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At least 19 recordsLinked to original sources
PubMed1986

Metabolic consequences of DNA damage: DNA damage induces alterations in glucose metabolism by activation of poly (ADP-ribose) polymerase.

In this communication we show that activation of poly(ADP-ribose) polymerase by DNA damage can produce drastic alterations in carbohydrate metabolism. We examined alterations in NAD+, NADP+, ATP and glucose-6-phosphate in L1210 murine leukemia cells, following exposure to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. Treatment of cells with 20 micrograms/ml MNNG produced rapid depletion of NAD+ and ATP. The G-6-P pool showed a biphasic change: first the pool size decreased, then increased to a level greater than that present in control cells. Nicotinamide treatment prevented the total depletion of NAD+ and this in turn helped preserve the ATP pools and prevented the biphasic alteration in G-6-P pool sizes.

Adenosine Triphosphate↗
PubMed2005

DNA damage, DNA susceptibility to oxidation and glutathione level in women with polycystic ovary syndrome.

Recent studies have addressed the possibility of an association between polycystic ovaries and ovarian cancer. DNA damage is the first step of the carcinogenesis, and susceptibility to cancer, in general, is characterized by high DNA damage. Free radical-mediated DNA damage and impaired antioxidant defence have been implicated as contributory factors for the development of cancer. This study evaluates DNA damage (strand breakage, base oxidation, formamidopyrimidine DNA glycosylase (Fpg) sensitive sites), H2O2-induced DNA damage, a marker of DNA susceptibility to oxidation and glutathione (GSH) level, a powerful antioxidant, in women with polycystic ovary syndrome (PCOS). Women with PCOS showed a significant decrease in GSH level, a significant increase in DNA strand breakage and H2O2-induced DNA damage. Although Fpg-sensitive sites were higher in the PCOS group compared to the control group, the difference did not reach a statistically significant level. Significant correlations were found between free testosterone and DNA strand breakage (r = 0.46, p<0.01) and free testosterone and H2O2-induced DNA damage (r = 0.41, p<0.05). The data indicate that DNA damage and susceptibility of DNA to oxidative stress are increased in women with PCOS and may explain the association between PCOS and ovarian cancer.

Adult↗
PubMed1992

Relationship between DNA damage, DNA repair, metabolic state and cell lethality.

Induction of unrepairable DNA damage, accumulation of misrepaired DNA damage, and generation of imbalances in competing biochemical and/or metabolic processes have been proposed to explain the relationship between radiation-induced DNA damage and cell lethality. Theoretically, the temperature dependence of the critical DNA repair process(es) should be 1) either independent of or identical to the temperature dependence of cell killing if the first two hypotheses are correct, and 2) different if the third hypothesis is correct. To test this, exponentially growing rat 9L brain tumor cells were left at 37 degrees C or equilibrated for 3-14 h at 20 degrees C before irradiation. Cells were irradiated and allowed to repair at either 20 degrees C or 37 degrees C. Alternatively, the cells were irradiated at one of these temperatures and immediately shifted to the other temperature for repair. DNA damage was assessed by the alkaline elution technique; cell kill was assessed by a clonogenic assay. 9L cells maintained at 20 degrees C or 37 degrees C sustained the same amount of DNA damage as measured by alkaline elution. DNA repair instantaneously assumed the rate characteristic of the postirradiation temperature. For 9L cells equilibrated, irradiated, and repaired at 20 degrees C, the half-time of the fast phase of the DNA repair decreased by a factor of approximately 2 and the half-time of the slow phase decreased by a factor of approximately 5 over that measured in cells incubated, irradiated and repaired at 37 degrees C. Although the rate of DNA repair decreased substantially at 20 degrees C, the survival of 9L cells that were equilibrated and irradiated at 20 degrees C was greater (p less than 10(-4)) than those incubated and irradiated at 37 degrees C, when assayed by an immediate plating protocol. In addition, the survival of 9L cells equilibrated and irradiated at 20 degrees C and then shifted to 37 degrees C immediately after irradiation was greater (p less than 10(-2)) than that obtained with any other delayed plating protocol. Thus, the temperature dependence of the DNA repair processes measured by alkaline elution was different from the temperature dependence of cell killing measured either by an immediate or delayed plating protocol. These data support the hypothesis that many irradiated 9L tumor cells die because of imbalances in sets of competing biochemical and/or metabolic processes.

Animals↗
PubMed2004

Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer.

Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.

Alkylating Agents↗
PubMed1995

In vivo tumor measurement of DNA damage, DNA repair and NAD pools as indicators of radiosensitization by metoclopramide.

Metoclopramide (MCA), a N-substituted benzamide, causes DNA strand breaks and inhibits DNA repair in vitro and sensitizes radiation and chemotherapeutic drugs in human squamous cell carcinomas when xenographed into nude mice or in a rat glioma model. Here we report on the evaluation of the mechanism behind the radiosensitizing effects of MCA. DNA damage was measured in vivo in a CBA-mouse tumor line (A12B3, sarcoma tumor) by using both alkaline elution and nucleoid sedimentation analysis of cell suspensions prepared from either resected tumor, spleen tissues or whole blood samples. The amount of DNA damage caused by radiation alone, measured 30 min after the irradiation was started, was dose dependent up to 18 Gy in all tissues. The radiation-induced DNA damage in tumor tissue was elevated compared to radiation alone in the presence of MCA, but the level was not higher at 18 Gy compared to 6 Gy in the presence of MCA, and it was still not fully repaired 12 h after irradiation. HPLC analysis of the NAD pools in tumor tissue after DNA damage induction showed a delay in the recovery of the NAD pools (presumably due to the presence of still unrepaired DNA) after exposure to MCA (2 mg/kg) + radiation (6 Gy) compared to tumors exposed to radiation (6 Gy) only, which were fully restored after 48 h. These data confirm earlier published in vitro data on MCA as an inducer of DNA damage and an effector of DNA repair. In addition, the in vivo measurement of radiation-induced DNA damage and DNA repair using the nucleoid sedimentation and alkaline elution assays together with NAD pool determinations may prove to be effective intermediate endpoints in the evaluation of drugs as potential radiosensitizers.

Animals↗
PubMed1997

DNA damage, DNA repair, and alcohol toxicity--a review.

Alcohol (ethanol) is clearly a toxic substance when consumed in excess. Chronic alcohol abuse results in a variety of pathological effects, including damage to the liver and brain, as well as other organs, and is associated with an increased risk of certain types of cancers. Alcohol consumption by pregnant women can result in fetal alcohol effects and fetal alcohol syndrome. All of these toxic effects are well documented. What is needed at present is a complete understanding of the molecular mechanisms by which alcohol causes these toxic effects. Such an understanding may lead to better treatments of some of these toxic effects. This review, focuses on the possibility that toxic effects of ethanol are mediated, at least in part, by damage to DNA. In particular, I emphasize data on the production of endogenous DNA-damaging molecules as a result of alcohol consumption and metabolism. Specific examples of DNA-damaging molecules to be considered are reactive oxygen species, including oxygen radicals, lipid peroxidation products, and acetaldehyde. The relevant DNA repair pathways that protect cells against DNA damage produced by these molecules will also be reviewed. The goal of this review is to integrate recent results from the fields of mutagenesis and DNA repair with the alcohol toxicity literature, with the aim of stimulating research into the role of DNA damage in different types of alcohol toxicity and the role of DNA repair in protecting cells from alcohol-related damage.

Animals↗
PubMed2005

Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.

The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.

Animals↗
PubMed1991

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli.

A study of the conjugal transfer of ColV,I-K94 tn 10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.

Conjugation, Genetic↗
PubMed2000

Antioxidant intervention studies related to DNA damage, DNA repair and gene expression.

In human cells oxidatively modified nucleobases can be measured in the DNA and strand breaks can be detected by the Comet assay, optionally with the use of repair enzymes introducing breaks at oxidized bases. Oxidized bases and nucleosides from DNA repair, the nucleotide pool and cell turnover can be measured in urine. The excretion rate represents the average rate of damage in the body, whereas the level of oxidized bases in DNA is a concentration measurement in the specific cells. The expression of genes relevant for the defence against oxidative DNA damage, antioxidant and DNA repair enzymes can be assessed at the mRNA, protein and activity level. Functional assays can involve susceptibility to, and disappearance of, damage induced ex vivo in lymphocytes. Vitamins C and E, beta-carotene, coenzyme Q as well as various vegetables, fruit and carotenoid rich products, have been assessed with biomarkers mainly including damage in lymphocytes DNA by Comet and chemical assays and urinary excretion of oxidized bases and nucleosides. The basal levels as well as the reported effects of the interventions have been rather variable, possibly reflecting differences in the populations, regimens, functional correlates of the biomarkers as well as between laboratories and assays. However, the data suggest that a depleted state due to nutritional deficiency and/or an increased oxidative stress might facilitate demonstration of protective effects of antioxidants with respect to DNA damage. The effect of antioxidants on gene expression has been little studied in humans and only at the activity and protein level.

Antioxidants↗
PubMed2005

2,4-Diaminotoluene (2,4-DAT)-induced DNA damage, DNA repair and micronucleus formation in the human hepatoma cell line HepG2.

2,4-Diaminotoluene (2,4-DAT) is a widely used industrial intermediate and human exposure is possible in the dye and plastics industries. We investigated the genotoxicity of the environmental pollutant, 2,4-DAT, in human HepG2 cells using the unscheduled DNA synthesis (UDS) test, the micronucleus (MN) assay and single-cell gel electrophoresis (SCGE). 2,4-DAT was first tested by the RNA synthesis inhibition test as a cytotoxicity assay: the IC(50) of 2,4-DAT was 5.2 mM after 20 h of exposure. The compound had a genotoxic effect at concentrations from 1.45 to 6.80 mM in both micronucleus and comet assays. In the micronucleus assay, the number of MN/1000 BNC was 3.5 times higher at a concentration of 6.80 mM 2,4-DAT than in the negative control. At the same concentration, DNA migration (SCGE) showed an Olive tail moment (OTM) of 3.56+/-0.45, as compared to 0.19+/-0.02 for the negative control. The UDS test detected genotoxic effects at lower concentrations than did the other assays (0.01-5 mM). The percentage of cells in repair increased in a concentration-dependent manner to a maximum of 57% at 1mM. At the highest concentration tested (5 mM), the NNG/cell score was 13.6+/-0.5 whereas it was -2.7+/-0.5 for the negative control. These data, based on various endpoints, show a midly genotoxic effect of 2,4-DAT in the HepG2 cells and confirm that this cell line is a suitable model to study the toxic effects of aromatic amines.

Carcinogens↗
PubMed2003

Mechanisms of DNA damage, DNA hypomethylation, and tumor progression in the folate/methyl-deficient rat model of hepatocarcinogenesis.

Using the folate/methyl-deficient rat model of hepatocarcinogenesis, we obtained evidence that may provide new insights into a major unresolved paradox in DNA methylation and cancer research: the mechanistic basis for genome-wide hypomethylation despite an increase in DNA methyltransferase activity and gene-specific regional hypermethylation. Previous studies revealed that the methyltransferase binds with higher affinity to DNA strand breaks, gaps, abasic sites, and uracil than it does to its cognate hemimethylated CpG sites, consistent with its ancestral function as a DNA repair enzyme. These same DNA lesions are an early occurrence in models of folate and methyl deficiency and are often present in human preneoplastic cells. We hypothesized that the high-affinity binding of the maintenance DNA methyltransferase to unrepaired lesions in DNA could sequester available enzyme away from the replication fork and promote passive replication-dependent demethylation. In support of this possibility, we found that lesion-containing DNA is less efficiently methylated than lesion-free DNA from folate/methyl-deficient rats and that an increase in DNA strand breaks precedes DNA hypomethylation. Despite an adaptive increase in DNA methyltransferase activity, hemimethylated DNA from folate/methyl-deficient rats is progressively replaced by double-stranded unmethylated DNA that is resistant to remethylation with dietary methyl repletion. In promoter regions, the inappropriate binding of the DNA methyltransferase to unrepaired lesions or mispairs may promote local histone deacetylation, methylation, and regional hypermethylation associated with tumor suppressor gene silencing. These insights in an experimental model are consistent with the possibility that DNA lesions may be a necessary prerequisite for the disruption of normal DNA methylation patterns in preneoplastic and neoplastic cells.

Animals↗
PubMed1987

In vivo effects of mercury (II) on deoxyuridine triphosphate nucleotidohydrolase, DNA polymerase (alpha, beta), and uracil-DNA glycosylase activities in cultured human cells: relationship to DNA damage, DNA repair, and cytotoxicity.

The effect of mercuric acetate on the activities of deoxyuridine triphosphate nucleotidohydrolase (dUTPase), DNA polymerase (alpha, beta), and uracil-DNA glycosylase has been studied in cultured human KB cells. There was a dose- and time-dependent inactivation of both dUTPase and DNA polymerase alpha activities by mercuric acetate. In cells exposed to low concentrations (10 microM) of mercuric acetate, dUTPase was most sensitive to inhibition with 30% of the activity being inhibited after a 1-hr exposure. At higher concentrations or for longer exposure times, DNA polymerase alpha was most sensitive to inhibition with greater than 60% of the activity being inhibited by 25 microM mercuric acetate after a 15-min exposure. There was no inhibition of DNA polymerase beta or uracil-DNA glycosylase activities in cells exposed to 50 microM mercuric acetate for 90 min. In fact, there was a time- and dose-dependent activation of uracil-DNA glycosylase activity with maximum activation occurring in cells exposed to 50 microM mercuric acetate. The inhibition of dUTPase and DNA polymerase alpha activities and the activation of uracil-DNA glycosylase activity correlated with the induction of single-strand breaks in DNA by mercuric acetate and with the decrease in cell viability.

Cations, Divalent↗
PubMed1992

The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis?

DNA breaks can arise from many sources after incubation of cells with toxic agents. Very few agents break DNA directly, rather most breaks occur as a result of metabolic participation by the cell, such as during attempts to repair the damage. It is now realized that many DNA breaks arise as a consequence of steps in the pathway of cell death. Upon reanalyzing the methodology commonly used to detect DNA breaks, it is evident that many studies would not have observed DNA breaks associated with cell death. Frequently experimental conditions have been used that are extremely toxic to cells with the justification that the cells were still viable as measured by their ability to exclude dyes such as trypan blue. However, the DNA digestion associated with cell death by apoptosis occurs prior to changes in membrane integrity. Because the possibility of endogenous endonuclease activity was not realized, many studies may have inaccurately assumed that DNA breaks arose during, for example, inhibition of DNA repair or as intermediates in recombination. In light of the new understanding of apoptosis and the formation of DNA breaks as an early event in cell death, it is important to both reevaluate past conclusions and to ensure that future studies fully consider the breaks derived from the cytotoxicity of every agent under investigation.

Animals↗