Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1989
We examined the effect of exposure to H 202 at 37 °C on Chinese hamster ovary cell survival, DNA single-strand break (SSB) induction and rejoining, and activation of poly(ADP-ribose) (ADPR) polymerase. The effect of the ADPR polymerase inhibitor 3.aminobenzamide on each of these processes was also determined. SSB induction increased progressively with increasing HzO 2 concentration. SSB levels were maximal after approx. 5 rain of exposure to H202 (100/tM) and then decreased at longer times. This decrease, which paralleled the time-dependent depletion of H 202, was due to the rejoining of SSBs. 3-Am|nobenzamide enhanced the level of SSBs at each time point. H 20, increased the level of both ADPR synthesis and NAD + depletion (both measures of ADPR polymerase activity) in a concentration-dependent fashion, with the maximum effect being reached after approx. 20 rain. After 100 pM HzO 2, the effects on both ADPR and NAD + were reversible. 3-Aminobenzamide completely blocked the effects of the oxidant on both NAD + and ADPR levels. Thus, SSB induction by H 202 at 37 °C was accompanied by a marked but reversible stimulation of ADPR polymerase. However, cell killing by H zO2 was only slightly enhanced in the presence of 3-aminobenzamide (5 raM), so the above-mentioned effects do not appear to be relevant to the cytotoxic effect of H 202 under these conditions. Comparing these results with data obtained previously for cells treated with H 202 at 4°C suggests that the mechanisms of DNA strand breakage and cell killing may be quite different at the two temperatures, and that DNA damage at 37 °C may be indirectly mediated by temperature-dependent metabolic events.
Proceedings of the National Academy of Sciences of the United States of America, 1986
H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in ...
Oxidant-induced DNA damage of target cells
Journal of Clinical Investigation, 1988
In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H202 added extracellularly in micromolar concentrations (10-100 MM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H202 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H202 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase.
Nuclear DNA damage during NAD(P)H oxidation by membrane redox chains
Free Radical Biology and Medicine, 1996
Nuclear DNA damage, as the result of active oxygen formation by NAD(P)H-dependent redox chains, was studied. Isolated rat liver nuclei were incubated in the presence of NAD(P)H and iron chelators. Nuclear DNA damage was analyzed by electrophoresis in alkaline agarose. DNA damage after the addition of electron donors alone or with FeCI3 or DFO-Fe 3÷ was not visualized. Dramatic decay of high molecular weight DNA was observed with EDTA-Fe 3÷ or DTPA-Fe 3÷ in the presence of NAD(P)H. SOD did not prevent DNA damage, whereas catalase was protective. DNA damage was revealed after the addition of cumene hydroperoxide with EDTA-Fe 3÷, and it was sharply increased in the presence of NADPH. It is suggested that alkoxyl radicals in addition to hydroxyl radicals are involved in DNA damage during NAD(P)H oxidation in the presence of iron chelators, which can be reduced by membrane redox chains.
Generation of oxidative stress by the respiratory chain following treatment with DNA damaging agents
1999
The author has granted a nonexclusive licence dowing the National Library of Canada to reproduce, loan, distri'bute or sell copies of this thesis in microform, paper or electronic formats. The author retains ownership of the copyright in this thesis. Neither the thesis nor substantiai extracts f?om it may be printed or otheMrise reproduced without the author's permission. L'auteur a accorde une licence non exclusive permettant a la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique. L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thése ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.
Biochemical and Biophysical Research Communications, 1996
When human respiratory tract epithelial cells were exposed to 100 mM H 2 O 2 , there was rapid induction of DNA strand breakage and chemical modifications to all 4 DNA bases suggestive of attack by OH •. The major products were FAPy-adenine, FAPy-guanine, and 8-OH-guanine. Some of the base modifications were removed very quickly from the DNA (e.g., 8-OH-guanine), whereas others persisted for longer (e.g., thymine glycol), probably due to differential activity of different repair enzymes. By contrast, strand breaks continued to increase over the time course of the experiment, perhaps because strand breakage is also implicated in the repair process. One should therefore be cautious in using strand breakage as a sole measure of oxidative DNA damage, and when drawing conclusions about the pattern and biological significance of oxidative DNA damage in cells the relative persistence of different lesions must be considered. ᭧ 1996 Academic Press, Inc. Oxygen derived species such as the superoxide radical (O • 0 2) and hydrogen peroxide (H 2 O 2) are produced in mammalian cells during normal aerobic metabolism [1,2]. Excess generation of these species in vivo results in damage to many biological molecules including lipids, protein and carbohydrates [2-4]. Reactive oxygen species (ROS) are also thought to contribute to the development of cancer by promoting chemical changes in DNA which are potentially mutagenic [5-9]. Both DNA strand breakage [2,10-13] and modification of DNA bases [14-16] are frequently observed in cells subjected to oxidative stress. Such damage may result by a variety of mechanisms including rises in intracellular free Ca 2/ that are sufficient to activate endonucleases [17] or from direct attack on DNA by highly reactive radicals, such as hydroxyl (OH •) [6,7,18,19]. It is well established that H 2 O 2 may react with transition metal ions bound to DNA to form OH • [10,20-22] and produce a pattern of base modification very similar to that produced by ionizing radiation, an established source of OH • [7,21,14,23,24]. Consistent with direct OH • radical attack on DNA, several groups have reported increases in products of base oxidation, particularly in 8-hydroxyguanine (or 8-hydroxydeoxyguanosine) in mammalian cells exposed to oxidative stress [8,11,12,25-27]. Chemical changes in the DNA bases are of considerable importance if repair of these changes does not occur, or if repair is such that the fidelity of the original code is lost [28]. For example, formation of thymine glycol or formamidopyrimidines in the DNA template is known to cause a block in DNA replication [29,30] and 8-hydroxyguanine causes miscoding (GrC r TrA transversion mutation) [31,32]. H 2 O 2-derived radicals have been shown to cause GrC r TrA transversions in the SupF gene of E. coli [33] and UV light can cause tandem double CC r TT mutations in the p53 gene, implicated in the development of squamous cell
Free Radical Biology and Medicine, 2002
Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H 2 O 2 by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H 2 O 2 was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 M H 2 O 2 . As the H 2 O 2 was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 l growth medium (containing 1.5 ϫ 10 5 cells) generated 2.0 Ϯ 0.2 M H 2 O 2 per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H 2 O 2 were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H 2 O 2 itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H 2 O 2 , it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1989
Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.
International Journal of Advances in Scientific Research and Engineering, 2022
Oxidative stress-induced reactive oxygen species induce DNA damages that may result in cell death and DNA mutations. In this study, a genotoxic and cytotoxic potential of hydrogen peroxide as a ROS generator, and the effect of cell repair of oxidative stress in TK6 cells were investigated by in vitro micronucleus assay. Cell repair was inhibited by using cytosine arabinoside. Cellular responses to different concentrations of hydrogen peroxide were evaluated by generating dose-response curves. The incidence of increased MN concentrations due to increased H 2 O 2 concentrations did not allow the identification of a plateau region. A similar curve pattern was also observed with the repair inhibited group. However, damage caused by H 2 O 2 was significantly higher (p<0.001) in the cells with repair inhibition. This demonstrates the importance of DNA repair in the observation of oxidative stress. Proliferative indexes did not change due to increasing H 2 O 2 concentrations in both groups. But the curve of repair inhibited cells was significantly higher (p<0.001) than the curve of repair efficient cells. BN and multinucleate cell counts showed decreases with increasing ROS levels generated by H 2 O 2. But these decreases were not reflected in proliferative index scores. This may be due to H 2 O 2-induced apoptosis, which results in the overestimation of PI values in increasing H 2 O 2 concentrations.