DNA Repair In Human Lymphocytes Research Articles

Low doses of ochratoxin A induce micronucleus formation and delay DNA repair in human lymphocytes

The contamination of food commodities by fungal toxins has attracted great interest because many of these mycotoxins are responsible for different diseases, including cancer and other chronic illnesses. Ochratoxin A (OTA) is a mycotoxin naturally present in food, and long-term exposure to food contaminated with low levels of OTA has been associated with renal cancer. In the present study, the cytotoxicity, cytostaticity, and genotoxicity of OTA (0.075–15 µM) in human lymphocytes were evaluated. A comet assay, a modified comet assay (DNA repair assay), which uses N-hydroxyurea (NHU) to detect non-repaired lesions produced by OTA, and a cytokinesis-blocked micronucleus assay were used. Treatments with OTA were not cytotoxic, but OTA caused a cytostatic effect in human lymphocytes at a concentration of 15 µM. OTA (0.075–5 µM) produced a slight increase in the percentage of DNA in the comets and a delay in the DNA repair capacity of the lymphocytes. Micronucleus (MN) induction was observed at OTA concentrations of 1.5 and 5 µM. Our results indicate that OTA induces DNA stable damage at low doses that are neither cytotoxic nor cytostatic, and OTA delays the DNA repair kinetics. These findings indicate that OTA affects two pivotal events in the carcinogenesis pathway.

Gentiana asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticles-induced DNA damage

Exposure to high levels of different environmental pollutants is known to be associated with induction of DNA damage in humans. Thus DNA repair is of great importance in preventing mutations and contributes crucially to the prevention of cancer. In our study we have focused on quantitative analysis of Gentiana asclepiadea aqueous or methanolic extracts obtained from flower and haulm, their antioxidant potency in ABTS post-column derivatisation, and their potential ability to enhance DNA repair in human lymphocytes after hydrogen peroxide (H2O2) treatment (250μM, 5min). We also studied DNA repair in human kidney HEK 293 cells after exposure to 20nm silver nanoparticles (AgNPs) (100μg/ml, 30min) in the presence and absence of the plant extract. We have found that mangiferin along with unidentified polar compounds are the most pronounced antioxidants in the studied extracts. Extract from haulm exhibited slightly stronger antioxidant properties compared to flower extracts. However, all four extracts showed significant ability to enhance DNA repair in both cell types after H2O2 and AgNP treatments.

DNA Repair in Human Lymphocytes by Sinigrin ‐ a Possible Mechanism of Action: Comet –FLARE Assay.

Chronic low level exposure to environmental pollutants such as synthetic pesticides has been implicated in many health conditions such as oxidative stress, leading to DNA damage, a cause behind many serious diseases such as cancer. The objective of the present study was to investigate the possible mode of action of Sinigrin (10–30μM) a bio‐active compound found in vegetables such as broccoli, cabbage, Brussels sprouts, in the DNA repair process, using Single Cell Gel Electrophoresis (Comet Assay) ‐ Fragment Length Analysis using Repair Enzymes (FLARE) assay. Human lymphocytes were exposed to various concentrations of Sinigrin (10–30 μM) along with (a) a mixture of most widely used pesticides and (b) 100 mM of H2O2 incubated simultaneously at 37° C, at 5% CO2 for 3 hours and DNA repair was evaluated using formamidopyrimidine FLARE‐Comet Assay. The efficiency of Sinigrin in protecting DNA damage was found at and above 20 μM, suggesting a possible role in the functioning of DNA glycosylase involved in DNA repair process.

DNA damage and repair in gastric cancer—A correlation with the hOGG1 and RAD51 genes polymorphisms

The cell's susceptibility to mutagens and its ability to repair DNA lesions are important for cancer induction, promotion and progression. Both the mutagens’ sensitivity and the efficacy of DNA repair may be affected by variation in several genes, including DNA repair genes. The hOGG1 gene encodes glycosylase of base excision repair and RAD51 specifies a key protein in homologues recombination repair. Both can be involved in the repair of oxidative DNA lesions, which can contribute to stomach cancer. In the present work we determined the level of basal and oxidative DNA damage and the kinetics of removal of DNA damage induced by hydrogen peroxide in peripheral blood lymphocytes of 30 gastric cancer patients and 30 healthy individuals. The metrics from DNA damage and repair study were correlated with the genotypes of common polymorphisms of the hOGG1 and RAD51 genes: a G → C transversion at 1245 position of the hOGG1 gene producing a Ser → Cys substitution at the codon 326 (the Ser326Cys polymorphism) and a G → C substitution at position 135 (5′-untranslated region) of the RAD51 gene (the G135C polymorphism). DNA damage and repair were evaluated by alkaline single cell gel electrophoresis (comet assay) assisted by DNA repair enzymes: endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), preferentially recognizing oxidized DNA bases. The genotypes of the polymorphism were determined by restriction fragment length polymorphism PCR. We observed a strong association between gastric cancer occurrence, impaired DNA repair in human lymphocytes and the G/C genotype of the G135C polymorphism of the RAD51 gene. Moreover, there was a strong correlation between that genotype and stomach cancer occurrence in subjects with high level of oxidatively damaged DNA. We did not observe any correlation between the Ser1245Cys polymorphism of the hOGG1 gene and gastric cancer, including subjects with impaired DNA repair and/or high levels of endogenous oxidative DNA lesions. Therefore, our result suggest that the G135C polymorphism of the RAD51 gene may be linked with gastric cancer by the modulation of the cellular response to oxidative stress and this polymorphism may be a useful additional marker in this disease along with the genetic or/and environmental indicators of oxidative stress.

How Strong Is the Evidence that Lycopene Supplementation Can Modify Biomarkers of Oxidative Damage and DNA Repair in Human Lymphocytes?

How Strong Is the Evidence that Lycopene Supplementation Can Modify Biomarkers of Oxidative Damage and DNA Repair in Human Lymphocytes?

The Effects of Niacin on DNA Repair afterN-Methyl-N′-nitro-N-nitrosoguanidine Treatment in Normal Human Lymphocytes

We have investigated the effects of niacin on NAD levels and on DNA repair in human lymphocytes. When lymphocytes were incubated in culture medium with various concentrations of niacin, incubation of lymphocytes with nicotinic acid at 5 microM or nicotinamide at 10 mM caused a 2-3 fold increase in NAD content. Under these conditions lymphocytes were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Interestingly, the rejoining of DNA strand breaks was promoted by nicotinic acid but nicotinamide inhibited the rejoining.

DNA repair in human lymphocytes treated in vitro with (±)-anti- and (±)-syn-benzol[a]pyrene diolepoxide

Human PBL were treated in vitro with the ultimate reactive metabolites of BaP anti- and syn-BaPDE and DNA damage and repair were measured. The incorporation of radioactivity into DNA due to UDS was higher after treatment with anti-BaPDE. Radioactive DNA adduct dosimetry applied to PBL treated with tritiated syn- and anti-BaPDE demonstrated that anti-BaPDE gave more DNA adducts, which were more efficiently removed than syn adducts in the 24 h following the treatment. HPLC analysis of deoxynucleosides obtained from the enzymatic digestion of DNA showed that in treated PBL the major DNA adduct involved deoxyguanosine. DNA strand breaks, detected by FADU, were induced at comparable levels by anti- and syn-BaPDE (0.1–0.4 μg/ml), and persisted after 20 h of post-treatment incubation. Only in the case of syn-BaPDE did the percentage of double-stranded DNA tend to increase with time after the treatment.

Radiation risk of tissue late effects, a net consequence of probabilities of various cellular responses

Late effects from the exposure to low doses of ionizing radiation are hardly or not at all observed in man probably due to the low values of risk coefficients that preclude statistical analyses of data from populations that are exposed to doses less than 0.2 Gy. In order to arrive at an assessment of potential risk from radiation exposure in the low dose range, the microdosimetry approach is essential. In the low dose range, ionizing radiation generates particle tracks, mainly electrons, which are distributed rather heterogenously within the exposed tissue. Taking the individual cell as the elemental unit of life, observations and calculations of cellular responses to being hit by energy deposition events from low LET type are analysed. It emerges that besides the probability of a hit cell to sustain a detrimental effect with the consequence of malignant transformation there are probabilities of various adaptive responses that equip the hit cell with a benefit. On the one hand, an improvement of cellular radical detoxification was observed in mouse bone marrow cells; another adaptive response pertaining to improved DNA repair, was reported for human lymphocytes. The improved radical detoxification in mouse bone marrow cells lasts for a period of 5-10 hours and improved DNA repair in human lymphocytes was seen for some 60 hours following acute irradiation. It is speculated that improved radical detoxification and improved DNA repair may reduce the probability of spontaneous carcinogenesis. Thus it is proposed to weigh the probability of detriment for a hit cell within a multicellular system against the probability of benefit through adaptive responses in other hit cells in the same system per radiation exposure. In doing this, the net effect of low doses of low LET radiation in tissue with individual cells being hit by energy deposition events could be zero or even beneficial. Since there was no simple additivity of equal effects from repeated exposures to equal doses and because of the potential effect of adaptive cell responses on the spontaneous evolution of malignancy in tissue, the extrapolation of risk with absorbed dose reaching down to zero, does not appear to be generally valid.

Role of intracellular deoxynucleoside triphosphate levels in DNA repair in human lymphocytes.

Resting peripheral blood lymphocytes (PBL) contain single strand breaks1,2 and are very sensitive to DNA damaging agents3. Resting PBL contain very low levels of intracellular deoxynucleoside triphosphates which can be disrupted by the addition of extracellular deoxyadenosine in the presence of the adenosine deaminase inhibitor deoxycoformycin4,5 thus creating imbalance between deoxyATP levels and other deoxynucleoside triphosphates. In the present work I have investigated the role of this deoxynucleoside triphosphate imbalance in the repair of DNA damage induced by deoxyadenosine6,7 and other DNA damaging agents.

Distress and DNA repair in human lymphocytes.

This research assessed differences in DNA repair in lymphocytes from high- and low-distressed individuals. A median split on Minnesota Multiphasic Personality Inventory (MMPI) Scale 2 divided 28 newly admitted nonpsychotic psychiatric inpatients into high- and low-distress subgroups. The high-distress subgroup had significantly poorer DNA repair in lymphocytes exposed to X-irradiation than low-distress subjects. We also found that lymphocytes obtained from this psychiatric sample had significantly poorer DNA repair than lymphocytes from nonpsychiatric control subjects when compared 5 hr after X-irradiation. A high level of distress therefore appears to be associated with significant dysfunctional differences at the molecular level which may have important implications for health. These data provide evidence for a direct pathway through which distress could influence the incidence of cancer.

The effect of aphidicolin on DNA repair in resting and mitogen stimulated human lymphocytes

Aphidicolin inhibits DNA repair in human lymphocytes as measured by unscheduled DNA synthesis and the rejoining of strand breaks. When the lymphocytes are mitogen stimulated, sensitivity of DNA repair towards aphidicolin decreases, possibly due to the induction of the beta DNA polymerase.

3-aminobenzamide stimulates unscheduled dna synthesis and rejoining of strand breaks in human lymphocytes

A comparative study of the effect of 3-aminobenzamide on the activity of DNA repair in human lymphocytes and in L 1210 cells has been made. Damage of DNA has been performed by treatment with UV light or with dimethylsulphate. DNA repair has been measured both as unscheduled DNA synthesis and by the rejoining of strand breaks. Both types of measurement have shown that 3-aminobenzamide stimulates DNA repair in human lymphocytes while inhibiting it in L 1210 cells.

Correlation of carcinogen-induced unscheduled DNA synthesis and NAD reduction in fresh human lymphocytes

Incorporation of [ 3H]thymidine into the DNA of fresh human lymphocytes, treated with various chemical mutagens, was measured and correlated with cellular NAD levels before and after treatment. NAD levels in lymphocytes were significantly reduced following treatment with mutagenic chemicals. Reduction of cellular NAD pools was directly correlated with [ 3H]thymidine incorporation. As NAD levels decreased, [ 3H]thymidine incorporation increased. Theophylline, a known inhibitor of poly(ADP-ribose)polymerase, inhibited both the NAD reduction in cells treated with DNA damaging agents and the incorporation of [ 3H]thymidine into DNA. The inhibitory effect of theophylline on NAD depletion and on [ 3H]thymidine incorporation was dose and cell number dependent. Near normal responses to carcinogen exposure could be restored to theophylline-treated cells following the removal of theophylline. These data suggest that conversion of NAD to poly(ADP-ribose) may be necessary, or at least closely associated with, DNA repair in human lymphocytes.