Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

PubMed

Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

2011-12-24

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation

PubMed Central

Zhang, Xurui; Ye, Caiyong; Sun, Fang; Wei, Wenjun; Hu, Burong; Wang, Jufang

2016-01-01

Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92–1 cells. By measuring senescence associated-β-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research. PMID:27187621

Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response.

PubMed

Luo, Man; Bao, Zhengqiang; Xu, Feng; Wang, Xiaohui; Li, Fei; Li, Wen; Chen, Zhihua; Ying, Songmin; Shen, Huahao

2018-04-14

The inflammatory cascade can be initiated with the recognition of damaged DNA. Macrophages play an essential role in particulate matter (PM)-induced airway inflammation. In this study, we aim to explore the PM induced DNA damage response of macrophages and its function in airway inflammation. The DNA damage response and inflammatory response were assessed using bone marrow-derived macrophages following PM treatment and mouse model instilled intratracheally with PM. We found that PM induced significant DNA damage both in vitro and in vivo and simultaneously triggered a rapid DNA damage response, represented by nuclear RPA, 53BP1 and γH2AX foci formation. Genetic ablation or chemical inhibition of the DNA damage response sensor amplified the production of cytokines including Cxcl1, Cxcl2 and Ifn-γ after PM stimulation in bone marrow-derived macrophages. Similar to that seen in vitro , mice with myeloid-specific deletion of RAD50 showed higher levels of airway inflammation in response to the PM challenge, suggesting a protective role of DNA damage sensor during inflammation. These data demonstrate that PM exposure induces DNA damage and activation of DNA damage response sensor MRN complex in macrophages. Disruption of MRN complex lead to persistent, unrepaired DNA damage that causes elevated inflammatory response.

Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans.

PubMed

Feng, Wei-Hong; Xue, Kathy S; Tang, Lili; Williams, Phillip L; Wang, Jia-Sheng

2016-12-26

Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB₁, using Caenorhabditis elegans as a study model. Results found that AFB₁ induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB₁ inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB₁-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans .

DNA Damage Response in Cisplatin-Induced Nephrotoxicity

PubMed Central

Zhu, Shiyao; Pabla, Navjotsingh; Tang, Chengyuan; He, Liyu; Dong, Zheng

2015-01-01

Cisplatin and its derivatives are widely used chemotherapeutic drugs for cancer treatment. However, they have debilitating side-effects in normal tissues and induce ototoxicity, neurotoxicity, and nephrotoxicity. In kidneys, cisplatin preferentially accumulates in renal tubular cells causing tubular cell injury and death, resulting in acute kidney injury (AKI). Recent studies have suggested that DNA damage and the associated DNA damage response (DDR) is an important pathogenic mechanism of AKI following cisplatin treatment. Activation of DDR may lead to cell cycle arrest and DNA repair for cell survival or, in the presence of severe injury, kidney cell death. Modulation of DDR may provide novel renoprotective strategies for cancer patients undergoing cisplatin chemotherapy. PMID:26564230

Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response.

PubMed

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Ciarapica, Veronica; Bracci, Massimo; Caboni, Maria Fiorenza; Pasini, Federica; Monaco, Federica; Amati, Monica; Borghi, Battista; Tomasetti, Marco

2016-10-01

Glyphosate (GLY) and organophosphorus insecticides such as chlorpyrifos (CPF) may cause DNA damage and cancer in exposed individuals through mitochondrial dysfunction. Polyphenols ubiquitously present in fruits and vegetables, have been viewed as antioxidant molecules, but also influence mitochondrial homeostasis. Here, honey containing polyphenol compounds was evaluated for its potential protective effect on pesticide-induced genotoxicity. Honey extracts from four floral organic sources were evaluated for their polyphenol content, antioxidant activity, and potential protective effects on pesticide-related mitochondrial destabilization, reactive oxygen and nitrogen species formation, and DNA damage response in human bronchial epithelial and neuronal cells. The protective effect of honey was, then evaluated in a residential population chronically exposed to pesticides. The four honey types showed a different polyphenol profile associated with a different antioxidant power. The pesticide-induced mitochondrial dysfunction parallels ROS formation from mitochondria (mtROS) and consequent DNA damage. Honey extracts efficiently inhibited pesticide-induced mtROS formation, and reduced DNA damage by upregulation of DNA repair through NFR2. Honey supplementation enhanced DNA repair activity in a residential population chronically exposed to pesticides, which resulted in a marked reduction of pesticide-induced DNA lesions. These results provide new insight regarding the effect of honey containing polyphenols on pesticide-induced DNA damage response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oxidative DNA damage induced by a hydroperoxide derivative of cyclophosphamide.

PubMed

Murata, Mariko; Suzuki, Toshinari; Midorikawa, Kaoru; Oikawa, Shinji; Kawanishi, Shosuke

2004-09-15

Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.

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

PubMed

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.

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

PubMed Central

Wang, Shu-Huei; Lin, Pei-Ya; Chiu, Ya-Chen; Huang, Ju-Sui; Kuo, Yi-Tsen; Wu, Jen-Chine; Chen, Chin-Chuan

2015-01-01

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity. PMID:26218133

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

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 6more » 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.« less

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

PubMed

Hall, Ashley; Ballantyne, Jack

2004-09-01

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

PubMed

Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

2017-12-07

Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mequindox induced cellular DNA damage via generation of reactive oxygen species.

PubMed

Liu, Jing; Ouyang, Man; Jiang, Jun; Mu, Peiqiang; Wu, Jun; Yang, Qi; Zhang, Caihui; Xu, Weiying; Wang, Lijuan; Huen, Michael S Y; Deng, Yiqun

2012-01-24

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS. © 2011 Elsevier B.V. All rights reserved.

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

PubMed

Ganesan, Shanthi; Keating, Aileen F

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 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h 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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed Central

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G.

2013-01-01

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. PMID:23523584

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis.

PubMed

Mavragani, Ifigeneia V; Nikitaki, Zacharenia; Souli, Maria P; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy; Georgakilas, Alexandros G

2017-07-18

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent "danger" signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

Tangeretin sensitizes SGS1-deficient cells by inducing DNA damage.

PubMed

Chong, Shin Yen; Wu, Meng-Ying; Lo, Yi-Chen

2013-07-03

Tangeretin, a polymethoxyflavone found in citrus peel, has been shown to have antiatherogenic, anti-inflammatory, and anticarcinogenic properties. However, the underlying target pathways are not fully characterized. We investigated the tangeretin sensitivity of yeast (Saccharomyces cerevisiae) mutants for DNA damage response or repair pathways. We found that tangeretin treatment significantly reduced (p < 0.05) survival rate, induced preferential G1 phase accumulation, and elevated the DNA double-strand break (DSB) signal γH2A in DNA repair-defective sgs1Δ cells, but had no obvious effects on wild-type cells or mutants of the DNA damage checkpoint (including tel1Δ, sml1Δ mec1Δ, sml1Δ mec1Δ tel1Δ, and rad9Δ mutants). Additionally, microarray data indicated that tangeretin treatment up-regulates genes involved in nutritional processing and down-regulates genes related to RNA processing in sgs1Δ mutants. These results suggest tangeretin may sensitize SGS1-deficient cells by increasing a marker of DNA damage and by inducing G1 arrest and possibly metabolic stress. Thus, tangeretin may be suitable for chemosensitization of cancer cells lacking DSB-repair ability.

[UV-induced DNA damage and protective effects of antioxidants on DNA damage in human lens epithelial cells studied with comet assay].

PubMed

Wu, Zhi-hong; Wang, Mian-rong; Yan, Qi-chang; Pu, Wei; Zhang, Jin-song

2006-11-01

To investigate the mechanism of UV-induced DNA damage and repair and the protective effects of antioxidants on DNA damage in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group), 2.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated group 1 - 4). The amounts of DNA single strand breaks (SSB) were measured with the alkaline comet assay (CA). The spontaneous repair of DNA SSB after exposure to UV at 10.0 mJ/cm(2) was also determined in human lens epithelial cells. Human lens epithelial cells were treated with different concentration of VitaminC (VitC), taurine, superoxide dismutase (SOD) and epigallocatechin gallate (EGCG) before and after ultraviolet radiation, the effects of antioxidants on DNA damage was examined with alkaline comet assay. The amount of DNA SSB in control group and treated groups 1 - 4 showed increased tendency, was dose-dependent to the dose of UV irradiation, the differences of DNA SSB in 5 group were significantly (P < 0.01). UV-induced DNA SSB at 10.0 mJ/cm(2) in human lens epithelial cells, the half repair time was 60 minutes. Human lens epithelial cells were treated with different concentrations of taurine, SOD and EGCG before ultraviolet radiation. The differences of DNA damage in control and various antioxidant treated groups was statistically significant (F = 6.591, 13.542, 4.626 in cells treated with taurine, SOD and EGCG, respectively, P < 0.01), the difference of VitC effect on DNA in control and treated group were not significantly (F = 1.451, P > 0.05). Human lens epithelial cells were treated with different concentration of VitC, taurine, SOD and EGCG after ultraviolet radiation. The differences of DNA damage between the control and treated group were statistically significant (F = 6.571, 4.810, 6.824, 9.182 in cells treated with VitC, taurine, SOD and EGCG, respectively, P < 0.01). The differences of protective effects on DNA damage in these four different kinds of antioxidants added before UV

Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Jiali; Hui, Pingping; Meng, Wenying

The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificatedmore » when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells. - Highlights: • Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells. • Ku70 knockdown facilitates gemcitabine-induced DNA damage and cell apoptosis. • Ku70 overexpression deceases gemcitabine's sensitivity in pancreatic cancer cells. • Ku70 knockdown sensitizes gemcitabine-induced anti-tumor activity in vivo.« less

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

PubMed Central

Mavragani, Ifigeneia V.; Nikitaki, Zacharenia; Souli, Maria P.; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy

2017-01-01

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair. PMID:28718816

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

PubMed Central

Ganapathy, Vengatesh; Manyanga, Jimmy; Brame, Lacy; McGuire, Dehra; Sadhasivam, Balaji; Floyd, Evan; Rubenstein, David A.; Ramachandran, Ilangovan; Wagener, Theodore

2017-01-01

Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public. PMID:28542301

House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs.

PubMed

Chan, Tze Khee; Loh, Xin Yi; Peh, Hong Yong; Tan, W N Felicia; Tan, W S Daniel; Li, Na; Tay, Ian J J; Wong, W S Fred; Engelward, Bevin P

2016-07-01

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) in vivo and in vitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX) foci in bronchial epithelium. BEAS-2B cells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fishermore » 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. - Highlights: • PM exposure induces DNA damage repair gene expression. • Inhibition of ATM prevented PM-induced follicle depletion. • PKCδ deficiency did not impact PM-induced ovotoxicity.« less

Nuclear aggregates of polyamines in a radiation-induced DNA damage model.

PubMed

Iacomino, Giuseppe; Picariello, Gianluca; Stillitano, Ilaria; D'Agostino, Luciano

2014-02-01

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of DNA damage induced by Auger electrons from 137Cs.

PubMed

Watanabe, Ritsuko; Hattori, Yuya; Kai, Takeshi

2016-11-01

To understand the biological effect of external and internal exposure from 137 Cs, DNA damage spectrum induced by directly emitted electrons (γ-rays, internal conversion electrons, Auger electrons) from 137 Cs was compared with that induced by 137 Cs γ-rays. Monte Carlo track simulation method was used to calculate the microscopic energy deposition pattern in liquid water. Simulation was performed for the two simple target systems in microscale. Radiation sources were placed inside for one system and outside for another system. To simulate the energy deposition by directly emitted electrons from 137 Cs placed inside the system, the multiple ejections of electrons after internal conversion were considered. In the target systems, induction process of DNA damage was modeled and simulated for both direct energy deposition and the water radical reaction on the DNA. The yield and spatial distribution of simple and complex DNA damage including strand breaks and base lesions were calculated for irradiation by electrons and γ-rays from 137 Cs. The simulation showed that the significant difference in DNA damage spectrum was not caused by directly ejected electrons and γ-rays from 137 Cs. The result supports the existing perception that the biological effects by internal and external exposure by 137 Cs are equivalent.

DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAY

EPA Science Inventory

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposur...

DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENNTIAL FLUORESENCE ASSAY

EPA Science Inventory

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures...

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

PubMed Central

Huang, Shar-yin N.; Murai, Junko; Dalla Rosa, Ilaria; Dexheimer, Thomas S.; Naumova, Alena; Gmeiner, William H.; Pommier, Yves

2013-01-01

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs. PMID:23775789

Occurrence, Biological Consequences, and Human Health Relevance of Oxidative Stress-Induced DNA Damage.

PubMed

Yu, Yang; Cui, Yuxiang; Niedernhofer, Laura J; Wang, Yinsheng

2016-12-19

A variety of endogenous and exogenous agents can induce DNA damage and lead to genomic instability. Reactive oxygen species (ROS), an important class of DNA damaging agents, are constantly generated in cells as a consequence of endogenous metabolism, infection/inflammation, and/or exposure to environmental toxicants. A wide array of DNA lesions can be induced by ROS directly, including single-nucleobase lesions, tandem lesions, and hypochlorous acid (HOCl)/hypobromous acid (HOBr)-derived DNA adducts. ROS can also lead to lipid peroxidation, whose byproducts can also react with DNA to produce exocyclic DNA lesions. A combination of bioanalytical chemistry, synthetic organic chemistry, and molecular biology approaches have provided significant insights into the occurrence, repair, and biological consequences of oxidatively induced DNA lesions. The involvement of these lesions in the etiology of human diseases and aging was also investigated in the past several decades, suggesting that the oxidatively induced DNA adducts, especially bulky DNA lesions, may serve as biomarkers for exploring the role of oxidative stress in human diseases. The continuing development and improvement of LC-MS/MS coupled with the stable isotope-dilution method for DNA adduct quantification will further promote research about the clinical implications and diagnostic applications of oxidatively induced DNA adducts.

Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

PubMed Central

Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

2011-01-01

Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

PubMed Central

Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

2015-01-01

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

Repair of DNA damage induced by accelerated heavy ions--a mini review.

PubMed

Okayasu, Ryuichi

2012-03-01

Increasing use of heavy ions for cancer therapy and concerns from exposure to heavy charged particles in space necessitate the study of the basic biological mechanisms associated with exposure to heavy ions. As the most critical damage induced by ionizing radiation is DNA double strand break (DSB), this review focuses on DSBs induced by heavy ions and their repair processes. Compared with X- or gamma-rays, high-linear energy transfer (LET) heavy ion radiation induces more complex DNA damage, categorized into DSBs and non-DSB oxidative clustered DNA lesions (OCDL). This complexity makes the DNA repair process more difficult, partially due to retarded enzymatic activities, leading to increased chromosome aberrations and cell death. In general, the repair process following heavy ion exposure is LET-dependent, but with nonhomologous end joining defective cells, this trend is less emphasized. The variation in cell survival levels throughout the cell cycle is less prominent in cells exposed to high-LET heavy ions when compared with low LET, but this mechanism has not been well understood until recently. Involvement of several DSB repair proteins is suggested to underlie this interesting phenomenon. Recent improvements in radiation-induced foci studies combined with high-LET heavy ion exposure could provide a useful opportunity for more in depth study of DSB repair processes. Accelerated heavy ions have become valuable tools to investigate the molecular mechanisms underlying repair of DNA DSBs, the most crucial form of DNA damage induced by radiation and various chemotherapeutic agents. Copyright © 2011 UICC.

The thyroid hormone receptor β induces DNA damage and premature senescence.

PubMed

Zambrano, Alberto; García-Carpizo, Verónica; Gallardo, María Esther; Villamuera, Raquel; Gómez-Ferrería, Maria Ana; Pascual, Angel; Buisine, Nicolas; Sachs, Laurent M; Garesse, Rafael; Aranda, Ana

2014-01-06

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

Escin-induced DNA damage promotes escin-induced apoptosis in human colorectal cancer cells via p62 regulation of the ATM/γH2AX pathway.

PubMed

Wang, Zhong; Chen, Qiang; Li, Bin; Xie, Jia-Ming; Yang, Xiao-Dong; Zhao, Kui; Wu, Yong; Ye, Zhen-Yu; Chen, Zheng-Rong; Qin, Zheng-Hong; Xing, Chun-Gen

2018-05-31

Escin, a triterpene saponin isolated from horse chestnut seed, has been used to treat encephaledema, tissue swelling and chronic venous insufficiency. Recent studies show that escin induces cell cycle arrest, tumor proliferation inhibition and tumor cell apoptosis. But the relationship between escin-induced DNA damage and cell apoptosis in tumor cells remains unclear. In this study, we investigated whether and how escin-induced DNA damage contributed to escin-induced apoptosis in human colorectal cancer cells. Escin (5-80 μg/mL) dose-dependently inhibited the cell viability and colony formation in HCT116 and HCT8 cells. Escin treatment induced DNA damage, leading to p-ATM and γH2AX upregulation. Meanwhile, escin treatment increased the expression of p62, an adaptor protein, which played a crucial role in controlling cell survival and tumorigenesis, and had a protective effect against escin-induced DNA damage: knockdown of p62 apparently enhanced escin-induced DNA damage, whereas overexpression of p62 reduced escin-induced DNA damage. In addition, escin treatment induced concentration- and time-dependent apoptosis. Similarly, knockdown of p62 significantly increased escin-induced apoptosis in vitro and produced en escin-like antitumor effect in vivo. Overexpression of p62 decreased the rate of apoptosis. Further studies revealed that the functions of p62 in escin-induced DNA damage were associated with escin-induced apoptosis, and p62 knockdown combined with the ATM inhibitor KU55933 augmented escin-induced DNA damage and further increased escin-induced apoptosis. In conclusion, our results demonstrate that p62 regulates ATM/γH2AX pathway-mediated escin-induced DNA damage and apoptosis.

The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis.

PubMed

Li, Diqiu; Huang, Qingchun; Lu, Miaoqing; Zhang, Lei; Yang, Zhichuan; Zong, Mimi; Tao, Liming

2015-09-01

The organophosphate insecticide chlorpyrifos (CPF) is known to induce neurological effects, malformation and micronucleus formation, persistent developmental disorders, and maternal toxicity in rats and mice. The binding of chlorpyrifos with DNA to produce DNA adducts leads to an increasing social concern about the genotoxic risk of CPF in human, but CPF-induced cytotoxicity through DNA damage and cell apoptosis is not well understood. Here, we quantified the cytotoxicity and potential genotoxicity of CPF using the alkaline comet assay, γH2AX foci formation, and the DNA laddering assay in order to detect DNA damage and apoptosis in human HeLa and HEK293 cells in vitro. Drosophila S2 cells were used as a positive control. The alkaline comet assay showed that sublethal concentrations of CPF induced significant concentration-dependent increases in single-strand DNA breaks in the treated cells compared with the control. The percentage of γH2AX-positive HeLa cells revealed that CPF also causes DNA double-strand breaks in a time-dependent manner. Moreover, DNA fragmentation analysis demonstrated that exposure to CPF induced a significant concentration- and time-dependent increase in cell apoptosis. We conclude that CPF is a strongly genotoxic agent that induces DNA damage and cell apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Is UV-induced DNA damage greater at higher elevation?

PubMed

Wang, Qing-Wei; Hidema, Jun; Hikosaka, Kouki

2014-05-01

• Although ultraviolet radiation (UV) is known to have negative effects on plant growth, there has been no direct evidence that plants growing at higher elevations are more severely affected by ultraviolet-B (UV-B) radiation, which is known to increase with elevation. We examined damage to DNA, a primary target of UV-B, in the widespread species Polygonum sachalinense (Fallopia sachalinensis) and Plantago asiatica at two elevations.• We sampled leaves of both species at 300 and 1700 m above sea level every 2 h for 11 d across the growing season and determined the level of cyclobutane pyrimidine dimer (CPD), a major product of UV damage to DNA.• The CPD level was significantly influenced by the time of day, date, elevation, and their interactions in both species. The CPD level tended to be higher at noon or on sunny days. DNA damage was more severe at 1700 m than at 300 m: on average, 8.7% greater at high elevation in P. asiatica and 7.8% greater in P. sachalinense Stepwise multiple regression analysis indicated that the CPD level was explained mainly by UV-B and had no significant relationship with other environmental factors such as temperature and photosynthetically active radiation.• UV-induced DNA damage in plants is greater at higher elevations. © 2014 Botanical Society of America, Inc.

Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

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 6more » (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.« less

Calculation on spectrum of direct DNA damage induced by low-energy electrons including dissociative electron attachment.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2017-03-01

In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40-70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20-40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A-T.

Plasma induced DNA damage: Comparison with the effects of ionizing radiation

NASA Astrophysics Data System (ADS)

Lazović, S.; Maletić, D.; Leskovac, A.; Filipović, J.; Puač, N.; Malović, G.; Joksić, G.; Petrović, Z. Lj.

2014-09-01

We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

A pathway of targeted autophagy is induced by DNA damage in budding yeast

PubMed Central

Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

2017-01-01

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

PubMed

Eapen, Vinay V; Waterman, David P; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G; Loewith, Robbie J; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J; Haber, James E

2017-02-14

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

DNA damage and polyploidization.

PubMed

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.

Clustered DNA damages induced by high and low LET radiation, including heavy ions

NASA Technical Reports Server (NTRS)

Sutherland, B. M.; Bennett, P. V.; Schenk, H.; Sidorkina, O.; Laval, J.; Trunk, J.; Monteleone, D.; Sutherland, J.; Lowenstein, D. I. (Principal Investigator)

2001-01-01

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- 20%, with the other clustered damages being at least 80%.

Rapid communications: antiperspirant induced DNA damage in canine cells by comet assay.

PubMed

Yiu, Gloria

2004-01-01

Abstract Millions of people around the world use antiperspirants to decrease or eliminate body odors. Most antiperspirants contain aluminum zirconium or another form of aluminum as its active ingredient. The present investigation applied Comet assay to detect if Secret Platinum for women, Old Spice for men, or Crystal Natural produced DNA damage in Madin-Darby canine kidney cells (MDCKII). This study has shown that antiperspirants cause DNA damage on a single-cell level. Additionally, our data showed us that in general, Secret Platinum for women and Old Spice for men, produced equivalent damage. Crystal Natural, marketed as being safer or less damaging, induced the most extensive damage of all three antiperspirants tested.

Plasma induced DNA damage: Comparison with the effects of ionizing radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazović, S.; Maletić, D.; Puač, N.

2014-09-22

We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of themore » treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.« less

DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-½ that promotes efficient DNA repair. [corrected].

PubMed

Belin, Brittany J; Lee, Terri; Mullins, R Dyche

2015-08-19

Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments-detectable by phalloidin and live-cell actin probes-with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.

Analysis of nicotine-induced DNA damage in cells of the human respiratory tract.

PubMed

Ginzkey, Christian; Stueber, Thomas; Friehs, Gudrun; Koehler, Christian; Hackenberg, Stephan; Richter, Elmar; Hagen, Rudolf; Kleinsasser, Norbert H

2012-01-05

Epithelium of the upper and lower airways is a common origin of tobacco-related cancer. The main tobacco alkaloid nicotine may be associated with tumor progression. The potential of nicotine in inducing DNA mutations as a step towards cancer initiation is still controversially discussed. Different subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in human nasal mucosa and a human bronchial cell line representing respiratory mucosa as a possible target for receptor-mediated pathways. In the present study, both cell systems were investigated with respect to DNA damage induced by nicotine and its mechanisms. Specimens of human nasal mucosa were harvested during surgery of the nasal air passage. After enzymatic digestion over night, single cells were exposed to an increasing nicotine concentration between 0.001 mM and 4.0mM. In a second step co-incubation was performed using the antioxidant N-acetylcysteine (NAC) and the nAChR antagonist mecamylamine. DNA damage was assessed using the alkali version of the comet assay. Dose finding experiments for mecamylamine to evaluate the maximal inhibitory effect were performed in the human bronchial cell line BEAS-2B with an increasing mecamylamine concentration and a constant nicotine concentration. The influence of nicotine in the apoptotic pathway was evaluated in BEAS-2B cells with the TUNEL assay combined with flow cytometry. After 1h of nicotine exposure with 0.001, 0.01, 0.1, 1.0 and 4.0mM, significant DNA damage was determined at 1.0mM. Further co-incubation experiments with mecamylamine and NAC were performed using 1.0mM of nicotine. The strongest inhibitory effect was measured at 1.0mM mecamylamine and this concentration was used for co-incubation. Both, the antioxidant NAC at a concentration of 1.0mM, based on the literature, as well as the receptor antagonist were capable of complete inhibition of the nicotine-induced DNA migration in the comet assay. A nicotine-induced increase or decrease in

Dietary spices protect against hydrogen peroxide-induced DNA damage and inhibit nicotine-induced cancer cell migration.

PubMed

Jayakumar, R; Kanthimathi, M S

2012-10-01

Spices are rich sources of antioxidants due to the presence of phenols and flavonoids. In this study, the DNA protecting activity and inhibition of nicotine-induced cancer cell migration of 9 spices were analysed. Murine fibroblasts (3T3-L1) and human breast cancer (MCF-7) cells were pre-treated with spice extracts and then exposed to H₂O₂ and nicotine. The comet assay was used to analyse the DNA damage. Among the 9 spices, ginger, at 50 μg/ml protected against 68% of DNA damage in 3T3-L1 cells. Caraway, cumin and fennel showed statistically significant (p<0.05) DNA protecting activity. Treatment of MCF-7 cells with nicotine induced cell migration, whereas pre-treatment with spices reduced this migration. Pepper, long pepper and ginger exhibited a high rate of inhibition of cell migration. The results of this study prove that spices protect DNA and inhibit cancer cell migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA,more » which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.« less

Role of platinum DNA damage-induced transcriptional inhibition in chemotherapy-induced neuronal atrophy and peripheral neurotoxicity.

PubMed

Yan, Fang; Liu, Johnson J; Ip, Virginia; Jamieson, Stephen M F; McKeage, Mark J

2015-12-01

Platinum-based anticancer drugs cause peripheral neurotoxicity by damaging sensory neurons within the dorsal root ganglia (DRG), but the mechanisms are incompletely understood. The roles of platinum DNA binding, transcription inhibition and altered cell size were investigated in primary cultures of rat DRG cells. Click chemistry quantitative fluorescence imaging of RNA-incorporated 5-ethynyluridine showed high, but wide ranging, global levels of transcription in individual neurons that correlated with their cell body size. Treatment with platinum drugs reduced neuronal transcription and cell body size to an extent that corresponded to the amount of preceding platinum DNA binding, but without any loss of neuronal cells. The effects of platinum drugs on neuronal transcription and cell body size were inhibited by blocking platinum DNA binding with sodium thiosulfate, and mimicked by treatment with a model transcriptional inhibitor, actinomycin D. In vivo oxaliplatin treatment depleted the total RNA content of DRG tissue concurrently with altering DRG neuronal size. These findings point to a mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. DRG neurons may be particularly vulnerable to this mechanism of toxicity because of their requirements for high basal levels of global transcriptional activity. Findings point to a new stepwise mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. Dorsal root ganglion neurons may be particularly vulnerable to this neurotoxicity because of their high global transcriptional outputs, demonstrated in this study by click chemistry quantitative fluorescence imaging. © 2015 International Society for Neurochemistry.

Stress-induced DNA Damage biomarkers: Applications and limitations

NASA Astrophysics Data System (ADS)

Nikitaki, Zacharenia; Hellweg, Christine; Georgakilas, Alexandros; Ravanat, Jean-Luc

2015-06-01

A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes like replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damages play a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g. X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e. single, complex DNA lesions etc. that can be used as biomarkers. We critically compare DNA damage detection methods and their limitations. In addition to such DNA damage products, we suggest possible gene inductions that can be used to characterize responses to different types of stresses i.e. radiation, oxidative and replication stress, based on bioinformatic approaches and stringent meta-analysis of literature data.

Molecular Regulation of DNA Damage-Induced Apoptosis in Neurons of Cerebral Cortex

PubMed Central

Liu, Zhiping; Pipino, Jacqueline; Chestnut, Barry; Landek, Melissa A.

2009-01-01

Cerebral cortical neuron degeneration occurs in brain disorders manifesting throughout life, but the mechanisms are understood poorly. We used cultured embryonic mouse cortical neurons and an in vivo mouse model to study mechanisms of DNA damaged-induced apoptosis in immature and differentiated neurons. p53 drives apoptosis of immature and differentiated cortical neurons through its rapid and prominent activation stimulated by DNA strand breaks induced by topoisomerase-I and -II inhibition. Blocking p53-DNA transactivation with α-pifithrin protects immature neurons; blocking p53-mitochondrial functions with μ-pifithrin protects differentiated neurons. Mitochondrial death proteins are upregulated in apoptotic immature and differentiated neurons and have nonredundant proapoptotic functions; Bak is more dominant than Bax in differentiated neurons. p53 phosphorylation is mediated by ataxia telangiectasia mutated (ATM) kinase. ATM inactivation is antiapoptotic, particularly in differentiated neurons, whereas inhibition of c-Abl protects immature neurons but not differentiated neurons. Cell death protein expression patterns in mouse forebrain are mostly similar to cultured neurons. DNA damage induces prominent p53 activation and apoptosis in cerebral cortex in vivo. Thus, DNA strand breaks in cortical neurons induce rapid p53-mediated apoptosis through actions of upstream ATM and c-Abl kinases and downstream mitochondrial death proteins. This molecular network operates through variations depending on neuron maturity. PMID:18820287

Black soybean seed coat polyphenols prevent AAPH-induced oxidative DNA-damage in HepG2 cells

PubMed Central

Yoshioka, Yasukiyo; Li, Xiu; Zhang, Tianshun; Mitani, Takakazu; Yasuda, Michiko; Nanba, Fumio; Toda, Toshiya; Yamashita, Yoko; Ashida, Hitoshi

2017-01-01

Black soybean seed coat extract (BE), which contains abundant polyphenols such as procyanidins, cyanidin 3-glucoside, (+)-catechin, and (−)­epicatechin, has been reported on health beneficial functions such as antioxidant activity, anti-inflammatory, anti-obesity, and anti-diabetic activities. In this study, we investigated that prevention of BE and its polyphenols on 2,2'-azobis(2-methylpropionamide) dihydrochloride (AAPH)-induced oxidative DNA damage, and found that these polyphenols inhibited AAPH-induced formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for oxidative DNA damage in HepG2 cells. Under the same conditions, these polyphenols also inhibited AAPH-induced accumulation of reactive oxygen species (ROS) in the cells. Inhibition of ROS accumulation was observed in both cytosol and nucleus. It was confirmed that these polyphenols inhibited formation of AAPH radical using oxygen radical absorbance capacity assay under the cell-free conditions. These results indicate that polyphenols in BE inhibit free radical-induced oxidative DNA damages by their potent antioxidant activity. Thus, BE is an effective food material for prevention of oxidative stress and oxidative DNA damages. PMID:28366989

Aeroallergens Induce Reactive Oxygen Species Production and DNA Damage and Dampen Antioxidant Responses in Bronchial Epithelial Cells.

PubMed

Chan, Tze Khee; Tan, W S Daniel; Peh, Hong Yong; Wong, W S Fred

2017-07-01

Exposure to environmental allergens is a major risk factor for asthma development. Allergens possess proteolytic activity that is capable of disrupting the airway epithelium. Although there is increasing evidence pointing to asthma as an epithelial disease, the underlying mechanism that drives asthma has not been fully elucidated. In this study, we investigated the direct DNA damage potential of aeroallergens on human bronchial epithelial cells and elucidated the mechanisms mediating the damage. Human bronchial epithelial cells, BEAS-2B, directly exposed to house dust mites (HDM) resulted in enhanced DNA damage, as measured by the CometChip and the staining of DNA double-strand break marker, γH2AX. HDM stimulated cellular reactive oxygen species production, increased mitochondrial oxidative stress, and promoted nitrosative stress. Notably, expression of nuclear factor erythroid 2-related factor 2-dependent antioxidant genes was reduced immediately after HDM exposure, suggesting that HDM altered antioxidant responses. HDM exposure also reduced cell proliferation and induced cell death. Importantly, HDM-induced DNA damage can be prevented by the antioxidants glutathione and catalase, suggesting that HDM-induced reactive oxygen and nitrogen species can be neutralized by antioxidants. Mechanistic studies revealed that HDM-induced cellular injury is NADPH oxidase (NOX)-dependent, and apocynin, a NOX inhibitor, protected cells from double-strand breaks induced by HDM. Our results show that direct exposure of bronchial epithelial cells to HDM leads to the production of reactive oxygen and nitrogen species that damage DNA and induce cytotoxicity. Antioxidants and NOX inhibitors can prevent HDM-induced DNA damage, revealing a novel role for antioxidants and NOX inhibitors in mitigating allergic airway disease. Copyright © 2017 by The American Association of Immunologists, Inc.

Effects of different levels of vitamin C on UV radiation-induced DNA damage

NASA Astrophysics Data System (ADS)

Zhou, Dianfeng; Heng, Hang; Ji, Kang; Ke, Weizhong

2005-05-01

The Raman spectra of DNA in different levels of vitamin C with 10- and 30-min ultraviolet (UV) radiations were reported. The intensity of UV radiation was 18.68 W/m2. The experimental results proved that vitamin C could alone prevent UV radiation from damaging DNA, but the effects depended on the concentration of vitamin C. When the concentration of vitamin C was about 0.08-0.4 mmol/L, vitamin C decreased UV radiation-induced DNA's damage. When the concentration of vitamin C exceeded 0.4 mmol/L, vitamin C accelerated DNA's damage instead. Maybe the reason is that when DNA in aqueous solution is radiated by UV, free radicals come into being, and vitamin C can scavenge free radicals, so vitamin C in lower concentration can protect DNA. The quantity of free radicals is finite, when vitamin C is superfluous, free radicals have been scavenged absolutely and vitamin C is residual. Vitamin C is a strong reductant. When the mixture of DNA and residual vitamin C is radiated by UV, vitamin C reacts with DNA. The more residual vitamin C and the longer time of UV radiation, the more DNA is damaged.

The production and repair of aflatoxin B sub 1 -induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leadon, S.A.

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of aflatoxin B{sub 1} (AFB{sub 1})-induced damage in the nontranscribed, heterochromatic alpha DNA of monkey cells and in the metallothionein genes of human cells. In confluent cells, AFB{sub 1} adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha DNA is severely deficient, however, removal of AFB{sub 1} adducts from alpha DNA is enhanced by small doses of UV. The repair deficiencies are not observed in actively growing cells. We havemore » also shown that there is preferential repair of AFB{sub 1} damage in active genes. AFB{sub 1} damage is efficiently repaired in the active human metallothionein (hMT) genes, but deficiently repaired in inactive hMT genes. 51 refs., 3 tabs.« less

DDB2 promotes chromatin decondensation at UV-induced DNA damage

PubMed Central

Lindh, Michael; Acs, Klara; Vrouwe, Mischa G.; Pines, Alex; van Attikum, Haico; Mullenders, Leon H.

2012-01-01

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains. PMID:22492724

Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio).

PubMed

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.

Buckwheat Honey Attenuates Carbon Tetrachloride-Induced Liver and DNA Damage in Mice

PubMed Central

Cheng, Ni; Wu, Liming; Zheng, Jianbin; Cao, Wei

2015-01-01

Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and p-coumaric acid were the main phenolic compounds present. Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity. PMID:26508989

Buckwheat Honey Attenuates Carbon Tetrachloride-Induced Liver and DNA Damage in Mice.

PubMed

Cheng, Ni; Wu, Liming; Zheng, Jianbin; Cao, Wei

2015-01-01

Buckwheat honey, which is widely consumed in China, has a characteristic dark color. The objective of this study was to investigate the protective effects of buckwheat honey on liver and DNA damage induced by carbon tetrachloride in mice. The results revealed that buckwheat honey had high total phenolic content, and rutin, hesperetin, and p-coumaric acid were the main phenolic compounds present. Buckwheat honey possesses super DPPH radical scavenging activity and strong ferric reducing antioxidant power. Administration of buckwheat honey for 10 weeks significantly inhibited serum lipoprotein oxidation and increased serum oxygen radical absorbance capacity. Moreover, buckwheat honey significantly inhibited aspartate aminotransferase and alanine aminotransferase activities, which are enhanced by carbon tetrachloride. Hepatic malondialdehyde decreased and hepatic antioxidant enzymes (superoxide dismutase and glutathione peroxidase) increased in the presence of buckwheat honey. In a comet assay, lymphocyte DNA damage induced by carbon tetrachloride was significantly inhibited by buckwheat honey. Therefore, buckwheat honey has a hepatoprotective effect and inhibits DNA damage, activities that are primarily attributable to its high antioxidant capacity.

The possible DNA damage induced by environmental organic compounds: The case of Nonylphenol.

PubMed

Noorimotlagh, Zahra; Mirzaee, Seyyed Abbas; Ahmadi, Mehdi; Jaafarzadeh, Neemat; Rahim, Fakher

2018-08-30

Human impact on the environment leads to the release of many pollutants that produce artificial compounds, which can have harmful effects on the body's endocrine system; these are known as endocrine disruptors (EDs). Nonylphenol (NP) is a chemical compound with a nonyl group that is attached to a phenol ring. NP-induced H 2 AX is a sensitive genotoxic biomarker for detecting possible DNA damage; it also causes male infertility and carcinogenesis. We attempt to comprehensively review all the available evidence about the different ways with descriptive mechanisms for explaining the possible DNA damage that is induced by NP. We systematically searched several databases, including PubMed, Scopus, Web of Science, and gray literature, such as Google Scholar by using medical subheading (MeSH) terms and various combinations of selected keywords from January 1970 to August 2017. The initial search identified 62,737 potentially eligible studies; of these studies, 33 were included according to the established inclusion criteria. Thirty-three selected studies, include the topics of animal model (n = 21), cell line (n = 6), human model (n = 4), microorganisms (n = 1), solid DNA (n = 1), infertility (n = 4), apoptosis (n = 6), and carcinogenesis (n = 3). This review highlighted the possible deleterious effects of NP on DNA damage through the ability to produce ROS/RNS. Finally, it is significant to observe caution at this stage with the continued use of environmental pollutants such as NP, which may induce DNA damage and apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

PubMed

Zhang, Guopei; Guan, Yangyang; Zhao, Yuejiao; van der Straaten, Tahar; Xiao, Sha; Xue, Ping; Zhu, Guolian; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

2017-02-01

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5 ERCC2 (AA) as compared to UV5 ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5 ERCC2 (AA) was significant decreased compared to UV5 ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5 ERCC2 (AA) alleviated the apoptosis compared to UV5 ERCC2 (CC) , meanwhile P53mRNA levels in UV ERCC2 (AA) was also lower when compared UV5 ERCC2 (CC) . It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism. Copyright © 2016 Elsevier Ireland Ltd. All

Mitochondria regulate DNA damage and genomic instability induced by high LET radiation

NASA Astrophysics Data System (ADS)

Zhang, Bo; Davidson, Mercy M.; Hei, Tom K.

2014-04-01

High linear energy transfer (LET) radiation including α particles and heavy ions is the major type of radiation found in space and is considered a potential health risk for astronauts. Even though the chance that these high LET particles traversing through the cytoplasm of cells is higher than that through the nuclei, the contribution of targeted cytoplasmic irradiation to the induction of genomic instability and other chromosomal damages induced by high LET radiation is not known. In the present study, we investigated whether mitochondria are the potential cytoplasmic target of high LET radiation in mediating cellular damage using a mitochondrial DNA (mtDNA) depleted (ρ0) human small airway epithelial (SAE) cell model and a precision charged particle microbeam with a beam width of merely one micron. Targeted cytoplasmic irradiation by high LET α particles induced DNA oxidative damage and double strand breaks in wild type ρ+ SAE cells. Furthermore, there was a significant increase in autophagy and micronuclei, which is an indication of genomic instability, together with the activation of nuclear factor kappa-B (NF-κB) and mitochondrial inducible nitric oxide synthase (iNOS) signaling pathways in ρ+ SAE cells. In contrast, ρ0 SAE cells exhibited a significantly lower response to these same endpoints examined after cytoplasmic irradiation with high LET α particles. The results indicate that mitochondria are essential in mediating cytoplasmic radiation induced genotoxic damage in mammalian cells. Furthermore, the findings may shed some light in the design of countermeasures for space radiation.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

Involvement of DNA polymerase beta in repairing oxidative damages induced by antitumor drug adriamycin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shukun; Wu Mei; Zhang Zunzhen, E-mail: zhangzunzhen@163.co

2010-08-01

Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol {beta} +/+) and homozygous pol {beta} null cell (pol {beta} -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here,more » cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol {beta} -/- cells were more sensitive to ADM compared with pol {beta} +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol {beta} -/- cells. ROS level in pol {beta} -/- cells increased along with decreased activity of SOD. These results demonstrated that pol {beta} deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol {beta} -/- cells to ADM. These findings suggested that pol {beta} is vital for repairing oxidative damage induced by ADM.« less

The effect of 2-[(aminopropyl)amino] ethanethiol on fission-neutron-induced DNA damage and repair.

PubMed Central

Grdina, D. J.; Sigdestad, C. P.; Dale, P. J.; Perrin, J. M.

1989-01-01

The effect(s) of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) on fission-neutron-induced DNA damage and repair in V79 Chinese hamster cells was determined by using a neutral filter elution procedure (pH 7.2). When required, WR1065, at a final working concentration of 4 mM, was added to the culture medium, either 30 min before and during irradiation with fission spectrum neutrons (beam energy of 0.85 MeV) from the JANUS research reactor, or for selected intervals of time following exposure. The frequency of neutron-induced DNA strand breaks as measured by neutral elution as a function of dose equalled that observed for 60Co gamma-ray-induced damage (relative biological effectiveness of one). In contrast to the protective effect exhibited by WR1065 in reducing 60Co-induced DNA damage, WR1065 was ineffective in reducing or protecting against induction of DNA strand breaks by JANUS neutrons. The kinetics of DNA double-strand rejoining were measured following neutron irradiation. In the absence of WR1065, considerable DNA degradation by cellular enzymes was observed. This process was inhibited when WR1065 was present. These results indicate that, under the conditions used, the quality (i.e. nature), rather than quantity, of DNA lesions (measured by neutral elution) formed by neutrons was significantly different from that formed by gamma-rays. PMID:2667608

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

PubMed Central

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-01-01

ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

PubMed

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-02-16

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.

Protective roles of single-wall carbon nanotubes in ultrasonication-induced DNA base damage.

PubMed

Petersen, Elijah J; Tu, Xiaomin; Dizdaroglu, Miral; Zheng, Ming; Nelson, Bryant C

2013-01-28

The overall level of ultrasonication-induced DNA damage is reduced in the presence of single-wall carbon nanotubes (SWCNTs), particularly for DNA lesions formed by one-electron reduction of intermediate radicals. The protective role of SWCNTs observed in this work suggests a contrary view to the general idea that carbon nanotubes have damaging effects on biomolecules. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

PubMed Central

Calvo, Jennifer A.; Moroski-Erkul, Catherine A.; Lake, Annabelle; Eichinger, Lindsey W.; Shah, Dharini; Jhun, Iny; Limsirichai, Prajit; Bronson, Roderick T.; Christiani, David C.; Meira, Lisiane B.; Samson, Leona D.

2013-01-01

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag −/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage. PMID:23593019

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS

EPA Science Inventory

Rapid, sensitive and simple assays for radiation- and chemically-induced DNA damage can be of significant benefit to a number of fields including radiation biology, clinical research, and environmental monitoring. Although temperature-induced DNA strand separation has been use...

Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

NASA Technical Reports Server (NTRS)

Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

2015-01-01

Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

NASA Technical Reports Server (NTRS)

Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

2015-01-01

Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on the cellular response to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

The DNA damage response during mitosis.

PubMed

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.

Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage – When is a DSB not a DSB?☆

PubMed Central

Reynolds, Pamela; Botchway, Stanley W.; Parker, Anthony W.; O’Neill, Peter

2013-01-01

The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair. PMID:23688615

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

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

PubMed

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.

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

PubMed Central

Koizume, S; Inoue, H; Kamiya, H; Ohtsuka, E

1998-01-01

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G. PMID:9671825

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage.

PubMed

Xavier, Miguel J; Nixon, Brett; Roman, Shaun D; Aitken, Robert John

2018-01-01

Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.

XRCC1 Arg399Gln was associated with repair capacity for DNA damage induced by occupational chromium exposure

PubMed Central

2012-01-01

Background Occupational chromium exposure may induce DNA damage and lead to lung cancer and other work-related diseases. DNA repair gene polymorphisms, which may alter the efficiency of DNA repair, thus may contribute to genetic susceptibility of DNA damage. The aim of this study was to test the hypothesis that the genetic variations of 9 major DNA repair genes could modulate the hexavalent chromium (Cr (VI))-induced DNA damage. Findings The median (P25-P75) of Olive tail moment was 0.93 (0.58–1.79) for individuals carrying GG genotype of XRCC1 Arg399Gln (G/A), 0.73 (0.46–1.35) for GA heterozygote and 0.50 (0.43–0.93) for AA genotype. Significant difference was found among the subjects with three different genotypes (P = 0.048) after adjusting the confounding factors. The median of Olive tail moment of the subjects carrying A allele (the genotypes of AA and GA) was 0.66 (0.44–1.31), which was significantly lower than that of subjects with GG genotype (P = 0.043). The A allele conferred a significantly reduced risk of DNA damage with the OR of 0.39 (95% CI: 0.15–0.99, P = 0.048). No significant association was found between the XRCC1Arg194Trp, ERCC1 C8092A, ERCC5 His1104Asp, ERCC6 Gly399Asp, GSTP1 Ile105Val, OGG1 Ser326Cys, XPC Lys939Gln, XPD Lys751Gln and DNA damage. Conclusion The polymorphism of Arg399Gln in XRCC1 was associated with the Cr (VI)- induced DNA damage. XRCC1 Arg399Gln may serve as a genetic biomarker of susceptibility for Cr (VI)- induced DNA damage. PMID:22642904

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

Protection of cadmium chloride induced DNA damage by Lamiaceae plants

PubMed Central

Thirugnanasampandan, Ramaraj; Jayakumar, Rajarajeswaran

2011-01-01

Objective To analyze the total phenolic content, DNA protecting and radical scavenging activity of ethanolic leaf extracts of three Lamiaceae plants, i.e. Anisomelos malabarica (A. malabarica), Leucas aspera (L. aspera) and Ocimum basilicum (O. basilicum). Methods The total polyphenols and flavonoids were analyzed in the ethanolic leaf extracts of the lamiaceae plants. To determine the DNA protecting activity, various concentrations of the plant extracts were prepared and treated on cultured HepG2 human lung cancer cells. The pretreated cells were exposed to H2O2 to induce DNA damage through oxidative stress. Comet assay was done and the tail length of individual comets was measured. Nitric oxide and superoxide anion scavenging activities of lamiaceae plants were analyzed. Results Among the three plant extracts, the highest amount of total phenolic content was found in O. basilicum (189.33 mg/g), whereas A. malabarica showed high levels of flavonoids (10.66 mg/g). O. basilicum also showed high levels of DNA protecting (85%) and radical scavenging activity. Conclusions The results of this study shows that bioactive phenols present in lamiaceae plants may prevent carcinogenesis through scavenging free radicals and inhibiting DNA damage. PMID:23569799

Optical control of filamentation-induced damage to DNA by intense, ultrashort, near-infrared laser pulses

PubMed Central

Dharmadhikari, J. A.; Dharmadhikari, A. K.; Kasuba, K. C.; Bharambe, H.; D’Souza, J. S.; Rathod, K. D.; Mathur, D.

2016-01-01

We report on damage to DNA in an aqueous medium induced by ultrashort pulses of intense laser light of 800 nm wavelength. Focusing of such pulses, using lenses of various focal lengths, induces plasma formation within the aqueous medium. Such plasma can have a spatial extent that is far in excess of the Rayleigh range. In the case of water, the resulting ionization and dissociation gives rise to in situ generation of low-energy electrons and OH-radicals. Interactions of these with plasmid DNA produce nicks in the DNA backbone: single strand breaks (SSBs) are induced as are, at higher laser intensities, double strand breaks (DSBs). Under physiological conditions, the latter are not readily amenable to repair. Systematic quantification of SSBs and DSBs at different values of incident laser energy and under different external focusing conditions reveals that damage occurs in two distinct regimes. Numerical aperture is the experimental handle that delineates the two regimes, permitting simple optical control over the extent of DNA damage. PMID:27279565

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

PubMed Central

Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

2017-01-01

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

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

PubMed

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.

Oleuropein Prevents Azoxymethane-Induced Colon Crypt Dysplasia and Leukocytes DNA Damage in A/J Mice.

PubMed

Sepporta, Maria Vittoria; Fuccelli, Raffaela; Rosignoli, Patrizia; Ricci, Giovanni; Servili, Maurizio; Fabiani, Roberto

2016-08-19

Previous studies have shown that the precursor of olive oil secoiridoids, Oleuropein (OL) has several in vitro chemopreventive properties. OL inhibits proliferation and induces apoptosis in breast, thyroid, prostate, and colorectal cancer (CRC) cells. Much less is known about the effects of OL on animal models of carcinogenesis. In this study, we investigated the ability of OL to prevent the azoxymethane (AOM)-induced colon cancer upset and DNA damage in mice. Animals, fed with a basal diet either enriched or not with OL (125 mg/kg), were injected with AOM (10 mg/kg, once a week for 6 weeks) and sacrificed after either 7 weeks for histological analysis of colon crypt dysplasia and evaluation of DNA damage in leukocytes or 17 weeks for counting the macroscopically observable colon tumors. An OL-enriched diet prevented the AOM-induced preneoplastic lesions in different colon segments, reducing the severity of crypt dysplasia and DNA damage in peripheral leukocytes. In addition, OL significantly reduced the AOM-induced tumor incidence from 57% to 14% (P < .05, chi-square test) in the medial colon segment. This study shows that OL is able to prevent CRC and DNA damage in mice treated with the carcinogen AOM. These results stimulate further human cancer prevention studies with OL-enriched food supplements that are actually available on the market.

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

PubMed

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.

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

PubMed Central

Donaldson, Janet R; Courcelle, Charmain T; Courcelle, Justin

2004-01-01

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo. PMID:15126385

Delayed repair of radiation induced clustered DNA damage: Friend or foe?

PubMed Central

Eccles, Laura J.; O’Neill, Peter; Lomax, Martine E.

2011-01-01

A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a “friend”, leading to cell killing in tumour cells or as a “foe”, resulting in the formation of mutations and genetic instability in normal tissue. PMID:21130102

Sleep loss and acute drug abuse can induce DNA damage in multiple organs of mice.

PubMed

Alvarenga, T A; Ribeiro, D A; Araujo, P; Hirotsu, C; Mazaro-Costa, R; Costa, J L; Battisti, M C; Tufik, S; Andersen, M L

2011-09-01

The purpose of the present study was to characterize the genetic damage induced by paradoxical sleep deprivation (PSD) in combination with cocaine or ecstasy (3,4-methylenedioxymethamphetamine; MDMA) in multiple organs of male mice using the single cell gel (comet) assay. C57BL/6J mice were submitted to PSD by the platform technique for 72 hours, followed by drug administration and evaluation of DNA damage in peripheral blood, liver and brain tissues. Cocaine was able to induce genetic damage in the blood, brain and liver cells of sleep-deprived mice at the majority of the doses evaluated. Ecstasy also induced increased DNA migration in peripheral blood cells for all concentrations tested. Analysis of damaged cells by the tail moment data suggests that ecstasy is a genotoxic chemical at the highest concentrations tested, inducing damage in liver or brain cells after sleep deprivation in mice. Taken together, our results suggest that cocaine and ecstasy/MDMA act as potent genotoxins in multiple organs of mice when associated with sleep loss.

Mechanism of UVA-dependent DNA damage induced by an antitumor drug dacarbazine in relation to its photogenotoxicity.

PubMed

Iwamoto, Takuya; Hiraku, Yusuke; Okuda, Masahiro; Kawanishi, Shosuke

2008-03-01

It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC. We examined DNA damage induced by UVA-irradiated DTIC using 32P-5'-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC. UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5'-GGT-3' sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC. Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

Effect of complex polyphenols and tannins from red wine (WCPT) on chemically induced oxidative DNA damage in the rat.

PubMed

Casalini, C; Lodovici, M; Briani, C; Paganelli, G; Remy, S; Cheynier, V; Dolara, P

1999-08-01

Flavonoids are polyphenolic antioxidants occurring in vegetables and fruits as well as beverages such as tea and wine which have been thought to influence oxidative damage. We wanted to verify whether a complex mixture of wine tannins (wine complex polyphenols and tannins, WCPT) prevent chemically-induced oxidative DNA damage in vivo. Oxidative DNA damage was evaluated by measuring the ratio of 8-hydroxy-2'-deoxyguanosine (80HdG)/ 2-deoxyguanosine (2dG) x 10(-6) in hydrolyzed DNA using HPLC coupled with electrochemical and UV detectors. We treated rats with WCPT (57 mg/kg p.o.) for 14 d, a dose 10-fold higher than what a moderate wine drinker would be exposed to. WCPT administration significantly reduced the ratio of 80HdG/2dG x 10(-6) in liver DNA obtained from rats treated with 2-nitropropane (2NP) relative to controls administered 2NP only (33. 3 +/- 2.5 vs. 44.9 +/- 3.2 x 10(-6) 2dG; micro +/- SE; p<0.05). On the contrary, pretreatment with WCPT for 10 d did not protect the colon mucosa from oxidative DNA damage induced by 1, 2-dimethylhydrazine (DMH). 2NP and DMH are hepatic and colon carcinogens, respectively, capable of inducing oxidative DNA damage. WCPT have protective action against some types of chemically-induced oxidative DNA damage in vivo.

Study on DNA Damage Induced by Neon Beam Irradiation in Saccharomyces Cerevisiae

NASA Astrophysics Data System (ADS)

Lu, Dong; Li, Wenjian; Wu, Xin; Wang, Jufang; Ma, Shuang; Liu, Qingfang; He, Jinyu; Jing, Xigang; Ding, Nan; Dai, Zhongying; Zhou, Jianping

2010-12-01

Yeast strain Saccharomyces cerevisiae was irradiated with different doses of 85 MeV/u 20Ne10+ to investigate DNA damage induced by heavy ion beam in eukaryotic microorganism. The survival rate, DNA double strand breaks (DSBs) and DNA polymorphic were tested after irradiation. The results showed that there were substantial differences in DNA between the control and irradiated samples. At the dose of 40 Gy, the yeast cell survival rate approached 50%, DNA double-strand breaks were barely detectable, and significant DNA polymorphism was observed. The alcohol dehydrogenase II gene was amplified and sequenced. It was observed that base changes in the mutant were mainly transversions of T→G and T→C. It can be concluded that heavy ion beam irradiation can lead to change in single gene and may be an effective way to induce mutation.

4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

PubMed

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.

Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

NASA Astrophysics Data System (ADS)

Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

2016-07-01

Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

Molecular and sensory mechanisms to mitigate sunlight-induced DNA damage in treefrog tadpoles.

PubMed

Schuch, André P; Lipinski, Victor M; Santos, Mauricio B; Santos, Caroline P; Jardim, Sinara S; Cechin, Sonia Z; Loreto, Elgion L S

2015-10-01

The increased incidence of solar ultraviolet B (UVB) radiation has been proposed as an environmental stressor, which may help to explain the enigmatic decline of amphibian populations worldwide. Despite growing knowledge regarding the UV-induced biological effects in several amphibian models, little is known about the efficacy of DNA repair pathways. In addition, little attention has been given to the interplay between these molecular mechanisms with other physiological strategies that avoid the damage induced by sunlight. Here, DNA lesions induced by environmental doses of solar UVB and UVA radiation were detected in genomic DNA samples of treefrog tadpoles (Hypsiboas pulchellus) and their DNA repair activity was evaluated. These data were complemented by monitoring the induction of apoptosis in blood cells and tadpole survival. Furthermore, the tadpoles' ability to perceive and escape from UV wavelengths was evaluated as an additional strategy of photoprotection. The results show that tadpoles are very sensitive to UVB light, which could be explained by the slow DNA repair rates for both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6,4) pyrimidone photoproducts (6,4PPs). However, they were resistant to UVA, probably as a result of the activation of photolyases during UVA irradiation. Surprisingly, a sensory mechanism that triggers their escape from UVB and UVA light avoids the generation of DNA damage and helps to maintain the genomic integrity. This work demonstrates the genotoxic impact of both UVB and UVA radiation on tadpoles and emphasizes the importance of the interplay between molecular and sensory mechanisms to minimize the damage caused by sunlight. © 2015. Published by The Company of Biologists Ltd.

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

PubMed

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.

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

PubMed Central

Jagot-Lacoussiere, Léonard; Faye, Audrey; Bruzzoni-Giovanelli, Heriberto; Villoutreix, Bruno O; Rain, Jean-Christophe; Poyet, Jean-Luc

2015-01-01

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response. PMID:25695197

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

PubMed

Pinto, A Viviana; Deodato, Elder L; Cardoso, Janine S; Oliveira, Eliza F; Machado, Sérgio L; Toma, Helena K; Leitão, Alvaro C; de Pádula, Marcelo

2010-06-01

Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine. Copyright 2010 Elsevier B.V. All rights reserved.

Inactivation of NADPH oxidases NOX4 and NOX5 protects human primary fibroblasts from ionizing radiation-induced DNA damage.

PubMed

Weyemi, Urbain; Redon, Christophe E; Aziz, Towqir; Choudhuri, Rohini; Maeda, Daisuke; Parekh, Palak R; Bonner, Michael Y; Arbiser, Jack L; Bonner, William M

2015-03-01

Human exposure to ionizing radiation from medical procedures has increased sharply in the last three decades. Recent epidemiological studies suggest a direct relationship between exposure to ionizing radiation and health problems, including cancer incidence. Therefore, minimizing the impact of radiation exposure in patients has become a priority in the development of future clinical practices. Crucial players in radiation-induced DNA damage include reactive oxygen species (ROS), but the sources of these have remained elusive. To the best of our knowledge, we show here for the first time that two members of the ROS-generating NADPH oxidase family (NOXs), NOX4 and NOX5, are involved in radiation-induced DNA damage. Depleting these two NOXs in human primary fibroblasts resulted in reduced levels of DNA damage as measured by levels of radiation-induced foci, a marker of DNA double-strand breaks (DSBs) and the comet assay coupled with increased cell survival. NOX involvement was substantiated with fulvene-5, a NOXs-specific inhibitor. Moreover, fulvene-5 mitigated radiation-induced DNA damage in human peripheral blood mononuclear cells ex vivo. Our results provide evidence that the inactivation of NOXs protects cells from radiation-induced DNA damage and cell death. These findings suggest that NOXs inhibition may be considered as a future pharmacological target to help minimize the negative effects of radiation exposure for millions of patients each year.

Inactivation of NADPH Oxidases NOX4 and NOX5 Protects Human Primary Fibroblasts from Ionizing Radiation-Induced DNA Damage

PubMed Central

Weyemi, Urbain; Redon, Christophe E.; Aziz, Towqir; Choudhuri, Rohini; Maeda, Daisuke; Parekh, Palak R.; Bonner, Michael Y.; Arbiser, Jack L.; Bonner, William M.

2015-01-01

Human exposure to ionizing radiation from medical procedures has increased sharply in the last three decades. Recent epidemiological studies suggest a direct relationship between exposure to ionizing radiation and health problems, including cancer incidence. Therefore, minimizing the impact of radiation exposure in patients has become a priority in the development of future clinical practices. Crucial players in radiation-induced DNA damage include reactive oxygen species (ROS), but the sources of these have remained elusive. To the best of our knowledge, we show here for the first time that two members of the ROS-generating NADPH oxidase family (NOXs), NOX4 and NOX5, are involved in radiation-induced DNA damage. Depleting these two NOXs in human primary fibroblasts resulted in reduced levels of DNA damage as measured by levels of radiation-induced foci, a marker of DNA double-strand breaks (DSBs) and the comet assay coupled with increased cell survival. NOX involvement was substantiated with fulvene-5, a NOXs-specific inhibitor. Moreover, fulvene-5 mitigated radiation-induced DNA damage in human peripheral blood mononuclear cells ex vivo. Our results provide evidence that the inactivation of NOXs protects cells from radiation-induced DNA damage and cell death. These findings suggest that NOXs inhibition may be considered as a future pharmacological target to help minimize the negative effects of radiation exposure for millions of patients each year. PMID:25706776

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

PubMed

Heenen, M; Giacomoni, P U; Golstein, P

2001-10-01

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an

Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells.

PubMed

Yao, Ke; Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

2008-05-19

The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (gammaH2AX) foci formation assay. After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by gammaH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 muT electromagnetic noise could block RF-induced ROS increase and DNA damage. DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage.

NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi

2015-06-05

Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated inmore » the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.« less

DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

PubMed

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza

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 peroxidasemore » (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

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

PubMed Central

Ferreira, S.G.; Peliciari-Garcia, R.A.; Takahashi-Hyodo, S.A.; Rodrigues, A.C.; Amaral, F.G.; Berra, C.M.; Bordin, S.; Curi, R.; Cipolla-Neto, J.

2013-01-01

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy. PMID:23471360

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

PubMed Central

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C.M.; Jansen, Jacob G.; Hogenbirk, Marc A.; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. PMID:25505145

DNA and chromosome damage induced by bleomycin in mammalian cells: An update.

PubMed

Bolzán, Alejandro D; Bianchi, Martha S

Bleomycin (BLM) is an antibiotic isolated from Streptomyces verticillus. It has radiomimetic actions on DNA thus it has been widely used in clinical chemotherapy for the treatment of different types of cancer, including head and neck tumors, lymphomas, squamous-cell carcinomas and germ-cell tumors. Because of this, the study of BLM genotoxicity is of practical interest. This antibiotic is an S-independent clastogen and an agent that generates free radicals and induces single- and double-strand breaks in DNA. In the present review, we will summarize our current knowledge concerning the DNA and chromosome damage induced by BLM in mammalian cells, with emphasis on new developments published since 1991. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

PubMed

Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

2016-03-01

Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

PubMed

Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

2015-09-30

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells

PubMed Central

Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N.; Guo, Lei; Mei, Nan

2015-01-01

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

Investigations of antioxidant-mediated protection and mitigation of radiation-induced DNA damage and lipid peroxidation in murine skin.

PubMed

Jelveh, Salomeh; Kaspler, Pavel; Bhogal, Nirmal; Mahmood, Javed; Lindsay, Patricia E; Okunieff, Paul; Doctrow, Susan R; Bristow, Robert G; Hill, Richard P

2013-08-01

Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions. DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay. None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation. Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm

Significant Suppression of CT Radiation-Induced DNA Damage in Normal Human Cells by the PrC-210 Radioprotector.

PubMed

Jermusek, Frank; Benedict, Chelsea; Dreischmeier, Emma; Brand, Michael; Uder, Michael; Jeffery, Justin J; Ranallo, Frank N; Fahl, William E

2018-05-21

While computed tomography (CT) is now commonly used and considered to be clinically valuable, significant DNA double-strand breaks (γ-H2AX foci) in white blood cells from adult and pediatric CT patients have been frequently reported. In this study to determine whether γ-H2AX foci and X-ray-induced naked DNA damage are suppressed by administration of the PrC-210 radioprotector, human blood samples were irradiated in a CT scanner at 50-150 mGy with or without PrC-210, and γ-H2AX foci were scored. X-ray-induced naked DNA damage was also studied, and the DNA protective efficacy of PrC-210 was compared against 12 other common "antioxidants." PrC-210 reduced CT radiation-induced γ-H2AX foci in white blood cells to near background ( P < 0.0001) at radiation doses of 50-150 mGy. PrC-210 was most effective among the 13 "antioxidants" in reducing naked DNA X-ray damage, and its addition at 30 s before an • OH pulse reduced to background the • OH insult that otherwise induced >95% DNA damage. A systemic PrC-210 dose known to confer 100% survival in irradiated mice had no discernible effect on micro-CT image signal-to-noise ratio and CT image integrity. PrC-210 suppressed DNA damage to background or near background in each of these assay systems, thus supporting its development as a radioprotector for humans in multiple radiation exposure settings.

Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

PubMed Central

Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

2008-01-01

Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

PubMed

Ververis, Katherine; Rodd, Annabelle L; Tang, Michelle M; El-Osta, Assam; Karagiannis, Tom C

2011-12-01

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

PubMed

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Evaluation of Cassia tora Linn. against Oxidative Stress-induced DNA and Cell Membrane Damage

PubMed Central

Kumar, R Sunil; Narasingappa, Ramesh Balenahalli; Joshi, Chandrashekar G; Girish, Talakatta K; Prasada Rao, Ummiti JS; Danagoudar, Ananda

2017-01-01

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. PMID:28584491

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

PubMed

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C M; Jansen, Jacob G; Hogenbirk, Marc A; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis

PubMed Central

Yusof, Yasmin Anum Mohd; Md. Saad, Suhana; Makpol, Suzana; Shamaan, Nor Aripin; Ngah, Wan Zurinah Wan

2010-01-01

OBJECTIVES: The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2. INTRODUCTION: The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail. METHODS: HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis. RESULTS: Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2. CONCLUSIONS: Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis. PMID:21340229

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

PubMed

Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

2017-11-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes

PubMed Central

Tran, Thai Q.; Ishak Gabra, Mari B.; Lowman, Xazmin H.; Yang, Ying; Reid, Michael A.; Pan, Min; O’Connor, Timothy R.

2017-01-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. PMID:29107960

Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode.

PubMed

Qiu, Yanyan; Qu, Xiangjin; Dong, Jing; Ai, Shiyun; Han, Ruixia

2011-06-15

A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide (AA) and its metabolite was presented in this work. The graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was prepared, and then horseradish peroxidase (HRP) and natural double-stranded DNA were alternately assembled on the modified electrode by the layer-by-layer method. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)(n) film were characterized by electrochemical impedance spectroscopy. With the guanine signal in DNA as an indicator, the damage of DNA was detected by differential pulse voltammetry after PGE/graphene-ionic liquid-Nafion/(HRP/DNA)(n) was incubated in AA solution or AA+H(2)O(2) solution at 37°C. This method provides a new model to mimic and directly detect DNA damage induced by chemical pollutants and their metabolites in vitro. The results indicated that, in the presence of H(2)O(2), HRP was activated and catalyzed the transformation of AA to glycidamide, which could form DNA adducts and induce more serious damage of DNA than AA. In order to further verify these results, UV-vis spectrophotometry was also used to investigate DNA damage induced by AA and its metabolites in solution and the similar results were obtained. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA Damage Induced Neuronal Death

DTIC Science & Technology

1999-10-01

heterozygous for the DNA repair genes Os-methylguanine methyltransferase (Mgmt), 3-methyladenine DNA glycosylase (Aag) , and xeroderma pigmentosum ...mice by human 06-alkylguanine-DNA alkyltransferase. Science 1993; 259: 219-222. 4. Enokido Y, Inamura N, Araki T, et al: Loss of the xeroderma ... pigmentosum group A gene (XPA) enhances apoptosis of cultured cerebellar neurons induced by UV but not by low-K+ medium. J Neurochem 199; 69: 246-251. 5

DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay.

PubMed

Topinka, Jan; Rossner, Pavel; Milcova, Alena; Schmuczerova, Jana; Svecova, Vlasta; Sram, Radim J

2011-05-10

The genotoxic activities of complex mixtures of organic extracts from the urban air particles collected in various localities of the Czech Republic, which differed in the extent and sources of air pollution, were compared. For this purpose, PM2.5 particles were collected by high volume samplers in the most polluted area of the Czech Republic--Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting a low level of air pollution--Trebon--a small town in the non-industrial region of Southern Bohemia. To prepare extractable organic matter (EOM), PM2.5 particles were extracted by dichloromethane and c-PAHs contents in the EOMs were determined. As markers of genotoxic potential, DNA adduct levels and oxidative DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG, levels) induced by EOMs in an acellular assay of calf thymus DNA coupled with ³²P-postlabeling (DNA adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct levels (17.20 adducts/10⁸ nucleotides/m³ vs. 8.49 adducts/10⁸ nucleotides/m³) were induced by EOM from Ostrava-Bartovice (immediate proximity of heavy industry) compared with that from Ostrava-Poruba (mostly traffic emissions). Oxidative DNA damage induced by EOM from Ostrava-Bartovice was more than fourfold higher than damage induced by EOM from Trebon (8-oxodG/10⁸ dG/m³: 0.131 vs. 0.030 for Ostrava-Bartovice vs. Trebon, respectively). Since PM2.5 particles collected in various localities differ with respect to their c-PAHs content, and c-PAHs significantly contribute to genotoxicity (DNA adduct levels), we suggest that monitoring of PM2.5 levels is not a sufficient basis to assess genotoxicity of respirable aerosols. It seems likely that the industrial emissions prevailing in Ostrava-Bartovice represent a substantially higher genotoxic risk than mostly traffic-related emissions in Ostrava-Poruba. B[a]P and c-PAH contents in EOMs are the most

Gene 33/Mig6 inhibits hexavalent chromium-induced DNA damage and cell transformation in human lung epithelial cells

PubMed Central

Park, Soyoung; Li, Cen; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

2016-01-01

Hexavalent Chromium [Cr(VI)] compounds are human lung carcinogens and environmental/occupational hazards. The molecular mechanisms of Cr(VI) carcinogenesis appear to be complex and are poorly defined. In this study, we investigated the potential role of Gene 33 (ERRFI1, Mig6), a multifunctional adaptor protein, in Cr(VI)-mediated lung carcinogenesis. We show that the level of Gene 33 protein is suppressed by both acute and chronic Cr(VI) treatments in a dose- and time-dependent fashion in BEAS-2B lung epithelial cells. The inhibition also occurs in A549 lung bronchial carcinoma cells. Cr(VI) suppresses Gene 33 expression mainly through post-transcriptional mechanisms, although the mRNA level of gene 33 also tends to be lower upon Cr(VI) treatments. Cr(VI)-induced DNA damage appears primarily in the S phases of the cell cycle despite the high basal DNA damage signals at the G2M phase. Knockdown of Gene 33 with siRNA significantly elevates Cr(VI)-induced DNA damage in both BEAS-2B and A549 cells. Depletion of Gene 33 also promotes Cr(VI)-induced micronucleus (MN) formation and cell transformation in BEAS-2B cells. Our results reveal a novel function of Gene 33 in Cr(VI)-induced DNA damage and lung epithelial cell transformation. We propose that in addition to its role in the canonical EGFR signaling pathway and other signaling pathways, Gene 33 may also inhibit Cr(VI)-induced lung carcinogenesis by reducing DNA damage triggered by Cr(VI). PMID:26760771

[Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

PubMed

Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

2014-03-01

The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P < 0.05), and the correlation between Olive TM and reactive oxygen species was better than other parameters (r = 0.77, P < 0.05). This study indicates that FPG-comet assay

Autoxidation and toxicant-induced oxidation of lipid and DNA in monkey liver: reduction of molecular damage by melatonin.

PubMed

Cabrer, J; Burkhardt, S; Tan, D X; Manchester, L C; Karbownik, M; Reiter, R J

2001-11-01

Melatonin, the main secretory product of the pineal gland, is a free radical scavenger and antioxidant which protects against oxidative damage due to a variety of toxicants. However, there is little information regarding melatonin's antioxidative capacity in tissues of primates. In this study we examined the protective effects of melatonin in monkey liver homogenates against lipid damage that occurred as a result of autoxidation or that induced by exogenous addition of H202 and ferrous iron (Fe2+). Additionally, we tested melatonin's protective effect against oxidative damage to DNA induced by chromium(III) (CrIII) plus H202. The levels of malondialdehyde and 4-hydroxyalkenals were assayed as an index of lipid peroxidation, and the concentrations of 8-hydroxydeoxyguanosine (8-OHdG) as an endpoint of oxidative DNA damage. The increases in malondialdehyde+4-hydroxyalkenals concentrations as a consequence of autoxidation or after the addition of H202 plus Fe2+ to the homogenates were time-dependent. The accumulation of these damaged products due to either auto-oxidative processes or induced by H202 and Fe2+ were significantly reduced by melatonin in a concentration-dependent-manner. The levels of 8-OHdG were elevated in purified monkey liver DNA incubated with a combination of CrCl3 plus H2O2. This rise in oxidatively damaged DNA was prevented by 10 microM concentration of melatonin. Also, melatonin reduced the damage to DNA that was caused by auto-oxidative processes. These findings in monkey liver tissue document the ability of melatonin to protect against oxidative damage to both lipid and DNA in primate tissue, as observed previously in rodent tissue. The findings provide support for the use of melatonin as suitable agent to reduce damage inflicted by free radical species in primates.

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

DNA Damage in Bone Marrow Cells Induced by Femtosecond and Nanosecond Ultraviolet Laser Pulses.

PubMed

Morkunas, Vaidotas; Gabryte, Egle; Vengris, Mikas; Danielius, Romualdas; Danieliene, Egle; Ruksenas, Osvaldas

2015-12-01

The purpose of this study was to investigate the possible genotoxic impact of new generation 205 nm femtosecond solid-state laser irradiation on the DNA of murine bone marrow cells in vitro, and to compare the DNA damage caused by both femtosecond and nanosecond UV laser pulses. Recent experiments of corneal stromal ablation in vitro and in vivo applying femtosecond UV pulses showed results comparable with or superior to those obtained using nanosecond UV lasers. However, the possible genotoxic effect of ultrashort laser pulses was not investigated. Mouse bone marrow cells were exposed to different doses of 205 nm femtosecond, 213 and 266 nm nanosecond lasers, and 254 nm UV lamp irradiation. The comet assay was used for the evaluation of DNA damage. All types of irradiation demonstrated intensity-dependent genotoxic impact. The DNA damage induced depended mainly upon wavelength rather than on other parameters such as pulse duration, repetition rate, or beam delivery to a target. Both 205 nm femtosecond and clinically applied 213 nm nanosecond lasers' pulses induced a comparable amount of DNA breakage in cells exposed to the same irradiation dose. To further evaluate the suitability of femtosecond UV laser sources for microsurgery, a separate investigation of the genotoxic and mutagenic effects on corneal cells in vitro and, particularly, in vivo is needed.

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

PubMed

El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

2018-05-01

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xuejiao; Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000; Zhang, Zhan

Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose-more » and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by

Mechanisms of neurotoxicity induced in the developing brain of mice and rats by DNA-damaging chemicals.

PubMed

Doi, Kunio

2011-01-01

It is not widely known how the developing brain responds to extrinsic damage, although the developing brain is considered to be sensitive to diverse environmental factors including DNA-damaging agents. This paper reviews the mechanisms of neurotoxicity induced in the developing brain of mice and rats by six chemicals (ethylnitrosourea, hydroxyurea, 5-azacytidine, cytosine arabinoside, 6-mercaptopurine and etoposide), which cause DNA damage in different ways, especially from the viewpoints of apoptosis and cell cycle arrest in neural progenitor cells. In addition, this paper also reviews the repair process following damage in the developing brain.

Study of terahertz-radiation-induced DNA damage in human blood leukocytes

NASA Astrophysics Data System (ADS)

Angeluts, A. A.; Gapeyev, A. B.; Esaulkov, M. N.; Kosareva, O. G.; Matyunin, S. N.; Nazarov, M. M.; Pashovkin, T. N.; Solyankin, P. M.; Cherkasova, O. P.; Shkurinov, A. P.

2014-03-01

We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 - 200 μW cm-2 within the frequency range of 0.1 - 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes.

Catch the live show: Visualizing damaged DNA in vivo.

PubMed

Oshidari, Roxanne; Mekhail, Karim

2018-06-01

The health of an organism is intimately linked to its ability to repair damaged DNA. Importantly, DNA repair processes are highly dynamic. This highlights the necessity of characterizing DNA repair in live cells. Advanced genome editing and imaging approaches allow us to visualize damaged DNA and its associated factors in real time. Here, we summarize both established and recent methods that are used to induce DNA damage and visualize damaged DNA and its repair in live cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

PubMed

You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

2014-03-01

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

PubMed Central

Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

2012-01-01

Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage.

PubMed

Marcelain, Katherine; De La Torre, Consuelo; González, Patricio; Pincheira, Juana

2005-01-01

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

PubMed Central

Chen, Pin-I; Cao, Aiqin; Miyagawa, Kazuya; Tojais, Nancy F.; Hennigs, Jan K.; Li, Caiyun G.; Sweeney, Nathaly M.; Inglis, Audrey S.; Wang, Lingli; Li, Dan; Ye, Matthew; Feldman, Brian J.

2017-01-01

Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress. PMID:28138562

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

PubMed

Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

2014-06-01

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry ofmore » γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.« less

[Blocking 1800 MHz mobile phone radiation-induced reactive oxygen species production and DNA damage in lens epithelial cells by noise magnetic fields].

PubMed

Wu, Wei; Yao, Ke; Wang, Kai-jun; Lu, De-qiang; He, Ji-liang; Xu, Li-hong; Sun, Wen-jun

2008-01-01

To investigate whether the exposure to the electromagnetic noise can block reactive oxygen species (ROS) production and DNA damage of lens epithelial cells induced by 1800 MHz mobile phone radiation. The DCFH-DA method and comet assay were used respectively to detect the intracellular ROS and DNA damage of cultured human lens epithelial cells induced by 4 W/kg 1800 MHz mobile phone radiation or/and 2 muT electromagnetic noise for 24 h intermittently. 1800 MHz mobile phone radiation at 4 W/kg for 24 h increased intracellular ROS and DNA damage significantly (P<0.05). However, the ROS level and DNA damage of mobile phone radiation plus noise group were not significant enhanced (P>0.05) as compared to sham exposure group. Electromagnetic noise can block intracellular ROS production and DNA damage of human lens epithelial cells induced by 1800 MHz mobile phone radiation.

XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

PubMed Central

Kaushik Tiwari, Meetu; Rogers, Faye A.

2013-01-01

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation. PMID:23913414

Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malloy, K.D.; Holman, M.A.; Mitchell, D.

The detrimental effects of elevated intensities of mid-UV radiation (UVB), a result of stratospheric ozone depletion during the austral spring, on the primary producers of the Antarctic marine ecosystem have been well documented. Here we report that natural populations of Antarctic zooplankton also sustain significant DNA damage [measured as cyclobutane pyrimidine dimers (CPDs)] during periods of increased UVB flux. This is the first direct evidence that increased solar UVB may result in damage to marine organisms other than primary producers in Antarctica. The extent of DNA damage in pelagic icefish eggs correlated with daily incident UVB irradiance, reflecting the differencemore » between acquisition and repair of CPDs. Patterns of DNA damage in fish larvae did not correlated with daily UVB flux, possibly due to different depth distributions and/or different capacities for DNA repair. Clearance of CPDs by Antarctic fish and krill was mediated primarily by the photoenzymatic repair system. Although repair rates were large for all species evaluated, they were apparently inadequate to prevent the transient accumulation of substantial CPD burdens. The capacity for DNA repair in Antarctic organisms was highest in those species whose early life history stages occupy the water column during periods of ozone depletion (austral spring) and lowest in fish species whose eggs and larvae are abundant during winter. Although the potential reduction in fitness of Antarctic zooplankton resulting from DNA damage is unknown, we suggest that increased solar UV may reduce recruitment and adversely affect trophic transfer of productivity by affecting heterotrophic species as well as primary producers. 54 refs., 4 figs., 2 tabs.« less

FIBER OPTIC BIOSENSOR FOR DNA DAMAGE

EPA Science Inventory

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...

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

PubMed

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p < 0.01); a significant reduction of genome-wide DNA MLs ( p < 0.01); and an increase in the methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p < 0.01). Our study indicated that a reduction in folic acid concentration promotes DNA damage and DNA methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

The repair of low dose UV light-induced damage to human skin DNA in condition of trace amount Mg 2+

NASA Astrophysics Data System (ADS)

Gao, Fang; Guo, Zhouyi; Zheng, Changchun; Wang, Rui; Liu, Zhiming; Meng, Pei; Zhai, Juan

2008-12-01

Ultraviolet light-induced damage to human skin DNA was widely investigated. The primary mechanism of this damage contributed to form cyclobutane pyrimidine dimmers (CPDs). Although the distribution of UV light-induced CPDs within a defined sequence is similar, the damage in cellular environment which shields the nuclear DNA was higher than that in organism in apparent dose. So we use low UVB light as main study agent. Low dose UV-irradiated HDF-a cells (Human Dermal Fibroblasts-adult cells) which is weaker than epidermic cells were cultured with DMEM at different trace amount of Mg2+ (0mmol/L , 0.1mmol/L , 0.2mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L) free-serum DMEM and the repair of DNA strands injured were observed. Treat these cells with DNA strand breaks detection, photoproducts detection and the repair of photoproducts detection. Then quantitate the role of trace amount Mg2+ in repair of UV light-induced damage to human skin. The experiment results indicated that epidermic cells have capability of resistance to UV-radiation at a certain extent. And Mg2+ can regulate the UV-induced damage repair and relative vitality. It can offer a rationale and experiment data to relieve UV light-induced skin disease.

Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Feiye, E-mail: zhizi0269@doc.medic.mie-u.ac.jp; Ma, Ning, E-mail: maning@suzuka-u.ac.jp; Horibe, Yoshiteru, E-mail: violinteru@yahoo.co.jp

Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20–30 nm (CNT20) and 40–70 nm (CNT40) significantly induced 8-nitroguanine formationmore » at 5 and 10 μg/ml (p < 0.05), which persisted for 24 h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p < 0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p < 0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis. Highlights: ►Multi-walled carbon nanotube (MWCNT) caused DNA damage in A549 cells. ►MWCNT formed 8-nitroguanine, a DNA

Chemotherapy-induced bystander effect in response to several chloroethylnitrosoureas: an origin independent of DNA damage?

PubMed

Merle, Patrick; Morvan, Daniel; Caillaud, Denis; Demidem, Aicha

2008-01-01

Chloroethylnitrosourea (CENU) chemotherapy is used for the treatment of melanoma tumors. The main mechanism of action of this anticancer agent is via DNA damage. We recently showed in murine experiments using a parental double B16 melanoma tumor model that, after treatment of primary tumors with cystemustine (CENU agent), untreated secondary tumors exhibited growth inhibition and metabolism disorders. The response of secondary untreated tumor was called the chemotherapy-induced bystander effect. To see whether chemotherapy-induced bystander effects were induced with other members of the CENU family, we compared three CENU(s) used in melanoma treatment: cystemustine, carmustine and fotemustine. Our results demonstrate that fotemustine, like cystemustine, but not carmustine induced a protective effect against secondary untreated tumors including alterations in phospholipid derivative and glutathione which are the metabolic signature of the bystander effect. From these data we may conclude that DNA damage to the primary tumor is not sufficient to explain chemotherapy-induced bystander effects.

Investigation on the correlation between energy deposition and clustered DNA damage induced by low-energy electrons.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2018-05-01

This study presents the correlation between energy deposition and clustered DNA damage, based on a Monte Carlo simulation of the spectrum of direct DNA damage induced by low-energy electrons including the dissociative electron attachment. Clustered DNA damage is classified as simple and complex in terms of the combination of single-strand breaks (SSBs) or double-strand breaks (DSBs) and adjacent base damage (BD). The results show that the energy depositions associated with about 90% of total clustered DNA damage are below 150 eV. The simple clustered DNA damage, which is constituted of the combination of SSBs and adjacent BD, is dominant, accounting for 90% of all clustered DNA damage, and the spectra of the energy depositions correlating with them are similar for different primary energies. One type of simple clustered DNA damage is the combination of a SSB and 1-5 BD, which is denoted as SSB + BD. The average contribution of SSB + BD to total simple clustered DNA damage reaches up to about 84% for the considered primary energies. In all forms of SSB + BD, the SSB + BD including only one base damage is dominant (above 80%). In addition, for the considered primary energies, there is no obvious difference between the average energy depositions for a fixed complexity of SSB + BD determined by the number of base damage, but average energy depositions increase with the complexity of SSB + BD. In the complex clustered DNA damage constituted by the combination of DSBs and BD around them, a relatively simple type is a DSB combining adjacent BD, marked as DSB + BD, and it is of substantial contribution (on average up to about 82%). The spectrum of DSB + BD is given mainly by the DSB in combination with different numbers of base damage, from 1 to 5. For the considered primary energies, the DSB combined with only one base damage contributes about 83% of total DSB + BD, and the average energy deposition is about 106 eV. However, the

Silymarin Protects Epidermal Keratinocytes from Ultraviolet Radiation-Induced Apoptosis and DNA Damage by Nucleotide Excision Repair Mechanism

PubMed Central

Katiyar, Santosh K.; Mantena, Sudheer K.; Meeran, Syed M.

2011-01-01

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells. PMID:21731736

Ursolic Acid-Regulated Energy Metabolism—Reliever or Propeller of Ultraviolet-Induced Oxidative Stress and DNA Damage?

PubMed Central

Lee, Yuan-Hao; Sun, Youping; Glickman, Randolph D.

2014-01-01

Ultraviolet (UV) light is a leading cause of diseases, such as skin cancers and cataracts. A main process mediating UV-induced pathogenesis is the production of reactive oxygen species (ROS). Excessive ROS levels induce the formation of DNA adducts (e.g., pyrimidine dimers) and result in stalled DNA replication forks. In addition, ROS promotes phosphorylation of tyrosine kinase-coupled hormone receptors and alters downstream energy metabolism. With respect to the risk of UV-induced photocarcinogenesis and photodamage, the antitumoral and antioxidant functions of natural compounds become important for reducing UV-induced adverse effects. One important question in the field is what determines the differential sensitivity of various types of cells to UV light and how exogenous molecules, such as phytochemicals, protect normal cells from UV-inflicted damage while potentiating tumor cell death, presumably via interaction with intracellular target molecules and signaling pathways. Several endogenous molecules have emerged as possible players mediating UV-triggered DNA damage responses. Specifically, UV activates the PIKK (phosphatidylinositol 3-kinase-related kinase) family members, which include DNA-PKcs, ATM (ataxia telangiectasia mutated) and mTOR (mammalian target of rapamycin), whose signaling can be affected by energy metabolism; however, it remains unclear to what extent the activation of hormone receptors regulates PIKKs and whether this crosstalk occurs in all types of cells in response to UV. This review focuses on proteomic descriptions of the relationships between cellular photosensitivity and the phenotypic expression of the insulin/insulin-like growth receptor. It covers the cAMP-dependent pathways, which have recently been shown to regulate the DNA repair machinery through interactions with the PIKK family members. Finally, this review provides a strategic illustration of how UV-induced mitogenic activity is modulated by the insulin sensitizer, ursolic

Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis

PubMed Central

Magni, Martina; Ruscica, Vincenzo; Buscemi, Giacomo; Kim, Ja-Eun; Nachimuthu, Benjamin Tamilselvan; Fontanella, Enrico; Delia, Domenico; Zannini, Laura

2014-01-01

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis. PMID:25361978

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

PubMed

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 < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.

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

PubMed

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.

The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

PubMed

Santos, Margarida A; Faryabi, Robert B; Ergen, Aysegul V; Day, Amanda M; Malhowski, Amy; Canela, Andres; Onozawa, Masahiro; Lee, Ji-Eun; Callen, Elsa; Gutierrez-Martinez, Paula; Chen, Hua-Tang; Wong, Nancy; Finkel, Nadia; Deshpande, Aniruddha; Sharrow, Susan; Rossi, Derrick J; Ito, Keisuke; Ge, Kai; Aplan, Peter D; Armstrong, Scott A; Nussenzweig, André

2014-10-02

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier

PubMed Central

Santos, Margarida A.; Faryabi, Robert B.; Ergen, Aysegul V.; Day, Amanda M.; Malhowski, Amy; Canela, Andres; Onozawa, Masahiro; Lee, Ji-Eun; Callen, Elsa; Gutierrez-Martinez, Paula; Chen, Hua-Tang; Wong, Nancy; Finkel, Nadia; Deshpande, Aniruddha; Sharrow, Susan; Rossi, Derrick J.; Ito, Keisuke; Ge, Kai; Aplan, Peter D.; Armstrong, Scott A.; Nussenzweig, André

2015-01-01

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells1. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks2–4, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma5,6, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL–AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4−/− MLL–AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL–AF9 blasts, which requires cyclin-dependent kinase inhibitor p21Cip1 (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia. PMID:25079327

REC-2006-A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo.

PubMed

Chaudhary, Pankaj; Shukla, Sandeep Kumar; Sharma, Rakesh Kumar

2011-01-01

Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg(-1) body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair.

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

PubMed

Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

2006-01-01

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan

Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lungmore » cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.« less

Age-dependent oxidative stress-induced DNA damage in Down's lymphocytes.

PubMed

Zana, Marianna; Szécsényi, Anita; Czibula, Agnes; Bjelik, Annamária; Juhász, Anna; Rimanóczy, Agnes; Szabó, Krisztina; Vetró, Agnes; Szucs, Péter; Várkonyi, Agnes; Pákáski, Magdolna; Boda, Krisztina; Raskó, István; Janka, Zoltán; Kálmán, János

2006-06-30

The aim of the present study was to investigate the oxidative status of lymphocytes from children (n=7) and adults (n=18) with Down's syndrome (DS). The basal oxidative condition, the vulnerability to in vitro hydrogen peroxide exposure, and the repair capacity were measured by means of the damage-specific alkaline comet assay. Significantly and age-independently elevated numbers of single strand breaks and oxidized bases (pyrimidines and purines) were found in the nuclear DNA of the lymphocytes in the DS group in the basal condition. These results may support the role of an increased level of endogenous oxidative stress in DS and are similar to those previously demonstrated in Alzheimer's disease. In the in vitro oxidative stress-induced state, a markedly higher extent of DNA damage was observed in DS children as compared with age- and gender-matched healthy controls, suggesting that young trisomic lymphocytes are more sensitive to oxidative stress than normal ones. However, the repair ability itself was not found to be deteriorated in either DS children or DS adults.

Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium.

PubMed

Dong, Zixun; Ran, Jianhua; Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.

Protection of radiation induced DNA and membrane damages by total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst.

PubMed

Smina, T P; Maurya, D K; Devasagayam, T P A; Janardhanan, K K

2015-05-25

The total triterpenes isolated from the fruiting bodies of Ganoderma lucidum was examined for its potential to prevent γ-radiation induced membrane damage in rat liver mitochondria and microsomes. The effects of total triterpenes on γ-radiation-induced DNA strand breaks in pBR 322 plasmid DNA in vitro and human peripheral blood lymphocytes ex vivo were evaluated. The protective effect of total triterpenes against γ-radiation-induced micronuclei formations in mice bone marrow cells in vivo were also evaluated. The results indicated the significant effectiveness of Ganoderma triterpenes in protecting the DNA and membrane damages consequent to the hazardous effects of radiation. The findings suggest the potential use of Ganoderma triterpenes in radio therapy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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.

Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

PubMed Central

Panda, Brahma B.; Achary, V. Mohan M.

2014-01-01

In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

Hereditary Disorders with Defective Repair of UV-Induced DNA Damage

PubMed Central

Moriwaki, Shinichi

2013-01-01

Nucleotide excision repair (NER) is an essential system for correcting ultraviolet (UV)—induced DNA damage. Lesions remaining in DNA due to reduced capacity of NER may result in cellular death, premature aging, mutagenesis and carcinogenesis of the skin. So, NER is an important protection against these changes. There are three representative genodermatoses resulting from genetic defects in NER: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). In Japan, CS is similarly rare but XP is more common and TTD is less common compared to Western countries. In 1998, we established the system for the diagnosis of these disorders and we have been performing DNA repair and genetic analysis for more than 400 samples since then. At present, there is no cure for any human genetic disorder. Early diagnosis and symptomatic treatment of neurological, ocular and dermatological abnormalities should contribute to prolonging life and elevating QOL in patients. PMID:23966815

DNA Damage Signals and Space Radiation Risk

NASA Technical Reports Server (NTRS)

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.

Induction of oxidative DNA damage in anaerobes.

PubMed

Takeuchi, T; Nakaya, Y; Kato, N; Watanabe, K; Morimoto, K

1999-05-07

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8-hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Hui; Shi, Qiong; Song, Xiufang

2015-07-01

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observedmore » phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.« less

Cellular responses to environmental DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

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.

Biomolecular damage induced by ionizing radiation: the direct and indirect effects of low-energy electrons on DNA.

PubMed

Alizadeh, Elahe; Orlando, Thomas M; Sanche, Léon

2015-04-01

Many experimental and theoretical advances have recently allowed the study of direct and indirect effects of low-energy electrons (LEEs) on DNA damage. In an effort to explain how LEEs damage the human genome, researchers have focused efforts on LEE interactions with bacterial plasmids, DNA bases, sugar analogs, phosphate groups, and longer DNA moieties. Here, we summarize the current understanding of the fundamental mechanisms involved in LEE-induced damage of DNA and complex biomolecule films. Results obtained by several laboratories on films prepared and analyzed by different methods and irradiated with different electron-beam current densities and fluencies are presented. Despite varied conditions (e.g., film thicknesses and morphologies, intrinsic water content, substrate interactions, and extrinsic atmospheric compositions), comparisons show a striking resemblance in the types of damage produced and their yield functions. The potential of controlling this damage using molecular and nanoparticle targets with high LEE yields in targeted radiation-based cancer therapies is also discussed.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression.

PubMed

Sen, Onur; Saurin, Adrian T; Higgins, Jonathan M G

2018-05-21

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10-25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Genistein protects hematopoietic stem cells against G-CSF-induced DNA damage.

PubMed

Souza, Liliana R; Silva, Erica; Calloway, Elissa; Kucuk, Omer; Rossi, Michael; McLemore, Morgan L

2014-05-01

Granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most striking example is in severe congenital neutropenia (SCN). Patients with SCN develop MDS/AML at a high rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and AML that arise in these settings are commonly associated with chromosomal deletions. We have demonstrated in this study that chronic G-CSF treatment in mice results in expansion of the hematopoietic stem cell (HSC) population. In addition, primitive hematopoietic progenitors from G-CSF-treated mice show evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromosomal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF-induced proliferation of HSCs. Importantly, genistein does not impair G-CSF-induced proliferation of committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of genistein was accomplished with plasma levels that are attainable through dietary supplementation.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy

PubMed Central

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog

2016-01-01

Abstract Aims: 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. Results: 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. Innovation: 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. Conclusion: 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. PMID:26935406

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

PubMed

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.

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

PubMed

Gavella, Mirjana; Garaj-Vrhovac, Verica; Lipovac, Vaskresenija; Antica, Mariastefania; Gajski, Goran; Car, Nikica

2010-06-01

We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.

Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

NASA Astrophysics Data System (ADS)

Ramesh, Govindarajan; Wu, Honglu

Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Xu; Ptasinska, Sylwia; Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556

2013-06-10

The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

NASA Astrophysics Data System (ADS)

Han, Xu; Klas, Matej; Liu, Yueying; Sharon Stack, M.; Ptasinska, Sylwia

2013-06-01

The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

Cigarette smoke-induced DNA damage and repair detected by the comet assay in HPV-transformed cervical cells.

PubMed

Moktar, Afsoon; Ravoori, Srivani; Vadhanam, Manicka V; Gairola, C Gary; Gupta, Ramesh C

2009-12-01

Human papillomavirus (HPV) is the causative factor in the development and progression of cervical cancers in >97% of the cases, although insufficient. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers; therefore, we examined cigarette smoke-induced DNA damage and repair in HPV16-transformed human ectocervical cells (ECT1/E6 E7). Cells were treated with cigarette smoke condensate (CSC) for 72 h to assess the formation of single- and double-strand DNA breaks, measured by alkaline and neutral single cell gel electrophoresis assays, respectively. The mean tail length of cells with single-strand breaks was increased by 1.8-, 2.7- and 3.7-fold (p<0.001) after treatment with 4, 8 and 12 microg/ml CSC, respectively. The tail length with double-strand breaks was also increased dose-dependently. These results were further supported by measurement of the mean tail moment: the increase in both single- and double-strand breaks were much more pronounced with increasing concentration of CSC, by up to 23.5-fold (p<0.0001 for both assays). To examine the DNA repair, cells were treated with CSC for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, or 72 h. Both single- and double-strand DNA breaks were removed during the initial 24 h but no further removal of the damage was observed. Up to 80% of residual single- and double-strand DNA breaks (p<0.05) were found to persist at all CSC concentrations examined. Ellagic acid, a known antioxidant and free-radical scavenger, was found to significantly inhibit DNA breaks induced by CSC. Thus, free radicals may be a plausible source of CSC-induced DNA damage. These data show that CSC-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk.

REC-2006—A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo

PubMed Central

Chaudhary, Pankaj; Shukla, Sandeep Kumar; Sharma, Rakesh Kumar

2011-01-01

Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg−1 body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair. PMID:20008078

DNA damage in blood cells exposed to low-level lasers.

PubMed

Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

2015-04-01

In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice.

PubMed

Yamamoto, Hiro-aki; Mohanan, Parayanthala V

2003-07-20

The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

Ex vivo study for the assessment of behavioral factor and gene polymorphisms in individual susceptibility to oxidative DNA damage metals-induced.

PubMed

Di Pietro, Angela; Baluce, Barbara; Visalli, Giuseppa; La Maestra, Sebastiano; Micale, Rosanna; Izzotti, Alberto

2011-06-01

Transition metals in fine particulate matter generated by combustion induce oxidative DNA damage and inflammation. However, there is remarkable inter-individual variability in susceptibility to these damages. To assess this variability, an ex vivo study was performed using lymphocytes of 47 Caucasian healthy subjects. Cell samples were exposed to a water solution of oil fly ash (OFA). This was formed by the distinctive transition metals vanadium, iron, and nickel. Oxidative DNA damage was evaluated by testing cell viability, intracellular ROS production and 8-oxo-dG. DNA fragmentation and DNA repair capacity were assessed by using the Alkaline-Halo assay. GSTM1, GSTT1, hOGG1, and C677T and A1298C MTHFR gene polymorphisms were tested. Demographic and behavioral factors, collected by questionnaire, were also considered. OFA induced damages showed: (a) a 20-fold variation in range among different subjects in ROS production, (b) a 7-fold variation in range of 8-oxo-dG, and (c) a 25-fold variation in range in DNA repair capacity. A significant increase in DNA damage was detected in GSTT1-deficent subjects compared with wild type genotype carriers. Increases in cytoplasmic ROS and decreases in DNA repair capacity (P<0.05) were observed in C677T and A1298C variants of MTHFR. A remarkable protective effect of high fruits and vegetable intake was observed for ROS production and DNA damage. Conversely, an adverse effect of meat intake was observed on ROS increase, DNA damage and repair capacity, probably due to the increased intake of bioavailable iron. Smoking decreased DNA repair capacity, while age increased OFA-induced DNA damage. The wide comparative analysis of the complex interactions network, between genetic and behavioral factors provides evidence of the remarkable role of several lifestyle factors. In comparison to genetic polymorphisms they seem to have a higher weight in determining individual susceptibility to the adverse effects of airborne pollutants as

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krüger, Katharina; Ziegler, Verena; Hartmann, Christina

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model.more » The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage

X-Ray Induced DNA Damage and Repair in Germ Cells of PARP1−/− Male Mice

PubMed Central

Villani, Paola; Fresegna, Anna Maria; Ranaldi, Roberto; Eleuteri, Patrizia; Paris, Lorena; Pacchierotti, Francesca; Cordelli, Eugenia

2013-01-01

Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1−/− and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1−/− and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1−/− cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1−/− mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX. PMID:24009020

Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium

PubMed Central

Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Background Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Methodology/Principal Findings Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. Conclusions/Significance UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders. PMID:24204711

Retinoic acid-related orphan receptor-α is induced in the setting of DNA damage and promotes pulmonary emphysema.

PubMed

Shi, Ying; Cao, Jiaofei; Gao, Jane; Zheng, Liang; Goodwin, Andrew; An, Chang Hyoek; Patel, Avignat; Lee, Janet S; Duncan, Steven R; Kaminski, Naftali; Pandit, Kusum V; Rosas, Ivan O; Choi, Augustine M K; Morse, Danielle

2012-09-01

The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. To determine the role of Rora in smoke-induced emphysema. Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals.

PubMed

Zhou, Juan; Li, Peng; Cheng, Ni; Gao, Hui; Wang, Bini; Wei, Yahui; Cao, Wei

2012-08-01

To understand the antioxidant properties of buckwheat honeys, we investigated their antioxidant effects on hydroxyl radical-induced DNA breaks in the non-site-specific and site-specific systems, the physicochemical properties, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity, chelating, and reducing power assays), total phenolic content and individual phenolic acids were also determined. Total phenolic content of buckwheat honeys ranged from 774 to 1694 mg PA/kg, and p-hydroxybenzoic and p-coumaric acids proved to be the main components in buckwheat honeys. All the buckwheat honey samples possess stronger capability to protect DNA in the non-site-specific systems than in the site-specific systems from being damaged by hydroxyl radicals. In the non-site-specific and site-specific system, buckwheat honeys samples prevented ()OH-induced DNA breaks by 21-78% and 5-31% over control value, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

NASA Technical Reports Server (NTRS)

Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

2002-01-01

Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

PubMed Central

Ciamporcero, Eric; Shen, He; Ramakrishnan, Swathi; Ku, Sheng Yu; Chintala, Sreenivasulu; Shen, Li; Adelaiye, Remi; Miles, Kiersten Marie; Ullio, Chiara; Pizzimenti, Stefania; Daga, Martina; Azabdaftari, Gissou; Attwood, Kris; Johnson, Candace; Zhang, Jianmin; Barrera, Giuseppina; Pili, Roberto

2015-01-01

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC. PMID:26119935

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage.

PubMed

Ciamporcero, E; Shen, H; Ramakrishnan, S; Yu Ku, S; Chintala, S; Shen, L; Adelaiye, R; Miles, K M; Ullio, C; Pizzimenti, S; Daga, M; Azabdaftari, G; Attwood, K; Johnson, C; Zhang, J; Barrera, G; Pili, R

2016-03-24

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knockdown sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC.

A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY STYRENE OXIDE

EPA Science Inventory

A rapid and simple assay to detect DNA damage to calf thymus DNA caused by styrene oxide (SO) is reported. This assay is based on changes observed in the melting and annealing behavior of the damaged DNA. The melting annealing process was monitored using a fluorescence indicat...

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

Toll-Like Receptor-4 deficiency enhances repair of ultraviolet radiation induced cutaneous DNA damage by nucleotide excision repair mechanism

PubMed Central

Ahmad, Israr; Simanyi, Eva; Guroji, Purushotham; Tamimi, Iman A; delaRosa, Hillary J; Nagar, Anusuiya; Nagar, Priyamvada; Katiyar, Santosh K; Elmets, Craig A; Yusuf, Nabiha

2014-01-01

UVB-induced DNA damage plays a critical role in development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4-/-) and TLR4 competent (TLR4+/+) mice were subjected to 90 mJ/cm2 UVB radiation locally, DNA damage in the form of CPD, were repaired more efficiently in the skin and bone marrow dendritic cells (BMDC) of TLR4-/- mice in comparison to TLR4+/+ mice. Expression of DNA repair gene XPA (Xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDC of TLR4+/+ mice than TLR4-/- mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDC from UV-irradiated TLR4-/- mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (p<0.05) than BMDC from TLR4+/+ mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDC of TLR4-/- mice (before UVB exposure) inhibited repair of CPD, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4+/+ BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer. PMID:24326454

Modelling and Holographic Visualization of Space Radiation-Induced DNA Damage

NASA Technical Reports Server (NTRS)

Plante, Ianik

2017-01-01

Space radiation is composed by a mixture of ions of different energies. Among these, heavy inos are of particular importance because their health effects are poorly understood. In. the recent years, a software named RITRACKS (Relativistic Ion Tracks) was developed to simulate the detailed radiation track structure, several DNA models and DNA damage. As the DNA structure is complex due to packing, it is difficult to the damage using a regular computer screen.

Measurement of changes in impedance of DNA nanowires due to radiation induced structural damage. A novel approach for a DNA-based radiosensitive device

NASA Astrophysics Data System (ADS)

Heimbach, Florian; Arndt, Alexander; Nettelbeck, Heidi; Langner, Frank; Giesen, Ulrich; Rabus, Hans; Sellner, Stefan; Toppari, Jussi; Shen, Boxuan; Baek, Woon Yong

2017-08-01

The ability of DNA to conduct electric current has been the topic of numerous investigations over the past few decades. Those investigations indicate that this ability is dependent on the molecular structure of the DNA. Radiation-induced damages, which lead to an alteration of the molecular structure, should therefore change the electrical impedance of a DNA molecule. In this paper, the damage due to ionising radiation is shown to have a direct effect on the electrical transport properties of DNA. Impedance measurements of DNA samples were carried out by an AC impedance spectrometer before, during and after irradiation. The samples comprised of DNA segments, which were immobilized between gold electrodes with a gap of 12 μm. The impedance of all DNA samples exhibited rising capacitive behaviour with increasing absorbed dose.

Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

PubMed Central

Panich, Uraiwan; Sittithumcharee, Gunya; Rathviboon, Natwarath

2016-01-01

Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been linked to impaired skin homeostasis (e.g., skin premature aging and skin cancers). Skin stem cells, with self-renewal capability and multipotency, are frequently affected by environment. Ultraviolet radiation (UVR), a major cause of stem cell DNA damage, can contribute to depletion of stem cells (ESCs and mesenchymal stem cells) and damage of stem cell niche, eventually leading to photoinduced skin aging. In this review, we discuss the role of UV-induced DNA damage and oxidative stress in the skin stem cell aging in order to gain insights into the pathogenesis and develop a way to reduce photoaging of skin cells. PMID:27148370

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

PubMed

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.

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

PubMed

Rodeiro, I; Delgado, R; Garrido, G

2014-02-01

Mangifera indica L. (mango) stem bark aqueous extract (MSBE) that has antioxidant, anti-inflammatory and immunomodulatory properties, can be obtained in Cuba. It is rich in polyphenols, where mangiferin is the main component. In this study, we have tested DNA damage and protection effects of MSBE and mangiferin on primary human lymphocytes and lymphoblastoid cells. Cell suspensions were incubated with the products (50-1000 μg/ml) for experiments on damage induction, and evaluation of any potential protective effects (5-100 μg/ml) for 60 min at 37 °C. Irradiation was performed using a γ-ray source, absorbed dose 5 Gy. At the end of exposure, DNA damage, protection and repair processes were evaluated using the comet assay. MSBE (100-1000 μg/ml) induced DNA damage in a concentration dependent manner in both cell types tested, primary cells being more sensitive. Mangiferin (200 μg/ml) only induced light DNA damage at higher concentrations. DNA repair capacity was not affected after MSBE or mangiferin exposure. On the other hand, MSBE (25 and 50 μg/ml) and mangiferin (5-25 ug/ml) protected against gamma radiation-induced DNA damage. These results show MSBE has protector or harmful effects on DNA in vitro depending on the experimental conditions, which suggest that the extract could be acting as an antioxidant or pro-oxidant product. Mangiferin was involved in protective effects of the extract. © 2013 John Wiley & Sons Ltd.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

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

PubMed

Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

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. Copyright © 2012 Elsevier Inc. All rights reserved.

Visualization of complex DNA damage along accelerated ions tracks

NASA Astrophysics Data System (ADS)

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.

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

PubMed Central

Arango, Daniel; Parihar, Arti; Villamena, Frederick A.; Wang, Liwen; Freitas, Michael A.; Grotewold, Erich; Doseff, Andrea I.

2014-01-01

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid. PMID:22985621

DNA damage in an animal model of maple syrup urine disease.

PubMed

Scaini, Giselli; Jeremias, Isabela C; Morais, Meline O S; Borges, Gabriela D; Munhoz, Bruna P; Leffa, Daniela D; Andrade, Vanessa M; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2012-06-01

Maple syrup urine disease is an inborn error of metabolism caused by a severe deficiency of the branched chain alpha-ketoacid dehydrogenase complex. Neurological dysfunction is a common finding in patients with maple syrup urine disease. However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly understood. In this study, we investigated whether acute or chronic administration of a branched chain amino acid pool (leucine, isoleucine and valine) causes transient DNA damage, as determined by the alkaline comet assay, in the brain and blood of rats during development and whether antioxidant treatment prevented the alterations induced by branched chain amino acids. Our results showed that the acute administration of branched chain amino acids increased the DNA damage frequency and damage index in the hippocampus. However, the chronic administration of branched chain amino acids increased the DNA damage frequency and damage index in both the hippocampus and the striatum, and the antioxidant treatment was able to prevent DNA damage in the hippocampus and striatum. The present study demonstrated that metabolite accumulation in MSUD induces DNA damage in the hippocampus and striatum and that it may be implicated in the neuropathology observed in the affected patients. We demonstrated that the effect of antioxidant treatment (N-acetylcysteine plus deferoxamine) prevented DNA damage, suggesting the involvement of oxidative stress in DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

Levetiracetam mitigates doxorubicin-induced DNA and synaptic damage in neurons.

PubMed

Manchon, Jose Felix Moruno; Dabaghian, Yuri; Uzor, Ndidi-Ese; Kesler, Shelli R; Wefel, Jeffrey S; Tsvetkov, Andrey S

2016-05-11

Neurotoxicity may occur in cancer patients and survivors during or after chemotherapy. Cognitive deficits associated with neurotoxicity can be subtle or disabling and frequently include disturbances in memory, attention, executive function and processing speed. Searching for pathways altered by anti-cancer treatments in cultured primary neurons, we discovered that doxorubicin, a commonly used anti-neoplastic drug, significantly decreased neuronal survival. The drug promoted the formation of DNA double-strand breaks in primary neurons and reduced synaptic and neurite density. Pretreatment of neurons with levetiracetam, an FDA-approved anti-epileptic drug, enhanced survival of chemotherapy drug-treated neurons, reduced doxorubicin-induced formation of DNA double-strand breaks, and mitigated synaptic and neurite loss. Thus, levetiracetam might be part of a valuable new approach for mitigating synaptic damage and, perhaps, for treating cognitive disturbances in cancer patients and survivors.

Levetiracetam mitigates doxorubicin-induced DNA and synaptic damage in neurons

PubMed Central

Manchon, Jose Felix Moruno; Dabaghian, Yuri; Uzor, Ndidi-Ese; Kesler, Shelli R.; Wefel, Jeffrey S.; Tsvetkov, Andrey S.

2016-01-01

Neurotoxicity may occur in cancer patients and survivors during or after chemotherapy. Cognitive deficits associated with neurotoxicity can be subtle or disabling and frequently include disturbances in memory, attention, executive function and processing speed. Searching for pathways altered by anti-cancer treatments in cultured primary neurons, we discovered that doxorubicin, a commonly used anti-neoplastic drug, significantly decreased neuronal survival. The drug promoted the formation of DNA double-strand breaks in primary neurons and reduced synaptic and neurite density. Pretreatment of neurons with levetiracetam, an FDA-approved anti-epileptic drug, enhanced survival of chemotherapy drug-treated neurons, reduced doxorubicin-induced formation of DNA double-strand breaks, and mitigated synaptic and neurite loss. Thus, levetiracetam might be part of a valuable new approach for mitigating synaptic damage and, perhaps, for treating cognitive disturbances in cancer patients and survivors. PMID:27168474

Chromosome territories reposition during DNA damage-repair response

PubMed Central

2013-01-01

Background Local higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored. Results Our report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells. Conclusions Thus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response. PMID:24330859

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts

PubMed Central

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-01-01

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention. PMID:26447614

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

PubMed

Guillermo-Lagae, Ruth; Deep, Gagan; Ting, Harold; Agarwal, Chapla; Agarwal, Rajesh

2015-11-24

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.

DNA vaccination protects mice against Zika virus-induced damage to the testes

PubMed Central

Griffin, Bryan D.; Muthumani, Kar; Warner, Bryce M.; Majer, Anna; Hagan, Mable; Audet, Jonathan; Stein, Derek R.; Ranadheera, Charlene; Racine, Trina; De La Vega, Marc-Antoine; Piret, Jocelyne; Kucas, Stephanie; Tran, Kaylie N.; Frost, Kathy L.; De Graff, Christine; Soule, Geoff; Scharikow, Leanne; Scott, Jennifer; McTavish, Gordon; Smid, Valerie; Park, Young K.; Maslow, Joel N.; Sardesai, Niranjan Y.; Kim, J. Joseph; Yao, Xiao-jian; Bello, Alexander; Lindsay, Robbin; Boivin, Guy; Booth, Stephanie A.; Kobasa, Darwyn; Embury-Hyatt, Carissa; Safronetz, David; Weiner, David B.; Kobinger, Gary P.

2017-01-01

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract. PMID:28589934

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 DNAmore » 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.« less

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

PubMed

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

DNA damage, DNA susceptibility to oxidation and glutathione redox status in patients with Alzheimer's disease treated with and without memantine.

PubMed

Akkaya, Çağlayan; Yavuzer, Serap Sahin; Yavuzer, Hakan; Erkol, Gökhan; Bozluolcay, Melda; Dinçer, Yıldız

2017-07-15

The aim of the current study was to compare oxidative DNA damage, DNA susceptibility to oxidation, and ratio of GSH/GSSG in patients with Alzheimer's disease (AD) treated with acetylcholinesterase inhibitor (AChEI) and combined AChEI+memantine. The study included 67 patients with AD and 42 volunteers as control. DNA damage parameters (strand breaks, oxidized purines, H 2 O 2 -induced DNA damage) in lymphocyte DNA and GSH/GSSG ratio in erythrocytes were determined by the comet assay and spectrophotometric assay, respectively. DNA damage was found to be higher, GSH/GSSG ratio was found to be lower in the AD group than those in the control group. DNA strand breaks and H 2 O 2 -induced DNA damage were lower in the patients taking AChEI+memantine than those in the patients taking AChEI but no significant difference was determined between the groups for oxidized purines and GSH/GSSG ratio. In conclusion, increased systemic oxidative DNA damage and DNA susceptibility to oxidation may be resulted from diminished GSH/GSSG ratio in AD patients. Although DNA strand breaks and H 2 O 2 -induced DNA damage are lower in the AD patients treated with combined AChEI and memantine, this may not indicate protective effect of memantine against DNA oxidation due to similar levels of oxidized purines in the patients treated with AChEI and AChEI+memantine. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

Sunlight damage to cellular DNA: Focus on oxidatively generated lesions.

PubMed

Schuch, André Passaglia; Moreno, Natália Cestari; Schuch, Natielen Jacques; Menck, Carlos Frederico Martins; Garcia, Camila Carrião Machado

2017-06-01

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All

DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

EPA Science Inventory

A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells.

PubMed

Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

2017-01-01

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N -acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.

Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

PubMed Central

Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

2017-01-01

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells. PMID:28936177

Metallothionein blocks oxidative DNA damage induced by acute inorganic arsenic exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu, Wei, E-mail: qu@niehs.nih.gov; Waalkes, Michael P.

We studied how protein metallothionein (MT) impacts arsenic-induced oxidative DNA damage (ODD) using cells that poorly express MT (MT-I/II double knockout embryonic cells; called MT-null cells) and wild-type (WT) MT competent cells. Arsenic (as NaAsO{sub 2}) was less cytolethal over 24 h in WT cells (LC{sub 50} = 11.0 ± 1.3 μM; mean ± SEM) than in MT-null cells (LC{sub 50} = 5.6 ± 1.2 μM). ODD was measured by the immuno-spin trapping method. Arsenic (1 or 5 μM; 24 h) induced much less ODD in WT cells (121% and 141% of control, respectively) than in MT-null cells (202% andmore » 260%). In WT cells arsenic caused concentration-dependent increases in MT expression (transcript and protein), and in the metal-responsive transcription factor-1 (MTF-1), which is required to induce the MT gene. In contrast, basal MT levels were not detectable in MT-null cells and unaltered by arsenic exposure. Transfection of MT-I gene into the MT-null cells markedly reduced arsenic-induced ODD levels. The transport genes, Abcc1 and Abcc2 were increased by arsenic in WT cells but either showed no or very limited increases in MT-null cells. Arsenic caused increases in oxidant stress defense genes HO-1 and GSTα2 in both WT and MT-null cells, but to much higher levels in WT cells. WT cells appear more adept at activating metal transport systems and oxidant response genes, although the role of MT in these responses is unclear. Overall, MT protects against arsenic-induced ODD in MT competent cells by potential sequestration of scavenging oxidant radicals and/or arsenic. - Highlights: • Metallothionein blocks arsenic toxicity. • Metallothionein reduces arsenic-induced DNA damage. • Metallothionein may bind arsenic or radicals produced by arsenic.« less

Day and night variations in the repair of ionizing-radiation-induced DNA damage in mouse splenocytes.

PubMed

Palombo, Philipp; Moreno-Villanueva, Maria; Mangerich, Aswin

2015-04-01

In mammals, biological rhythms synchronize physiological and behavioral processes to the 24-h light-dark (LD) cycle. At the molecular level, self-sustaining processes, such as oscillations of transcription-translation feedback loops, control the circadian clock, which in turn regulates a wide variety of cellular processes, including gene expression and cell cycle progression. Furthermore, previous studies reported circadian oscillations in the repair capacity of DNA lesions specifically repaired by nucleotide excision repair (NER). However, it is so far only poorly understood if DNA repair pathways other than NER are under circadian control, in particular base excision and DNA strand break repair. In the present study, we analyzed potential day and night variations in the repair of DNA lesions induced by ionizing radiation (i.e., mainly oxidative damage and DNA strand breaks) in living mouse splenocytes using a modified protocol of the automated FADU assay. Our results reveal that splenocytes isolated from mice during the light phase (ZT06) displayed higher DNA repair activity than those of the dark phase (ZT18). As analyzed by highly sensitive and accurate qPCR arrays, these alterations were accompanied by significant differences in expression profiles of genes involved in the circadian clock and DNA repair. Notably, the majority of the DNA repair genes were expressed at higher levels during the light phase (ZT06). This included genes of all major DNA repair pathways with the strongest differences observed for genes of base excision and DNA double strand break repair. In conclusion, here we provide novel evidence that mouse splenocytes exhibit significant differences in the repair of IR-induced DNA damage during the LD cycle, both on a functional and on a gene expression level. It will be interesting to test if these findings could be exploited for therapeutic purposes, e.g. time-of-the-day-specific application of DNA-damaging treatments used against blood

Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

PubMed Central

Cline, Susan D.

2012-01-01

How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance

PubMed Central

Seager, Anna L.

2012-01-01

Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H2O2), potassium bromate (KBrO3), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H2O2 and KBrO3, but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair–focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H2O2-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H2O2 dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in “genotoxic tolerance.” PMID:22539617

Time-Restricted Feeding Shifts the Skin Circadian Clock and Alters UVB-Induced DNA Damage.

PubMed

Wang, Hong; van Spyk, Elyse; Liu, Qiang; Geyfman, Mikhail; Salmans, Michael L; Kumar, Vivek; Ihler, Alexander; Li, Ning; Takahashi, Joseph S; Andersen, Bogi

2017-08-01

The epidermis is a highly regenerative barrier protecting organisms from environmental insults, including UV radiation, the main cause of skin cancer and skin aging. Here, we show that time-restricted feeding (RF) shifts the phase and alters the amplitude of the skin circadian clock and affects the expression of approximately 10% of the skin transcriptome. Furthermore, a large number of skin-expressed genes are acutely regulated by food intake. Although the circadian clock is required for daily rhythms in DNA synthesis in epidermal progenitor cells, RF-induced shifts in clock phase do not alter the phase of DNA synthesis. However, RF alters both diurnal sensitivity to UVB-induced DNA damage and expression of the key DNA repair gene, Xpa. Together, our findings indicate regulation of skin function by time of feeding and emphasize a link between circadian rhythm, food intake, and skin health. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

DNA damage and oxidative stress induced by imidacloprid exposure in the earthworm Eisenia fetida.

PubMed

Wang, Juan; Wang, Jinhua; Wang, Guangchi; Zhu, Lusheng; Wang, Jun

2016-02-01

To investigate the soil ecological effect of imidacloprid, earthworm Eisenia fetida was exposed to various concentrations of imidacloprid (0.10, 0.50, and 1.00 mg kg(-1) soil) respectively after 7, 14, 21, and 28 d. The effect of imidacloprid on reactive oxygen species (ROS) generation, antioxidant enzymes activity [superoxide dismutase (SOD) and catalase (CAT), glutathione S-transferase enzyme (GST)], malondialdehyde (MDA) content and DNA damage of the E. fetida was investigated. Significant increase of the ROS level was observed. The SOD and GST activity were significantly induced at most exposure intervals. CAT activity was inhibited and reflected a dose-dependent relationship on days 7, 14 and 21. High MDA levels were observed and the olive tail moment (OTM) as well as the percentage of DNA in the comet tail (tail DNA%) in comet assay declined with increasing concentrations and exposure time after 7 d. Our results suggested that the sub-chronic exposure of imidacloprid caused DNA damage and lipid peroxidation (LPO) leading to antioxidant responses in earthworm E. fetida. Copyright © 2015 Elsevier Ltd. All rights reserved.

BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes.

PubMed

Sun, Yang; Wang, Peiling; Li, Hongyu; Dai, Jun

2018-06-26

A diverse array of biological processes are under circadian controls. In mouse skin, ultraviolet ray (UVR)-induced apoptosis and DNA damage responses are time-of-day dependent, which are controlled by core clock proteins. This study investigates the roles of clock proteins in regulating UVB responses in human keratinocytes (HKCs). We found that the messenger RNA expression of brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) genes is altered by low doses (5 mJ/cm 2 ) of UVB in the immortalized HaCat HKCs cell line. Although depletion of BMAL1 or CLOCK has no effect on the activation of Rad3-related protein kinases-checkpoint kinase 1-p53 mediated DNA damage checkpoints, it leads to suppression of UVB-stimulated apoptotic responses, and downregulation of UVB-elevated expression of DNA damage marker γ-H2AX and cell cycle inhibitor p21. Diminished apoptotic responses are also observed in primary HKCs depleted of BMAL1 or CLOCK after UVB irradiation. While CLOCK depletion shows a suppressive effect on UVB-induced p53 protein accumulation, depletion of either clock gene triggers early keratinocyte differentiation of HKCs at their steady state. These results suggest that UVB-induced apoptosis and DNA damage responses are controlled by clock proteins, but via different mechanisms in the immortalized human adult low calcium temperature and primary HKCs. Given the implication of UVB in photoaging and photocarcinogenesis, mechanistic elucidation of circadian controls on UVB effects in human skin will be critical and beneficial for prevention and treatment of skin cancers and other skin-related diseases. © 2018 Wiley Periodicals, Inc.

DNA damage-inducible genes as biomarkers for exposures to environmental agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, N.F.; Carpenter, T.R.; Jaramillo, R.J.

1997-06-01

A biodosimetric approach to determine alpha-particle dose to the respiratory tract epithelium from known exposures to radon has been developed in the rat. Cytotoxicity assays have been used to obtain dose-conversion factors for cumulative exposures typical of those encountered by underground uranium miners. However, this approach is not sensitive enough to derive close-conversion factors for indoor radon exposures. The expression of DNA damage-inducible genes is being investigated as a biomarker of exposure to radon progeny. Exposure of cultures of A549 cells to alpha particles resulted in an increase in the protein levels of the DNA damage-inducible genes, p53, Cip 1,more » and Gadd45. These protein changes were associated with a transient arrest of cells passing through the cell cycle. This arrest was typified by an increase in the number of cells in the G{sub 1} and G{sub 2} phases and a decrease in the number of cells in the S phase. The effect of inhaled alpha particles (radon progeny) in rats was examined in the epithelial cells of the lateral wall of the anterior nasal cavity. Exposures to radon progeny resulted in a significant increase in the number of cells in the G{sub 1} phase and a decrease in the number of cells in the S phase. These cell-cycle changes were concomitant with an increase in the number of cells containing DNA strand breaks. In addition to ionizing radiation, A549 cells were exposed to 4-nitroquinoline-1-oxide, methyl methanesulphonate, crocidolite asbestos, and glass microfiber. These studies showed that physical and chemical agents induce different expression patterns of p53, Cip 1, and Gadd153 proteins and they could be used to discriminate between toxic and nontoxic materials such as asbestos and glass microfiber. The measurement of gene expression in A549 cells may provide a means to identify a broad spectrum of physical and chemical toxicants encountered in the environment. 9 figs., 42 refs.« less

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

EPA Science Inventory

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...

Evaluation of γ-radiation-induced DNA damage in two species of bivalves and their relative sensitivity using comet assay.

PubMed

Praveen Kumar, M K; Shyama, S K; Sonaye, B S; Naik, U Roshini; Kadam, S B; Bipin, P D; D'costa, A; Chaubey, R C

2014-05-01

Ionizing radiation is known to induce genetic damage in diverse groups of organisms. Under accidental situations, large quantities of radioactive elements get released into the environment and radiation emitted from these radionuclides may adversely affect both the man and the non-human biota. The present study is aimed (a) to know the genotoxic effect of gamma radiation on aquatic fauna employing two species of selected bivalves, (b) to evaluate the possible use of 'Comet assay' for detecting genetic damage in haemocytes of bivalves as a biomarker for environmental biomonitoring and also (c) to compare the relative sensitivity of two species of bivalves viz. Paphia malabarica and Meretrix casta to gamma radiation. The comet assays was optimized and validated using different concentrations (18, 32 and 56 mg/L) of ethyl methanesulfonate (EMS), a direct-acting reference genotoxic agent, to which the bivalves were exposed for various times (24, 48 and 72 h). Bivalves were irradiated (single acute exposure) with 5 different doses (viz. 2, 4, 6, 8 and 10 Gy) of gamma radiation and their genotoxic effects on the haemocytes were studied using the comet assay. Haemolymph was collected from the adductor muscle at 24, 48 and 72 h of both EMS-exposed and irradiated bivalves and comet assay was carried out using standard protocol. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA damage at different concentrations of EMS and all the doses of gamma radiation as compared to controls in both bivalve species. This showed a dose-dependent increase of genetic damage induced in bivalves by EMS as well as gamma radiation. Further, the highest DNA damage was observed at 24h. The damage gradually decreased with time, i.e. was smaller at 48 and 72 h than at 24h post irradiation in both species of bivalves. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the post irradiation time advanced. The present study

ORGANIC AND INORGANIC ARSENICALS SENSITIZE HUMAN BRONCHIAL EPITHELIAL CELLS TO HYDROGEN PEROXIDE-INDUCED DNA DAMAGE

EPA Science Inventory

The lungs are a target organ for arsenic carcinogenesis, however, its mechanism of action remains unclear. Furthermore, it has been suggested that inorganic arsenic (iAs) can potentiate DNA damage induced by other agents. Once inside the human body iAs generally undergoes two ...

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis

PubMed Central

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-01-01

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways. PMID:28277539

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

PubMed

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-03-09

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

Lead induced oxidative DNA damage in battery-recycling child workers from Bangladesh.

PubMed

Arif, Mohammad; Islam, Mm Towhidul; Shekhar, Hossain Uddin

2018-04-01

Lead exposure can damage cells directly by effecting DNA or indirectly by modifying proteins and enzymes. In Bangladesh, many working children are exposed to a very high level of lead during their early life due to their involvement with lead-oriented professions. This imposes a severe threat to the growth and development of the children. Therefore to study the effect of lead, we enrolled 60 age-matched male children, from an area of old Dhaka city, where battery-recycling shops are located, depending on their blood lead concentration. If the children had a plasma lead concentration above the WHO recommended threshold level of 10 µg/dl, we grouped them as test subjects and others as control subjects to determine the effect of lead on different biochemical parameters of the body. Compared to the controls, acculumlation of the lipid peroxidation product, malondialdehyde, increased significantly in test subjects ( p < 0.01). Lead exposure also increased the protein carbonyl content ( p < 0.05) and significantly decreased the plasma glutathione levels of test subjects compared to the controls ( p < 0.05). While comparing the lead-exposed group against controls, it was found that the percentage of damaged DNA, as measured using the Comet assay, significantly increased in tail ( p < 0.01) and decreased in head regions. All of these results suggest that high-plasma lead content may induce an oxidative stress to the study population, which may lead to DNA damage.

Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

PubMed

Singh, Deepti; Narayanamoorthy, Shwetha; Gamre, Sunita; Majumdar, Ananda Guha; Goswami, Manish; Gami, Umesh; Cherian, Susan; Subramanian, Mahesh

2018-05-20

Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

PubMed Central

Singh, Mayank; Hunt, Clayton R.; Pandita, Raj K.; Kumar, Rakesh; Yang, Chin-Rang; Horikoshi, Nobuo; Bachoo, Robert; Serag, Sara; Story, Michael D.; Shay, Jerry W.; Powell, Simon N.; Gupta, Arun; Jeffery, Jessie; Pandita, Shruti; Chen, Benjamin P. C.; Deckbar, Dorothee; Löbrich, Markus; Yang, Qin; Khanna, Kum Kum; Worman, Howard J.

2013-01-01

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair. PMID:23319047

Ameliorating reactive oxygen species-induced in vitro lipid peroxidation in brain, liver, mitochondria and DNA damage by Zingiber officinale Roscoe.

PubMed

Ajith, T A

2010-01-01

Iron is an essential nutrient for a number of cellular activities. However, excess cellular iron can be toxic by producing reactive oxygen species (ROS) such as superoxide anion (O(2) (-)) and hydroxyl radical (HO(·)) that damage proteins, lipids and DNA. Mutagenic and genotoxic end products of lipid peroxidation can induce the decline of mitochondrial respiration and are associated with various human ailments including aging, neurodegenerative disorders, cancer etc. Zingiber officinale Roscoe (ginger) is a widely used spice around the world. The protective effect of aqueous ethanol extract of Z. officinale against ROS-induced in vitro lipid peroxidation and DNA damage was evaluated in this study. The lipid peroxidation was induced by hydroxyl radical generated from Fenton's reaction in rat liver and brain homogenates and mitochondrial fraction (isolated from rat liver). The DNA protection was evaluated using H(2)O(2)-induced changes in pBR-322 plasmid and Fenton reaction-induced DNA fragmentation in rat liver. The results indicated that Z. officinale significantly (P<0.001) protected the lipid peroxidation in all the tissue homogenate/mitochondria. The extract at 2 and 0.5 mg/ml could protect 92 % of the lipid peroxidation in brain homogenate and liver mitochondria respectively. The percent inhibition of lipid peroxidation at 1mg/ml of Z. officinale in the liver homogenate was 94 %. However, the extract could partially alleviate the DNA damage. The protective mechanism can be correlated to the radical scavenging property of Z. officinale. The results of the study suggest the possible nutraceutical role of Z. officinale against the oxidative stress induced human ailments.

Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage.

PubMed

Cui, Hongmei; Gu, Xinsheng; Chen, Jingshu; Xie, Ying; Ke, Sui; Wu, Jing; Golovko, Andrei; Morpurgo, Benjamin; Yan, Chunhong; Phillips, Timothy D; Xie, Wen; Luo, Jianyuan; Zhou, Zhijun; Tian, Yanan

2017-06-05

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation. Copyright © 2017 Elsevier B.V. All rights reserved.

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

PubMed

Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive

2015-06-01

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The Effect of a Grape Seed Extract on Radiation-Induced DNA Damage in Human Lymphocytes

NASA Astrophysics Data System (ADS)

Dicu, Tiberius; Postescu, Ion D.; Foriş, Vasile; Brie, Ioana; Fischer-Fodor, Eva; Cernea, Valentin; Moldovan, Mircea; Cosma, Constantin

2009-05-01

Plant-derived antioxidants due to their phenolic compounds content are reported as potential candidates for reducing the levels of oxidative stress in living organisms. Grape seed extracts are very potent antioxidants and exhibit numerous interesting pharmacologic activities. Hydroethanolic (50/50, v/v) standardized extract was obtained from red grape seed (Vitis vinifera, variety Burgund Mare—BM). The total polyphenols content was evaluated by Folin-Ciocalteu procedure and expressed as μEq Gallic Acid/ml. The aim of this study was to evaluate the potential antioxidant effects of different concentrations of BM extract against 60Co γ-rays induced DNA damage in human lymphocytes. Samples of human lymphocytes were incubated with BM extract (12.5, 25.0 and 37.5 μEq GA/ml, respectively) administered at 30 minutes before in vitro irradiation with γ-rays (2 Gy). The DNA damage and repair in lymphocytes were evaluated using alkaline comet assay. Using the lesion score, the radiation-induced DNA damage was found to be significantly different (p<0.05) from control, both in the absence and presence of BM extract (except the lymphocytes treated with 37.5 μEq GA/ml BM extract). DNA repair analyzed by incubating the irradiated cells at 37° C and 5% CO2 atmosphere for 2 h, indicated a significant difference (p<0.05) in the lymphocytes group treated with 25.0 μEq GA/ml BM extract, immediately and two hours after irradiation. These results suggest radioprotective effects after treatment with BM extract in human lymphocytes.

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelali, Ala

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional proteinmore » 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis. PMID:26273425

OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

PubMed Central

Liu, Zhong-Jie; Zhao, Wei; Zhang, Qing-Guo; Li, Le; Lai, Lu-Ying; Jiang, Shan; Xu, Shi-Yuan

2015-01-01

Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use. PMID:26161242

Interplay of space radiation and microgravity in DNA damage and DNA damage response.

PubMed

Moreno-Villanueva, María; Wong, Michael; Lu, Tao; Zhang, Ye; Wu, Honglu

2017-01-01

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose constant threat to the DNA integrity of living organisms. Specifically, space radiation can cause damage to DNA directly, through the interaction of charged particles with the DNA molecules themselves, or indirectly through the production of free radicals. Although organisms have evolved strategies on Earth to confront such damage, space environmental conditions, especially microgravity, can impact DNA repair resulting in accumulation of severe DNA lesions. Ultimately these lesions, namely double strand breaks, chromosome aberrations, micronucleus formation, or mutations, can increase the risk for adverse health effects, such as cancer. How spaceflight factors affect DNA damage and the DNA damage response has been investigated since the early days of the human space program. Over the years, these experiments have been conducted either in space or using ground-based analogs. This review summarizes the evidence for DNA damage induction by space radiation and/or microgravity as well as spaceflight-related impacts on the DNA damage response. The review also discusses the conflicting results from studies aimed at addressing the question of potential synergies between microgravity and radiation with regard to DNA damage and cellular repair processes. We conclude that further experiments need to be performed in the true space environment in order to address this critical question.

Alterations of GSH and MDA levels and their association with bee venom-induced DNA damage in human peripheral blood leukocytes.

PubMed

Gajski, Goran; Domijan, Ana-Marija; Garaj-Vrhovac, Vera

2012-07-01

Bee venom (BV) has toxic effects in a variety of cell systems and oxidative stress has been proposed as a possible mechanism of its toxicity. This study investigated the in vitro effect of BV on glutathione (GSH) and malondialdehyde (MDA) levels, and their association with BV-induced DNA strand breaks and oxidative DNA damage in human peripheral blood leukocytes (HPBLs). Blood samples were treated with BV at concentrations ranging from 0.1 to 10 μg/ml over different lengths of time, and DNA damage in HPBLs was monitored with the alkaline and formamidopyrimidine glycoslyase (FPG)-modified comet assays, while GSH and MDA levels were determined in whole blood. Results showed a significant increase in overall DNA damage and FPG-sensitive sites in DNA of HPBLs exposed to BV compared with HPBLs from controls. An increase in DNA damage (assessed with both comet assays) was significantly associated with changes in MDA and GSH levels. When pretreated with N-acetyl-L-cysteine, a source of cysteine for the synthesis of the endogenous antioxidant GSH, a significant reduction of the DNA damaging effects of BV in HPBLs was noted. This suggests that oxidative stress is at least partly responsible for the DNA damaging effects of BV. Copyright © 2012 Wiley Periodicals, Inc.

Mimosa (Mimosa caesalpiniifolia) prevents oxidative DNA damage induced by cadmium exposure in Wistar rats.

PubMed

Silva, Marcelo Jose Dias; Vilegas, Wagner; da Silva, Marcelo Aparecido; de Moura, Carolina Foot Gomes; Ribeiro, Flávia Andressa Pidone; da Silva, Victor Hugo Pereira; Ribeiro, Daniel Araki

2014-12-01

The Mimosa (Mimosa caesalpiniifolia) is a plant native from South America; it is used in the traditional medicine systems for treating bacterial, fungal, parasitic and inflammatory conditions. The aim of this study was to evaluate the antigenotoxic and antioxidant activities induced by mimosa (M. caesalpiniifolia) in multiple rodent organs subjected to intoxication with cadmium chloride. A total of 40 Wistar rats (8 weeks old, 250 g) were distributed into eight groups (n = 5), as follows: Control group (non-treated group, CTRL); Cadmium exposed group (Cd); cadmium exposure and treated with extract at 62.5 mg/kg/day; cadmium exposure and treated with extract at 125 mg/kg/day; cadmium exposure and treated with extract at 250 mg/kg/day; cadmium exposure and treated with ethyl acetate fraction at 62.5 mg/kg/day. For evaluating the toxicogenetic potential of mimosa, two groups were included in the study being treated with extract at 250 mg/kg/day and acetate fraction of mimosa at 62 mg/kg/day, only. Extract of mimosa at concentrations of 62.5 and 125 mg decreased DNA damage in animals intoxicated with cadmium when compared to cadmium group. In a similar manner, treatment with ethyl acetate fraction of mimosa at 62.5 mg concentration in animals previously exposed to cadmium reduced genetic damage in peripheral blood cells. In a similar manner, the treatment with ethyl acetate fraction reduced DNA damage in liver cells. Oxidative DNA damage was reduced to animals exposed to cadmium and treated with 125 mg of extract as well as those intoxicated to cadmium and treated with 62.5 of acetate fraction of mimosa. Taken together, our results indicate that mimosa prevents genotoxicity induced by cadmium exposure in liver and peripheral blood cells of rats as a result of antioxidant activity.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage in a skin cell model.

PubMed

Pérez-Sánchez, Almudena; Barrajón-Catalán, Enrique; Herranz-López, María; Castillo, Julián; Micol, Vicente

2016-11-01

Solar ultraviolet (UV) radiation is one of the main causes of a variety of cutaneous disorders, including photoaging and skin cancer. Its UVB component (280-315nm) leads to oxidative stress and causes inflammation, DNA damage, p53 induction and lipid and protein oxidation. Recently, an increase in the use of plant polyphenols with antioxidant and anti-inflammatory properties has emerged to protect human skin against the deleterious effects of sunlight. This study evaluates the protective effects of lemon balm extract (LBE) (Melissa Officinalis, L) and its main phenolic compound rosmarinic acid (RA) against UVB-induced damage in human keratinocytes. The LBE composition was determined by HPLC analysis coupled to photodiode array detector and ion trap mass spectrometry with electrospray ionization (HPLC-DAD-ESI-IT-MS/MS). Cell survival, ROS generation and DNA damage were determined upon UVB irradiation in the presence of LBE. The melanogenic capacity of LBE was also determined. RA and salvianolic acid derivatives were the major compounds, but caffeic acid and luteolin glucuronide were also found in LBE. LBE and RA significantly increased the survival of human keratinocytes upon UVB radiation, but LBE showed a stronger effect. LBE significantly decreased UVB-induced intracellular ROS production. Moreover, LBE reduced UV-induced DNA damage and the DNA damage response (DDR), which were measured as DNA strand breaks in the comet assay and histone H2AX activation, respectively. Finally, LBE promoted melanogenesis in the cell model. These results suggest that LBE may be considered as a candidate for the development of oral/topical photoprotective ingredients against UVB-induced skin damage. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Hypochlorite-induced damage to DNA, RNA, and polynucleotides: formation of chloramines and nitrogen-centered radicals.

PubMed

Hawkins, Clare L; Davies, Michael J

2002-01-01

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes, to give nucleoside-derived, nitrogen-centered, radicals. The latter have been characterized by EPR spin trapping. The propensity for radical formation with polynucleotides is cytidine > adenosine = guanosine > uridine = thymidine. The rates of decay, and yield of radicals formed, are dependent on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA gives rise to single- and double-strand breaks via chloramine-mediated reactions. Preformed nucleoside chloramines also induce plasmid cleavage, though this only occurs to a significant extent with unstable thymidine- and uridine-derived chloramines, where radical formation is rapid. Overall the data rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence

DNA Damage, DNA Repair, Aging, and Neurodegeneration

PubMed Central

Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

2015-01-01

Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis

PubMed Central

Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Mehta, Jawahar L.

2013-01-01

Our studies in HUVECs show that ox-LDL induced autophagy and damaged mtDNA leading to TLR9 expression. LOX-1 antibody or the ROS inhibitor apocynin attenuated ox-LDL-mediated autophagy, mtDNA damage and TLR9 expression, suggesting that these events are LOX-1 and ROS-dependent phenomena. Experiments using siRNA to DNase II indicated that DNase II digests mtDNA to protect the tissue from inflammation. Next, we studied and found intense autophagy, TLR9 expression and inflammatory signals (CD45 and CD68) in the aortas of LDLR knockout mice fed high cholesterol diet. Deletion of LOX-1 (LDLR/LOX-1 double knockout mice) attenuated autophagy, TLR9 expression as well as CD45 and CD68. Damaged mtDNA signal was also very high in LDLR knockout mice aortas, and this signal was attenuated by LOX-1 deletion. Thus, it appears that oxidative stress-mediated damaged mtDNA that escapes autophagy induces a potent inflammatory response in atherosclerosis. PMID:23326634

Bisphenol A Promotes Cell Survival Following Oxidative DNA Damage in Mouse Fibroblasts

PubMed Central

Gassman, Natalie R.; Coskun, Erdem; Stefanick, Donna F.; Horton, Julie K.; Jaruga, Pawel; Dizdaroglu, Miral; Wilson, Samuel H.

2015-01-01

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway. PMID:25693136

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells.

PubMed

Zhang, Benping; Zhao, Jie; Li, Shanshan; Zeng, Linglan; Chen, Yan; Fang, Jun

2015-04-01

Mangiferin (2-C-β-d-gluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) is a well-known natural antioxidant distributed in various plants of the Anacardiaceae and Gentianaceae families. Mangiferin can inhibit carcinogen-induced lung or colon tumor formation in experimental animals. However, the molecular mechanisms of its chemopreventive activity remain unexplored. This study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-ARE signaling in hematopoietic cells. Mononuclear cells (MNCs) were isolated from human umbilical cord blood (hUCB). DNA damage was evaluated by comet and micronucleus assays. The expression of Nrf2 and NQO1 was examined by immunofluorescence and western blotting. An electrophoretic mobility shift assay (EMSA) was used to detect the binding activity of Nrf2 with NQO1-ARE sequences. We found that mangiferin treatment significantly reduced DNA damage in etoposide-treated MNCs, which was verified by decreased olive tail moment (OTM) and micronucleus (MN) frequency. Mangiferin treatment significantly promoted Nrf2 translocation into the nucleus and increased nuclear Nrf2 expression. Moreover, NQO1, an Nrf2 signaling target, was significantly upregulated by mangiferin treatment, and the binding activity of Nrf2 with NQO1-ARE sequences was elevated after mangiferin treatment. Mangiferin activated Nrf2 signaling, upregulated NQO1 expression, and significantly reduced etoposide-induced DNA damage. Thus, mangiferin is a potential cytoprotective agent for hematopoietic cells.

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

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

Diphenylmethyl selenocyanate attenuates malachite green induced oxidative injury through antioxidation & inhibition of DNA damage in mice

PubMed Central

Das, Jayanta Kumar; Sarkar, Sibani; Hossain, Sk Ugir; Chakraborty, Pramita; Das, Rajat Kumar; Bhattacharya, Sudin

2013-01-01

Background & objectives: Malachite green (MG), an environmentally hazardous material, is used as a non permitted food colouring agent, especially in India. Selenium (Se) is an essential nutritional trace element required for animals and humans to guard against oxidative stress induced by xenobiotic compounds of diverse nature. In the present study, the role of the selenium compound diphenylmethyl selenocyanate (DMSE) was assessed on the oxidative stress (OS) induced by a food colouring agent, malachite green (MG) in vivo in mice. Methods: Swiss albino mice (Mus musculus) were intraperitoneally injected with MG at a standardized dose of 100 μg/ mouse for 30 days. DMSE was given orally at an optimum dose of 3 mg/kg b.w. in pre (15 days) and concomitant treatment schedule throughout the experimental period. The parameters viz. ALT, AST, LPO, GSH, GST, SOD, CAT, GPx, TrxR, CA, MN, MI and DNA damage have been evaluated. Results: The DMSE showed its potential to protect against MG induced hepatotoxicity by controlling the serum alanine aminotransferase and aspartate amino transferase (ALT and AST) levels and also ameliorated oxidative stress by modulating hepatic lipid peroxidation and different detoxifying and antioxidative enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and also the selenoenzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and reduced glutathione level which in turn reduced DNA damage. Interpretation & conclusions: The organo-selenium compound DMSE showed significant protection against MG induced heptotoxicity and DNA damage in murine model. Better protection was observed in pretreatment group than in the concomitant group. Further studies need to be done to understand the mechanism of action. PMID:23852297

Direct observation of ultrafast-electron-transfer reactions unravels high effectiveness of reductive DNA damage

PubMed Central

Nguyen, Jenny; Ma, Yuhan; Luo, Ting; Bristow, Robert G.; Jaffray, David A.; Lu, Qing-Bin

2011-01-01

Both water and electron-transfer reactions play important roles in chemistry, physics, biology, and the environment. Oxidative DNA damage is a well-known mechanism, whereas the relative role of reductive DNA damage is unknown. The prehydrated electron (), a novel species of electrons in water, is a fascinating species due to its fundamental importance in chemistry, biology, and the environment. is an ideal agent to observe reductive DNA damage. Here, we report both the first in situ femtosecond time-resolved laser spectroscopy measurements of ultrafast-electron-transfer (UET) reactions of with various scavengers (KNO3, isopropanol, and dimethyl sulfoxide) and the first gel electrophoresis measurements of DNA strand breaks induced by and OH• radicals co-produced by two-UV-photon photolysis of water. We strikingly found that the yield of reductive DNA strand breaks induced by each is twice the yield of oxidative DNA strand breaks induced by each OH• radical. Our results not only unravel the long-standing mystery about the relative role of radicals in inducing DNA damage under ionizing radiation, but also challenge the conventional notion that oxidative damage is the main pathway for DNA damage. The results also show the potential of femtomedicine as a new transdisciplinary frontier and the broad significance of UET reactions of in many processes in chemistry, physics, biology, and the environment. PMID:21730183

Radioprotective effects of honeybee venom (Apis mellifera) against 915-MHz microwave radiation-induced DNA damage in wistar rat lymphocytes: in vitro study.

PubMed

Gajski, Goran; Garaj-Vrhovac, Vera

2009-01-01

The aim of this study is to investigate the radioprotective effect of bee venom against DNA damage induced by 915-MHz microwave radiation (specific absorption rate of 0.6 W/kg) in Wistar rats. Whole blood lymphocytes of Wistar rats are treated with 1 microg/mL bee venom 4 hours prior to and immediately before irradiation. Standard and formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays are used to assess basal and oxidative DNA damage produced by reactive oxygen species. Bee venom shows a decrease in DNA damage compared with irradiated samples. Parameters of Fpg-modified comet assay are statistically different from controls, making this assay more sensitive and suggesting that oxidative stress is a possible mechanism of DNA damage induction. Bee venom is demonstrated to have a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom is not genotoxic and does not produce oxidative damage in the low concentrations used in this study.

Linking JNK Activity to the DNA Damage Response

PubMed Central

Picco, Vincent

2013-01-01

The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents. PMID:24349633

NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

PubMed Central

Sarker, Altaf H.; Chatterjee, Arpita; Williams, Monique; Lin, Sabrina; Havel, Christopher; Jacob III, Peyton; Boldogh, Istvan; Hazra, Tapas K.; Talbot, Prudence; Hang, Bo

2014-01-01

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. PMID:24595271

[Study on three kinds of gasoline oxygenates-induced DNA damage in mice fibroblasts].

PubMed

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.

DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages.

PubMed

Fatehi, A N; Bevers, M M; Schoevers, E; Roelen, B A J; Colenbrander, B; Gadella, B M

2006-01-01

The main goal of this study was to investigate whether and at what level damage of paternal DNA influences fertilization of oocytes and early embryonic development. We hypothesized that posttesticular sperm DNA damage will only marginally affect sperm physiology due to the lack of gene expression, but that it will affect embryo development at the stage that embryo genome (including the paternal damaged DNA) expression is initiated. To test this, we artificially induced sperm DNA damage by irradiation with x- or gamma rays (doses of 0-300 Gy). Remarkably, sperm cells survived the irradiation quite well and, when compared with nonirradiated cells, sperm motility and integrity of plasma membrane, acrosome, and mitochondria were not altered by this irradiation treatment. In contrast, a highly significant logarithmic relation between irradiation dose and induced DNA damage to sperm cells was found by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and the acridin orange assay. Despite the DNA damage, irradiated sperm cells did not show any sign of apoptosis (nuclear fragmentation, depolarization of inner mitochondrial membranes, or phospholipid scrambling) and were normally capable of fertilizing oocytes, as there was no reduction in cleavage rates when compared with nonirradiated sperm samples up to irradiation doses of less than 10 Gy. Further embryonic development was completely blocked as the blastocyst rates at days 7 and 9 dropped from 28% (nonirradiated sperm) to less than 3% by greater than 2.5-Gy-irradiated sperm. This block in embryonic development was accompanied with the initiation of apoptosis after the second or third cleavage. Specific signs of apoptosis, such as nuclear fragmentation and aberrations in spindle formation, were observed in all embryos resulting from in vitro fertilization with irradiated sperm (irradiation doses >1.25 Gy). The results show that sperm DNA damage does not impair fertilization of the

Nickel-Refining Fumes Induced DNA Damage and Apoptosis of NIH/3T3 Cells via Oxidative Stress

PubMed Central

Wang, Yue; Wang, Sheng-Yuan; Jia, Li; Zhang, Lin; Ba, Jing-Chong; Han, Dan; Yu, Cui-Ping; Wu, Yong-Hui

2016-01-01

Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. PMID:27347984

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

PubMed

Kulms, D; Pöppelmann, B; Schwarz, T

2000-05-19

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression.

PubMed

Zager, Valerija; Cemazar, Maja; Hreljac, Irena; Lah, Tamara T; Sersa, Gregor; Filipic, Metka

2010-03-01

Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

PubMed

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dizdaroglu, Miral

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 successmore » 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

Modulation of inflammation and disease tolerance by DNA damage response pathways.

PubMed

Neves-Costa, Ana; Moita, Luis F

2017-03-01

The accurate replication and repair of DNA is central to organismal survival. This process is challenged by the many factors that can change genetic information such as replication errors and direct damage to the DNA molecule by chemical and physical agents. DNA damage can also result from microorganism invasion as an integral step of their life cycle or as collateral damage from host defense mechanisms against pathogens. Here we review the complex crosstalk of DNA damage response and immune response pathways that might be evolutionarily connected and argue that DNA damage response pathways can be explored therapeutically to induce disease tolerance through the activation of tissue damage control processes. Such approach may constitute the missing pillar in the treatment of critical illnesses caused by multiple organ failure, such as sepsis and septic shock. © 2016 Federation of European Biochemical Societies.

Mitochondrial ROS Induces Cardiac Inflammation via a Pathway through mtDNA Damage in a Pneumonia-Related Sepsis Model.

PubMed

Yao, Xiao; Carlson, Deborah; Sun, Yuxiao; Ma, Lisha; Wolf, Steven E; Minei, Joseph P; Zang, Qun S

2015-01-01

We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL-1β. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL-1β in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis.

Sodium fluoride induces apoptosis in the kidney of rats through caspase-mediated pathways and DNA damage.

PubMed

Song, Guo Hua; Gao, Ji Ping; Wang, Chun Fang; Chen, Chao Yang; Yan, Xiao Yan; Guo, Min; Wang, Yu; Huang, Fu Bing

2014-09-01

Long-term excessive sodium fluoride (NaF) intake can cause many bone diseases and nonskeletal fluorosis. The kidneys are the primary organs involved in the excretion and retention of NaF. The objective of the present study was to determine the effects of NaF treatment on renal cell apoptosis, DNA damage, and the protein expression levels of cytosolic cytochrome C (Cyt C) and cleaved caspases 9, 8, and 3 in vivo. Male Sprague-Dawley rats were divided randomly into four groups (control, low fluoride, medium fluoride, and high fluoride) and administered 0, 50, 100, and 200 mg/L of NaF, respectively, via drinking water for 120 days. Histopathological changes in the kidneys were visualized using hematoxylin and eosin staining. Renal cell apoptosis was examined using flow cytometry, and renal cell DNA damage was detected using the comet assay. Cytosolic Cyt C and cleaved caspases 9, 8, and 3 protein expression levels were visualized using immunohistochemistry and Western blotting. The results showed that NaF treatment increased apoptosis and DNA damage. In addition, NaF treatment increased the protein expression levels of cytosolic Cyt C and cleaved caspases 9, 8, and 3. These results indicated that NaF induces apoptosis in the kidney of rats through caspase-mediated pathway, and DNA damage may be involved in this process.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

PubMed

Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

2017-01-01

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.