In vivo ozone exposure does not increase DNA single-strand breaks in human peripheral lymphocytes

2013 ◽  
Vol 33 (5) ◽  
pp. 517-521 ◽  
Author(s):  
C Finkenwirth ◽  
B Roßbach ◽  
HC Schröder ◽  
A Muttray
Keyword(s):  
Dna Damage ◽  
Human Lymphocytes ◽  
Ozone Exposure ◽  
Detectable Effect ◽  
Parallel Study ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Peripheral Lymphocytes ◽  
Dna Strand

In this randomized parallel study, we examined whether an acute ozone (O3) exposure leads to increased DNA strand breaks in human lymphocytes. The groups were exposed to 0.21 ppm O3 or filtered air for two hours. 30min and 4.5 h after exposure, DNA damage was determined in isolated lymphocytes using the Fast Micromethod. There was no detectable effect after O3 exposure. We conclude that an acute O3 exposure at the tested concentration does not lead to persistent DNA damage.

scholarly journals Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates

2019 ◽  
Vol 48 (3) ◽  
pp. e13-e13 ◽  
Author(s):  
Le P Ngo ◽  
Norah A Owiti ◽  
Carol Swartz ◽  
John Winters ◽  
Yang Su ◽  
...  
Keyword(s):  
Comet Assay ◽  
Dna Adducts ◽  
Excision Repair ◽  
Environmental Chemicals ◽  
Single Strand ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Bulky Dna Adducts ◽  
Dna Strand

Abstract Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

scholarly journals Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats

Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 348 ◽  
Author(s):  
Leticia Díez-Quijada ◽  
Concepción Medrano-Padial ◽  
María Llana-Ruiz-Cabello ◽  
Giorgiana M. Cătunescu ◽  
Rosario Moyano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Fatty Degeneration ◽  
Dna Strand Breaks ◽  
Histopathological Study ◽  
Strand Breaks ◽  
Dna Strand

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

Ascorbate potentiates DNA damage by 1-methyl-1-nitrosourea in vivo and generates DNA strand breaks in vitro

1987 ◽  
Vol 8 (11) ◽  
pp. 1657-1662 ◽  
Author(s):  
Paul V. Woolley ◽  
Shailendra Kumar ◽  
Peter Fitzgerald ◽  
Robert T. Simpson
Keyword(s):  
Dna Damage ◽  
Dna Strand Breaks ◽  
Strand Breaks ◽  
Dna Strand

scholarly journals Analysis of DNA damage and repair accompanying differentiation in the intestinal crypt

1998 ◽  
Vol 353 (1370) ◽  
pp. 895-902 ◽  
Author(s):  
Douglas J. Winton ◽  
Roger A. Brooks
Keyword(s):  
Dna Damage ◽  
Dna Strand Breaks ◽  
Dna Damage And Repair ◽  
Strand Breaks ◽  
Differentiated Cells ◽  
Cell Mutation ◽  
Spatial Differences ◽  
Repair Pathways ◽  
Dna Strand

The ability to process damaged DNA may vary between cells depending on their differentiated status. However, there is little in vivo data available and it is not intuitively obvious how the activity of specific repair pathways may vary between different subpopulations (e.g. stem cells and proliferative, committed and differentiated cells) of a particular tissue. To obtain such information for the intestinal epithelium, we have developed an assay that detects differences in the way different regions of the crypt (stem, proliferative and maturation zones) respond to DNA damage. The assay is a variant of the ‘comet’ assay, which detects DNA strand breaks by measuring the proportion of DNA migrating from individual cells, or in this case intact isolated crypts, in an electrophoretic field. The method is quantitative, with the amount of migrating DNA being proportional to the number of strand breaks. Isolated crypts are repair competent and spatial differences are apparent with some agents. The assay has the potential to characterize the repair properties of cells at different stages of differentiation within the crypt, determine the characteristics that might predispose them to damage and may help in understanding the route of stem cell mutation.

scholarly journals Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

2015 ◽  
Vol 291 (4) ◽  
pp. 1789-1802 ◽  
Author(s):  
Hilmar Strickfaden ◽  
Darin McDonald ◽  
Michael J. Kruhlak ◽  
Jean-Francois Haince ◽  
John P. H. Th'ng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Histone H1 ◽  
Parp Inhibition ◽  
Chromatin Decondensation ◽  
Strand Breaks ◽  
Photoactivatable Gfp ◽  
Dna Strand ◽  
Kinetics Of ◽  
Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

scholarly journals Lymphocyte dysfunction after DNA damage by toxic oxygen species. A model of immunodeficiency.

1986 ◽  
Vol 163 (3) ◽  
pp. 746-751 ◽  
Author(s):  
D A Carson ◽  
S Seto ◽  
D B Wasson
Keyword(s):  
Dna Damage ◽  
Adenosine Deaminase ◽  
Inflammatory Diseases ◽  
Human Lymphocytes ◽  
Dna Strand Breaks ◽  
Oxygen Species ◽  
Strand Breaks ◽  
Immune Impairment ◽  
Dna Strand ◽  
Resting Lymphocytes

The metabolic causes for immune impairment in patients with severe chronic inflammatory diseases have not been clearly defined. Recently, the overproduction of poly(ADP-ribose) in resting lymphocytes with unrepaired DNA strand breaks has been suggested to contribute to immune dysfunction in adenosine deaminase-deficient patients. Our experiments have determined to what extent DNA damage and poly(ADP-ribose) synthesis might also explain the impaired mitogen responsiveness of PBL exposed to toxic oxygen species. Treatment of normal resting human lymphocytes with xanthine oxidase and hypoxanthine dose-dependently induced DNA strand breaks and triggered the rapid synthesis of poly(ADP-ribose). Subsequently, NAD+ and ATP pools decreased precipitously. Lymphocytes exposed previously to the enzymatic oxidizing system did not synthesize DNA after stimulation with PHA. However, if the medium was supplemented with 3-aminobenzamide or nicotinamide, two compounds that inhibit poly(ADP-ribose) formation, cellular NAD+ and ATP pools were preserved, and the lymphocytes responded vigorously to a mitogenic challenge. Excessive poly(ADP-ribose) synthesis, provoked by DNA strand breakage, may represent a common pathway that connects the immunodeficiency syndromes associated with (a) exposure of lymphocytes to toxic oxygen species during chronic inflammatory states, (b) adenosine deaminase deficiency, and (c) certain DNA repair disorders.

scholarly journals Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

1997 ◽  
Vol 17 (9) ◽  
pp. 5437-5452 ◽  
Author(s):  
J Legault ◽  
A Tremblay ◽  
D Ramotar ◽  
M E Mirault
Keyword(s):  
Dna Damage ◽  
Single Strand ◽  
S1 Nuclease ◽  
Strand Breaks ◽  
Abasic Sites ◽  
Mouse Tissues ◽  
Hypersensitive Sites ◽  
Dna Strand ◽  
Gamma Irradiated

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.

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