scholarly journals Automodified Poly(ADP-Ribose) Polymerase Analysis to monitor DNA Damage in Peripheral Lymphocytes of Floricoltorists occupationally exposed to Pesticides

2019 ◽  
Author(s):  
Serena Imperato ◽  
Carmela Mistretta ◽  
Maria Marone ◽  
Ilaria Migliaccio ◽  
Ilaria Pulcinelli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genotoxic Stress ◽  
Polymerase Activity ◽  
Peripheral Blood Lymphocytes ◽  
Activity Levels ◽  
Strand Breaks ◽  
Non Invasive ◽  
Occupationally Exposed ◽  
Parp Activity ◽  
Dna Strand

Background: Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Methods: Peripheral lymphocyte lysates were analysed for Poly(ADP-Ribose) Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibody to measure automodified anti-Poly(ADP-Ribose) Polymerase levels by densitometry. Results: Poly(ADP-Ribose)Polymerase activity levels were consistent with those of enzyme auto-modification. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. Conclusions: PARP activity and auto-modification in peripheral blood lymphocytes are possible,non-invasive, and routinar tools to monitor the healthy conditions of floricoltorists.

scholarly journals Automodified Poly(ADP-Ribose) Polymerase Analysisto Monitor DNA Damagein Peripheral Lymphocytes of Floriculturists Occupationally Exposed to Pesticides

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 137 ◽  
Author(s):  
Serena Imperato ◽  
Carmela Mistretta ◽  
Maria Marone ◽  
Ilaria Migliaccio ◽  
Ilaria Pulcinelli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genotoxic Stress ◽  
Polymerase Activity ◽  
Peripheral Blood Lymphocytes ◽  
Strand Breaks ◽  
Non Invasive ◽  
Sds Page ◽  
Occupationally Exposed ◽  
Parp Activity ◽  
Dna Strand

Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Peripheral lymphocyte lysates were analyzed for Poly(ADP-Ribose)Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibodyto measure automodified Poly(ADP-Ribose)Polymerase levels bydensitometry. Poly(ADP-Ribose)Polymerase activity and PARP automodification followed the same trend. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, androutinartools to monitor the healthy conditions of floricoltorists.

scholarly journals Mechanistic insight into the role of Poly(ADP-ribosyl)ation in DNA topology modulation and response to DNA damage

Mutagenesis ◽  
2019 ◽  
Vol 35 (1) ◽  
pp. 107-118
Author(s):  
Bakhyt T Matkarimov ◽  
Dmitry O Zharkov ◽  
Murat K Saparbaev
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genotoxic Stress ◽  
Strand Break ◽  
Dna Supercoiling ◽  
Dna Strand Breaks ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Strand

Abstract Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.

Evaluation of genotoxic potential of commonly used organophosphate pesticides in peripheral blood lymphocytes of rats

2014 ◽  
Vol 34 (4) ◽  
pp. 390-400 ◽  
Author(s):  
A Ojha ◽  
YK Gupta
Keyword(s):  
Dna Damage ◽  
Damage Index ◽  
Fluorescence Emission ◽  
Strand Break ◽  
Peripheral Blood Lymphocytes ◽  
Dependent Manner ◽  
Strand Breaks ◽  
Dna Strand Break ◽  
Cross Links ◽  
Dna Strand

Chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are among the most extensively used organophosphate (OP) pesticides in India. DNA protein cross-links (DPC) and DNA strand breaks are toxic lesions associated with the mechanism(s) of toxicity of carcinogenic compounds. In the present study, we examined the hypothesis that individual and interactive genotoxic effects of CPF, MPT, and MLT are involved in the formation of DPC and DNA strand break. The DNA strand break was measured by comet assay and expressed as DNA damage index, while DPC estimation was carried out by fluorescence emission assay. The results showed that exposure of rat lymphocytes with CPF, MPT, and MLT caused significantly marked increase in DNA damage and DPC formation in time-dependent manner. MPT caused the highest damage, and these pesticides do not potentiate the toxicity of each other.

scholarly journals Increased activity of poly(ADP-ribose)-polymerase in PML-depleted cells - novel perspectives for cancer therapy

2013 ◽  
Vol 94 (1) ◽  
pp. 75-79
Author(s):  
S V Boichuk ◽  
B R Ramazanov ◽  
I G Mustafin ◽  
Gjoerup O
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Genotoxic Stress ◽  
Dna Double Strand Breaks ◽  
Compensatory Response ◽  
Strand Breaks ◽  
Fetal Bovine ◽  
Parp Activity ◽  
The Relationship ◽  
Increased Activity

Aim. To investigate the relationship between PML expression and poly(ADP-ribose)-polymerase (PARP) activity in physiological conditions and at genotoxic stress induced by chemotherapy and ionizing radiation. Methods. The study was conducted on BJ fibroblasts cultured in DMEM/199 medium supplemented with fetal bovine serum, L-glutamine and antibiotics. PML down-regulation was achieved by short interfering ribonucleic acid transfection. To induce deoxyribonucleic acid (DNA) damage in BJ fibroblasts, doxorubicin and hydroxyurea or ionizing radiation were used. PARP activity, formation of DNA double-strand breaks and DNA damage response were examined by Western blotting and immunofluorescence microscopy. Results. PML knockdown was accomplished with an increased PARP activity, confirmed by an increased expression of poly-ADP-ribose (PAR) polymers. At PML knockdown ant DNA damage caused by chemotherapy and ionizing radiation, there is a significant increase in PAR polymers expression as well as increase in the number of cells containing PAR foci. Conclusion. Increased activity of poly(ADP-ribose)-polymerase at PML knockdown and DNA damaging conditions indicates the compensatory response due to insufficiency of the homologous recombination mechanisms. The phenomenon found widens the spectrum of malignancies that might be potentially sensitive to the therapy with poly(ADP-ribose)-polymerase inhibitors.

scholarly journals Inhibition of poly(ADP-ribose) polymerase activity affects its subcellular localization and DNA strand break rejoining.

2009 ◽  
Vol 56 (2) ◽  
Author(s):  
Nadezhda I Ryabokon ◽  
Artur Cieślar-Pobuda ◽  
Joanna Rzeszowska-Wolny
Keyword(s):  
Dna Repair ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Parp Inhibitors ◽  
Polymerase Activity ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Dna Strand Break ◽  
Parp Activity ◽  
Dna Strand

Poly(ADP-ribose) polymerase (PARP) plays a crucial role in DNA repair. Modulation of its activity by stimulation or inhibition is considered as a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radiotherapy through inhibition of DNA repair. Here we studied the effect of the three PARP inhibitors, 5-iodo-6-amino-benzopyrone (INH(2)BP), 1,5-isoquinolinediol (1,5-dihydroxyisoquinolinediol (1,5-IQD) and 8-hydroxy-2-methylquinazolin-4-[3H]one (NU1025), and for two of them the efficiency in slowing the rejoining of DNA strand breaks induced by H(2)O(2) was compared. Inhibition of PARP changed its intranuclear localization markedly; cells exposed to the inhibitor NU1025 showed a significant tendency to accumulate PARP in large foci, whereas in untreated cells its distribution was more uniform. The speed and efficiency of rejoining of H(2)O(2)-induced DNA strand breaks were lower in cells incubated with a PARP inhibitor, and the kinetics of rejoining were modulated in a different manner by each inhibitor. At a concentration of 100 microM the efficiency of the inhibitors could be ranked in the order NU1025 > IQD > INH(2)BP. The two first compounds were able to decrease the overall PARP activity below the level detected in control cells, while INH(2)BP showed up to 40% PARP activity after exposure to H(2)O(2).

scholarly journals Repair of DNA Strand Breaks by the Overlapping Functions of Lesion-Specific and Non-Lesion-Specific DNA 3′ Phosphatases

2001 ◽  
Vol 21 (21) ◽  
pp. 7191-7198 ◽  
Author(s):  
John R. Vance ◽  
Thomas E. Wilson
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Synthetic Lethality ◽  
Dna Strand Breaks ◽  
Triple Mutant ◽  
Phosphatase Domain ◽  
Strand Breaks ◽  
Alternative Pathways ◽  
Processing Enzymes ◽  
Dna Strand

ABSTRACT In Saccharomyces cerevisiae, the apurinic/apyrimidinic (AP) endonucleases Apn1 and Apn2 act as alternative pathways for the removal of various 3′-terminal blocking lesions from DNA strand breaks and in the repair of abasic sites, which both result from oxidative DNA damage. Here we demonstrate that Tpp1, a homologue of the 3′ phosphatase domain of polynucleotide kinase, is a third member of this group of redundant 3′ processing enzymes. Unlike Apn1 and Apn2, Tpp1 is specific for the removal of 3′ phosphates at strand breaks and does not possess more general 3′ phosphodiesterase, exonuclease, or AP endonuclease activities. Deletion ofTPP1 in an apn1 apn2 mutant background dramatically increased the sensitivity of the double mutant to DNA damage caused by H2O2 and bleomycin but not to damage caused by methyl methanesulfonate. The triple mutant was also deficient in the repair of 3′ phosphate lesions left by Tdp1-mediated cleavage of camptothecin-stabilized Top1-DNA covalent complexes. Finally, the tpp1 apn1 apn2 triple mutation displayed synthetic lethality in combination with rad52, possibly implicating postreplication repair in the removal of unrepaired 3′-terminal lesions resulting from endogenous damage. Taken together, these results demonstrate a clear role for the lesion-specific enzyme, Tpp1, in the repair of a subset of DNA strand breaks.

scholarly journals Engineered Nanoparticles Induce DNA Damage in Primary Human Skin Cells, Even at Low Doses

Nano LIFE ◽  
2014 ◽  
Vol 04 (01) ◽  
pp. 1440001 ◽  
Author(s):  
Amelia A. Romoser ◽  
Michael F. Criscitiello ◽  
Christie M. Sayes
Keyword(s):  
Dna Damage ◽  
Stress Protein ◽  
Dermal Fibroblasts ◽  
Engineered Nanoparticles ◽  
Ros Generation ◽  
Heme Oxygenase 1 ◽  
Dna Double Strand Breaks ◽  
Biological Reduction ◽  
Strand Breaks ◽  
Dna Strand

It is well documented that various particulate matter — either incidental or engineered — are known to generate reactive oxygen species (ROS) in living cells. In circumstances where these reactive species are generated, antioxidant production is often increased. This balance in the biological reduction/oxidation (a.k.a. redox) state within the cell has not been thoroughly studied in exposures involving engineered nanoparticles. However, nanoparticle exposure has been postulated to induce a DNA damage cascade. In this study, we examined primary human dermal fibroblasts (HDF) exposed to three different, but commonly used engineered nanoparticles (i.e., cerium dioxide ( CeO 2), titanium dioxide ( TiO 2) and zinc oxide ( ZnO )) in an attempt to determine the potential DNA damaging effects through the analysis of ROS generation, relevant protein upregulation response and single and double DNA strand breaks. Cell death was most elevated with exposure to ZnO , followed by TiO 2 and CeO 2. ROS generation was measured at 1 h, 6 h and 24 h after exposure to particles via a cell-based DCFH-DA (2′, 7′-dichlorfluorescein-diacetate) assay and indicated that ZnO generated the most significant amount of ROS. ZnO also caused upregulation of oxidative stress protein, heme oxygenase-1 and phosphorylation of p38; whereas CeO 2 caused upregulation of superoxide dismutase. Results from the comet assay indicated that ZnO triggered significant DNA damage in cells at relatively low dosing concentrations (20 ppm). Immunocytochemistry with ZnO -treated cells revealed notable DNA double strand breaks evidenced by a marked increase in the presence of γ-H2AX foci. This finding was also indicated by western blot, as well as cell cycle arrest by the phosphorylation of cyclin-dependent kinase 1. These data suggest that the three particle-types induce different degrees of DNA damage. And, of the three particle-types tested, exposure to ZnO nanoparticles may cause the most significant DNA damage.

scholarly journals Evaluation of the Protective Effects of Quercetin, Rutin, Naringenin, Resveratrol and Trolox Against Idarubicin-Induced DNA Damage

2010 ◽  
Vol 13 (2) ◽  
pp. 231 ◽  
Author(s):  
Haydar Çelik ◽  
Emel Arinç
Keyword(s):  
Dna Damage ◽  
Cytochrome P450 ◽  
Mitomycin C ◽  
Dna Strand Breaks ◽  
Protective Effects ◽  
Cytochrome P450 Reductase ◽  
Strand Breaks ◽  
P450 Reductase ◽  
Nadph Cytochrome P450 Reductase ◽  
Dna Strand

PURPOSE. Idarubicin is a synthetic anthracycline anticancer drug widely used in the treatment of some hematological malignancies. The studies in our laboratory have clearly demonstrated that idarubicin can undergo reductive bioactivation by NADPH-cytochrome P450 reductase to free radicals with resulting formation of DNA strand breaks, which can potentially contribute to its genotoxic effects [Çelik, H., Arinç, E., Bioreduction of idarubicin and formation of ROS responsible for DNA cleavage by NADPH-cytochrome P450 reductase and its potential role in the antitumor effect. J Pharm Pharm Sci, 11(4):68-82, 2008]. In the current study, our aim was to investigate the possible protective effects of several phenolic antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, against the DNA-damaging effect of idarubicin originating from its P450 reductase-catalyzed bioactivation. METHODS. DNA damage was measured by detecting single-strand breaks in plasmid pBR322 DNA using a cell-free agarose gel method. RESULTS. Our results indicated that, among the compounds tested, quercetin was the most potent antioxidant in preventing DNA damage. Quercetin significantly decreased the extent of DNA strand breaks in a dose-dependent manner; 100 μM of quercetin almost completely inhibited the DNA strand breakage. Unlike quercetin, its glycosidated conjugate rutin, failed to provide any significant protection against idarubicin-induced DNA strand breaks except at the highest concentration tested (2 mM). The protective effects of other antioxidants were significantly less than that of quercetin even at high concentrations. Quercetin was found to be also an effective protector against DNA damage induced by mitomycin C. CONCLUSION. We conclude that quercetin, one of the most abundant flavonoids in the human diet, is highly effective in reducing the DNA damage caused by the antitumor agents, idarubicin and mitomycin C, following bioactivation by P450 reductase.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1174-L1180 ◽  
Author(s):  
M. Takeoka ◽  
W. F. Ward ◽  
H. Pollack ◽  
D. W. Kamp ◽  
R. J. Panos
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Epithelial Cells ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Alveolar Epithelial ◽  
Radiation Induced ◽  
Dna Strand

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

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