Detect DNA Single-strand Breaks Research Articles

Cooperative dynamics of PARP-1 zinc-finger domains in the detection of DNA single-strand breaks

The DNA single-strand break (SSB) repair pathway is initiated by the multifunctional enzyme PARP-1, which recognizes the broken DNA ends by its two zinc-finger domains, Zn1 and Zn2. Despite a number of experiments performed with different DNA configurations and reduced fragments of PARP-1, many details of this interaction that is crucial to the correct initiation of the repair chain are still unclear. We performed Molecular Dynamics (MD) computer simulations of the interaction between the Zn1/Zn2 domains of PARP-1 and a DNA hairpin including a missing nucleotide to simulate the presence of an SSB, a construct used in recent experiments. The role of Zn1 and Zn2 interacting with the SSB ends is studied in detail, both independently and cooperatively. We also explored, PARP-1 operating as a dimer, with the two Zn-fingers coming from two separate copies of the enzyme. By an extensive set of all-atom molecular simulations employing state-of-the art force fields, assisted by empirical docking and free-energy calculations, we conclude that the particular conformation of the DNA hairpin can indeed spontaneously open up by thermal fluctuations, up to extremely kinked deformations. However, such extreme localized deformations are rarely observed in free, long DNA fragments. Protein side-loops make contact with the DNA hairpin grooves, and help Zn2 to penetrate deep in the SSB gap. In this way, Zn2 can interact with the nucleotides opposite to the missing base. Overall, Zn1 plays a secondary role: the crucial factor for the interaction is rather the relative arrangement of the Zn1/Zn2 couple, and their mutual orientation with respect to the 3′\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$3'$$\\end{document} and 5′\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$5'$$\\end{document} SSB end terminals. This helps to obtain an early interacting configuration, which ultimately leads to molecular PARP-1-DNA structures similar to those observed experimentally. Such findings represent an important step toward defining the detailed function of PARP-1 in the early stages of SSB recognition.

Genotoxic changes in peripheral lymphocytes after therapeutic exposure to crude coal tar and ultraviolet radiation

Goeckerman therapy is based on combined exposure to UV radiation (UVA, UVB) and crude coal tar (PAHs). Some indicators suggest a genotoxic hazard, however, the level of genotoxic risk of the therapy has not yet been investigated sufficiently. This study aims to assesss the genotoxic risk. The studied group consisted of patients with chronic stable plaque psoriasis treated by Goeckerman therapy (n = 29). Heparin-treated peripheral blood samples were collected one day before the first treatment and immediately after the last procedure. The lymphocytes were isolated from the blood. The level of genotoxicity was evaluated using an alkaline version of the Comet assay which detects DNA single strand breaks (DNA-SSBs), a neutral version of the Comet assay which detects DNA double strand breaks (DNA-DSBs), and using chromosomal aberrations. The level of DNA-SSBs increased insignificantly (median; Q1-Q3): 1.4 (0.4; 0.1-1.4) vs. 2.5 (0.6; 0.3-2.7) %tDNA (P = 0.11) and the level of DNA-DSBs increased significantly: 7.8 (6.5; 3.4-10.5) vs. 20.7 (19.3; 14.2-24.6) % DNA (P < 0.001). The total number of aberrated cells (P < 0.001) and structurally aberrated cells (P < 0.001) increased significantly. The elevated levels of the DNA-DSBs and the chromosomal aberrations in the peripheral lymphocytes indicated a genotoxic hazard. However, the elevated level of the chromosomal abnormalities was below the upper level of the reference range for healthy Czech adults. While, the genotoxic risk appears to be low, Goeckerman treatment represents a further contribution to the lifetime load of genotoxic factors.

Abstract 3785: Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer

Abstract Purpose: Poly (ADP-ribose) polymerase (PARP) detects DNA single strand breaks and facilitates their repair. Recently, PARP inhibitors have been shown to potentiate radiotherapy in mouse models of human non-small-cell lung cancer (NSCLC) by compromising DNA repair and increasing tumor vascular perfusion. This study examined radiosensitization by PARP inhibition in xenograft models of NSCLC with differing levels of hypoxia. Methods: For in vitro studies, human NSCLC cell lines Calu-6 and Calu-3 were treated with olaparib (5 µM) or vehicle prior to ionizing radiation (IR). Cytotoxicity was evaluated by clonogenic survival assays and DNA damage repair was assessed by γH2AX foci immunofluorescence staining. For in vivo efficacy studies, Calu-6 or Calu-3 subcutaneous xenografts (100 mm3) were established in 6-8 week old female BALB/c nude mice. Animals (n=48) were then randomized into four groups (i) control, (ii) olaparib (50 mg/kg, d1-3), (iii) IR (10 Gy, d1) and (iv) combination treatment. Subsequent tumor growth was monitored for up to 42 days. In parallel, for pharmacodynamic studies, tumor bearing animals were sacrificed 24h and 72h after treatment (n=3 for each treatment and time point). Tumor sections were evaluated by immunohistochemistry (IHC). Results: In vitro, both NSCLC cell lines were significantly radiosensitized by olaparib. Sensitization enhancement ratios were 1.9 (Calu-6) and 2.2 (Calu-3). Furthermore, olaparib markedly inhibited DNA damage repair in both Calu-6 and Calu-3 cells 24h post-IR (p&amp;lt;0.01). Growth of Calu-6 and Calu-3 xenografts was reduced by IR alone, but unaffected by olaparib treatment alone. Surprisingly, although olaparib significantly increased radiation-induced growth delay in Calu-6 xenografts (p&amp;lt;0.01), there was no significant effect in Calu-3 xenografts, in contrast to the in vitro data. IHC staining for vascularity (CD31) and hypoxia (CA9) showed that Calu-3 xenografts were well-oxygenated without evidence of necrotic tissue, whereas Calu-6 xenografts had large areas of hypoxia surrounding regions of necrosis. RAD51 expression (a protein involved in homologous recombination, HR) was selectively down-regulated in hypoxic regions of Calu-6 tumors. 24h and 72h after IR, olaparib treated Calu-6 xenografts displayed significantly higher levels of γH2AX (DNA double strand breaks) and cleaved caspase 3 (apoptosis) staining in CA9 positive (hypoxic) cells compared with the hypoxic regions in xenografts treated with IR alone. Conclusion: Our data identify a novel mechanism that contributes to radiosensitization by PARP inhibition in NSCLC. Olaparib selectively increased DNA damage and apoptosis in radioresistant hypoxic tumor cells with decreased HR potential. Our data also suggest that tumor hypoxia may be a potential biomarker of differential benefit from the combination of olaparib with radiotherapy. Citation Format: Yanyan Jiang, Tom Verbiest, Aoife M. Devery, Sivan M. Bokobza, Anika M. Weber, Anderson J. Ryan. Olaparib increases the effectiveness of radiation in hypoxic tumor cells in xenograft models of human non-small-cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2014-3785

Genotoxicidad de tres plaguicidas utilizados en la actividad bananera de Costa Rica

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro

Background aimsAdipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. MethodsAfter isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. ResultsDuring in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. ConclusionsThe study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell–based therapy.

3′-5′ Phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

pAp (3′-5′ phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.

Role of benzalkonium chloride in DNA strand breaks in human corneal epithelial cells

To investigate the toxic effects of benzalkonium chloride (BAC), a preservative commonly used in ophthalmic preparations, on DNA single- and double-strand breaks in immortalized human corneal epithelial cells (HCEs). HCEs were treated with BAC in concentrations ranging from 0.00005% to 0.001% for 30 min. Cells were examined immediately after BAC exposure and after 24-h recovery. Alkaline comet assay was used to detect DNA single-strand breaks (SSBs). Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci indicated DNA double-strand breaks (DSBs). Cell viability was measured by the MTT test. A significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with BAC exposure in HCEs at concentrations of 0.00005% and higher. Such BAC treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of γH2AX foci. In addition, a significant change in the relative cell survival rate of HCEs was observed after exposure to 0.001% BAC for 30 min. Although the toxic effects of BAC could be partly repaired after 24 h of cell recovery, SSBs and DSBs in HCEs were still present after BAC removal. The results demonstrated that exposure to BAC in HCEs, even at low concentrations, could induce DNA strand breaks, which were present after BAC removal. Cell survival analysis indicated that BAC-induced DNA damage was correlated with the cytotoxic effects.

Comparative Genotoxicity of Cadmium and Lead in Earthworm Coelomocytes

To determine genotoxicity to coelomocytes,Pheretima peguanaearthworms were exposed in filter paper studies to cadmium (Cd) and lead (Pb) for 48 h, at concentrations less than the LC10—Cd: 0.09, 0.19, 0.38, 0.75, and 1.50 μg cm−2; Pb: 1.65, 3.29, 6.58, 13.16, and 26.32 μg cm−2. For Cd at 0.75 μg cm−2, in the micronucleus test (detects chromosomal aberrations), significant increases () in micronuclei and binucleate cells were observed, and in the comet assay (detects DNA single-strand breaks), tail DNA% was significantly increased. Lead was less toxic with minimal effects on DNA, but the binucleates were significantly increased by Pb at 3.29 μg cm−2. This study shows that Cd is more acutely toxic and sublethally genotoxic than Pb toP. peguana. Cadmium caused chromosomal aberrations and DNA single-strand breaks at 45% of the LC10concentration. Lead, in contrast, did not induce DNA damage but caused cytokinesis defects.

Aprataxin, poly-ADP ribose polymerase 1 (PARP-1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1 (AOA1), is a DNA repair protein that processes the product of abortive ligations, 5' adenylated DNA. In addition to its interaction with the single-strand break repair protein XRCC1, aprataxin also interacts with poly-ADP ribose polymerase 1 (PARP-1), a key player in the detection of DNA single-strand breaks. Here, we reveal reduced expression of PARP-1, apurinic endonuclease 1 (APE1) and OGG1 in AOA1 cells and demonstrate a requirement for PARP-1 in the recruitment of aprataxin to sites of DNA breaks. While inhibition of PARP activity did not affect aprataxin activity in vitro, it retarded its recruitment to sites of DNA damage in vivo. We also demonstrate the presence of elevated levels of oxidative DNA damage in AOA1 cells coupled with reduced base excision and gap filling repair efficiencies indicative of a synergy between aprataxin, PARP-1, APE-1 and OGG1 in the DNA damage response. These data support both direct and indirect modulating functions for aprataxin on base excision repair.

Detection of DNA Single Strand Breaks Induced by Chemical Mutagens Using the Acellular Comet Assay

In the acellular comet assay, slides with gels prepared from untreated cells are exposed after lysis to the test agents and then processed according to the standard comet assay protocol. The sensitivity of acellular comet assay was compared with that of standard assay using human lymphoblastoid WTK1 cells. Selected model mutagens were N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), bleomycin (BLM), and UVC. In the acellular assay, lysed slides were exposed to model chemical mutagens for 2 h or irradiated to UVC, and then electrophoresed immediately after chemical mutagen-treatment or 2 h after UVC-irradiation. The slides for standard assay were prepared immediately after 2 h-exposure to each chemical mutagen or 2 h after UVC-irradiation. In both assays, slides were electrophoresed at pH>13 or pH 12 for 20 min after 20 min unwinding. BLM was positive at pH>13 and pH 12 in both assays. UVC was positive in the standard assay but not in the acellular assay. In spite of positive responses of alkylating agents in the acellular assay at pH>13 and pH 12, they were positive at pH>13 but not at pH 12 in the standard assay. The positive responses in the acellular assay were greater than those in the standard assay. Our present results suggest that acellular comet assay can detect DNA single strand breaks (SSBs) as initial lesions but not alkali-labile sites generated from DNA lesion such as alkylated bases and that the sensitivity to detect SSBs as initial lesions is lower in the standard than in the acellular assay.

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.

Enhancement effect of polychlorinated biphenyl on benzo (a) pyrene-induced DNA damage in HepG2 cells

To study the effect of polychlorinated biphenyl, Aroclor1254 on benzo (a) pyrene [B (a) P]-induced DNA damage in HepG2 cells. HepG2 cells were pretreated with Aroclor1254 (11.5, 23 and 46 micromol/L) for 24 hours and then exposed to B (a) P (50 micromol/L). DMSO (10 ml/L) was used as solvent control. Single cell gel electrophoresis (SCGE) and high-performance liquid chromatography-electrochemical detection (HPLC-EC) assays were applied to detect DNA single-strand breaks and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in HepG2 cells, respectively. Average Oliver tail moment (OTM) and 8-OHdG level in HepG2 cells were significantly increased in B (a) P treated group (1.66 +/- 0.21), (23.31 +/- 6.02) 8-OHdG/10(6)dG than that in solvent control (0.79 +/- 0.15), (12.31 +/- 3.24) 8-OHdG/10(6)dG, respectively. In Aroclor 1254 treated group (11.5, 23.0, 46.0 micromol/L), average OTM were 0.88 +/- 0.20, 1.01 +/- 0.15 and 1.10 +/- 0.16, and 8-OHdG levels were (19.57 +/- 7.57), (22.80 +/- 9.16) and (31.74 +/- 9.25) 8-OHdG/10(6)dG, respectively. A concentration of 46 micromol/L Aroclor1254 caused a significant increase of 8-OHdG level as compared with the solvent control. After pretreatment of HepG2 cells with Aroclor1254 (11.5, 23.0 and 46.0 micromol/L), B (a) P induced more DNA strand breaks (OTM: 2.14 +/- 0.22, 2.43 +/- 0.32 and 2.71 +/- 0.31) and 8-OHdG [(32.50 +/- 3.81), (49.23 +/- 16.66) and (60.36 +/- 18.04) 8-OHdG/10(6)dG] in HepG2 cells than B (a) P alone. Aroclor1254 might enhance B (a) P-induced DNA damage in HepG2 cells, which should imply a synergistic effect of Aroclor1254 on the genotoxicity of B (a) P.

Effect of low doses and low dose-rates of gamma radiation on DNA damage in human peripheral blood leukocytes using comet assay

Comet assay is a sensitive technique and it provides a unique opportunity to investigate intercellular differences in DNA damage and repair in any eukaryotic cell population. This assay can be used to detect DNA single strand breaks, alkali-labile sites and incomplete DNA repair sites, under alkaline conditions and DNA double strand breaks under neutral condition. We report here our observations on the low-dose (1.25, 2.50, 5.0 and 10.0 cGy) effects of gamma rays on DNA damage in human peripheral blood leukocytes. In a separate experiment on dose-rate, human blood samples were exposed to a total dose of 10.0 cGy at different dose-rates (0.46, 0.93 and 1.85 cGy/ min) of gamma rays using an industrial radiographic camera. Cells were processed by alkaline comet assay, stained in SYBR Green II and analysed using an imaging software SCGE-Pro, developed in our laboratory for quantification of DNA strand breaks using comet assay. Various comet parameters, e.g., tail length, % DNA in tail and tail moment, were measured for each comet. Analysis of data revealed significant increase in DNA damage even at the lowest dose of 1.25 cGy. Lower dose-rate produced higher DNA damage as compared to the higher dose-rate.

Comparison of Genotoxicity of Textile Dyestuffs in Salmonella Mutagenicity Assay, In Vitro Micronucleus Assay, and Single Cell Gel/Comet Assay

The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

Effects of ozone on DNA single-strand breaks and 8-oxoguanine formation in A549 cells

Animal studies have demonstrated that ozone exposure can induce lung tumors. Recent epidemiological studies have also shown that increased ozone exposure is associated with a greater risk of lung cancer. This study used single-cell gel electrophoresis (the Comet assay) and flow cytometry to investigate DNA damage in A549 cells exposed to ozone levels below the current ambient standard. Cells were exposed to ozone at levels of 0, 60, 80, and 120 ppb, and then DNA single-strand breaks and 8-oxoguanine levels were measured. Additionally, the formamidopyrimidine glycosylase (Fpg) repair enzyme was added to the Comet assay to enhance detection of oxidative damage. Vitamins C and E were also added to determine their inhibitory effects on ozone-induced 8-oxoguanine. Measurements of tail length, tail intensity, and tail moment of the Comet assay were shown to correlate with each other. However, tail moment appeared to be more sensitive than the other two indicators in detecting DNA single-strand breaks. Tail moments of cells exposed to 80 and 120 ppb of ozone were significantly higher than those exposed to 0 ppb ( P<0.05). These three indicators of DNA single-strand breaks with Fpg were shown to be increased and more sensitive than those without Fpg. After Fpg was introduced, the tail moments at ozone levels of 60, 80, and 120 ppb were significantly higher than those at 0 ppb ( P<0.05). Furthermore, 8-oxoguanine levels, determined by fluorescence intensity, at 80 and 120 ppb of ozone exposure were significantly higher than the level at 0 ppb. Pretreatment with vitamins C and E reduced the 8-oxoguanine levels caused by ozone. We conclude that ozone levels below current ambient standards may induce DNA breaks and oxidative DNA damage. Moreover, the Fpg repair enzyme in the Comet assay can increase the sensitivity of oxidative damage detection in vitro.

Detection of DNA single-strand breaks and glutathione in mononuclear blood cells of radiotherapy technicians.

We wanted to investigate the effects of gamma radiation on DNA single-strand breaks (SSB) and glutathione (GSH) levels in mononuclear blood cells (MNC) of radiotherapy technicians. DNA SSB in MNC of radiotherapy technicians who use (60)Co-gamma source in their works were detected by alkaline filter elution and compared to control subjects. In addition, GSH levels were measured using the enzymatic method in MNC. Blood samples were collected from radiotherapy technicians on Monday and Friday. DNA SSB levels were found to be significantly higher in smoking controls compared to non-smoking controls. Significant increases of 36% and 49% in DNA SSB were detected from Monday to Friday for non-smoking and smoking radiotherapy technicians, respectively. GSH levels were found to be decreased significantly from Monday to Friday. Gamma-radiation resulted in increased DNA SSB levels of MNC in radiotherapy technicians throughout the working week and these breaks have been observed to be repaired at the weekend. Smoking habit caused an additional increase in the SSBs observed in radiotherapy technicians.

Breast 06

Folate is required for DNA synthesis, repair and methylation, and low folate status has been implicated in carcinogenesis possibly as a result of higher rate of genetic damage. To investigate the relationship between DNA damage and folate status in breast cancer and benign breast disease control patients. Fasting blood samples from 52 histologically confirmed untreated breast cancer patients (mean age: 57 years) and 30 benign breast disease control patients (mean age: 51 years) were obtained. Mononuclear cells were isolated for genetic damage analysis using basic alkaline comet assay (which detects DNA single-strand breaks) and modified comet assay (using endonuclease III and formamidopyrimidine glycosylase (FPG) to recognize oxidatively damaged pyrimidines and purines, respectively). Results are expressed as tail moment. Results were log transformed to normalize the data and presented as geometric mean (95 per cent confidence interval). DNA damage was found to be significantly higher in mononuclear cells of breast cancer patients compared to benign breast disease control patients. Breast cancer patients tended to have lower red cell folate and higher plasma homocysteine concentrations, but these differences were not significant. A mechanistic role for low folate status in the aetiology of breast cancer is under further investigation.

Analysis of DNA damage in individual cells

DNA is the most common intracellular target for the action of agents used to treat malignant disease, and it is an essential target for mutagenic agents. The comet or single-cell gel electrophoresis method can detect, either directly or indirectly, a variety of DNA lesions, including single-strand breaks, doublestrand breaks, interstrand cross-links, and damage to the DNA bases. Apoptotic cells can be easily identified because highly fragmented DNA in apoptotic cells migrates efficiently. As few cells are required for this method, fluorescence-activated cell sorting becomes a practical way to provide purified, populations for analysis of DNA damage. Comets can be stained with antibodies against incorporated bromodeoxyuridine (BrdUrd) or against specific DNA adducts. Much of the popularity of the comet assay is due to the ability of this method to measure DNA damage in virtually any single cell. A more unique aspect of this method is the ability to detect heterogeneity in response within different cells of a single population. Under alkaline conditions, the comet assay detects DNA single-strand breaks, double-strand breaks, and alkali labile lesions.

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX) in mammalian cells in vitro and in mice

3-Chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX) formed during chlorination of water containing natural organic substances, is a very potent bacterial mutagen. Recently, tumours at multiple sites were reported in rats given MX-containing drinking water. We have investigated the genotoxicity of MX in mammalian cells exposed in vitro and in vivo using alkaline filter elution to detect DNA single-strand breaks and/or alkali-labile sites (SSBs). Concentrations as high as 100 and 300 μM MX were required to induce detectable levels of SSBs in the HL-60 cells. If MX treatment was carried out in the presence of DNA repair inhibitors (AraC plus hydroxyurea), the sensitivity of the assay to detect MX-induced SSBs was increased by a factor of 100. The presence of serum proteins during exposure resulted in a minor reduction of the MX-induced DNA damage in HL-60 cells at the lowest MX concentrations. In primary cultures of testicular cells as well as in resting human peripheral blood mononuclear cells (PBMC), a slightly increased level of SSBs was observed at MX-concentrations above 30 μM, this effect was not further increased by repair inhibitors. In LLC-PK 1 renal proximal tubular epithelial cells and in growth stimulated human peripheral PBMC, increased SSBs were detected at MX concentrations as low as low as 3–10 μM and higher using repair inhibitors, and at 10 times higher concentrations without repair inhibitors. No dose dependent DNA damage was detected in the liver, kidney, spleen and colon of male B6C3F1 mice administrated high doses of MX (40 and 80 mg kg −1). Moderately increased and dose dependent SSBs were detected in the liver and kidney in the presence of DNA repair inhibitors during MX treatment, but no such increase was observed in the spleen and colon.