ataxia telangiectasia and Rad3-related protein (ATR, the main kinase mediating the response to RS, using the specific inhibitor VE-821 induced more cell death in HMCLs than in control lymphoblastoid cells and U266, an HMCL with a low level of DNA damage. The absence of ATR was partially compensated by ataxia telangiectasia-mutated protein (ATM, since chemical inhibition of both kinases using VE-821 and KU-55933 significantly increased the death of MM cells with DNA damage. We found that ATM and ATR are involved in DSB repair by homologous recombination (HR in MM. Inhibition of both kinases resulted in a stronger inhibition that may underlie cell death induction, since abolition of HR using two different inhibitors severely reduced survival of HMCLs that exhibit DNA damage. On the other hand, inhibition of the other route involved in DSB repair, non-homologous end joining (NHEJ, using the DNA-PK inhibitor NU7441, did not affect MM cell viability. Interestingly, we found that NHEJ inhibition did not increase cell death when HR was simultaneously inhibited with the RAD51 inhibitor B02, but it clearly increased the level of cell death when HR was inhibited with the MRE11 inhibitor mirin, which interferes with recombination before DNA resection takes place. Taken together, our results demonstrate for the first time that MM cells with ongoing DNA damage rely on an intact HR pathway, which thereby suggests therapeutic opportunities. We also show that inhibition of HR after the initial step of end resection might be more appropriate for inducing MM cell death, since it

  3. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

    Science.gov (United States)

    Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

    2015-09-01

    Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence

  4. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    International Nuclear Information System (INIS)

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

  5. Oxidative damage of DNA in subjects occupationally exposed to lead.

    Science.gov (United States)

    Pawlas, Natalia; Olewińska, Elżbieta; Markiewicz-Górka, Iwona; Kozłowska, Agnieszka; Januszewska, Lidia; Lundh, Thomas; Januszewska, Ewa; Pawlas, Krystyna

    2017-09-01

    Exposure to lead (Pb) in environmental and occupational settings continues to be a serious public health problem and may pose an elevated risk of genetic damage. The aim of this study was to assess the level of oxidative stress and DNA damage in subjects occupationally exposed to lead. We studied a population of 78 male workers exposed to lead in a lead and zinc smelter and battery recycling plant and 38 men from a control group. Blood lead levels were detected by graphite furnace atomic absorption spectrophotometry and plasma lead levels by inductively coupled plasma-mass spectrometry. The following assays were performed to assess the DNA damage and oxidative stress: comet assay, determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG), lipid peroxidation and total antioxidant status (TAS). The mean concentration of lead in the blood of the exposed group was 392 ± 103 μg/L and was significantly higher than in the control group (30.3 ± 29.4 μg/L, p lead exposure [lead in blood, lead in plasma, zinc protoporphyrin (ZPP)] and urine concentration of 8-OHdG. The level of oxidative damage of DNA was positively correlated with the level of lipid peroxidation (TBARS) and negatively with total anti-oxidative status (TAS). Our study suggests that occupational exposure causes an increase in oxidative damage to DNA, even in subjects with relatively short length of service (average length of about 10 years). 8-OHdG concentration in the urine proved to be a sensitive and non-invasive marker of lead induced genotoxic damage.

  6. Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

    Directory of Open Access Journals (Sweden)

    Robert L. Eoff

    2010-01-01

    Full Text Available DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

  7. Taking a Bad Turn: Compromised DNA Damage Response in Leukemia

    Directory of Open Access Journals (Sweden)

    Nadine Nilles

    2017-05-01

    Full Text Available Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2, key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS, and isocitrate dehydrogenase 1 and 2 (IDH1/2. A detailed understanding of the basis for defective DNA damage response (DDR mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients.

  8. Crosstalk between the nucleolus and the DNA damage response.

    Science.gov (United States)

    Ogawa, L M; Baserga, S J

    2017-02-28

    Nucleolar function and the cellular response to DNA damage have long been studied as distinct disciplines. New research and a new appreciation for proteins holding multiple functional roles, however, is beginning to change the way we think about the crosstalk among distinct cellular processes. Here, we focus on the crosstalk between the DNA damage response and the nucleolus, including a comprehensive review of the literature that reveals a role for conventional DNA repair proteins in ribosome biogenesis, and conversely, ribosome biogenesis proteins in DNA repair. Furthermore, with recent advances in nucleolar proteomics and a growing list of proteins that localize to the nucleolus, it is likely that we will continue to identify new DNA repair proteins with a nucleolar-specific role. Given the importance of ribosome biogenesis and DNA repair in essential cellular processes and the role that they play in diverse pathologies, continued elucidation of the overlap between these two disciplines will be essential to the advancement of both fields and to the development of novel therapeutics.

  9. Micronutrients intake associated with DNA damage assessed by in a human biomonitoring study

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-05-01

    ingestion decreases DNA damage and DNA oxidative damage (Hart et al., 1999; Heilbronn & Ravussin, 2003. A significant negative correlation was found between folate and % DNA in tail. Courtemanche et al. (2004 also found that folate deficiency leads to increased DNA damage in primary lymphocytes, and that deficiency in the physiological level of folate caused more DNA damage than low-dose radiation in primary T lymphocytes. A significant negative correlation between vitamin B12 and DNA oxidative damage (FPG was found, suggesting that vitamin B12 acts like a protective factor (Ames, 2001; Ames & Wakimoto, 2002; Ames, 2006. Minnet et al. (2011 also found a negative correlation between DNA damage and vitamin B12 levels, meaning that higher levels of vitamin B12 decrease DNA damage, in good agreement with the results herein. Comet assay allows for the study of the effects of nutrients with known anti- or pro-oxidant capacities on different cell types and in different concentrations. These studies have revealed an apparent paradox, or at least a hormetic effect, whereby many of these antioxidant compounds seem to protect against DNA damage at low doses while actually causing DNA damage at higher doses (Wasson et al., 2008. There are several possible reasons why significant associations are difficult to find. First, samples usually comprise mostly healthy persons; second, it is possible that a synergistic effect exists involving all antioxidants which is not seen for each individual nutrient (Watters et al., 2007. Third, it is plausible that associations between some of the antioxidants examined and oxidative DNA damage may be better captured using other measures of oxidative DNA damage. Fourth, it is possible that the range of antioxidant concentrations and/or oxidative DNA damage in this study was not wide enough to detect associations or that the associations simply do not exist (Watters et al., 2007. Previous studies have suggested a significant moderating effect of long

  10. DNA repair and the evolution of transformation in Bacillus subtilis. 3. Sex with damaged DNA

    International Nuclear Information System (INIS)

    Hoelzer, M.A.; Michod, R.E.

    1991-01-01

    Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. The authors document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome

  11. Un-repairable DNA damage in cell due to irradiation

    International Nuclear Information System (INIS)

    Yoshii, Giichi

    1992-01-01

    Radiation-induced cell reproductive deactivation is caused by damage to DNA. In a cell, cellular DNA radical reacts with diffusion controlled rate and generates DNA peroxide radical. The chemical repair of DNA radical with hydrogen donation by thiol competes with the reaction of oxygen with same radicals in the DNA molecules. From the point reaction rates, the prolongation of radical life time is not as great as expected from the reduction in the glutathione content of the cell. This indicates that further reducting compounds (protein bound thiol) are present in the cell. The residual radicals are altered to strand breaks, base damages and so on. The effective lesions for a number of endpoints is un-repaired double strand break, which has been discovered in a cluster. This event gives risk to high LET radiation or to a track end of X-rays. For X- or electron irradiations the strand breaks are frequently induced by the interactions between sublesions on two strands in DNA. A single strand break followed by radical action may be unstable excited state, because of remaining sugar radical action and of having negative charged phosphates, in which strands breaks will be rejoined in a short time to stable state. On the same time, a break in the double helix will be immediately produced if two breaks are on either or approximately opposite locations. The formation of a double strand break in the helix depends on the ion strength of the cell. The potassium ions are largely released from polyanionic strand during irradiation, which results in the induction of denatured region. Double strand break with the denatured region seems to be un-repairable DNA damage. (author)

  12. Endogenous melatonin and oxidatively damaged guanine in DNA

    Directory of Open Access Journals (Sweden)

    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  13. Repair of DNA damage in the human metallothionein gene family

    International Nuclear Information System (INIS)

    Leadon, S.A.; Snowden, M.M.

    1987-01-01

    In order to distinguish enhanced repair of a sequence due to its transcriptional activity from enhanced repair due to chromatin alterations brought about by integration of a sequence into the genome, we have investigated the repair of damage both in endogenous genes and in cell lines that contain an integrated gene with an inducible promoter. The endogenous genes we are studying are the metallothioneins (MTs), a multigene family in man consisting of about 10-12 members. Cultured cells were exposed to 10-J/m 2 uv light and allowed to repair in the presence of bromodeoxyuridine. The DNA was then isolated, digested with Eco RI, and fully hybrid density DNA made by semiconservative synthesis was separated from unreplicated DNA by centrifugation in CsCl density gradients. Unreplicated, parental-density DNA was then reacted with a monoclonal antibody against bromouracil. 1 ref., 1 fig., 1 tab

  14. DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)

    International Nuclear Information System (INIS)

    Ammermann, D.

    1988-01-01

    Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6- 3 H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei

  15. The AID-induced DNA damage response in chromatin

    DEFF Research Database (Denmark)

    Daniel, Jeremy A; Nussenzweig, André

    2013-01-01

    Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity...... with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation...... of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles...

  16. Situation-dependent repair of DNA damage in yeast

    International Nuclear Information System (INIS)

    von Borstel, R.C.; Hastings, P.J.

    1985-01-01

    The concept of channelling of lesions in DNA into defined repair systems has been used to explain many aspects of induced and spontaneous mutation. The channelling hypothesis states that lesions excluded from one repair process will be taken up by another repair process. This is a simplification. The three known modes of repair of damage induced by radiation are not equivalent modes of repair; they are, instead, different solutions to the problem of replacement of damaged molecules with new molecules which have the same informational content as those that were damaged. The mode of repair that is used is the result of the response to the situation in which the damage takes place. Thus, when the most likely mode of repair does not take place, then the situation changes with respect to the repair of the lesion; the lesion may enter the replication fork and be reparable by another route

  17. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    Science.gov (United States)

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Damage-induced DNA repair processes in Escherichia coli cells

    International Nuclear Information System (INIS)

    Slezarikova, V.

    1986-01-01

    The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

  19. Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

    Energy Technology Data Exchange (ETDEWEB)

    LaRiviere, Frederick J. [Department of Chemistry, Washington and Lee University, Lexington, VA 24450 (United States); Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E. [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States); Greenwood, Paul G. [Department of Biology, Colby College, Waterville, ME 04901 (United States); Millard, Julie T., E-mail: jtmillar@colby.edu [Department of Chemistry, Colby College, 5757 Mayflower Hill Drive, Waterville, ME 04901 (United States)

    2009-05-12

    The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

  20. Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

    International Nuclear Information System (INIS)

    LaRiviere, Frederick J.; Newman, Adam G.; Watts, Megan L.; Bradley, Sharonda Q.; Juskewitch, Justin E.; Greenwood, Paul G.; Millard, Julie T.

    2009-01-01

    The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

  1. DNA turnover in buffer-held Escherichia coli and its effect on repair of UV damage

    International Nuclear Information System (INIS)

    Tang, M.S.; Wang, T.C.V.; Patrick, M.H.

    1979-01-01

    Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions it appears that at any given time there is a minimum of one repair site per 6.5 x 10 6 daltons DNA, each of which is at least 160 nucleotides long. While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. It is suggested that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, a model is proposed to explain the limit repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery). (author)

  2. Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage

    International Nuclear Information System (INIS)

    Park, Jung Wook; Nickel, Kwangok P.; Torres, Alexandra D.; Lee, Denis; Lambert, Paul F.; Kimple, Randall J.

    2014-01-01

    Background and purpose: Patients with human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV−) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV− HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. Material and methods: Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using γ-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. Results: HPV16 E7 increased retention of γ-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after γ-radiation. Conclusions: Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV− HNC

  3. Flow cytometric assessment of DNA damage in the fish Catla catla (Ham.) exposed to gamma radiation

    International Nuclear Information System (INIS)

    Anbumani, S.; Mohankumar, Mary N.; Selvanayagam, M.

    2012-01-01

    Environmental mutagens such as ionizing radiation and chemicals induce DNA damage in a wide variety of organisms. The International Commission on Radiological Protection (lCRP) has recently emphasized the need to protect non-human biota from the potential effects of ionizing radiation. Radiation exposures to non-humans can occur as a result of low-level radioactive discharges into the environment. Molecular genetic effects at low-level radiation exposures are largely unexplored and systematic studies using sensitive biomarkers are required to assess DNA damage in representative non-human species. The objective of the study was to detect DNA damage in the fish Catla catla exposed to gamma radiation using flow cytometry at different time intervals. Increases in the coefficient of variation (CV) of the G 0 /G 1 peak, indicating abnormal DNA distributions were observed in fish exposed to gamma radiation than in controls. Significant increase in the CV was observed from day 12-90 and thereafter decreased. This increase in CV might be due to DNA damage in the cell populations at G 0 /G 1 phase or deletions and duplications caused by improper repair of chromosomes in the cell-cycle machinery. Ionizing radiation induced cell-cycle perturbations and apoptosis were also observed after gamma radiation exposure. (author)

  4. DNA Damage Repair System in Plants: A Worldwide Research Update.

    Science.gov (United States)

    Gimenez, Estela; Manzano-Agugliaro, Francisco

    2017-10-30

    Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

  5. 32P-labeling test for DNA damage

    International Nuclear Information System (INIS)

    Randerath, K.; Reddy, M.V.; Gupta, R.C.

    1981-01-01

    Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32 P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, β-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [γ- 32 P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'- 32 P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyl-eneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10 5 DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding

  6. X-Ray induced DNA damage – why use plants?

    Directory of Open Access Journals (Sweden)

    John William Einset

    2015-06-01

    Full Text Available The comet assay was used to monitor DNA repair after X-ray exposures caused by 0.2-15 Gy. A clear distinction in the time course of DNA repair after 2 Gy was observed with an early ‘rapid phase’, lasting 20-40 minutes, being followed by a ‘slow phase’ which actually consists of a period of negligible repair and then rapid repair during 140-160 minutes. The fact that homozygous mutants for both ATM and BRCA1 fail to repair DNA completely during 3 hours after 2 Gy exposures indicates that repair processes occurring during the ‘slow phase’ involve ds breaks in DNA. Both BRCA1 and Rad51 expression are strongly upregulated by X-rays in Arabidopsis. Rye grass, Norway spruce and Sawara cypress also have ‘slow phase’ repair similar to Arabidopsis, suggesting that the requisite enzymes have to be induced in these plants as well. To look at the effect of genome size in relation to sensitivity to DNA damage, we exposed isolated nuclei from Norway spruce (19.2 Gbp genome, celery (14.1 Gbp, spinach (12.6 Gbp Sawara cypress (8.9 Gbp, lettuce (2.6 Gbp and Arabidopsis (0.135 Gbp to X-rays. After a 1 Gy exposure, a linear relationship was seen between % tails and genome size, confirming the idea that larger genomes are more sensitive to X-ray damage.

  7. Primary DNA damage in chrome-plating workers.

    Science.gov (United States)

    Gambelunghe, A; Piccinini, R; Ambrogi, M; Villarini, M; Moretti, M; Marchetti, C; Abbritti, G; Muzi, G

    2003-06-30

    In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).

  8. The Inhibition Effect of Cell DNA Oxidative Damage and LDL Oxidation by Bovine Colostrums

    Directory of Open Access Journals (Sweden)

    Chih-Wei Chen

    2016-10-01

    Full Text Available In the present study, we investigated the effect of bovine colostrums on inhibition of DNA oxidative damage and low density lipoprotein (LDL oxidation in vitro. Results showed that whey and skimmed milk exhibited not only higher inhibitory activities of oxidative damage of deoxyribose but also an inhibitory effect on the breakdown of supercoiled DNA into open circular DNA and linear DNA. The quantities of 8-OH-2′-dG formed under whey, caseins and skimmed milk treatment were 0.24, 0.24 and 1.24 μg/mL, respectively. The quantity of malondialdehyde formed through LDL oxidation induced by copprous ion was significantly decreased as colostrums protein solutions were added, in which whey and caseins led to a more significant decrease than skimmed milk. The formation of conjugated dienes could be inhibited by treatment with colostrums protein solutions. Whey exhibited the longest lag time of conjugated dienes formation among the colostrums proteins. The lag time of the whey was 2.33 times that of the control. From the results of foregoing, the bovine colostrums protein has potential value in the inhibition of DNA oxidation damage and LDL oxidation.

  9. Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder.

    Science.gov (United States)

    Ceylan, Deniz; Tuna, Gamze; Kirkali, Güldal; Tunca, Zeliha; Can, Güneş; Arat, Hidayet Ece; Kant, Melis; Dizdaroglu, Miral; Özerdem, Ayşegül

    2018-05-01

    Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO......-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4....

  11. The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

    Science.gov (United States)

    Khoe, Clairine V; Chung, Long H; Murray, Vincent

    2018-06-01

    The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

  12. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

    2008-02-21

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  13. DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

    2007-12-01

    The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-induced heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.

  14. Oxidative damage of mitochondrial and nuclear DNA induced by ionizing radiation in human hepatoblastoma cells

    International Nuclear Information System (INIS)

    Morales, Albert; Miranda, Merce; Sanchez-Reyes, Alberto; Biete, Alberto; Fernandez-Checa, Jose C.

    1998-01-01

    Purpose: Since reactive oxygen species (ROS) act as mediators of radiation-induced cellular damage, the aim of our studies was to determine the effects of ionizing radiation on the regulation of hepatocellular reduced glutathione (GSH), survival and integrity of nuclear and mitochondrial DNA (mtDNA) in human hepatoblastoma cells (Hep G2) depleted of GSH prior to radiation. Methods and Materials: GSH, oxidized glutathione (GSSG), and generation of ROS were determined in irradiated (50-500 cGy) Hep G2 cells. Clonogenic survival, nuclear DNA fragmentation, and integrity of mtDNA were assessed in cells depleted of GSH prior to radiation. Results: Radiation of Hep G2 cells (50-400 cGy) resulted in a dose-dependent generation of ROS, an effect accompanied by a decrease of reduced GSH, ranging from a 15% decrease for 50 cGy to a 25% decrease for 400 cGy and decreased GSH/GSSG from a ratio of 17 to a ratio of 7 for controls and from 16 to 6 for diethyl maleate (DEM)-treated cells. Depletion of GSH prior to radiation accentuated the increase of ROS by 40-50%. The depletion of GSH by radiation was apparent in different subcellular sites, being particularly significant in mitochondria. Furthermore, depletion of nuclear GSH to 50-60% of initial values prior to irradiation (400 cGy) resulted in DNA fragmentation and apoptosis. Consequently, the survival of Hep G2 to radiation was reduced from 25% of cells not depleted of GSH to 10% of GSH-depleted cells. Fitting the survival rate of cells as a function of GSH using a theoretical model confirmed cellular GSH as a key factor in determining intrinsic sensitivity of Hep G2 cells to radiation. mtDNA displayed an increased susceptibility to the radiation-induced loss of integrity compared to nuclear DNA, an effect that was potentiated by GSH depletion in mitochondria (10-15% intact mtDNA in GSH-depleted cells vs. 25-30% of repleted cells). Conclusion: GSH plays a critical protective role in maintaining nuclear and mtDNA functional

  15. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  16. Ultraviolet radiation-mediated damage to cellular DNA

    International Nuclear Information System (INIS)

    Cadet, Jean; Sage, Evelyne; Douki, Thierry

    2005-01-01

    Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1 O 2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals

  17. DNA-repair, cell killing and normal tissue damage

    International Nuclear Information System (INIS)

    Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

    1998-01-01

    Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

  18. Radiation induced DNA damage and repair in mutagenesis

    International Nuclear Information System (INIS)

    Strniste, G.F.; Chen, D.J.; Okinaka, R.T.

    1987-01-01

    The central theme in cellular radiobiological research has been the mechanisms of radiation action and the physiological response of cells to this action. Considerable effort has been directed toward the characterization of radiation-induced DNA damage and the correlation of this damage to cellular genetic change that is expressed as mutation or initiating events leading to cellular transformation and ultimately carcinogenesis. In addition, there has been a significant advancement in their understanding of the role of DNA repair in the process of mutation leading to genetic change in cells. There is extensive literature concerning studies that address radiation action in both procaryotic and eucaryotic systems. This brief report will make no attempt to summarize this voluminous data but will focus on recent results from their laboratory of experiments in which they have examined, at both the cellular and molecular levels, the process of ionizing radiation-induced mutagenesis in cultured human cells

  19. Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1982-10-01

    The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

  20. Nickel exposure induces oxidative damage to mitochondrial DNA in Neuro2a cells: the neuroprotective roles of melatonin.

    Science.gov (United States)

    Xu, Shang-Cheng; He, Min-Di; Lu, Yong-Hui; Li, Li; Zhong, Min; Zhang, Yan-Wen; Wang, Yuan; Yu, Zheng-Ping; Zhou, Zhou

    2011-11-01

    Recent studies suggest that oxidative stress and mitochondrial dysfunction play important roles in the neurotoxicity of nickel. Because mitochondrial DNA (mtDNA) is highly vulnerable to oxidative stress and melatonin can efficiently protect mtDNA against oxidative damage in various pathological conditions, the aims of this study were to determine whether mtDNA oxidative damage was involved in the neurotoxicity of nickel and to assay the neuroprotective effects of melatonin in mtDNA. In this study, we exposed mouse neuroblastoma cell lines (Neuro2a) to different concentrations of nickel chloride (NiCl(2), 0.125, 0.25, and 0.5 mm) for 24 hr. We found that nickel significantly increased reactive oxygen species (ROS) production and mitochondrial superoxide levels. In addition, nickel exposure increased mitochondrial 8-hydroxyguanine (8-OHdG) content and reduced mtDNA content and mtDNA transcript levels. Consistent with this finding, nickel was found to destroy mtDNA nucleoid structure and decrease protein levels of Tfam, a key protein component for nucleoid organization. However, all the oxidative damage to mtDNA induced by nickel was efficiently attenuated by melatonin pretreatment. Our results suggest that oxidative damage to mtDNA may account for the neurotoxicity of nickel. Melatonin has great pharmacological potential in protecting mtDNA against the adverse effects of nickel in the nervous system. © 2011 John Wiley & Sons A/S.

  1. Radioprotection against DNA damage by an extract of Indian green mussel, Perna viridis (L.)

    Digital Repository Service at National Institute of Oceanography (India)

    Kumaran, S.P.; Kutty, B.C.; Chatterji, A.; Parameswaran, P.S.; Mishra, K.P.

    -irradiation Prevention of DNA damage both in plasmid and lymphocytes and cell death in lymphocytes appears correlated with reduction of oxidatively generated free radicals It is concluded that protection against radiation-induced cell death and DNA damage by MH...

  2. Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals

    DEFF Research Database (Denmark)

    Møller, Peter; Jensen, Ditte Marie; Christophersen, Daniel Vest

    2015-01-01

    -reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations...... of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure...... and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential. Environ. Mol. Mutagen., 2014. © 2014 Wiley Periodicals, Inc....

  3. Proton-induced direct and indirect damage of plasmid DNA

    Czech Academy of Sciences Publication Activity Database

    Vyšín, Luděk; Pachnerová Brabcová, Kateřina; Štěpán, V.; Moretto-Capelle, P.; Bugler, B.; Legube, G.; Cafarelli, P.; Casta, R.; Champeaux, J. P.; Sence, M.; Vlk, M.; Wagner, Richard; Štursa, Jan; Zach, Václav; Incerti, S.; Juha, Libor; Davídková, Marie

    2015-01-01

    Roč. 54, č. 3 (2015), s. 343-352 ISSN 0301-634X R&D Projects: GA ČR GA13-28721S; GA MŠk LD12008; GA MŠk LM2011019 Institutional support: RVO:68378271 ; RVO:61389005 Keywords : proton radiation * DNA plasmid * direct and indirect effects * clustered damage * repair enzymes Subject RIV: BO - Biophysics Impact factor: 1.923, year: 2015

  4. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  5. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  6. Repair of oxidative DNA damage by amino acids.

    Science.gov (United States)

    Milligan, J R; Aguilera, J A; Ly, A; Tran, N Q; Hoang, O; Ward, J F

    2003-11-01

    Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

  7. HSV-I and the cellular DNA damage response.

    Science.gov (United States)

    Smith, Samantha; Weller, Sandra K

    2015-04-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al . that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

  8. Repair of uv damaged DNA in systemic lupus erythematosus. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Beighlie, D J; Teplitz, R L

    1975-06-01

    The NZB NZW hybrid mouse is an animal model of human systemic lupus erythematosus (SLE). Two breeding schemes were devised using NZB, NZW, B/W, and CBA mice, which permit definitive decisions regarding genetic and/or viral origin of the disease. It is proposed that at least two factors must be involved: a genetic abnormality producing hyper-responsiveness to nucleic acid antigens, and a DNA repair defect which results in liberation of DNA and RNA when cells are lethally injured. Evidence is presented for a DNA repair deficit in human SLE lymphocytes following in vitro irradiation with ultraviolet (uv) light. Lymphocytes from adult New Zealand and control mice were found to lack normal amounts of endonuclease necessary for repairing uv damage.

  9. Single Nucleotide Polymorphisms in Noncoding Regions of Rad51C Do Not Change the Risk of Unselected Breast Cancer but They Modulate the Level of Oxidative Stress and the DNA Damage Characteristics

    DEFF Research Database (Denmark)

    Gresner, Peter; Gromadzinska, Jolanta; Jablonska, Ewa

    2014-01-01

    affect the unselected BrC risk. Contrary to this, carriers of rs12946522, rs16943176, rs12946397 and rs17222691 rare-alleles were found to present significantly increased level of blood plasma TBARS compared to respective wild-type homozygotes (p... decreased fraction of oxidatively generated DNA damage (34% of total damaged DNA) in favor of DNA strand breakage, with no effect on total DNA damage, unlike respective wild-types, among which more evenly distributed proportions between oxidatively damaged DNA (48% of total DNA damage) and DNA strand...

  10. rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint

    NARCIS (Netherlands)

    Walworth, N.C.; Bernards, R.A.

    1996-01-01

    Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet

  11. DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

    Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

  12. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

  13. Heavy ion induced damage to plasmid DNA : plateau region vs. spread out Bragg-peak

    NARCIS (Netherlands)

    Dang, H.M.; van Goethem, M.J.; van der Graaf, E.R.; Brandenburg, S.; Hoekstra, R.A.; Schlathölter, T.A.

    We have investigated the damage of synthetic plasmid pBR322 DNA in dilute aqueous solutions induced by fast carbon ions. The relative contribution of indirect damage and direct damage to the DNA itself is expected to vary with linear energy transfer along the ion track, with the direct damage

  14. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    International Nuclear Information System (INIS)

    Roper, Katherine; Coverley, Dawn

    2012-01-01

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naïve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: ► A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ► Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ► PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ► Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ► LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  15. Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

    DEFF Research Database (Denmark)

    Møller, P; Loft, S; Lundby, C

    2001-01-01

    ; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage...... oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity...

  16. Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

    International Nuclear Information System (INIS)

    Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

    2003-01-01

    To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of γ-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of γ-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced γ-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression

  17. Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

    2003-11-27

    To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of {gamma}-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of {gamma}-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced {gamma}-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.

  18. Capturing Snapshots of APE1 Processing DNA Damage

    Science.gov (United States)

    Freudenthal, Bret D.; Beard, William A.; Cuneo, Matthew J.; Dyrkheeva, Nadezhda S.; Wilson, Samuel H.

    2015-01-01

    DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage. PMID:26458045

  19. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  20. In vitro studies of cellular response to DNA damage induced by boron neutron capture therapy

    International Nuclear Information System (INIS)

    Perona, M.; Pontiggia, O.; Carpano, M.; Thomasz, L.; Thorp, S.; Pozzi, E.; Simian, M.; Kahl, S.; Juvenal, G.; Pisarev, M.; Dagrosa, A.

    2011-01-01

    The aim of these studies was to evaluate the mechanisms of cellular response to DNA damage induced by BNCT. Thyroid carcinoma cells were incubated with 10 BPA or 10 BOPP and irradiated with thermal neutrons. The surviving fraction, the cell cycle distribution and the expression of p53 and Ku70 were analyzed. Different cellular responses were observed for each irradiated group. The decrease of Ku70 in the neutrons +BOPP group could play a role in the increase of sensitization to radiation.

  1. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  2. DNA damage and repair activity after broccoli intake in young healthy smokers

    DEFF Research Database (Denmark)

    Riso, Patrizia; Martini, Daniela; Møller, Peter

    2010-01-01

    compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period...... mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72......%) and the resistance to H(2)O(2)-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli...

  3. The repair of damage to DNA in different cell types

    International Nuclear Information System (INIS)

    Karran, P.

    1974-01-01

    DNA single strand breaks induced by either X-ray irradiation or by methyl methanesulphonate (MMS) were studied in different lymphoid cell populations directly taken from the animal and maintained in tissue culture merely for the duration of the experiment. The results obtained from these cell populations were compared with those obtained with L5178Y cells maintained in tissue culture. All cell types studied were found to possess at least one class of enzymes required for repair of DNA damage, namely those enzymes involved in the rejoining of X-ray induced by MMS is different in each cell type. Repair replication was at much reduced levels and the endonucleolytic degradation was at much reduced levels and the endonucleolytic degradation was initiated at lower MMS concentration in the lymphoid cells as compared to L5178Y cells. It is suggested that the overall ''repair capacity'' of a population may be related to the number of cells in a cycle which, moreover, might be the only ones to have the ability to repair damage to DNA induced by MMS (G.G.)

  4. Radiation damage to DNA: The importance of track structure

    International Nuclear Information System (INIS)

    Hill, M.A.

    1999-01-01

    A wide variety of biological effects are induced by ionizing radiation, from cell death to mutations and carcinogenesis. The biological effectiveness is found to vary not only with the absorbed dose but also with the type of radiation and its energy, i.e., with the nature of radiation tracks. An overview is presented of some of the biological experiments using different qualities of radiation, which when compared with Monte Carlo track structure studies, have highlighted the importance of the localized spatial properties of stochastic energy deposition on the nanometer scale at or near DNA. The track structure leads to clustering of damage which may include DNA breaks, base damage etc., the complexity of the cluster and therefore its biological repairability varying with radiation type. The ability of individual tracks to produce clustered damage, and the subsequent biological response are important in the assessment of the risk associated with low-level human exposure. Recent experiments have also shown that biological response to radiation is not always restricted to the 'hit' cell but can sometimes be induced in 'un-hit' cells near by

  5. Maintaining Genome Stability in Defiance of Mitotic DNA Damage

    Science.gov (United States)

    Ferrari, Stefano; Gentili, Christian

    2016-01-01

    The implementation of decisions affecting cell viability and proliferation is based on prompt detection of the issue to be addressed, formulation and transmission of a correct set of instructions and fidelity in the execution of orders. While the first and the last are purely mechanical processes relying on the faithful functioning of single proteins or macromolecular complexes (sensors and effectors), information is the real cue, with signal amplitude, duration, and frequency ultimately determining the type of response. The cellular response to DNA damage is no exception to the rule. In this review article we focus on DNA damage responses in G2 and Mitosis. First, we set the stage describing mitosis and the machineries in charge of assembling the apparatus responsible for chromosome alignment and segregation as well as the inputs that control its function (checkpoints). Next, we examine the type of issues that a cell approaching mitosis might face, presenting the impact of post-translational modifications (PTMs) on the correct and timely functioning of pathways correcting errors or damage before chromosome segregation. We conclude this essay with a perspective on the current status of mitotic signaling pathway inhibitors and their potential use in cancer therapy. PMID:27493659

  6. Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model

    Directory of Open Access Journals (Sweden)

    Nadine Schulz

    2017-06-01

    Full Text Available Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX immunohistochemistry. In vivo results were then compared (in silico with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15–60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

  7. Yields of clustered DNA damage induced by charged-particle radiations of similar kinetic energy per nucleon: LET dependence in different DNA microenvironments

    International Nuclear Information System (INIS)

    Keszenman, D.J.; Sutherland, B.M.

    2010-01-01

    To determine the linear energy transfer (LET) dependence of the biological effects of densely ionizing radiation in relation to changes in the ionization density along the track, we measured the yields and spectrum of clustered DNA damages induced by charged particles of different atomic number but similar kinetic energy per nucleon in different DNA microenvironments. Yeast DNA embedded in agarose in solutions of different free radical scavenging capacity was irradiated with 1 GeV protons, 1 GeV/nucleon oxygen ions, 980 MeV/nucleon titanium ions or 968 MeV/nucleon iron ions. The frequencies of double-strand breaks (DSBs), abasic sites and oxypurine clusters were quantified. The total DNA damage yields per absorbed dose induced in non-radioquenching solution decreased with LET, with minor variations in radioquenching conditions being detected. However, the total damage yields per particle fluence increased with LET in both conditions, indicating a higher efficiency per particle to induce clustered DNA damages. The yields of DSBs and non-DSB clusters as well as the damage spectra varied with LET and DNA milieu, suggesting the involvement of more than one mechanism in the formation of the different types of clustered damages.

  8. 2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

    International Nuclear Information System (INIS)

    Wang Shuchi; Chung, Jing-Gung; Chen, C.-H.; Chen, S.-C.

    2006-01-01

    4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 μM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-α-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H 2 O 2 is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1 mM) and with the copper chelator neocupronine (NC) (100 μM) also decreased DNA damage in cells treated with 200 μM 2-ABP or 200 μM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 μM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both

  9. Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism.

    Science.gov (United States)

    Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

    2016-06-01

    The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2 (-) scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays. Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2(-) scavenging, DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3',4'-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form.

  10. Voltammetric Detection of Damage to DNA by Arsenic Compounds at a DNA Biosensor

    Directory of Open Access Journals (Sweden)

    R. Wennrich

    2005-11-01

    Full Text Available DNA biosensor can serve as a powerfull tool for simple in vitro tests of chemicaltoxicity. In this paper, damage to DNA attached to the surface of screen-printed carbonelectrode by arsenic compounds in solution is described. Using the Co(III complex with1,10-phenanthroline, [Co(phen3]3+ , as an electrochemical DNA marker and the Ru(IIcomplex with bipyridyne, [Ru(bipy3]2+ , as a DNA oxidation catalyst, the portion of originaldsDNA which survives an incubation of the biosensor in the cleavage medium was evaluated.The model cleavage mixture was composed of an arsenic compound at 10-3 mol/Lconcentration corresponding to real contaminated water, 2x10-4 mol/L Fe(II or Cu(II ions asthe redox catalyst, and 1.5x10-2 mol/L hydrogen peroxide. DNA damage by arsenite,dimethylarsinic acid as the metabolic product of inorganic arsenic and widely used herbicide,as well as phenylarsonic acid and p-arsanilic acid as the representatives of feed additives wasfound in difference to arsenate.

  11. Online imaging of initial DNA damages at the PTB microbeam

    International Nuclear Information System (INIS)

    Giesen, U.; Langner, F.; Mielke, C.; Mosconi, M.; Dirks, W. G.

    2011-01-01

    In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine Univ., live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions. (authors)

  12. Personal exposure to ultrafine particles and oxidative DNA damage

    DEFF Research Database (Denmark)

    Vinzents, Peter S; Møller, Peter; Sørensen, Mette

    2005-01-01

    10), nitrous oxide, nitrogen dioxide, carbon monoxide, and/or number concentration of UFPs at urban background or busy street monitoring stations was not a significant predictor of DNA damage, although personal UFP exposure was correlated with urban background concentrations of CO and NO2...... the morning after exposure measurement. Cumulated outdoor and cumulated indoor exposures to UFPs each were independent significant predictors of the level of purine oxidation in DNA but not of strand breaks. Ambient air concentrations of particulate matter with an aerodynamic diameter of ..., particularly during bicycling in traffic. The results indicate that biologic effects of UFPs occur at modest exposure, such as that occurring in traffic, which supports the relationship of UFPs and the adverse health effects of air pollution....

  13. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

    Energy Technology Data Exchange (ETDEWEB)

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Shahverdi, Ahmad Reza [Department of Pharmaceutical Biotechnology and Biotechnology Research Centre, Faculty of Pharmacy, TUMS, Tehran (Iran, Islamic Republic of); Ahmadi, Abbas [Department of Histology and Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia (Iran, Islamic Republic of); Baeeri, Maryam; Mohammadirad, Azadeh [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Abdollahi, Mohammad, E-mail: mohammad.abdollahi@utoronto.ca [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of)

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA damage

  14. Chemotherapeutic Drugs: DNA Damage and Repair in Glioblastoma.

    Science.gov (United States)

    Annovazzi, Laura; Mellai, Marta; Schiffer, Davide

    2017-05-26

    Despite improvements in therapeutic strategies, glioblastoma (GB) remains one of the most lethal cancers. The presence of the blood-brain barrier, the infiltrative nature of the tumor and several resistance mechanisms account for the failure of current treatments. Distinct DNA repair pathways can neutralize the cytotoxicity of chemo- and radio-therapeutic agents, driving resistance and tumor relapse. It seems that a subpopulation of stem-like cells, indicated as glioma stem cells (GSCs), is responsible for tumor initiation, maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity. Recently, attention has been focused on the pivotal role of the DNA damage response (DDR) in tumorigenesis and in the modulation of therapeutic treatment effects. In this review, we try to summarize the knowledge concerning the main molecular mechanisms involved in the removal of genotoxic lesions caused by alkylating agents, emphasizing the role of GSCs. Beside their increased DNA repair capacity in comparison with non-stem tumor cells, GSCs show a constitutive checkpoint expression that enables them to survive to treatments in a quiescent, non-proliferative state. The targeted inhibition of checkpoint/repair factors of DDR can contribute to eradicate the GSC population and can have a great potential therapeutic impact aiming at sensitizing malignant gliomas to treatments, improving the overall survival of patients.

  15. Systemic oxidatively generated DNA/RNA damage in clinical depression

    DEFF Research Database (Denmark)

    Jorgensen, Anders; Krogh, Jesper; Miskowiak, Kamilla

    2013-01-01

    oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy...... for trend=0.004). The 8-oxoGuo excretion was further increased after clinically effective ECT compared with pre-ECT values (P=0.006). There were no differences in 8-oxodG excretion between the groups or pre- vs. post-ECT. LIMITATIONS: Small sample size and the inclusion of both unipolar and bipolar patients...

  16. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning...... in genotyping procedures and investigated SNPs, potentially inducing differences in the coverage of gene regions. Specifically, five genes were not covered at all in the German data. Therefore, investigations in additional study populations are needed before final conclusion can be drawn....

  17. Oxidatively damaged DNA in animals exposed to particles

    DEFF Research Database (Denmark)

    Møller, Peter; Danielsen, Pernille Høgh; Jantzen, Kim

    2013-01-01

    on optimal methods. The majority of studies have used single intracavitary administration or inhalation with dose rates exceeding the pulmonary overload threshold, resulting in cytotoxicity and inflammation. It is unclear whether this is relevant for the much lower human exposure levels. Still...... not be equivocally determined. Roles of cytotoxicity or inflammation for oxidatively induced DNA damage could not be documented or refuted. Studies on exposure to particles in the gastrointestinal tract showed consistently increased levels of 8-oxo-7,8-dihydroguanine in the liver. Collectively, there is evidence...

  18. Lymphocyte DNA damage in elevator manufacturing workers in Guangzhou, China.

    Science.gov (United States)

    Lam, Tai Hing; Zhu, Chang Qi; Jiang, Chao Qiang

    2002-03-25

    To study the effect of smoking, passive smoking, alcohol drinking, and occupational exposure to low level of benzene on DNA strand breaks in elevator manufacturing workers in Guangzhou, China. Three hundred and fifty-nine workers (252 men and 107 women) of a modern elevator manufacturing factory, 205 were from production departments and 154 from managerial department. Information on the workers' health conditions, smoking, passive smoking, alcohol consumption and occupational exposure history was collected by personal interview. Lymphocyte DNA damage was measured by the Comet assay. None of the women smoked and 20.6% of the men were daily smokers. In non-smokers, the prevalence of passive smoking at work was 25% for men and 11.2% for women, and at home, 37.8 and 48.6%, respectively. Smoking significantly increased tail moment (P<0.001). Daily smokers had the largest tail moment (geometric mean, 95% CI) (0.93 microm (0.81-0.94)), followed by occasional smokers (0.76 microm (0.59-0.95)), ex-smokers (0.70 microm (0.58-0.85)), and never smokers (0.56 microm (0.53-0.60)). Tail moment increased significantly with daily tobacco consumption (cigarettes per day) (r=0.26, P<0.001) after adjusting for age, gender, occupational exposure, passive smoking, and drinking. Analysis of covariance (ANCOVA) showed that smoking (P<0.001), passive smoking at home (P=0.026), occupational exposure (P<0.001), male gender (P<0.001), and age (P=0.001) had independent effects on tail moment, whereas passive smoking at work and alcohol drinking had no significant effect. Smoking, passive smoking at home, male gender, age and occupational exposure independently increased lymphocyte DNA strand breaks. The presence of excess DNA damage under low level of occupational exposure to benzene or other solvents suggest that the current allowance concentrations may not be safe to prevent genotoxicity.

  19. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. [DNA damage in human pleural mesothelial cells induced by exposure to carbon nanotubes].

    Science.gov (United States)

    Ogasawara, Yuki; Umezu, Noriaki; Ishii, Kazuyuki

    2012-01-01

    Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

  1. NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells.

    Directory of Open Access Journals (Sweden)

    Altaf H Sarker

    Full Text Available Secondhand smoke (SHS is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS, the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.

  2. Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

    Directory of Open Access Journals (Sweden)

    Annemarie Grindel

    Full Text Available Diabetes mellitus type 2 (T2DM is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.Female T2DM patients (n = 146 were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72. In addition, tertiles according to diabetes duration (DD were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49. Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical

  3. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...

  4. Dietary flavonoid derivatives enhance chemotherapeutic effect by inhibiting the DNA damage response pathway

    International Nuclear Information System (INIS)

    Kuo, Ching-Ying; Zupkó, István; Chang, Fang-Rong; Hunyadi, Attila; Wu, Chin-Chung; Weng, Teng-Song; Wang, Hui-Chun

    2016-01-01

    Flavonoids are the most common group of polyphenolic compounds and abundant in dietary fruits and vegetables. Diet high in vegetables or dietary flavonoid supplements is associated with reduced mortality rate for patients with breast cancer. Many studies have been proposed for mechanisms linking flavonoids to improving chemotherapy efficacy in many types of cancers, but data on this issue is still limited. Herein, we report on a new mechanism through which dietary flavonoids inhibit DNA damage checkpoints and repair pathways. We found that dietary flavonoids could inhibit Chk1 phosphorylation and decrease clonogenic cell growth once breast cancer cells receive ultraviolet irradiation, cisplatin, or etoposide treatment. Since the ATR-Chk1 pathway mainly involves response to DNA replication stress, we propose that flavonoid derivatives reduce the side effect of chemotherapy by improving the sensitivity of cycling cells. Therefore, we propose that increasing intake of common dietary flavonoids is beneficial to breast cancer patients who are receiving DNA-damaging chemotherapy, such as cisplatin or etoposide-based therapy. - Highlights: • First report on inhibition of both DNA damage and repair by dietary flavonoids • Dietary flavonoids inhibit cisplatin- and UV-induced Chk1 phosphorylation. • Flavonoids combined with cisplatin or UV treatment show notable growth inhibition. • Promising treatment proposal for patients who are receiving adjuvant chemotherapy

  5. Dietary flavonoid derivatives enhance chemotherapeutic effect by inhibiting the DNA damage response pathway

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Ching-Ying [Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Zupkó, István [Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös Utca 6, Szeged H-6720 (Hungary); Chang, Fang-Rong [Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hunyadi, Attila [Institute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, Eötvös Utca 6, Szeged H-6720 (Hungary); Wu, Chin-Chung; Weng, Teng-Song [Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Wang, Hui-Chun, E-mail: wanghc@kmu.edu.tw [Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); PhD Program in Translational Medicine, College of Medicine and PhD Program in Toxicology, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Research Center for Natural Product and Drug Development, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Translational Research Center and Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan (China); Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan (China); Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2016-11-15

    Flavonoids are the most common group of polyphenolic compounds and abundant in dietary fruits and vegetables. Diet high in vegetables or dietary flavonoid supplements is associated with reduced mortality rate for patients with breast cancer. Many studies have been proposed for mechanisms linking flavonoids to improving chemotherapy efficacy in many types of cancers, but data on this issue is still limited. Herein, we report on a new mechanism through which dietary flavonoids inhibit DNA damage checkpoints and repair pathways. We found that dietary flavonoids could inhibit Chk1 phosphorylation and decrease clonogenic cell growth once breast cancer cells receive ultraviolet irradiation, cisplatin, or etoposide treatment. Since the ATR-Chk1 pathway mainly involves response to DNA replication stress, we propose that flavonoid derivatives reduce the side effect of chemotherapy by improving the sensitivity of cycling cells. Therefore, we propose that increasing intake of common dietary flavonoids is beneficial to breast cancer patients who are receiving DNA-damaging chemotherapy, such as cisplatin or etoposide-based therapy. - Highlights: • First report on inhibition of both DNA damage and repair by dietary flavonoids • Dietary flavonoids inhibit cisplatin- and UV-induced Chk1 phosphorylation. • Flavonoids combined with cisplatin or UV treatment show notable growth inhibition. • Promising treatment proposal for patients who are receiving adjuvant chemotherapy.

  6. Producing standard damaged DNA samples by heating: pitfalls and suggestions.

    Science.gov (United States)

    Fattorini, Paolo; Marrubini, Giorgio; Bonin, Serena; Bertoglio, Barbara; Grignani, Pierangela; Recchia, Elisa; Pitacco, Paola; Procopio, Francesca; Cantoni, Carolina; Pajnič, Irena Zupanič; Sorçaburu-Cigliero, Solange; Previderè, Carlo

    2018-05-15

    Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0-18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ("Protocol P") and in Eppendorf tubes provided with screwcaps ("Protocol S"). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the "Protocol S" affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2-3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO 2 , ROS, NO x and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of

  7. Replication of chromosomal and episomal DNA in X-ray-damaged human cells: A cis- or trans-acting mechanism

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Rose, R.; Mitchell, D.L.

    1990-01-01

    Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors

  8. Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity.

    Science.gov (United States)

    Pokrzywinski, Kaytee L; Biel, Thomas G; Kryndushkin, Dmitry; Rao, V Ashutosh

    2016-01-01

    Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.

  9. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  10. Electrolytic reduction of nitroheterocyclic drugs leads to biologically important damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, M.V.M.; Pluijmackers-Westmijze, E.J.; Loman, H.

    1985-01-01

    The effects of electrolytic reduction of nitroimidazole drugs on biologically active DNA was studied. The results show that reduction of the drugs in the presence of DNA affects inactivation for both double-stranded (RF) and single-stranded phiX174 DNA. However, stable reduction products did not make a significant contribution to the lethal damage in DNA. This suggests that probably a short-lived intermediate of reduction of nitro-compounds is responsible for damage to DNA. (author)

  11. The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

    Science.gov (United States)

    Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

    2012-09-01

    We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

  12. DNA Damage by Radiation in Tradescantia Leaf Cells

    International Nuclear Information System (INIS)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu; Nili, Mohammad

    2010-01-01

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  13. Damage of plasmid DNA by high energy ions

    International Nuclear Information System (INIS)

    Michaelidesova, A.; Pachnerova Brabcova, K.; Davidkova, M.

    2018-01-01

    The aim of the study was to determine the degree of direct DNA damage by high-energy ions, which are one of the components of cosmic rays, and therefore the knowledge of the biological effects of these ions is key to long-term space missions with human crew. The pBR322 plasmid containing 4361 base pairs was used in this study. The aqueous solution of plasmid pBR322 was transferred on ice to Japan to the Heavy Ion Medical Accelerator in Chiba, the Research Center for Charged Particle Therapy. Just before the experiment, the droplets of solution of known concentration were applied to the slides and the water was allowed to evaporate to produce dry DNA samples. Half of the slides were irradiated with 290 MeV/u of carbon ions and a dose rate of 20 Gy/min. The other half of the slides were irradiated with helium nuclei of 150 MeV/hr and a dose rate of 12.6 Gy/min. Both sets of slides were irradiated with doses of 0-1,400 Gy with a 200 Gy step. After irradiation, the samples were re-dissolved in distilled water, frozen and transported on ice to the Czech Republic for processing. Samples were analyzed by agarose gel electrophoresis. The plasmid was evaluated separately to determine the degree of radiation induced lesions and further to incubation with enzymes recognizing basal damage. (authors)

  14. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-04-15

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  15. Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52

    Directory of Open Access Journals (Sweden)

    Chin-Chuan Chen

    2017-12-01

    Full Text Available Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC, a flavoring agent, causes deficiencies in double-stand break (DSB repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC, was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

  16. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

    Science.gov (United States)

    One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

  17. Role of Rad54, Rad54b and Snm1 in DNA damage repair

    NARCIS (Netherlands)

    J. Wesoly (Joanna)

    2003-01-01

    textabstractThe aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If

  18. DNA damage by the cobalt (II) and zinc (II) complexes of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-03

    Sep 3, 2008 ... distributed in grade 3. The results indicated that Co(II)-L induced a relatively high level of DNA damage in comparison with the level of damage induced by Zn(II)-L. Key words: Tetraazamacrocycle Zn(II) complex, tetraazamacrocycle Co(II) complex, Tetrahymena thermophila, DNA damage, the comet assay.

  19. DNA Damage Induced by Alkylating Agents and Repair Pathways

    Science.gov (United States)

    Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

  20. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  1. How Does Thymine DNA Survive Ultrafast Dimerization Damage?

    Directory of Open Access Journals (Sweden)

    Hongjuan Wang

    2016-12-01

    Full Text Available The photodimerization reaction between the two adjacent thymine bases within a single strand has been the subject of numerous studies due to its potential to induce DNA mutagenesis and possible tumorigenesis in human skin cells. It is well established that the cycloaddition photoreaction takes place on a picosecond time scale along barrierless or low barrier singlet/triplet pathways. However, the observed dimerization quantum yield in different thymine multimer is considerable lower than might be expected. A reasonable explanation is required to understand why thymine in DNA is able to survive ultrafast dimerization damage. In this work, accurate quantum calculations based on the combined CASPT2//CASSCF/AMBER method were conducted to map the excited state relaxation pathways of the thymine monomer in aqueous solution and of the thymine oligomer in DNA. A monomer-like decay pathway, induced by the twisting of the methyl group, is found to provide a bypass channel to ensure the photostability of thymine in single-stranded oligomers. This fast relaxation path is regulated by the conical intersection between the bright SCT(1ππ* state with the intra-base charge transfer character and the ground state to remove the excess excitation energy, thereby achieving the ground-state recovery with high efficiency.

  2. The effect of ancient DNA damage on inferences of demographic histories

    DEFF Research Database (Denmark)

    Axelsson, Erik; Willerslev, Eske; Gilbert, Marcus Thomas Pius

    2008-01-01

    The field of ancient DNA (aDNA) is casting new light on many evolutionary questions. However, problems associated with the postmortem instability of DNA may complicate the interpretation of aDNA data. For example, in population genetic studies, the inclusion of damaged DNA may inflate estimates o...... for a change in effective population size in this data set vanishes once the effects of putative damage are removed. Our results suggest that population genetic analyses of aDNA sequences, which do not accurately account for damage, should be interpreted with great caution....

  3. Effects of motexafin gadolinium on DNA damage and X-ray-induced DNA damage repair, as assessed by the Comet assay

    International Nuclear Information System (INIS)

    Donnelly, Erling T.; Liu Yanfeng; Paul, Tracy K.; Rockwell, Sara

    2005-01-01

    Purpose: To investigate the effects of motexafin gadolinium (MGd) on the levels of reactive oxygen species (ROS), glutathione (GSH), and DNA damage in EMT6 mouse mammary carcinoma cells. The ability of MGd to alter radiosensitivity and to inhibit DNA damage repair after X-ray irradiation was also evaluated. Methods and Materials: Reactive oxygen species and GSH levels were assessed by 2,7-dichlorofluorescein fluorescence flow cytometry and the Tietze method, respectively. Cellular radiosensitivity was assessed by clonogenic assays. Deoxyribonucleic acid damage and DNA damage repair were assessed in plateau-phase EMT6 cells by the Comet assay and clonogenic assays. Results: Cells treated with 100 μmol/L MGd plus equimolar ascorbic acid (AA) had significantly increased levels of ROS and a 58.9% ± 3.4% decrease in GSH levels, relative to controls. Motexafin gadolinium plus AA treatment increased the hypoxic, but not the aerobic, radiosensitivity of EMT6 cells. There were increased levels of single-strand breaks in cells treated with 100 μmol/L MGd plus equimolar AA, as evidenced by changes in the alkaline tail moment (MGd + AA, 6 h: 14.7 ± 1.8; control: 2.8 ± 0.9). The level of single-strand breaks was dependent on the length of treatment. Motexafin gadolinium plus AA did not increase double-strand breaks. The repair of single-strand breaks at 2 h, but not at 4 h and 6 h, after irradiation was altered significantly in cells treated with MGd plus AA (MGd + AA, 2 h: 15.8 ± 3.4; control: 5.8 ± 0.6). Motexafin gadolinium did not alter the repair of double-strand breaks at any time after irradiation with 10 Gy. Conclusions: Motexafin gadolinium plus AA generated ROS, which in turn altered GSH homeostasis and induced DNA strand breaks. The MGd plus AA-mediated alteration of GSH levels increased the hypoxic, but not aerobic, radiosensitivity of EMT6 cells. Motexafin gadolinium altered the kinetics of single-strand break repair soon after irradiation but did not

  4. Reduction of DNA damage induced by titanium dioxide nanoparticles through Nrf2 in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Zhiqin [Department of Toxicology, Hebei Medical University, Shijiazhuang (China); Department of Laboratory Diagnosis, Hebei Medical University, Shijiazhuang (China); Niu, Yujie [Department of Occupational Health and Environmental Health, Hebei Medical University, Shijiazhuang (China); Wang, Qian [Department of Toxicology, Hebei Medical University, Shijiazhuang (China); Shi, Lei [Department of Occupational Health and Environmental Health, Hebei Medical University, Shijiazhuang (China); Guo, Huicai; Liu, Yi; Zhu, Yue [Department of Toxicology, Hebei Medical University, Shijiazhuang (China); Liu, Shufeng; Liu, Chao [Hebei Keylab of Laboratory Animal Science, Shijiazhuang (China); Chen, Xin [Xiumen Community Health Service Centre, Shijiazhuang (China); Zhang, Rong, E-mail: rongzhang@hebmu.edu.cn [Department of Toxicology, Hebei Medical University, Shijiazhuang (China); Hebei Keylab of Laboratory Animal Science, Shijiazhuang (China)

    2015-11-15

    Highlights: • Nrf2 signals were partly responsible for the DNA damage induced by Nano-TiO{sub 2}. • Nrf2 loss could aggravate the DNA damage induced by Nano-TiO{sub 2}. • Acquired Nrf2 decreased the susceptibility to DNA damage induced by Nano-TiO{sub 2}. - Abstract: Titanium dioxide nanoparticles (Nano-TiO{sub 2}) are widely used to additives in cosmetics, pharmaceutical, paints and foods. Recent studies have demonstrated that Nano-TiO{sub 2} induces DNA damage and increased the risk of cancer and the mechanism might relate with oxidative stress. The aim of this study was to evaluate the effects of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), an anti-oxidative mediator, on DNA damage induced by Nano-TiO{sub 2}. Wildtype, Nrf2 knockout (Nrf2(-/-)) and tert-butylhydroquinone (tBHQ) pre-treated HepG2 cells and mice were treated with Nano-TiO{sub 2}. And then the oxidative stress and DNA damage were evaluated. Our data showed that DNA damage, reactive oxygen species (ROS) generation and MDA content in Nano-TiO{sub 2} exposed cells were significantly increased than those of control in dose dependent manners. Nrf2/ARE droved the downstream genes including NAD(P)H dehydrogenase [quinine] 1(NQO1), heme oxygenase 1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) expression were significantly higher in wildtype HepG2 cells after Nano-TiO{sub 2} treatment. After treatment with Nano-TiO{sub 2}, the DNA damages were significantly increased in Nrf(-/-) cells and mice whereas significantly decreased in tBHQ pre-treatment cells and mice, compared with the wildtype HepG2 cells and mice, respectively. Our results indicated that the acquired of Nrf2 leads to a decreased susceptibility to DNA damages induction by Nano-TiO{sub 2} and decreasing of risk of cancer which would provide a strategy for a more efficacious sensitization of against of Nano-TiO{sub 2} toxication.

  5. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    Science.gov (United States)

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  6. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji; An, You Sun; Yi, Jae Youn, E-mail: yjy_71@kcch.re.kr

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.

  7. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  8. Plasmid DNA damage caused by stibine and trimethylstibine

    International Nuclear Information System (INIS)

    Andrewes, Paul; Kitchin, Kirk T.; Wallace, Kathleen

    2004-01-01

    Antimony is classified as 'possibly carcinogenic to humans' and there is also sufficient evidence for antimony carcinogenicity in experimental animals. Stibine is a volatile inorganic antimony compound to which humans can be exposed in occupational settings (e.g., lead-acid battery charging). Because it is highly toxic, stibine is considered a significant health risk; however, its genotoxicity has received little attention. For the work reported here, stibine was generated by sodium borohydride reduction of potassium antimony tartrate. Trimethylstibine is a volatile organometallic antimony compound found commonly in landfill and sewage fermentation gases at concentrations ranging between 0.1 and 100 μg/m 3 . Trimethylstibine is generally considered to pose little environmental or health risk. In the work reported here, trimethylstibine was generated by reduction of trimethylantimony dichloride using either sodium borohydride or the thiol compounds, dithioerythritol (DTE), L-cysteine, and glutathione. Here we report the evaluation of the in vitro genotoxicities of five antimony compounds--potassium antimony tartrate, stibine, potassium hexahydroxyantimonate, trimethylantimony dichloride, and trimethylstibine--using a plasmid DNA-nicking assay. Of these five antimony compounds, only stibine and trimethylstibine were genotoxic (significant nicking to pBR 322 plasmid DNA). We found stibine and trimethylstibine to be about equipotent with trimethylarsine using this plasmid DNA-nicking assay. Reaction of trimethylantimony dichloride with either glutathione or L-cysteine to produce DNA-damaging trimethylstibine was observed with a trimethylantimony dichloride concentration as low as 50 μM and L-cysteine or glutathione concentrations as low as 500 and 200 μM, respectively, for a 24 h incubation

  9. Association of DNA damage and dyslipidemia with polycystic ovarian syndrome

    Directory of Open Access Journals (Sweden)

    Manikkumar R

    2013-02-01

    Full Text Available Polycystic ovary syndrome (PCOS is associated with hyperinsuli-nemia and insulin resistance which may lead to cardiovascular diseases. Evidence for cardiovascular events in women who were affected by PCOS during fertile age is limited. The pathogenesis is unknown; however, it is a complex multigenetic disorder. The present study was undertaken to evaluate the various cardiovas-cular risk factors and their DNA repair efficiency in women with PCOS by investigating the biochemical, endocrinological and mo-lecular cytogenetic alterations. These investigations were carried out in 116 women in the age group of 15-35 years clinically diag-nosed with PCOS. Data were compared with that of 50 age-matched healthy normal women. Fasting blood sugar (FBS, Lipid profile, Follicle-Stimulating Hormone (FSH and Luteinizing Hor-mone (LH, Prolactin and Estradiol were estimated after getting the informed consent. Mutagen induced chromosome sensitivity analysis was carried out in the lymphocytes of the subjects to as-sess the DNA repair proficiency. Fasting Blood Sugar, total cho-lesterol and LDL cholesterol were found to be elevated whereas HDL cholesterol was found to be lowered in the test subjects. FSH, LH and prolactin were also found to be significantly elevated in the test subjects. Change in the estradiol concentration in the test subjects was not significant. The mutagen sensitivity analysis revealed a significant elevation in break per cell (b/c values indi-cating a deficiency in the DNA repair mechanism / DNA damage in PCOS patients. Modification of life style by changing the dietary habit and sedentary life style will help to reduce the oxidative stress and may increase the ovarian function and a sensible life-style management is recommended for reducing the risk for CVD.

  10. MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Khan, Shafqat A.; Sharda, Asmita; Reddy, Divya V; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-08-15

    Highlights: • Reversible reduction of H3S10 phosphorylation after DNA damage is G1 phase specific. • Dynamic balance between MAP kinases, MKP1 and MSK1 regulate H3S10P during DDR. • MKP1 associates with chromatin bearing γH2AX in response to DNA damage. • Inhibition of MKP1 activity with specific inhibitor promotes radiation-induced cell death. - Abstract: Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

  11. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  12. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

    International Nuclear Information System (INIS)

    Cooke, Marcus S.; Evans, Mark D.; Dove, Rosamund; Rozalski, Rafal; Gackowski, Daniel; Siomek, Agnieszka; Lunec, Joseph; Olinski, Ryszard

    2005-01-01

    The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15 N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair

  13. Evaluation of DNA damage induced by gamma radiation in gill and muscle tissues of Cyprinus carpio and their relative sensitivity.

    Science.gov (United States)

    M K, Praveen Kumar; Shyama, Soorambail K; D'Costa, Avelyno; Kadam, Samit B; Sonaye, Bhagatsingh Harisingh; Chaubey, Ramesh Chandra

    2017-10-01

    The effect of radiation on the aquatic environment is of major concern in recent years. Limited data is available on the genotoxicity of gamma radiation on different tissues of aquatic organisms. Hence, the present investigation was carried out to study the DNA damage induced by gamma radiation in the gill and muscle tissues and their relative sensitivity using the comet assay in the freshwater teleost fish, common carp (Cyprinus carpio). The comet assay was optimized and validated in common carp using cyclophosphamide (CP), a reference genotoxic agent. The fish were exposed (acute) to various doses of gamma radiation (2, 4, 6, 8 and 10Gy) and samplings (gill and muscle tissue) were done at regular intervals (24, 48 and 72h) to assess the DNA damage. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA for all doses of gamma radiation in both tissues. We also observed a dose-related increase and a time-dependent decrease of DNA damage. In comparison, DNA damage showed different sensitivity among the tissues at different doses. This shows that a particular dose may have different effects on different tissues which could be due to physiological factors of the particular tissue. Our study also suggests that the gills and muscle of fish are sensitive and reliable tissues for evaluating the genotoxic effects of reference and environmental agents, using the comet assay. Copyright © 2017. Published by Elsevier Inc.

  14. Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

    Directory of Open Access Journals (Sweden)

    Scott Cukras

    Full Text Available Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

  15. Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

    Science.gov (United States)

    Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

    2014-01-01

    Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

  16. Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

    Science.gov (United States)

    Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih-Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung-Jong; Chen, David J; Harrison, Lynn

    2011-11-01

    Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

  17. The 2100MHz radiofrequency radiation of a 3G-mobile phone and the DNA oxidative damage in brain.

    Science.gov (United States)

    Sahin, Duygu; Ozgur, Elcin; Guler, Goknur; Tomruk, Arın; Unlu, Ilhan; Sepici-Dinçel, Aylin; Seyhan, Nesrin

    2016-09-01

    We aimed to evaluate the effect of 2100MHz radiofrequency radiation emitted by a generator, simulating a 3G-mobile phone on the brain of rats during 10 and 40 days of exposure. The female rats were randomly divided into four groups. Group I; exposed to 3G modulated 2100MHz RFR signal for 6h/day, 5 consecutive days/wk for 2 weeks, group II; control 10 days, were kept in an inactive exposure set-up for 6h/day, 5 consecutive days/wk for 2 weeks, group III; exposed to 3G modulated 2100MHz RFR signal for 6h/day, 5 consecutive days/wk for 8 weeks and group IV; control 40 days, were kept in an inactive exposure set-up for 6h/day, 5 consecutive days/wk for 8 weeks. After the genomic DNA content of brain was extracted, oxidative DNA damage (8-hydroxy-2'deoxyguanosine, pg/mL) and malondialdehyde (MDA, nmoL/g tissue) levels were determined. Our main finding was the increased oxidative DNA damage to brain after 10 days of exposure with the decreased oxidative DNA damage following 40 days of exposure compared to their control groups. Besides decreased lipid peroxidation end product, MDA, was observed after 40 days of exposure. The measured decreased quantities of damage during the 40 days of exposure could be the means of adapted and increased DNA repair mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio).

    Science.gov (United States)

    Ge, Weili; Yan, Saihong; Wang, Jinhua; Zhu, Lusheng; Chen, Aimei; Wang, Jun

    2015-02-18

    Imidacloprid is a neonicotinoid insecticide that can have negative effects on nontarget animals. The present study was conducted to assess the toxicity of various imidacloprid doses (0.3, 1.25, and 5 mg/mL) on zebrafish sampled after 7, 14, 21, and 28 days of exposure. The levels of catalase (CAT), superoxide dismutase (SOD), reactive oxygen species (ROS), glutathione-S-transferase (GST), and malondialdehyde (MDA) and the extent of DNA damage were measured to evaluate the toxicity of imidacloprid on zebrafish. SOD and GST activities were noticeably increased during early exposure but were inhibited toward the end of the exposure period. In addition, the CAT levels decreased to the control level following their elevation during early exposure. High concentrations of imidacloprid (1.25 and 5 mg/L) induced excessive ROS production and markedly increased MDA content on the 21st day of exposure. DNA damage was dose- and time-dependent. In conclusion, the present study showed that imidacloprid can induce oxidative stress and DNA damage in zebrafish.

  19. Evaluation of Cassia tora Linn. against oxidative stress-induced DNA and cell membrane damage

    Directory of Open Access Journals (Sweden)

    R Sunil Kumar

    2017-01-01

    Full Text Available Objective: The present study aims to evaluate antioxidants and protective role of Cassia tora Linn. against oxidative stress-induced DNA and cell membrane damage. Materials and Methods: The total and profiles of flavonoids were identified and quantified through reversed-phase high-performance liquid chromatography. In vitro antioxidant activity was determined using standard antioxidant assays. The protective role of C. tora extracts against oxidative stress-induced DNA and cell membrane damage was examined by electrophoretic and scanning electron microscopic studies, respectively. Results: The total flavonoid content of CtEA was 106.8 ± 2.8 mg/g d.w.QE, CtME was 72.4 ± 1.12 mg/g d.w.QE, and CtWE was 30.4 ± 0.8 mg/g d.w.QE. The concentration of flavonoids present in CtEA in decreasing order: quercetin >kaempferol >epicatechin; in CtME: quercetin >rutin >kaempferol; whereas, in CtWE: quercetin >rutin >kaempferol. The CtEA inhibited free radical-induced red blood cell hemolysis and cell membrane morphology better than CtME as confirmed by a scanning electron micrograph. CtEA also showed better protection than CtME and CtWE against free radical-induced DNA damage as confirmed by electrophoresis. Conclusion: C. tora contains flavonoids and inhibits oxidative stress and can be used for many health benefits and pharmacotherapy.

  20. An alkaline separation method for detection of small amount of DNA damage

    International Nuclear Information System (INIS)

    Sakai, Kazuo; Okada, Shigefumi

    1981-01-01

    An alkaline separation technique originally established by Ahnstroem is modified to detect small amount of DNA damage in X-irradiated mouse leukemic L5178Y cells. It is made quantitative by calibration with an alkaline sucrose gradient centrifugation. The present method would make it possible to study DNA damage and its repair within a dose range of X-rays where cell survival and mutation are usually investigated. It is also useful for detecting DNA damage caused by chemicals. (author)

  1. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage binding protein.

    NARCIS (Netherlands)

    S. Keeney; A.P.M. Eker (André); T. Brody; W. Vermeulen (Wim); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); S. Linn

    1994-01-01

    textabstractCells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells

  2. Investigation of DNA damage and repair mechanism using deinococcus radiodurans

    International Nuclear Information System (INIS)

    Lau How Mooi; Kikuchi, M.; Kobayashi, Y.; Narumi, I.; Watanabe, H.

    1997-01-01

    Deninococcus Radiodurans, formerly known as Micrococcus Radiodurans, is a popular bacterium because of its high resistance to damage by carcinogens such as ionizing radiation (Dean et. al. 1966; Kitayama and Matsuyama 1968) and UV radiation (Gasvon et. al., 1995; Arrange et. al. 1993). In this report, we investigated the high resistance to ionizing radiation by this bacterium. The bacteria had been exposed from I to 5 kGy of gamma radiation and then incubated in TGY medium to study their ability to repair the broken DNA. The repair time was measured by Pulse Field Gel Electrophoresis (PFGE) method. The repair time for each dose was determined. Also in order to ensure that the repair was perfect, the bacterium was subjected to a second exposure of ionizing radiation after it has fully repaired. It was found that the 'second' repair characteristic was similar to the first repair. This confirmed that the repair after the exposure to the ionizing radiation was perfect

  3. Mitochondrial DNA Damage and Diseases [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Gyanesh Singh

    2015-07-01

    Full Text Available Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments.

  4. Gene polymorphisms and increased DNA damage in morbidly obese women.

    Science.gov (United States)

    Luperini, B C O; Almeida, D C; Porto, M P; Marcondes, J P C; Prado, R P; Rasera, I; Oliveira, M R M; Salvadori, D M F

    2015-06-01

    Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring

  5. Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation

    International Nuclear Information System (INIS)

    Tokuyama, Yuka; Terato, Hiroaki; Furusawa, Yoshiya; Ide, Hiroshi; Yasui, Akira

    2015-01-01

    Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm -1 , respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process. (author)

  6. HeLa DNA damage response induced by 12C6+ ions

    International Nuclear Information System (INIS)

    Chen Jidong; Li Ning; Zhang Hong; Wu Zhenhua

    2009-01-01

    The aim of this study is to explore the DNA damage response of HeLa irradiated by 12 C 6+ beam and the mechanism of the p53 activation change in this response.In our present study, double strands break(DSB)of HeLa cells irradiated with 12 C 6+ beam were detected through neutral single cell gel electrophoresis, and AO/EB staining was used to detect the apoptosis of irradiated HeLa in 24h irradiation. Moreover, HeLa was pre-treated with caffeine (ATM and ATR inhibiting) or wormannin with certain concentrations (20 μmol/L, ATM and DNA-PK inhibiting) and irradiated with 1Gy of 12 C 6+ beam,and the expression of p53 was detected with Western blot analysis. The results show that DSB of HeLa caused by 12 C 6+ beam increases with absorbed doses and decreases with the time after irradiation. The apoptosis percentage of irradiated HeLa increases with absorbed doses. It has been found that the p53 expression increases after irradiation, but has not significant increment with caffeine or wortmannin pre-treatment in cells.It can be deduced that the p53 activation is ATM-dependent, but not ATR and DNA-PK-dependent in HeLa DNA damage response induced by 12 C 6+ beam. (authors)

  7. DNA damage in grasshopper Chorthippus brunneus (Orthoptera) hatchlings following paraquat exposure.

    Science.gov (United States)

    Augustyniak, M; Nocoń, Ł; Kędziorski, A; Łaszczyca, P; Sawczyn, T; Tarnawska, M; Zawisza-Raszka, A

    2015-04-01

    Comet assay was applied to study genotoxic damage induced by paraquat (PQ) in brain cells of Chorthippus brunneus (Insecta: Orthoptera) hatchlings. Percentage of the comet fluorescence in the tail (TDNA), length of the comet tail (TL) and Olive tail moment (OTM) were used for quantitative assessment of the DNA damage. Multiple regression analysis supplemented standard statistical elaboration of the results. Increasing PQ concentrations applied either directly to the brain cells suspension (10, 50, and 250 μM PQ final concentration--in vitro protocol) or indirectly (50, 250, and 1250 μM PQ final concentration--in vivo protocol) provoked significant increase of oxidative damage to DNA (higher median TDNA and OTM values). The damage increased with time of exposure (0, 5, 15, and 30 min) following in vitro application, but decreased in longer interval (3 vs 24 h) after in vivo administration of paraquat. On contrary, median TL values did not correlate with paraquat concentration irrespectively of the exposure protocol. Possible reason of this discrepancy in light of paraquat toxicity is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D.

    2012-01-01

    Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial ...

  9. SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

    2012-01-01

    Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial fo...

  10. Retinoblastoma loss modulates DNA damage response favoring tumor progression.

    Directory of Open Access Journals (Sweden)

    Marcos Seoane

    Full Text Available Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS, crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

  11. DNA damage by smoke: Protection by turmeric and other inhibitors of ROS

    Energy Technology Data Exchange (ETDEWEB)

    Srinivas, L.; Shalini, V.K. (Department of Nutrition and Food Safety, Central Food Technological Research Institute, Mysore (India))

    1991-01-01

    Twigs-dry leaves smoke condensate (TDS), as a source of clastogenic ROS and carcinogenic PAH, was investigated for its in vitro DNA-damaging effect in calf thymus DNA and human peripheral lymphocytes. An aqueous turmeric component--Aq.T--with an established antioxidant activity, was tested as a DNA protectant. TDS induced 13-fold damage to calf thymus DNA as judged by the emergence of a DNA damage specific, fluorescent product (em: 405 nm). Aq.T at 800 ng/microL extended 69% protection to calf thymus DNA and was comparable to the other protectants such as curcumin, BHA, vitamin E, SOD, and CAT. In human peripheral lymphocytes, TDS induced extensive DNA damage in comparison with the tumor promoter TPA, as judged by FADU. Aq.T at 300 ng/microL extended 90% protection to human lymphocyte DNA against TDS-induced damage, and was more effective than the other protectants--DABCO, D-mannitol, sodium benzoate, vitamin E (ROS quenchers), SOD, CAT (antioxidant enzymes), tannic acid, flufenamic acid, BHA, BHT, n-PG, curcumin and quercetin (antioxidants). Aq.T offered 65% protection to human lymphocyte DNA against TPA-induced damage and was comparable to SOD. The above results indicate that TDS induces substantial DNA damage in calf thymus DNA and human lymphocytes and Aq.T is an efficient protectant.

  12. Radiation and non-radiation damage to DNA. Onset of molecular instability and carcinogenesis. Theoretical explorations on DNA damage and repair

    International Nuclear Information System (INIS)

    Pinak, Miroslay; Bunta, J.K.

    2006-01-01

    The current work is focused on results of molecular dynamics simulations performed on two DNA damages: 8-oxoguanine as the most significant oxidative damage leading to transversion mutation cytosine-guanine→adenine-thymine', which is common mutation found in human cancer cells; and on the DNA strand break, the type of damage that is considered to be one of the most significant damage leading to genetic instability that may result in enhanced cell proliferation or carcinogenesis. Except the structural changes induced by these two lesions the role and importance of electrostatic energy in recognition process in which a respective repair enzyme recognizes damaged DNA site is also described. Among the significant results can be included the fact, that most of the damages on DNA alternate locally electronic state by modifying chemical and electron orbital configuration. This modified configuration may be represented outside DNA molecule as an enhanced electrostatic interaction with surrounding environment, that may signal the presence of the damaged site toward the repair enzyme. Work on the DNA strand break shows that open valences at broken strand ends are quickly filled by the electrons generated during radiolysis. Results of simulation indicate a local instability of hydrogen bonds between complementary bases. (author)

  13. DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

    2017-07-01

    Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Participation of ATM in cellular response to DNA damage induced by ionizing radiation

    International Nuclear Information System (INIS)

    Meng Xiangbing; Song Yi; Mao Jianping; Gong Bo; Dong Yan; Liu Bin; Sun Zhixian

    2000-01-01

    Objective: To clone ATM full length cDNA and cDNA fragments containing some functional domains and to identify proteins that interact with ATM and mediate DNA damage signal transduction in cellular response to DNA damage. Methods: ATM cDNA was amplified from MarthomTM-Ready cDNA kit of human leukocytes by LD-PCR. ATM-interacting proteins were screened by yeast two hybrid system. Results: ATM full-length cDNA and cDNA fragments containing PI3K kinase domain, leucine zipper and proline rich region were amplified from human cDNAs. Several candidate clones that interacted with ATM PI3K domain were identified. Conclusion: ATM mediates DNA damage signal transduction by interacting with many proteins

  15. Umbelliferone suppresses radiation induced DNA damage and apoptosis in hematopoietic cells of mice

    International Nuclear Information System (INIS)

    Jayakumar, S.; Bhilwade, H.N.; Chaubey, R.C.

    2012-01-01

    Radiotherapy is one of the major modes of treatment for different types of cancers. But the success of radiotherapy is limited by injury to the normal cells. Protection of the normal cells from radiation damage by radioprotectors can increase therapeutic efficiency. These radioprotectors can also be used during nuclear emergency situations. Umbelliferone (UMB) is a wide spread natural product of the coumarin family. It occurs in many plants from the Apiaceae family. In the present study radioprotective effect of UMB was investigated in vitro and in vivo. Anti genotoxic effect of Umbelliferone was tested by treating the splenic lymphocytes with various doses of UMB (6.5 μM - 50 μM) prior to radiation (6Gy) exposure. After the radiation exposure, extent of DNA damage was assessed by comet assay at 5 mm and two hours after radiation exposure. At both the time points, it was observed that the pretreatment of UMB reduced the radiation induced DNA damage to a significant extent in comparison to radiation control. UMB pretreatment also significantly reduced the radiation induced apoptosis enumerated by propidium iodide staining assay. Results of clonogenic survival assay using intestinal cell line showed that pretreatment with UMB significantly protected against radiation induced loss of colony forming units. To assess the anti genotoxic role of umbelliferone in vivo two different doses of UMB (20 mg/Kg and 40 mg/Kg of body weight) were injected into Swiss mice or with vehicle and exposed to radiation. Thirty minutes after the radiation comet assay was performed in peripheral leukocytes. Frequency of micro nucleated erythrocytes was scored in bone marrow cells. It was observed that UMB alone did not cause any significant increase in DNA damage in comparison to control. Animals which are exposed to radiation alone showed significant increase in DNA damage and micronuclei frequency. But animals treated with UMB prior to the radiation exposure showed significant decrease

  16. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    International Nuclear Information System (INIS)

    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F.

    2014-01-01

    7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity

  17. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-12-01

    7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AX (γH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity.

  18. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

    International Nuclear Information System (INIS)

    Sebastian, J.; Sancar, G.B.

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

  19. Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

    Science.gov (United States)

    Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog; Jurkunas, Ula V

    2016-06-20

    Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

  20. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  1. ATP-dependent chromatin remodeling in the DNA-damage response

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  2. Repair of endogenous and ionizing radiation-induced DNA damages: mechanisms and biological functions

    International Nuclear Information System (INIS)

    Boiteux, S.

    2002-01-01

    The cellular DNA is continuously exposed to endogenous and exogenous stress. Oxidative stress due to cellular metabolism is the major cause of endogenous DNA damage. On the other hand, ionizing radiation (IR) is an important exogenous stress. Both induce similar DNA damages: damaged bases, abasic sites and strand breakage. Most of these lesions are lethal and/or mutagenic. The survival of the cell is managed by efficient and accurate DNA repair mechanisms that remove lesions before their replication or transcription. DNA repair pathways involved in the removal of IR-induced lesions are briefly described. Base excision repair (BER) is mostly involved in the removal of base damage, abasic sites and single strand breaks. In contrast, DNA double strand breaks are mostly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). How DNA repair pathways prevent cancer process is also discussed. (author)

  3. Estrogen signalling and the DNA damage response in hormone dependent breast cancers

    Directory of Open Access Journals (Sweden)

    C Elizabeth Caldon

    2014-05-01

    Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

  4. Effect of methionine load on homocysteine levels, lipid peroxidation and DNA damage in rats receiving ethanol

    Directory of Open Access Journals (Sweden)

    Alceu Afonso Jordao Júnior

    2009-12-01

    Full Text Available Changes in the metabolism of methionine can cause hyperhomocysteinemia, inducing a triad of atherosclerosis, hypertension, and increased oxidative stress. The generation of free radicals and oxidative damage to DNA is important in the liver damage caused by ethanol. In this study, the effect of methionine overload associated or otherwise with acute administration of ethanol on homocysteine values, damage to DNA, lipoperoxidation and vitamin E was evaluated. Thirty rats were divided into 3 groups: Group Ethanol 24 hours (EG24, Group Methionine 24 hours (MG24, and Group Methionine and Ethanol 24 hours (MEG24. TBARS, vitamin E, GS and, homocysteine values were determined and the Comet assay was carried out. Increased GSH, vitamin E and homocysteine levels were observed for MEG24, and increased TBARS were observed in EG24. The Comet assay showed an increase in DNA damage in EG24 and DNA protection in MEG24. The administration of ethanol decreased antioxidant levels and increased TBARS, indicating the occurrence of oxidative stress with possible DNA damage. The combination of methionine and ethanol had a protective effect against the ethanol-induced damage, but increased the levels of homocysteine.Alterações no metabolismo da metionina podem ocasionar hiper-homocisteinemia, quadro indutivo de aterosclerose, hipertensão e aumento do estresse oxidativo. A geração de radicais livres e dano oxidativo ao DNA são importantes na injúria hepática provocada pelo etanol. Neste estudo avaliaram-se os efeitos da sobrecarga de metionina associada ou não à administração aguda de etanol sobre valores de homocisteína, dano ao DNA, lipoperoxidação e vitamina E. Foram utilizados 30 ratos Wistar distribuídos em 3 Grupos: Grupo Etanol 24 horas (GE24, Grupo Metionina 24 horas (GM24 e Grupo Metionina e Etanol 24 horas (GME24. Realizaram-se determinações hepáticas de SRATB, vitamina E, GSH, homocisteína e Teste do Cometa e determinações plasm

  5. Decrease of FIB-induced lateral damage for diamond tool used in nano cutting

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei [State Key Laboratory of Precision Measuring Technology and Instruments, Centre of MicroNano Manufacturing Technology, Tianjin University, Tianjin 300072 (China); Xu, Zongwei, E-mail: zongweixu@163.com [State Key Laboratory of Precision Measuring Technology and Instruments, Centre of MicroNano Manufacturing Technology, Tianjin University, Tianjin 300072 (China); Fang, Fengzhou, E-mail: fzfang@gmail.com [State Key Laboratory of Precision Measuring Technology and Instruments, Centre of MicroNano Manufacturing Technology, Tianjin University, Tianjin 300072 (China); Liu, Bing; Xiao, Yinjing; Chen, Jinping [State Key Laboratory of Precision Measuring Technology and Instruments, Centre of MicroNano Manufacturing Technology, Tianjin University, Tianjin 300072 (China); Wang, Xibin [School of Mechanical Engineering, Beijing Institute of Technology, Beijing 100081 (China); Liu, Hongzhong [State Key Laboratory for Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an 710049 (China)

    2014-07-01

    Highlights: • We mainly aim to characterize and decrease the FIB-induced damage on diamond tool. • Raman and XPS methods were used to characterize the nanoscale FIB-induced damage. • Lower energy FIB can effectively lessen the FIB-induced damage on diamond tool. • The diamond tools’ performance was greatly improved after FIB process optimization. • 6 nm chip thickness of copper was achieved by diamond tool with 22 nm edge radius. - Abstract: Diamond cutting tools with nanometric edge radius used in ultra-precision machining can be fabricated by focused ion beam (FIB) technology. However, due to the nanoscale effects, the diamond tools performance and the cutting edge lifetime in nano cutting would be degraded because of the FIB-induced nanoscale lateral damage. In this study, the methods of how to effectively characterize and decrease the FIB-induced lateral damage for diamond tool are intensively studied. Based on the performance optimization diamond machining tools, the controllable chip thickness of less than 10 nm was achieved on a single-crystal copper in nano cutting. In addition, the ratio of minimum thickness of chip (MTC) to tool edge radius of around 0.3–0.4 in nano cutting is achieved. Methods for decreasing the FIB-induced damage on diamond tools and adding coolant during the nano cutting are very beneficial in improving the research of nano cutting and MTC. The nano cutting experiments based on the sharp and high performance of diamond tools would validate the nano cutting mechanisms that many molecular dynamic simulation studies have put forward and provide new findings for nano cutting.

  6. Mutagenic and DNA damaging activity in muscle of trout exposed in vivo to nitrite

    Energy Technology Data Exchange (ETDEWEB)

    De Flora, S; Arillo, A

    1983-09-01

    Muscle ether extracts of rainbow trout (Salmo gairdneri) exposed to lake water enriched with nitrite (450 micrograms/l) reverted plasmid-containing his- strains of Salmonella typhimurium, mainly eliciting frameshift mutations, and induced a DNA damage in Escherichia coli reparable through the recA/lexA-dependent SOS functions. The number of revertants was related to their content in nitroso-derivatives and to the physiological condition of the fish. Mutagenicity was efficiently decreased, through NADPH-requiring pathways, by liver S-9 fractions from rats or rainbow trout, while it was not affected by preliminary heating nor by pre-incubation with human gastric juice.

  7. Could humic acid relieve the biochemical toxicities and DNA damage caused by nickel and deltamethrin in earthworms (Eisenia foetida)?

    Science.gov (United States)

    Shen, Chen-Chao; Shen, Dong-Sheng; Shentu, Jia-Li; Wang, Mei-Zhen; Wan, Ming-Yang

    2015-12-01

    The aim of the study was to determine whether humic acid (HA) prevented gene and biochemical toxic effects in earthworms (Eisenia foetida) exposed to nickel and deltamethrin (at 100 and 1 mg kg(-1), respectively) in soil. Cellular- and molecular-level toxic effects of nickel and deltamethrin in earthworms were evaluated by measuring damage to lipid membranes and DNA and the production of protein carbonyls over 42 days of exposure. Nickel and deltamethrin induced significant levels of oxidative stress in earthworms, increasing the production of peroxidation products (malondialdehyde and protein carbonyls) and increasing the comet assay tail DNA% (determined by single-cell gel electrophoresis). DNA damage was the most sensitive of the three indices because it gave a higher sample/control ratio than did the other indices. The presence of HA alleviated (in decreasing order of effectiveness) damage to DNA, proteins, and lipid membranes caused by nickel and deltamethrin. A low HA dose (0.5-1% HA in soil) prevented a great deal of lipid membrane damage, but the highest HA dose (3% HA in soil) prevented still more DNA damage. However, the malondialdehyde concentrations in earthworms were higher at the highest HA dose than at the lower HA doses. The amounts of protein carbonyls produced at different HA doses were not significantly different. The toxic effects to earthworms caused by increased oxidizable nickel concentrations could be relieved by adding HA.

  8. Quantitative analysis of gene-specific DNA damage in human spermatozoa

    International Nuclear Information System (INIS)

    Sawyer, Dennis E.; Mercer, Belinda G.; Wiklendt, Agnieszka M.; Aitken, R. John

    2003-01-01

    Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H 2 O 2 ; 0-5 mM) or iron (as Fe(II)SO 4 , 0-500 μM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, β-pol and β-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H 2 O 2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H 2 O 2 . The mitochondrial genome of human spermatozoa was significantly (P 2 O 2 -induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage

  9. Radiation-induced DNA damage and cellular lethality

    International Nuclear Information System (INIS)

    Sakai, K.; Okada, S.

    1984-01-01

    Radiation-induced DNA scissions and their repair were investigated in mammalian cells using an alkaline separation method. DNA breaks in mouse L5178Y cells and Chinese hamster V79 cells were grouped into three in terms of their repair profile; fast-reparable breaks (FRBs; T1/2 = 5 min), slow-reparable breaks (SRBs; T1/2 = 70 min) and non-reparable breaks (NRBs). The three types of DNA lesions were studied under conditions where cellular radiosensitivity was modified. The authors obtained the following results: 1. Cell cycle fluctuation: L5178Y showed maximum sensitivity at M and G/sub 1/-S boundary, and minimum sensitivity at G/sub 1/ and late S. Cycle dependency was not found for FRBs or SRBs, but NRBs showed bimodal fluctuation with peaks at M and G/sub 1/-S, and with bottoms at G/sub 1/ and late S. 2. Different sensitivity of L5178Y and V79: L5178Y cells were more sensitive to X-rays (D/sub ο/ = 0.9 Gy) than V79 (D/sub ο/ = 1.8 Gy). The amount of FRBs or SRBs was identical in the two cell lines. However, the amount of NRBs in L5178Y was greater than that in V79. 3. Split dose irradiation: The time interval between two doses resulted in a gradual decrease of NRBs. The time course of the decrease was similar to the split dose recovery in terms of cell death. The parallel relationship between NRBs and cell killing implies that NRBs could play an important role in radiation-induced cell death

  10. Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Normal Human Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Vazhappilly Cijo George

    2017-01-01

    Full Text Available Scope. Human neoplastic transformation due to DNA damage poses an increasing global healthcare concern. Maintaining genomic integrity is crucial for avoiding tumor initiation and progression. The present study aimed to investigate the efficacy of an apple flavonoid fraction (AF4 against various carcinogen-induced toxicity in normal human bronchial epithelial cells and its mechanism of DNA damage response and repair processes. Methods and Results. AF4-pretreated cells were exposed to nicotine-derived nitrosamine ketones (NNK, NNK acetate (NNK-Ae, methotrexate (MTX, and cisplatin to validate cytotoxicity, total reactive oxygen species, intracellular antioxidants, DNA fragmentation, and DNA tail damage. Furthermore, phosphorylated histone (γ-H2AX and proteins involved in DNA damage (ATM/ATR, Chk1, Chk2, and p53 and repair (DNA-PKcs and Ku80 mechanisms were evaluated by immunofluorescence and western blotting, respectively. The results revealed that AF4-pretreated cells showed lower cytotoxicity, total ROS generation, and DNA fragmentation along with consequent inhibition of DNA tail moment. An increased level of γ-H2AX and DNA damage proteins was observed in carcinogen-treated cells and that was significantly (p≤0.05 inhibited in AF4-pretreated cells, in an ATR-dependent manner. AF4 pretreatment also facilitated the phosphorylation of DNA-PKcs and thus initiation of repair mechanisms. Conclusion. Apple flavonoids can protect in vitro oxidative DNA damage and facilitate repair mechanisms.

  11. Comparison of antioxidant enzyme activities and DNA damage in chickpea (Cicer arietinum L.) genotypes exposed to vanadium.

    Science.gov (United States)

    Imtiaz, Muhammad; Mushtaq, Muhammad Adnan; Rizwan, Muhammad Shahid; Arif, Muhammad Saleem; Yousaf, Balal; Ashraf, Muhammad; Shuanglian, Xiong; Rizwan, Muhammad; Mehmood, Sajid; Tu, Shuxin

    2016-10-01

    The present study was done to elucidate the effects of vanadium (V) on photosynthetic pigments, membrane damage, antioxidant enzymes, protein, and deoxyribonucleic acid (DNA) integrity in the following chickpea genotypes: C-44 (tolerant) and Balkasar (sensitive). Changes in these parameters were strikingly dependent on levels of V, at 60 and 120 mg V L(-1) induced DNA damage in Balkasar only, while photosynthetic pigments and protein were decreased from 15 to 120 mg V L(-1) and membrane was also damaged. It was shown that photosynthetic pigments and protein production declined from 15 to 120 mg V L(-1) and the membrane was also damaged, while DNA damage was not observed at any level of V stress in C-44. Moreover, the antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were increased in both genotypes of chickpea against V stress; however, more activities were observed in C-44 than Balkasar. The results suggest that DNA damage in sensitive genotypes can be triggered due to exposure of higher vanadium.

  12. SIRT3 mediates decrease of oxidative damage and prevention of ageing in porcine fetal fibroblasts.

    Science.gov (United States)

    Xie, Xiaoxian; Wang, Liangliang; Zhao, Binggong; Chen, Yangyang; Li, Jiaqi

    2017-05-15

    Sirtuin 3 (SIRT3) is a mitochondria-specific protein required for the deacetylation of metabolic enzymes and the action of oxidative phosphorylation by acting as a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase. SIRT3 increases oxidative stress resistance and prevents mitochondrial decay associated with ageing in response to caloric restriction. However, the effects of SIRT3 on oxidative damage and ageing are not well understood. We investigated the physiological functions of porcine SIRT3 on the damage and ageing in porcine fetal fibroblasts (PFFs). Overexpression and knockdown of SIRT3 were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. All cells were treated with three different stress reagents 12-o-tetradecanoylphorbol-13-acetate (TPA), methanesulfonic acid methylester (MMS), and tert-butylhydroperoxide (t-BHP), respectively, and then examined by flow cytometry following JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazol-carbocyanine iodide) staining. SIRT3 overexpression enhanced the ability of superoxide dismutase 2 (SOD2) to reduce cellular reactive oxygen species (ROS), which further decreased the damage to the membranes and the organelles of the cells, especially to mitochondria. It inhibited the initial decrease of mitochondrial membrane potential, and prevented the decrease of adenosine triphosphate (ATP) production and activity of Nampt. In contrast, SIRT3 knockdown reduced the ability of SOD2 to increase cellular ROS which was directly correlated with stress-induced oxidative damage and ageing in PFFs. Our findings identify one function of SIRT3 in PFFs was to dampen cytotoxicity, and, therefore, to decrease oxidative damage and attenuate ageing possibly by enhancing the activity of SOD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

    Science.gov (United States)

    Gibson, Shannon L; Narayanan, Latha; Hegan, Denise Campisi; Buermeyer, Andrew B; Liskay, R Michael; Glazer, Peter M

    2006-12-08

    Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

  14. The protective effect of DNA on the rat cell membrane damage induced by ultraviolet radiation

    International Nuclear Information System (INIS)

    Ma Shouxiang; Zhong Jinyan

    1988-01-01

    The protective effect of DNA on the cell membrane damage induced by ultra-violet radiation was studied. Rat erythrocytes were used as experimental materials. Blood samples were taken from the rat, and centrifuged to separate the plasma. The cells were washed twice with isotonic saline, resuspended in normal saline solution and then irradiated by ultra-violet radiation. The DNA was added before or after irradiation. THe cell suspensions were kept at 5 deg C for 20 hours after irradiation, and then centrifuged. The supernatants were used for hemoglobin determination. The main results obtained may summarized as follows: the cell suspension of erythrocytes were irradiated for 5, 10 and 20 min. The amount of hemolysis induced by irradiation dosage revealed a direct proportional relationship. If DNA (20-40μg/ml) was applied before irradiation, the amount of hemolysis induced apparently decreased. The differences between the control and DNA treated were statistically significant, P<0.01, but insignificant for DNA added after irradiation

  15. Decreased viscosity of rat-liver DNA treated by 3'-methyl-4-dimethylaminoazobenzene, detected with a new viscometric approach.

    Science.gov (United States)

    Parodi, S; Balbi, C; Taningher, M; Pala, M; Russo, P; Abelmoschi, M L; Santi, L

    1982-11-01

    DNA damage induced in vivo by 3'-methyl-4-dimethylaminoazobenzene (3'CH3DAB) was investigated with 2 differently sensitive techniques: the alkaline elution assay and the viscometric measurement of DNA damage. 3'CH3DAB appeared to be falsely negative with the alkaline elution assay, whereas with the viscometric approach, which is about 30-50 times more sensitive, it appeared positive, and the DNA damage was dose-dependent.

  16. Method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin

    International Nuclear Information System (INIS)

    Singh, G.; Hauswirth, W.W.; Ross, W.E.; Neims, A.H.

    1985-01-01

    This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [ 32 P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion

  17. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden); Glas, Rickard, E-mail: rickard.glas@ki.se [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden)

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  18. Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer

    International Nuclear Information System (INIS)

    Xia, Peng; An, Han-Xiang; Dang, Cheng-Xue; Radpour, Ramin; Kohler, Corina; Fokas, Emmanouil; Engenhart-Cabillic, Rita; Holzgreve, Wolfgang; Zhong, Xiao Yan

    2009-01-01

    Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters. Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein. The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed. Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer

  19. Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia.

    Science.gov (United States)

    Aslan, Mehmet; Horoz, Mehmet; Kocyigit, Abdurrahim; Ozgonül, Saadet; Celik, Hakim; Celik, Metin; Erel, Ozcan

    2006-10-10

    Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (ptotal antioxidant capacity was significantly lower (ptotal antioxidant capacity and hemoglobin levels (r=0.706, ptotal antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.

  20. Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

    Science.gov (United States)

    Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

    2017-11-22

    Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. DNA-damage response during mitosis induces whole-chromosome missegregation.

    Science.gov (United States)

    Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

    2014-11-01

    Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

  2. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status

    Energy Technology Data Exchange (ETDEWEB)

    Lyckesvärd, Madeleine Nordén, E-mail: madeleine.lyckesvard@oncology.gu.se [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden); Delle, Ulla; Kahu, Helena [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden); Lindegren, Sture [Department of Radiation Physics, Sahlgrenska Academy, University of Gothenburg (Sweden); Jensen, Holger [The PET and Cyclotron Unit Copenhagen University Hospital, Rigshospitalet (Denmark); Bäck, Tom [Department of Radiation Physics, Sahlgrenska Academy, University of Gothenburg (Sweden); Swanpalmer, John [Department of Medical Physics and Biomedical Engineering, Sahlgrenska University Hospital, Gothenburg (Sweden); Elmroth, Kecke [Department of Oncology, Sahlgrenska Academy, University of Gothenburg (Sweden)

    2014-07-15

    Highlights: • We study DNA damage response to low-LET photons and high-LET alpha particles. • Cycling primary thyrocytes are more sensitive to radiation than stationary cells. • Influence of radiation quality varies due to cell cycle status of normal cells. • High-LET radiation gives rise to a sustained DNA damage response. - Abstract: Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ({sup 211}At), concentrated in the thyroid by the same mechanism as {sup 131}I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ({sup 60}Co) and alpha particles from {sup 211}At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24 h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to {sup 211}At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1 Gy {sup 211}At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative

  3. A Green's Function Approach to Simulate DNA Damage by the Indirect Effect

    Science.gov (United States)

    Plante, Ianik; Cicinotta, Francis A.

    2013-01-01

    The DNA damage is of fundamental importance in the understanding of the effects of ionizing radiation. DNA is damaged by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research, many questions on the DNA damage by ionizing radiation remains. In the recent years, the Green's functions of the diffusion equation (GFDE) have been used extensively in biochemistry [1], notably to simulate biochemical networks in time and space [2]. In our future work on DNA damage, we wish to use an approach based on the GFDE to refine existing models on the indirect effect of ionizing radiation on DNA. To do so, we will use the code RITRACKS [3] developed at the NASA Johnson Space Center to simulate the radiation track structure and calculate the position of radiolytic species after irradiation. We have also recently developed an efficient Monte-Carlo sampling algorithm for the GFDE of reversible reactions with an intermediate state [4], which can be modified and adapted to simulate DNA damage by free radicals. To do so, we will use the known reaction rate constants between radicals (OH, eaq, H,...) and the DNA bases, sugars and phosphates and use the sampling algorithms to simulate the diffusion of free radicals and chemical reactions with DNA. These techniques should help the understanding of the contribution of the indirect effect in the formation of DNA damage and double-strand breaks.

  4. Colorimetric detection of DNA damage by using hemin-graphene nanocomposites

    Science.gov (United States)

    Wei, W.; Zhang, D. M.; Yin, L. H.; Pu, Y. P.; Liu, S. Q.

    2013-04-01

    A colorimetric method for detection of DNA damage was developed by using hemin-graphene nanosheets (H-GNs). H-GNs were skillfully synthesized by adsorping of hemin on graphene through π-π interactions. The as-prepared H-GNs possessed both the ability of graphene to differentiate the damage DNA from intact DNA and the catalytic action of hemin. The damaged DNA made H-GNs coagulated to different degrees from the intact DNA because there were different amount of negative charge exposed on their surface, which made a great impact on the solubility of H-GNs. As a result, the corresponding centrifugal supernatant of H-GNs solution showed different color in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, which could be discriminated by naked eyes or by ultraviolet (UV)-visible spectrometer. Based on this, the damaged effects of styrene oxide (SO), NaAsO2 and UV radiation on DNA were studied. Results showed that SO exerted most serious damage effect on DNA although all of them damaged DNA seriously. The new method for detection of DNA damage showed good prospect in the evaluation of genotoxicity of new compounds, the maximum limit of pesticide residue, food additives, and so on, which is important in the fields of food science, pharmaceutical science and pesticide science.

  5. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

    2014-03-03

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

  6. Genomics and radical mediated DNA damage: major differences between ionizing radiation and DNA-cleaving enediynes

    International Nuclear Information System (INIS)

    Cosgrove, J.P.; Begley, T.J.; Samson, L.D.; Dedon, P.C.

    2003-01-01

    While the evidence is strong for radical-mediated oxidative processes in the pathophysiology of cancer and aging, the mechanisms by which cells respond to oxidative stress have eluded definition. To this end, we have undertaken genomic studies comparing the response of S. cerevisiae to DNA-specific oxidizing agents, the enediynes calicheamicin (CAL), esperamicin (ESP), and neocarzinostatin (NCS), and the non-specific gamma-radiation (RAD). While RAD results in relatively indiscriminate oxidation of cellular molecules, the enediynes are highly specific to DNA and produce damage by a common mechanism involving radical-mediated oxidation of deoxyribose. Transcriptional profiling in response to these agents (80% survival; 15 min exposure; Affymetrix) revealed unexpected differences between RAD and the enediynes and among the three enediynes. Only 2 genes responded in common to all agents, while 9 genes were regulated in common for the 3 enediynes (no DNA repair genes altered in common). The limited common gene expression changes for the 3 enediynes may result from differences in deoxyribose oxidation chemistry, DNA and chromatin targets or the proportions of single- and double-strand DNA lesions. RAD produced a more robust response than the enediynes, altering expression of 195 and 52 genes by more than 2- and 5-fold, respectively, compared to 16-44 and *2 genes, respectively, for the enediynes. This suggests that the transcriptional response varies in intensity according to the number of cellular features affected by the toxin. Genes showing the strongest up-regulation with RAD: ribonucleotide reductase, multidrug resistance, DS break repair/RAD51, GSH transferase; strongly reduced gene expression: TEL1 (damage signaling), NAT2 (acetyltransferase). Genomic phenotyping studies, using a subset of the Research Genetics deletion library, revealed that loss of apn1, the major AP endonuclease, caused resistance to NCS, possibly due to reduced formation of protein-DNA cross

  7. Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage

    International Nuclear Information System (INIS)

    Stapleton, A.E.; Walbot, V.

    1994-01-01

    Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage. (author)

  8. Hide and seek: How do DNA glycosylases locate oxidatively damaged DNA bases amidst a sea of undamaged bases?

    Science.gov (United States)

    Lee, Andrea J; Wallace, Susan S

    2017-06-01

    The first step of the base excision repair (BER) pathway responsible for removing oxidative DNA damage utilizes DNA glycosylases to find and remove the damaged DNA base. How glycosylases find the damaged base amidst a sea of undamaged bases has long been a question in the BER field. Single molecule total internal reflection fluorescence microscopy (SM TIRFM) experiments have allowed for an exciting look into this search mechanism and have found that DNA glycosylases scan along the DNA backbone in a bidirectional and random fashion. By comparing the search behavior of bacterial glycosylases from different structural families and with varying substrate specificities, it was found that glycosylases search for damage by periodically inserting a wedge residue into the DNA stack as they redundantly search tracks of DNA that are 450-600bp in length. These studies open up a wealth of possibilities for further study in real time of the interactions of DNA glycosylases and other BER enzymes with various DNA substrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. [Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

    Science.gov (United States)

    Song, Chonglin; Zhang, Zhifu; Chen, Xue; Zhang, Yanfeng; Wang, Chunhua; Liu, Keming

    2002-10-01

    To study DNA damage of three kinds of gasoline oxygenates. Single cell gel electrophoresis assay(Comet assay) was used to detect the damage effects of three gasoline oxygenates[methyl tertiary butyl ether(MTBE), ethanol anhydrous(EA) and dimethyl carbonate(DMC)] on DNA in L-929 mice fibroblasts. In certain concentation(37.500-150.000 mg/ml), MTBE could directly cause DNA damage of L-929 mice fibroblasts. There was obvious dose-effect relationship, i.e. when the concentration of MTBE was increased from 9.375 to 150.000 mg/ml, the comet rate also increased from 4% to 85%, and the length of comet tail changed correspondingly. The results of EA and DMC were negative. Under the condition of this experiment(150.000 mg/ml), MTBE could directly cause DNA damage while the effect of EA and DMC on DNA damage was not found.

  10. Gymnemagenin-a triterpene saponin prevents γ-radiation induced cellular DNA damage

    International Nuclear Information System (INIS)

    Arunachalam, Kantha Deivi; Arun, Lilly Baptista; Annamalai, Sathesh Kumar; Hari, Shanmugasundaram

    2014-01-01

    Gymnema sylvestre an ethno-medicinally important plant was investigated for its protecting activity against radiation induced DNA damage. The major bioactive component present in Gymnema sylvestre such as gymnemic acid and gymnemagenin a triterpene saponin, were tested for its radioprotective effects against 60 Co irradiation induced DNA damage in fish model using fresh water fish Pangasius sutchi. Fishes subjected to a dose of 133 Gy of gamma radiation and observed for eight days. The genotoxic assessment by micronucleus assay showed us that that the plant extract helped in reducing the frequency of micronucleated and binucleated erythrocytes compared to the irradiated control group. The genotoxic assessment by alkaline comet assay by single gel electrophoresis shows that pretreatment with the plant extract appreciably decreased the percentage of tail DNA towards the levels close to those of normal control group. The gradual increase in the level of the antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) during the course of the experiment indicates that the antioxidant enzyme activities play an important role in protecting organisms against gamma radiation-induced cellular oxidative stress. In conclusion the leaf extracts of Gymnema sylvstre exerts its radio protective potential by suppressing the toxic assault of ROS generated by the ionizing radiation through its ability to boost the levels of antioxidant enzymes (CAT and SOD) due to the presence of its phytochemicals like gymnemgenenin- a Triterpene Saponin. (author)

  11. The Fanconi anemia pathway: Repairing the link between DNA damage and squamous cell carcinoma

    International Nuclear Information System (INIS)

    Romick-Rosendale, Lindsey E.; Lui, Vivian W.Y.; Grandis, Jennifer R.; Wells, Susanne I.

    2013-01-01

    Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today's bone marrow failure treatments on tomorrow's solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility

  12. Antioxidant and DNA Damage Protecting Activity of Exopolysaccharides from the Endophytic Bacterium Bacillus cereus SZ1

    Directory of Open Access Journals (Sweden)

    Li Ping Zheng

    2016-02-01

    Full Text Available An endophytic bacterium was isolated from the Chinese medicinal plant Artemisia annua L. The phylogenetic and physiological characterization indicated that the isolate, strain SZ-1, was Bacillus cereus. The endophyte could produce an exopolysaccharide (EPS at 46 mg/L. The 1,1-diphenyl-2-picrylhydracyl (DPPH radical scavenging activity of the EPS reached more than 50% at 3–5 mg/mL. The EPS was also effective in scavenging superoxide radical in a concentration dependent fashion with an EC50 value of 2.6 mg/mL. The corresponding EC50 for scavenging hydroxyl radical was 3.1 mg/mL. Moreover, phenanthroline-copper complex-mediated chemiluminescent emission of DNA damage was both inhibited and delayed by EPS. The EPS at 0.7–1.7 mg/mL also protected supercoiled DNA strands in plasmid pBR322 against scission induced by Fenton-mediated hydroxyl radical. The preincubation of PC12 cells with the EPS prior to H2O2 exposure increased the cell survival and glutathione (GSH level and catalase (CAT activities, and decreased the level of malondialdehyde (MDA and lactate dehydrogenase (LDH activity in a dose-dependent manner, suggesting a pronounced protective effect against H2O2-induced cytotoxicity. Our study indicated that the EPS could be useful for preventing oxidative DNA damage and cellular oxidation in pharmaceutical and food industries.

  13. Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

    Science.gov (United States)

    Chen, Liming; Liu, Yinghui; Dong, Liangliang; Chu, Xiaoxia

    2015-03-01

    Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p edaravone offers protection from radiation-induced cytogenetic alterations.

  14. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

    DEFF Research Database (Denmark)

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke

    2010-01-01

    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due...... to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet...... assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA...

  15. Multi-scale approach to radiation damage induced by ion beams: complex DNA damage and effects of thermal spikes

    International Nuclear Information System (INIS)

    Surdutovich, E.; Yakubovich, A.V.; Solov'yov, A.V.; Surdutovich, E.; Yakubovich, A.V.; Solov'yov, A.V.

    2010-01-01

    We present the latest advances of the multi-scale approach to radiation damage caused by irradiation of a tissue with energetic ions and report the calculations of complex DNA damage and the effects of thermal spikes on biomolecules. The multi-scale approach aims to quantify the most important physical, chemical, and biological phenomena taking place during and following irradiation with ions and provide a better means for clinically-necessary calculations with adequate accuracy. We suggest a way of quantifying the complex clustered damage, one of the most important features of the radiation damage caused by ions. This quantification allows the studying of how the clusterization of DNA lesions affects the lethality of damage. We discuss the first results of molecular dynamics simulations of ubiquitin in the environment of thermal spikes, predicted to occur in tissue for a short time after an ion's passage in the vicinity of the ions' tracks. (authors)

  16. Personal exposure to ultrafine particles and oxidative DNA damage

    DEFF Research Database (Denmark)

    Vinzents, Peter S; Møller, Peter; Sørensen, Mette

    2005-01-01

    Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable instr......, particularly during bicycling in traffic. The results indicate that biologic effects of UFPs occur at modest exposure, such as that occurring in traffic, which supports the relationship of UFPs and the adverse health effects of air pollution.......Exposure to ultrafine particles (UFPs) from vehicle exhaust has been related to risk of cardiovascular and pulmonary disease and cancer, even though exposure assessment is difficult. We studied personal exposure in terms of number concentrations of UFPs in the breathing zone, using portable...... instruments in six 18-hr periods in 15 healthy nonsmoking subjects. Exposure contrasts of outdoor pollution were achieved by bicycling in traffic for 5 days and in the laboratory for 1 day. Oxidative DNA damage was assessed as strand breaks and oxidized purines in mononuclear cells isolated from venous blood...

  17. 2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    El-Yazbi, Amira F.; Loppnow, Glen R.

    2012-01-01

    Highlights: ► Molecular beacon with 2AP bases detects DNA damage in a simple mix-and-read assay. ► Molecular beacons with 2AP bases detect damage at a 17.2 nM limit of detection. ► The 2AP molecular beacon is linear over a 0–3.5 μM concentration range for damage. - Abstract: Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R 2 = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.

  18. DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage.

    Science.gov (United States)

    Mueller, Michael M; Castells-Roca, Laia; Babu, Vipin; Ermolaeva, Maria A; Müller, Roman-Ulrich; Frommolt, Peter; Williams, Ashley B; Greiss, Sebastian; Schneider, Jennifer I; Benzing, Thomas; Schermer, Bernhard; Schumacher, Björn

    2014-12-01

    Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

  19. DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis

    OpenAIRE

    Durbach, S I; Springer, B; Machowski, E E; North, R J; Papavinasasundaram, K G; Colston, M J; Böttger, E C; Mizrahi, V

    2003-01-01

    One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N′-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is res...

  20. Study of genetic damage in the Japanese oyster induced by an environmentally-relevant exposure to diuron: evidence of vertical transmission of DNA damage.

    Science.gov (United States)

    Barranger, A; Akcha, F; Rouxel, J; Brizard, R; Maurouard, E; Pallud, M; Menard, D; Tapie, N; Budzinski, H; Burgeot, T; Benabdelmouna, A

    2014-01-01

    Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 μg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster

  1. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

  2. Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

    International Nuclear Information System (INIS)

    Muniz, Juan F.; McCauley, Linda; Scherer, J.; Lasarev, M.; Koshy, M.; Kow, Y.W.; Nazar-Stewart, Valle; Kisby, G.E.

    2008-01-01

    Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers and applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects

  3. Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Luca A Petruccelli

    Full Text Available Histone deacetylase inhibitors (HDACi are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

  4. Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

    Science.gov (United States)

    Pettersson, Filippa; Retrouvey, Hélène; Skoulikas, Sophia; Miller, Wilson H.

    2011-01-01

    Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. PMID:21695163

  5. The cell-free fetal DNA fraction in maternal blood decreases after physical activity

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Hatt, Lotte; Bach, Cathrine

    2014-01-01

    of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p decrease varying......OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has...... from 1-17 percentage points. This was due to a significant increase in the concentration of cfDNA (p physical activity. CONCLUSION: When planning the timing of noninvasive...

  6. Imitation of radiation-induced damages to DNA with a radionuclide incorporated into polynucleotides

    International Nuclear Information System (INIS)

    Korolev, V.G.

    1984-01-01

    Because of a great variety and different reparability of radiation-induced DNA lesions it is difficult to evaluate the radiobiologacal significance of certain individual alterations. It is suggested that the radionuclides incorporated anto DNA can be used to imitate different types of radiation damages to DNA. Both qualitative and quantitative aspects of the problem are discussed

  7. Clusters of DNA damage induced by ionizing radiation: Formation of short DNA fragments. I. Theoretical modeling

    International Nuclear Information System (INIS)

    Holley, W.R.; Chatterjee, A.

    1996-01-01

    We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber composed of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and δ rays due to knock-on collisions involving energy transfers > 100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of circ OH, circ H, e aq , etc.; circ OH attack on sugar molecules leading to strand breaks; circ OH attack on bases; direct ionization of the sugar molecules leading to strand breaks; direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 hp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the chromatin fibers in mammalian DNA. 27 refs., 7 figs

  8. Long non-coding RNAs as novel expression signatures modulate DNA damage and repair in cadmium toxicology

    Science.gov (United States)

    Zhou, Zhiheng; Liu, Haibai; Wang, Caixia; Lu, Qian; Huang, Qinhai; Zheng, Chanjiao; Lei, Yixiong

    2015-10-01

    Increasing evidence suggests that long non-coding RNAs (lncRNAs) are involved in a variety of physiological and pathophysiological processes. Our study was to investigate whether lncRNAs as novel expression signatures are able to modulate DNA damage and repair in cadmium(Cd) toxicity. There were aberrant expression profiles of lncRNAs in 35th Cd-induced cells as compared to untreated 16HBE cells. siRNA-mediated knockdown of ENST00000414355 inhibited the growth of DNA-damaged cells and decreased the expressions of DNA-damage related genes (ATM, ATR and ATRIP), while increased the expressions of DNA-repair related genes (DDB1, DDB2, OGG1, ERCC1, MSH2, RAD50, XRCC1 and BARD1). Cadmium increased ENST00000414355 expression in the lung of Cd-exposed rats in a dose-dependent manner. A significant positive correlation was observed between blood ENST00000414355 expression and urinary/blood Cd concentrations, and there were significant correlations of lncRNA-ENST00000414355 expression with the expressions of target genes in the lung of Cd-exposed rats and the blood of Cd exposed workers. These results indicate that some lncRNAs are aberrantly expressed in Cd-treated 16HBE cells. lncRNA-ENST00000414355 may serve as a signature for DNA damage and repair related to the epigenetic mechanisms underlying the cadmium toxicity and become a novel biomarker of cadmium toxicity.

  9. Regulation of DNA Damage Response by Estrogen Receptor β-Mediated Inhibition of Breast Cancer Associated Gene 2

    Directory of Open Access Journals (Sweden)

    Yuan-Hao Lee

    2015-04-01

    Full Text Available Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2, correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

  10. No association between alcohol supplementation and autoantibodies to DNA damage in postmenopausal women in a controlled feeding study.

    Science.gov (United States)

    Mahabir, S; Baer, D J; Johnson, L L; Frenkel, K; Dorgan, J F; Cambell, W; Hartman, T J; Clevidence, B; Albanes, D; Judd, J T; Taylor, P R

    2005-08-01

    Alcohol consumption is linked to increased breast cancer risk. Since oestrogens increase breast cancer risk, possibly through oxidative damage, and we have shown that alcohol consumption increases serum oestrogens, we tested whether moderate alcohol supplementation increased oxidative DNA damage among healthy postmenopausal women not on hormone replacement therapy in a randomized controlled crossover study. We used serum 5-hydroxymethyl-2-deoxyuridine (5-HMdU) autoantibodies (aAbs) as a marker of oxidative DNA damage. The results showed no evidence for increased or decreased levels of oxidative DNA damage among women who consumed 15 g or 30 g alcohol per day for 8 weeks compared with women in the 0 g alcohol group. We conclude that among healthy women, it is possible that an 8-week trial of moderate alcohol supplementation might be too short to make enough 5-HMdU aAbs to compare differences by alcohol dose. In future studies, a panel of biomarkers for DNA damage should be used.

  11. Symposium cellular response to DNA damage the role of poly(ADP-ribose) poly(ADP-ribose) in the cellular response to DNA damage

    International Nuclear Information System (INIS)

    Berger, N.A.

    1985-01-01

    Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Inhibitors of Poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of Poly(ADP-ribose) persists and the activated enzyme is capable of totaly consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of Poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest

  12. The influence of combined treatment of Cd, and γ-irradiation on DNA damage and repair in lymphoid tissues of mice

    International Nuclear Information System (INIS)

    Privezentsev, K.V.; Sirota, N.P.; Gaziev, A.I.

    1996-01-01

    The effect of combined treatment of Cd and γ-irradiation on DNA damage and repair was studied in lymphoid tissues of mice using single-cell gel assay. Single i.p. injection of CdCl 2 (1 mg Cd/kg body wt), 2 h prior to irradiation resulted in increasing of DNA lesions in peripheral blood lymphocytes (PBL) when compared to non-injected animals. However, the same treatment, 48 h prior to irradiation is shown to decrease DNA damage in PBL and splenocytes in comparison with untreated mice. In thymocytes maximal protective effect of Cd was determined when mice were irradiated in 24 h after injection. The protective effect observed is due to decreasing of initial level of DNA damage in thymocytes as well as acceleration of DNA repair in PBL and splenocytes. 28 refs.; 2 figs

  13. 4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

    Science.gov (United States)

    Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

    2018-03-01

    Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

  14. DNA Damages and White Blood Cell Death Processes in Victims with Severe Injury

    Directory of Open Access Journals (Sweden)

    V. V. Moroz

    2014-01-01

     pase3, caspase9, superoxide dismutase, and sAPO1/FAS were measured by enzyme immunoassay using test systems(Bender MedSystems, Austria.Results. There were significantly higher plasma levels of free DNA in the victims than in the controls throughout the followup, which is due to its exit from the cells damaged from tissue injury. In the first two weeks after injury, there were increases in DNA damages and white blood cell alteration processes by a necrotic and apoptotic mechanism in the victims with different types of injury, which may be associated with the active participation of leuko cytes in the processes of cellular breakdown product removal in the tissues damaged during injury and the in those of prevention of infectious complications. In the victims, white blood cell alteration in the necrotic pathway does not depend on BLV and hypoxia degree while that in the apoptotic pathway showed a relationship of leukocyte alteration to hypoxia in these patients. The sum of the values of necrotic DNA comets, apoptotic DNA comets, and single and doublestrand breaks on day 3 postinjury may serve as a predictor of the likely development of infectious complications in victims with injury, blood loss, and marked hypoxia. There were differences in the levels of DNA damages and white blood cell apoptosis and necrosis in the victims with BI and SSI. The injury victims showed a threefold decrease in plasma 8hydroxy2 deoxyguanosine concentrations, which was accompanied and, possibly, caused by an increase in the amount of superoxide dismutase.Conclusion. There was a relationship between the degree of DNA damages, apoptosis, and necrosis in the white blood cells of victims with injury and hypoxiainduced blood loss.

  15. HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

    Science.gov (United States)

    Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

    2008-01-01

    Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

  16. UV light induced DNA damages and the radiation protection effects of Lingzi mushroom extract

    International Nuclear Information System (INIS)

    Vo Thi Thuong Lan; Dinh Ba Tuan; Ta Bich Thuan; Tran Bang Diep; Tran Minh Quynh

    2016-01-01

    UV light has strongly influenced on the growth of E. coli as well as caused DNA damages. Configurations of both genomic DNA and pUC 19 plasmids extracted from E. coli were significantly changed by the exposure to UV light of 254 nm and DLT, an extract of Ganoderma lucidum Lingzi mushroom. The results also revealed the radio-protective effects of DLT to UV radiation. By adding 2% DLT to its culturing suspension, the growth of E. coli was significantly decreased, whereas a low DLT amount of about 0.5% slightly improved its growth, indicated that the DLT extract can be used as a promising protective substance against UV radiation. At the molecular level, the radio-protective effects of DLT were observed for both UV treated DNA and protein. Thus, DLT can protect DNA in vivo, but not in vitro. This effect was also observed for Taq polymerase, suggested that the radioprotection effect of DLT may due to it accelerated the degradation of radicals or species that produced in the suspensions during UV exposure. (author)

  17. An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands.

    Science.gov (United States)

    Weisel, Tamara; Baum, Matthias; Eisenbrand, Gerhard; Dietrich, Helmut; Will, Frank; Stockis, Jean-Pierre; Kulling, Sabine; Rüfer, Corinna; Johannes, Christian; Janzowski, Christine

    2006-04-01

    Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.

  18. Photoelectrochemical Sensors for the Rapid Detection of DNA Damage Induced by Some Nanoparticles

    Directory of Open Access Journals (Sweden)

    M. Jamaluddin Ahmed

    2010-06-01

    Full Text Available Photoelectrochemcal sensors were developed for the rapid detection of oxidative DNA damage induced by titanium dioxide and polystyrene nanoparticles. Each sensor is a multilayer film prepared on a tin oxide nanoparticle electrode using layer- by-layer self assembly and is composed of separate layer of a photoelectrochemical indicator, DNA