5-氮胞苷对贵州小型猪淋巴细胞DNA损伤及修复的影响

目的　研究贵州小型猪淋巴细胞对化学物或药物引起的DNA损伤及修复影响的反应。方法　用单细胞凝胶电泳技术检测比较 5 氮胞苷对PHA刺激和未刺激淋巴细胞的DNA损伤及其修复过程。结果　 5 氮胞苷引起未刺激淋巴细胞明显的DNA泳动 (彗星尾 ) ,经修复孵育 2h后 ,DNA泳动与孵育前比较无显著差异 ,而 5 氮胞苷引起的刺激细胞DNA泳动经 2h修复孵育后与孵育前比较显著减少。结论　 5 氮胞苷引起贵州小型猪未刺激淋巴细胞DNA损伤经 2h孵育未能修复 ,而刺激细胞的DNA损伤明显修复。     

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.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Quantification of DNA repair capacity in whole blood of patients with head and neck cancer and healthy donors by comet assay

Comet assay has been used to estimate cancer risk by quantification of DNA damage and repair in response to mutagen challenge. Our goal was to adopt best practices for the alkaline comet assay to measure DNA repair capacity of white blood cells in whole blood of patients with squamous cell carcinoma of the head and neck (HNSCC). The results show that initial damage by 10 Gy of gamma radiation expressed as percent DNA in comet tail was higher in stimulated lymphocytes (61.1+/-11.8) compared to whole blood (43.0+/-12.1) but subsequent repair was similar with comet tail of approximately 20% at 15 min and 13% at 45 min after exposure. Exposure of whole blood embedded in agarose from 5 to 10 Gy gamma radiation was followed by an approximately 70% repair of the DNA damage within 45 min with a faster repair phase in the first 15 min. Variability of the measurement was lower within repeated measurements of the same person compared to measurement of different healthy individuals. The repair during first 15 min was slower (p=0.01) in ex-/non-smokers (41.0+/-2.1%) compared to smokers (50.3+/-2.7%). This phase of repair was also slower (p=0.02) in HNSCC patients (36.8+/-2.1%) compared to controls matched on age and smoking (46.4+/-3.0%). The results of this pilot study suggest that quantification of repair in whole blood following a gamma radiation challenge is feasible. Additional method optimization would be helpful to improve the assay for a large population screening.     

DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the 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. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR

The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

D-ribose inhibits DNA repair synthesis in human lymphocytes

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.     

DNA damage and repair in Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a slowly progressive eye disease leading to blindness, mostly affecting people above 40 years old. The only known method of curing FECD is corneal transplantation. The disease is characterized by the presence of extracellular deposits called “cornea guttata”, apoptosis of corneal endothelial cells, dysfunction of Descement’s membrane and corneal edema. Oxidative stress is suggested to play a role in FECD pathogenesis. Reactive oxygen species produced during the stress may damage biomolecules, including DNA. In the present study we evaluated the extent of endogenous DNA damage, including oxidatively modified DNA bases, and damage induced by hydrogen peroxide as well as the kinetics of DNA repair in peripheral blood mononuclear cells of 50 patients with FECD and 43 age-matched controls without visual disturbances. To quantify DNA damage and repair we used the alkaline comet assay technique with the enzymes recognizing oxidative DNA damage, hOGG1 and EndoIII. We did not observe differences in the extent of endogenous and hydrogen peroxide-induced DNA damage between FECD patients and controls. However, we found a lower efficacy of DNA repair in FECD patients as compared with control individuals. The results obtained suggest that the lowering of the DNA repair capacity may be one of the mechanisms underlying the role of oxidative stress in the FECD pathology.     

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.     

Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.     

DNA damage induced by paclitaxel and DNA repair capability of peripheral blood lymphocytes as evaluated by the alkaline comet assay

This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.     

A modified alkaline comet assay for measuring DNA repair capacity in human populations

Trzeciak, A. R., Barnes, J. and Evans, M. K. A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations. Radiat. Res. 169, 110-121 (2008). Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.     

In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint, and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To test this hypothesis, we performed the host-cell reactivation assay for DRC by transfecting stimulated peripheral blood lymphocytes from 120 normal donors with transient expression plasmids damaged by benzo[a]pyrene diol epoxide (BPDE). The same cells were assessed for protein expression induction of eight cell-cycle checkpoint-related genes using the reverse-phase protein lysate microarray assay. In multivariate linear regression analysis adjusting for age, sex, blastogenic rate, and sample storage duration, the association between DRC and expression levels of cell-cycle checkpoint-related proteins induced by BPDE-adducts was statistically significant for p27, CCND1, ATM, and MDM2 (P = 0.00, 0.03, 0.03, and 0.03, respectively), borderline for p73 and p21 (P = 0.07 and 0.09, respectively), but not for p53 and p16 (P = 0.13 and 0.18, respectively). Because the relative expression levels of all these eight proteins were highly correlated, we further performed the principal component analysis and identified ATM as the most important predictor of DRC, followed by MDM2 and p27. Our results provide population-based in vitro evidence demonstrating that cell-cycle checkpoint-related proteins play essential roles in regulating DNA repair, at least in unaffected human peripheral blood lymphocytes. Further studies are warranted to investigate the role of interindividual variation in the expression levels of these proteins in cancer susceptibility.     

Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.     

The relationship between DNA repair after alkylation damage and in vitro aging in human T-lymphocytes

In vitro cultures of human peripheral blood lymphocytes, both unstimulated G0 cells as well as phytohemagglutinin (PHA) stimulated cells, have been used in the investigation of DNA repair following different types of damage, including that induced by ultraviolet light, ionizing radiation, and chemical agents. We report here repair of DNA damage in cultured normal human T-lymphocytes after treatment with the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methanesulfonate (MMS), as measured by the alkaline elution technique. T-lymphocytes cultured with different sources of T-cell growth factors (TCGFs) were shown to have similar repair levels, as measured by loss of single-strand breaks. However, diminished repair was observed with in vitro culture age when T-cells were cultured with PHA and TCGF for a 3-week period. Cell-cycle analysis performed on asynchronously growing cultures indicated that differential repair with in vitro aging was not cell-cycle-related. Fluorescence Activated Cell Sorting (FACS) was used to determine the percentages of CD4+ and CD8+ T-cell subsets present during the in vitro culture periods. Cultures consisted primarily of CD4+ cells until day 20 when the percentage of CD8+ cells increased to approximately 50% of the T-cell population. Neither the absolute percentages of CD4+ and CD8+ cells not the CD4+/CD8+ ratios correlated with repair rates of cultured T-cells. Therefore, the observed decreases in the repair of alkylating agent-induced damage are not due solely to the change in the CD4+/CD8+ ratio.