Affect DNA Damage Research Articles

Inhibitory effect of Eucommia ulmoides Oliv. on oxidative DNA damage in lymphocytes induced by H2O2.

This study used the alkaline single cell gel electrophoresis assay (comet assay) to investigate the effect of water extracts of roasted cortex and leaves from Du-zhong on DNA damage in lymphocytes induced by H(2)O(2). The results showed that the DNA damage in human lymphocytes increased with an increase in the concentration of H(2)O(2) (0-200 micro;M), but that the water extracts from Du-zhong (0-2 g l(-1)) only slightly affected DNA damage. The inhibitory effect of leaf extract on DNA damage induced by H(2)O(2) in lymphocytes was more significant (P<0.05) than that of roasted cortex. Leaf extract showed a rather significant inhibitory effect in a concentration-dependent manner. At a concentration of 2 g l(-1), the leaf extract inhibited 37.9% DNA oxidative damage in human lymphocytes. In order to elucidate the mechanism of the leaf extract suppression effect on DNA damage induced by H(2)O(2) in lymphocytes, an experiment was divided with six groups (A-F). Group A was used to evaluate the repair ability of the leaf extract for DNA damage; Group B was employed to determine the scavenging ability on H(2)O(2); and Group C was studied to assess the ability of leaf extract to increase the defense capability. Groups D-F were negative controls and blank. The results showed that group B had the best inhibitory effect. Also, leaf extract had significant ability to scavenge H(2)O(2) in an in vitro HRP-phenol red test. Thus, it appears that H(2)O(2) scavenging potency may be the major mechanism whereby leaf extract inhibits oxidative DNA damage induced by H(2)O(2).

P62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

Antioxidant and pro-oxidant effects of lycopene in comparison with β-carotene on oxidant-induced damage in Hs68 cells

Lycopene has become a focal point in recent research following clinical trials that suggest that β-carotene may promote lung cancer in smokers. Because lycopene only differs from β-carotene in lacking the β-ionone structure, and β-carotene is known to have pro-oxidant activity in vitro, we sought to determine whether lycopene might also have pro-oxidant activity in vitro in comparison with β-carotene. Human foreskin fibroblasts (Hs68 cells) were first enriched with 10 and 20 μM lycopene or β-carotene for 1 hr followed by incubation with various oxidants. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium and DNA damage was measured as formation of comet and 8-hydroxy-2′-deoxyguanosine. The results showed that lycopene at 20 μM significantly decreased levels of TBARS induced by ferric nitrilotriacetate (Fe/NTA) but enhanced levels of TBARS induced by a lipid-soluble radical generator (2,2′-azobis[2,4-dimethylvaleronitrile]; AMVN). Both the antioxidant and pro-oxidant effects of lycopene tended to be dose-dependent. β-Carotene at 20 μM did not significantly decrease TBARS induced by Fe/NTA but significantly increased TBARS induced by AMVN. Lipid peroxidation induced by a water-soluble radical generator 2,2’-azobis(2-amidinopropane)dihydrochloride was not significantly affected by either lycopene or β-carotene. Neither lycopene nor β-carotene affected DNA damage or changes in cell morphology induced by any of the three oxidants tested. The present study in Hs68 cells demonstrates that lycopene can be either an antioxidant or a pro-oxidant depending on the oxidants used, and that lycopene and β-carotene behave similarly under the in vitro oxidative conditions. Although it is unclear whether lycopene may have pro-oxidant activity in vivo, our results caution that it may be premature to undertake clinical trials with lycopene.

Homologous and non-homologous recombination differentially affect DNA damage repair in mice

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.

36] Gas chromatography-mass spectrometry analysis of DNA: Optimization of protocols for isolation and analysis of DNA from human blood

This chapter shows that 5 ml of whole blood, or packed cells from this volume, is required to extract 100μg DNA, which allows easy analysis of base damage by GC/MS, and storing blood at -20 ° for up to 1 year does not affect DNA damage. Although the DNA extraction protocol followed in this study requires removal of RNA to prevent artifacts in DNA base damage determination, showing a lack of effect with all the other conditions tested, it is not necessary that every DNA extraction procedure follows this pattern. The optimal protocol may depend on sources of reagents and the general level of contamination with prooxidants (e.g., transition metal ions in the water and reagents) in the laboratory concerned. It has subsequently applied this protocol to the investigation of factors affecting oxidative DNA damage in the human body. It is found that steady-state levels of oxidative DNA damage varied widely among healthy individuals. This may be related to a number of endogenous and exogenous factors, including genetic predisposition, exposure to environmental toxins, and antioxidant intake. Levels of DNA damage and repair is also influenced by endogenous rates of free radical production and level of antioxidant defenses, as well as the activity of repair enzymes and the ability to respond positively to increased levels of oxidative stress, including the absorption, processing, and transport of dietary antioxidants to the site of oxidative stress. Collectively, such factors contribute to the overall levels of oxidative stress in the human body and thus DNA damage and, in the long term, perhaps susceptibility to cancer.

Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay

Objective: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. Design: In vitro study. Setting: Academic medical center. Patient(s): None. Intervention(s): None. Main Outcome Measure(s): Plasmid relaxation. Result(s): Ham’s F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. Conclusion(s): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Induction of DNA breakage in X-irradiated nucleoids selectively stripped of nuclear proteins in two mouse lymphoma cell lines differing in radiosensitivity.

The role of nuclear proteins in protection of DNA against ionizing radiation and their contribution to the radiation sensitivity was examined by an alkaline version of comet assay in two L5178Y (LY) mouse lymphoma cell lines differing in sensitivity to ionizing radiation. LY-S cells are twice more sensitive to ionizing radiation than LY-R cells (D0 values of survival curves are 0.5 Gy and 1 Gy, respectively). Sequential removal of nuclear proteins by extraction with NaCl of different concentrations increased the X-ray induced DNA damage in LY-R nucleoids. In contrast, in the radiation sensitive LY-S cell line, depletion of nuclear proteins practically did not affect DNA damage. Although there is no doubt that the main cause of LYS cells' sensitivity to ionizing radiation is a defect in the repair of double-strand breaks, our data support the concept that nuclear matrix organisation may contribute to the cellular susceptibility to DNA damaging agents.

P4C30 - Paternal exposure to cyclophosphamide (CPA) affects DNA damage response/repair gene expression in rat embryos

P4C30 - Paternal exposure to cyclophosphamide (CPA) affects DNA damage response/repair gene expression in rat embryos

Mechanism of the antimycin A-mediated enhancement of t-butylhydroperoxide-induced single-strand breakage in DNA.

Inhibitors of complex III increased the DNA strand scission induced by t-butylhydroperoxide (tB-OOH) and cumene hydroperoxide but did not affect DNA damage induced by H2O2. The hypothesis that these effects are selectively linked to inhibition of the electron transport from cytochrome b to cytochrome c1 is validated by the following observations: (1) two complex III inhibitors, antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, enhanced the tB-OOH-induced DNA cleavage over the same concentration range as that in which inhibition of oxygen consumption was observed; (2) the complex III inhibitor-mediated enhancement of tB-OOH-induced DNA damage was abolished by the complex I inhibitor rotenone or by glucose omission, and (3) the enhancing effects of antimycin A were not observed in respiration-deficient cells. The mechanism whereby the complex III inhibitors potentiate DNA cleavage promoted by tB-OOH was subsequently investigated with intact as well as permeabilized cells. H2O2, produced at the level of mitochondria via a Ca2+-dependent process, was found to account for the enhancing effects of antimycin A.

Transient increase in DNA strand breaks in car refinishing spray painters.

Genotoxic risk was evaluated for spray painters possibly exposed to polyester resins and acrylic enamel-based paints in automotive body repair shops. DNA (deoxyribonucleic acid) strand breaks and alkali-labile sites were measured in peripheral mononuclear blood cells ex vivo using the alkaline elution method. Samples of venous blood were taken on Monday after a free weekend and again on Friday from 38 male and 1 female spray painters and compared with the blood samples from 36 male and 3 female referents. The elution rate of each DNA sample was standardized by dividing it by the elution rate obtained from simultaneously sampled untreated Chinese hamster V79 cells. The spray painters showed a significantly (P < 0.001) higher mean level of strand breaks and alkali-labile sites in the Friday samples [2.05 (SE 0.17)] compared with the Monday samples [1.38 (SE 0.07)]. The Monday results of the spray painters were not distinguishable from the referents' [1.41 (SE 0.10)]. The increase in DNA damage was numerically higher, but only weakly significant (use of masks, P < 0.05) or nonsignificant (use of spray booths), when fewer safety provisions were taken. A significant increase in DNA strand breaks and alkali-labile sites was found in spray painters after a week's work. However, DNA damage seems to be reversible. The use of modern safety equipment seems to affect DNA damage only marginally. There is an urgent need to identify the genotoxic chemicals in the occupational environment of spray painters and to develop corresponding satisfactory safety measures.

The respiratory-chain poison antimycin A promotes the formation of DNA single-strand breaks and reduces toxicity in U937 cells exposed to t-butylhydroperoxide

Antimycin A at levels that abolish oxygen consumption had a slight, although statistically significant, inhibitory effect on the toxicity elicited by t-butylhydroperoxide in U937 cells. The protective effect was observed after 6 h of post-treatment incubation, but was no longer apparent after 24 h. Unexpectedly, these events were associated with a marked accumulation of DNA single-strand breaks produced by low levels of t-butylhydroperoxide. Both an oxygen- and a carbon-centred radical were found to arise during treatment with t-butylhydroperoxide, and their formation was significantly lowered by antimycin A. Thus inhibition of electron transport at the level of complex III appears (a) to decrease the formation of toxic species which mediate, at least partially, the lethal effects elicited by t-butylhydroperoxide, and (b) to enhance the formation of DNA-damaging species generated at low concentrations of t-butylhydroperoxide. Rotenone and cyanide, which respectively inhibit complexes I and IV, did not affect DNA damage elicited by t-butylhydroperoxide. These results suggest that DNA single-strand breaks do not mediate the toxicity of t-butylhydroperoxide, and that specific mitochondrial functions might modulate the formation of the toxic and of DNA-damaging species generated by organic hydroperoxides.

Factors affecting DNA damage caused by lipid hydroperoxides and aldehydes

Single (SSB) and double strand breaks (DSB) in supercoiled plasmid DNA pBR322 reacted with linoleic acid hydroperoxides (LOOH) were followed by agarose gel electrophoresis to obtain definitive information about factors affecting LOOH interaction with DNA. In water, LOOH induced extensive DSB, which were metal mediated and increased with incubation time. Adventitious metal bound to DNA was sufficient to decompose LOOH to reactive radicals, activity that was not readily inhibited by chelators DTPA and desferrioxamine. Added Fe 2+ and Fe 3+ increased SSB and DSB, although the effects of Fe 2+ were more extensive. Above 100 μM both valences inhibited DNA damage. Strand breakage by LOOH proceeded via lipid alkoxyl and peroxyl radicals. Aldehydic lipid peroxidation products induced strand breaks via oxidation of double bonds, not by reactions of the carbonyl groups. Lipophilic antioxidants BHA, BHT, and a-tocopherol were about 20 times more effective than hydrophilic free radical scavengers sodium benzoate, inositol, DMSO, and mannitol in preventing LOOH-induced strand breaks, supporting lipid phase localization of the damage.

The sensitivity of chronic lymphocytic leukaemia lymphocytes to irradiation in vitro

The inhibition of [ 3H]-thymidine incorporation into the DMA of mitogen-stimulated chronic lymphocytic leukaemia lymphocytes by chlorambucil or γ-irradiation in vitro was measured in a series of patients, some of whom were untreated, some treated and some who were showing resistance to first-line or second-line treatment. There was evidence of resistance to irradiation developing in parallel with that to chlorambucil. The resistance to chlorambucil in chronic lymphocytic leukaemia (CLL) is not necessarily due to altered drug transport or metabolism but to a more fundamental process affecting DNA damage.

13-cis-retinoic acid affects oxidation and DNA damage in oxidative-positive SLE lymphocytes but may not be useful for therapy.

13-cis-Retinoic acid (13-CRA), a water-soluble vitamin A analog and 5'-lipoxygenase inhibitor, was tested in vitro for effects on excess oxidative metabolism and DNA damage in mitogen-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE), because other 5'-lipoxygenase enzyme inhibitors were shown to lower the excess oxidative metabolism in SLE cells. Excess chemiluminescence (CL) was abolished within minutes after the addition of 1 x 10(-6) M 13-CRA in five of five CL-positive mitogen-stimulated SLE lymphocytes, and was lowered in five of eight samples after 48 to 72 h culture. Similarly, low concentrations of 13-CRA for 48-72 h largely prevented the S1 nuclease-sensitive DNA changes/DNA damage observed in CL-positive lupus lymphocytes in vitro. However, 13-CRA did not affect DNA damage in four of four CL-negative lymphocyte samples. 13-CRA, like other retinoic acid compounds, was known to stimulate B-cell activities in vivo and in vitro but effects on dividing lupus T cells had not been studied. 13-CRA further inhibited the diminished PHA-stimulated lupus T-cell growth in tissue culture at a concentration of 9 x 10(-6) M in three of five lupus lymphocyte samples. 13-CRA has positive and negative effects on multiple aspects of the immune system and it is not clear whether 13-CRA will have positive or adverse clinical effects on SLE patients. Close attention to vitamin A and vitamin "supplements" in patients with SLE may answer this question.