Factors which affect DNA strand breakage in human leukocytes exposed to a tumor promoter, phorbol myristate acetate

1982 ◽  
Vol 60 (11) ◽  
pp. 1359-1366 ◽  
Author(s):  
H. C. Birnboim
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Phorbol Myristate Acetate ◽  
Respiratory Burst ◽  
Superoxide Anion ◽  
Tumor Promoter ◽  
Myristate Acetate ◽  
Strand Breakage ◽  
Dna Strand ◽  
Dna Strand Breakage

We have recently reported that phorbol myristate acetate (PMA) induces extensive DNA strand break damage in human peripheral blood leukocytes. The mechanism of action involves superoxide anion and hydrogen peroxide which are generated by phagocytes during the "respiratory burst." In this report, we describe the effect of various inhibitors and scavengers on PMA-induced DNA damage. Azide and cyanide greatly increased the level of damage; sulfhydryl compounds (glutathione, cysteine, and cysteamine) and ascorbate markedly decreased the level of damage. Hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and glycerol also decreased the level of damage but apparently did so by inhibiting the respiratory burst. Diethyldithiocarbamate (DDC) increased the level of DNA damage at low concentrations (<1 mM), but decreased DNA damage at ≥1 mM. The results are consistent with a mechanism involving superoxide anion and hydrogen peroxide, but the precise reaction (free radical or enzymatic) responsible for DNA strand breakage has not been determined. The PMA-stimulated phagocyte is an interesting model system for looking at "active oxygen" mediated DNA damage and factors which influence it.

Oxidative DNA Damage in Human Respiratory Tract Epithelial Cells. Time Course in Relation to DNA Strand Breakage

1996 ◽  
Vol 224 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Jeremy P.E. Spencer ◽  
Andrew Jenner ◽  
Okezie I. Aruoma ◽  
Carroll E. Cross ◽  
Reen Wu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Epithelial Cells ◽  
Respiratory Tract ◽  
Time Course ◽  
Oxidative Dna Damage ◽  
Human Respiratory Tract ◽  
Strand Breakage ◽  
Dna Strand ◽  
Dna Strand Breakage

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

1989 ◽  
Vol 67 (2) ◽  
pp. 556-562 ◽  
Author(s):  
D. W. Kamp ◽  
K. D. Bauer ◽  
A. Knap ◽  
M. M. Dunn
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Myristate Acetate ◽  
Superoxide Anion ◽  
Leukocyte Adherence ◽  
Myristate Acetate ◽  
Kinase C ◽  
Monocyte Adherence

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Comet Assay: A Strong Tool for Evaluating DNA Damage and Comprehensive Guidelines for Plant Cells

2017 ◽  
Vol 3 (02) ◽  
pp. 65-72
Author(s):  
Ashish Agnihotri ◽  
Chandra Shekhar Seth
Keyword(s):  
Heavy Metals ◽  
Dna Damage ◽  
Comet Assay ◽  
Single Cells ◽  
Strand Breakage ◽  
Dna Strand ◽  
Environmental Genotoxicity ◽  
Genetic Ecotoxicology ◽  
Detailed Protocol ◽  
Dna Strand Breakage

Heavy metals affect plant system in various toxic ways including morphophysiological alterations and genotoxic damages inside a plant cell. The extent of DNA damage under any genotoxic agents can be effectively measured in single cells applying comet assay approach. Comet assay primarily measures DNA strand breakage in single cells and its use has increased in different areas: clinical applications, human monitoring, radiation biology and genetic ecotoxicology. This paper is a review of the detailed protocol and precautions to be taken while performing comet assay and may have been slightly modified from other original protocols according to the plant, organ, cell type, etc. In conclusion, the study reviewed in this paper demonstrate that the comet assay application in plants provides a reliable, sensitive and rapid system for the study of environmental genotoxicity caused by heavy metals.

scholarly journals DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells

FEBS Letters ◽  
1995 ◽  
Vol 374 (2) ◽  
pp. 233-236 ◽  
Author(s):  
Jeremy P.E. Spencer ◽  
Andrew Jenner ◽  
Ken Chimel ◽  
Okezie I. Aruoma ◽  
Carroll E. Cross ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Epithelial Cells ◽  
Respiratory Tract ◽  
Human Respiratory Tract ◽  
Base Modification ◽  
Hydrogen Peroxide Treatment ◽  
Strand Breakage ◽  
Dna Strand ◽  
Dna Strand Breakage

scholarly journals Priming of macrophages for enhanced oxidative metabolism by exposure to proteolytic enzymes.

1981 ◽  
Vol 153 (6) ◽  
pp. 1678-1683 ◽  
Author(s):  
R B Johnston ◽  
D A Chadwick ◽  
Z A Cohn
Keyword(s):  
Hydrogen Peroxide ◽  
Phorbol Myristate Acetate ◽  
Oxidative Metabolism ◽  
Tissue Damage ◽  
Superoxide Anion ◽  
Candida Parapsilosis ◽  
Proteolytic Enzymes ◽  
Myristate Acetate ◽  
Oxygen Metabolites

Preincubation for 10-30 min with trypsin, pronase, chymotrypsin, or papain primed macrophages to undergo a twofold to sixfold increase in oxidative metabolism, measured as release of superoxide anion or hydrogen peroxide, during stimulation by phorbol myristate acetate or ingestion of Candida parapsilosis. Preincubation of macrophages with inactivated proteases, nonenzyme proteins, or neuraminidase did not affect their oxidase response. Exposure of macrophages to proteases generated at sites of inflammation could prime these cells for a more effective oxidase response to phagocytosis or for greater tissue damage from release of toxic oxygen metabolites.

scholarly journals Hydrogen peroxide-induced DNA damage is independent of nuclear calcium but dependent on redox-active ions

1998 ◽  
Vol 335 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Lan JORNOT ◽  
Hilke PETERSEN ◽  
Alain F. JUNOD
Keyword(s):  
Dna Damage ◽  
Oh Radicals ◽  
Free System ◽  
Nuclear Calcium ◽  
Iron Copper ◽  
Strand Breakage ◽  
Copper Chelator ◽  
Dna Strand ◽  
Dna Strand Breakage ◽  
In Cells

In cells undergoing oxidative stress, DNA damage may result from attack by •OH radicals produced by the Fenton reaction, and/or by nucleases activated by nuclear calcium. In the present study, the participation of these two mechanisms was investigated in HeLa cells. Nuclear-targeted aequorin was used for selectively monitoring Ca2+ concentrations within the nuclei ([Ca2+]n), in conjunction with the cell-permeant calcium chelator bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetraacetic acid acetoxymethyl ester (BAPTA/AM), the lipid-soluble broad-spectrum metal chelator with low affinity for Ca2+ and Mg2+ N,N,N´,N´-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and the high-affinity iron/copper chelator 1,10-phenanthroline (PHE). In Ca2+-containing medium, H2O2 induced extensive DNA strand breaks and an increase in [Ca2+]n that was almost identical to that observed in the cytosol ([Ca2+]c). In cells bathed in Ca2+-free/EGTA medium, in which the increases in [Ca2+]n and [Ca2+]c due to H2O2 were significantly reduced, similar levels of DNA fragmentation also occurred. In cells preloaded with BAPTA/AM or TPEN, the small increase of [Ca2+]n normally elicited by H2O2 in Ca2+-free medium was completely buffered, and DNA damage was largely prevented. On the other hand, pretreatment with PHE did not affect the calcium response in the nuclei, but completely prevented DNA strand breakage induced by H2O2. Re-addition of 100 µM CuSO4 and 100 µM FeSO4 to TPEN- and PHE-treated cells prior to H2O2 challenge reversed the effect of TPEN and PHE, whereas 1 mM was necessary to negate the effect of BAPTA/AM. The levels of DNA strand breakage observed, however, did not correlate with the amounts of 8-hydroxy 2´-deoxyguanosine (8-OHdG): H2O2 did not produce 8-OHdG, whereas PHE alone slightly increased 8-OHdG levels. CuSO4 and FeSO4 enhanced the effects of PHE, particularly in the presence of H2O2. Exposure of cells to a mixture of CuSO4/FeSO4 also resulted in a significant increase in 8-OHdG levels, which was prevented in cells preloaded with BAPTA/AM. Similar results were obtained in a cell-free system using isolated calf thymus DNA exposed to CuSO4/FeSO4, regardless of whether H2O2 was present or not. These results suggest that BAPTA/AM prevents H2O2-induced DNA damage by acting as an iron/copper chelator. These data also indicate that caution must be exercised in using Ca2+ chelating agents as evidence for a role in cellular Ca2+ levels in experimental conditions in which transition-metal-ion-mediated oxidant production is also occurring.

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