Mutagenesis Advance Access published online on October 3, 2008
Mutagenesis, doi:10.1093/mutage/gen052
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DNA damage and repair of leukocytes from Fanconi anaemia patients, carriers and healthy individuals as measured by the alkaline comet assay
Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, PO Box 14115-111, Tehran, Iran 1Department of Cellular and Molecular Biology, School of Science, Parand Islamic Azad University, Tehran, Iran
Fanconi anaemia (FA) patients show cellular sensitivity to a variety of clastogens and prominently to cross-linking agents. Although there is a long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. In this study, initial radiation-induced DNA damage and kinetics of DNA repair in 60Co gamma-irradiated leukocytes from healthy volunteers, FA patients and heterozygotes were assessed using alkaline comet assay. Results showed higher levels of baseline DNA damage in leukocytes of patients and heterozygotes than in controls. Gamma-ray-induced initial DNA damage in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. However, after a repair time of 4 h, following irradiation, samples from the healthy individuals and carriers showed less residual DNA damage in their leukocytes, whereas FA patients revealed more DNA damages than their baseline. Although similar initial induced DNA damage was observed for all groups, the repair kinetics of radiation-induced DNA damage of leukocytes from FA patients was statistically different from healthy and carrier subjects. These findings may suggest that hypersensitivity of FA cells to cross-linking and clastogenic agents might be due to inefficient and delayed repair machinery of these cells. Also, the amount of residual DNA damage after irradiation could be used as a putative predictor of FA screening and cellular radiosensitivity.
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Fanconi anaemia (FA) is a rare autosomal recessive genetic disease characterized by increased spontaneous and induced chromosome instability, a diverse assortment of congenital malformations, progressive pancytopenia and cancer susceptibility, especially acute myeloid leukaemia, but also solid tumours. This clinical heterogeneity is in part explained by genetic heterogeneity, where FA patients are now distributed into at least 12 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L and M) defined by cell fusion studies and 11 of the 12 responsible genes have been identified [see for review Taniguchi and D'Andrea (1
)]. FA cells are hypersensitive to cross-linking agents and, to a lesser extent, to ionizing radiation. Other features of FA are abnormal cell cycle regulation, oversensitivity to oxidative stress, high level of apoptosis, overproduction of tumour necrosis factor, deficient induction of P53, and genomic instability (2). In addition, FA cells have a cell cycle disturbance (3–5) and increased susceptibility to oxygen-induced damage (6,7). Importantly, defects of the FA genes have been found in a wide variety of cancer in the general population (8–10). The hallmark of the FA cellular phenotype is a high level of spontaneous chromosomal breakage (11) that is enhanced by exposure of the cells to DNA cross-linking agents such as mitomycin C (MMC) (12) and diepoxybutane (13). Because of this hypersensitivity to DNA cross-linking agents, many researchers have speculated that the primary defect of FA cells is in DNA damage response or DNA repair. The 11 identified FA proteins appear to cooperate in a common pathway, regulating the repair of interstrand DNA cross links (1). A less-known feature of FA cells is their radiosensitivity. Radiosensitivity of FA cells is a controversial issue so that some reports have shown a high sensitivity to X- or gamma irradiation (14–20), and others have found that FA peripheral blood lymphocytes displayed similar frequencies of chromosomal aberrations (12,21,22) as normal cells. In this respect, in vitro studies using different assays (16), cell lineage (23) or complementation groups (24) have led to different conclusion. In a study with DNA-damaging agents other than cross-linking agents, Carreau et al. (25) have shown that from eight FA complementation groups, groups D to H showed sensitivity to the bleomycin, a radiomimetic drug. This report may suggest that FA proteins may have different roles in various cellular pathways. Previously, impaired DNA repair has been reported for FA fibroblasts and FA peripheral blood mononuclear cells (26,27). However, the incubation time allowed for rejoining of X-ray induced DNA damages was only 50 min which might only include rejoining of fast repair component of DNA damage. Although obligate heterozygous individuals from FA families have not been extensively researched, all available evidences suggest that these individuals are free from major clinical symptoms, have a normal life expectancy and have only a minor, if any, elevation of cancer risk (28). It is recognized that individual risk of cancer may be related to genetically determined differences in the ability of a cell to identify and repair DNA damage and it has been suggested that individuals who are genetically susceptible to cancer manifest this by exhibiting increased DNA radiosensitivity (29). In light of our recent report (22) and conflicting data on radiosensitivity of FA and FA carriers, re-examination of the response of FA cells to gamma ray at DNA level has been done in this study. Furthermore, we have extended the incubation time for repair of gamma-ray-induced DNA damage up to 4 h because the process of DNA break rejoining contains a slow component needing a half-time of 1–2 h for rejoining. (30). Up to now, an optimum method for the prediction of radiosensitivity and the best parameter has not been found. This has largely been in the context of assessing inherent cellular radiosensitivity through damage induction or repair parameters. Various experimental approaches were used for the detection of DNA strand breaks; for example, filter elution, constant field gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and the comet assay (31). The parameters most frequently analysed with these test systems are the initial DNA damage, the residual DNA damage remaining after a period of repair and the repair rate (32).
The comet assay is already recognized as being among the most sensitive methods available for measuring DNA strand breaks; it has further advantages of speed, simplicity and the fact that observations are made at the level of single cells. It is also an invaluable tool for investigating fundamental aspects of DNA damage and cellular responses to this damage (32,33). The alkaline comet assay resolves break frequencies up to a few thousand per cell, so the distances between breaks are of the order of 109 Da, definitely well beyond the range of fragment size for which conventional electrophoresis is suitable. The alkaline comet assay detects single and double DNA strand breaks and has been used to assess the rejoining of double-strand break (DSB) and single-strand break (SSB) (33).
In the present study, induction of DNA damage and its repair in leukocytes of FA patients, obligate carriers and healthy volunteers were compared after in vitro exposure with gamma rays using alkaline comet assay.
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Study subjects, blood cells and irradiation
Ten FA patients, aged between 4.5 and 16 years (mean age = 9.6 ± 3.8), were recruited at Ali Asghar Children Hospital in Tehran, Iran. None of them had been treated with chemotherapy or radiotherapy. The clinical details of these patients are summarized in Table I. On the basis of chromosome breakage after mitomycin C treatment (40 ng/ml), the clinical diagnosis of FA was confirmed in all 10 patients (see Results, Table II). Thirteen FA obligate carriers, aged between 33 and 45 years (mean age = 38.7 ± 4.3), were also examined. Ten healthy volunteers aged between 28 and 44 years (mean age = 35 ± 4.9) were selected as a matching group. The study was approved by the Ethical Committee of the School of Medical Sciences of the Tarbiat Modares University. All donors gave their informed written consent and completed a written questionnaire to obtain information related to their lifestyle, such as dietary habits, medical history and exposure to chemical and physical agents.
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Sample preparation and irradiation
In total, 7.5 µl of heparinized venous blood was added to the 1.5-ml microcentrifuge tube containing 1 ml RPMI 1640 medium (Gibco, BRL, Long Island, NY, USA) supplemented with 10% foetal calf serum (Gibco, BRL). Diluted blood was irradiated on ice with 4 Gy gamma rays (at source to sample distance = 80 cm, room temperature 23 ± 2°C) with a dose rate of 2.75 Gy/min generated from a 60Co source (Theratron II, 780C, AECL, Kanata, Ontario, Canada). This radiation dose was selected based on our recent study on radiosensitivity of FA cells at chromosomal level (22
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Slide preparation
Samples were centrifuged at 0°C. The supernatant was poured off and the pelleted cells were mixed with 100 µl of 37°C low melting point agarose (Fermentas LQ, 0.75% agarose in phosphate-buffered saline, Ca2+, Mg2+ free). The cell mixture was added to the windows made on frosted slides with clear windows, precoated with 1% normal agarose (Merck, Darmstadt, Germany) and was immediately covered with a coverslip. The slides were placed on a tray kept for 10 min on ice to solidify. After solidification, the coverslip was removed.
Alkaline comet assay
Slides were submersed in an alkaline lysis solution (2.5 M sodium chloride, 100 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris base, 10% dimethylsulphoxide, 1% sodium N-lauroyl sarcosinate and 1% Triton X-100, Merck, pH = 10) for 1 h at 4°C. Lysis was followed by unwinding step by immersing the slides in a freshly prepared alkaline solution (0.3 M NaOH and 1 mM EDTA, Merck, pH > 13) in a horizontal gel electrophoresis tank (SEU-7305, Paya Pajouhesh, Iran) for 40 min at 4°C. Electrophoresis was done at 0.75 V/cm for 30 min at 4°C. The slides were washed three times in neutralization buffer (400 mM Tris buffer, pH = 7.5) and rinsed in ethanol for 5 min and air dried.
Staining, microscopic analysis and experimental parameters
Cells were stained with 20 µl ethidium bromide (Merck, 2 µg/ml) under a coverslip. Observations were made at a magnification of x200 using a Nikon E800 epifluorescence microscope (Japan) equipped with 546-516 wavelength band and a 590-nm barrier filter attached to a Charged Coupled Device (CCD) camera. The comets were analysed by visual classification (34) and for each sample 1000 cells were scored. Damage was assigned to five classes (0–4) based on the visual aspect of the comets, considering the extent of DNA migration according to the established criteria (35–37). Comets with a bright head and no tail were classified as class 0 (cells with no DNA migration) and comets with a small head and a long diffuse tail were classified as class 4 (severely damaged cells). Comets with intermediate appearance were classified into classes 1, 2 and 3. Damage scores were calculated based on the following equation adopted from Jaloszynski et al. (34) that ranged from 0 to 400 arbitrary units, corresponding to situations ranging from no damaged comets to all comets extremely damaged:
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n: total number of scored comets.
Coefficients 0–4 are weighting factors for each class of comet. One may suspect that the visual classification may be inferior to computerized analyses, such as tail moment analysis of images captured by CCD camera. However, it has been clearly shown that there is no statistical difference between visual quantification and image analysis by computer for tail moment quantification (35,38). However, to verify the validity of results obtained by visual analysis, pictures of 100 cells, for each incubation time after irradiation, captured by CCD camera for repair study, were analysed automatically using COMET IV software. COMET is a tool to image analysis in comet assay and has been developed to work with either colour or greyscale images of fluorescence-stained comets. Tail migration (TM) and Olive tail moment (OTM) were calculated by software. TM shows the rate of DNA migration from head to tail and OTM shows the amount of DNA in the tail of each comet. As shown in Figure 2B and C, the kinetics of repair is similar and not significantly different from that obtained by visual analysis of comets (Figure 2A).
Four experimental parameters were evaluated to characterize cellular radiation effects: (i) baseline DNA damage detectable in cells that had not been irradiated (DD0); (ii) induced DNA damage measured directly after irradiation (DD); (iii) net DNA damage which is calculated by subtracting the baseline DNA damage from DNA damage measured directly after irradiation; (iv) repair capacity was also estimated quantitatively at 0, 2 and 4 h after irradiation after standardization using the following equation adopted from Bergqvist et al. (39):
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Statistical analysis
All statistical analyses were carried out using SPSS software version 15. Differences between means of initial radio-induced DNA damage between groups were tested for significance with the two-sided, unpaired Student's t-test. To analyse the results of residual DNA damage between groups, the non-parametric Mann–Whitney U-test and one-way analysis of variance were also used and summarized by means, standard deviation and 95% limits of agreement. P-value of <0.05 was considered as significant level. Figures were drawn using Sigma Plot 2004 for Windows, version 10.
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Chromosome breakage analysis after MMC treatment
On the basis of chromosome breakage after mitomycin C treatment, the clinical diagnosis of FA was confirmed in all 10 patients. The results are summarized in Table 2. The frequency of spontaneous as well as MMC-induced chromosomal aberrations between controls and heterozygotes was not significantly different but frequency of spontaneous aberrations in lymphocytes of FA patients was
20 times and MMC-induced aberrations 58 times more than control values, statistically significant from each other (P < 0.0001). Therefore, samples used for DNA damage repair study were from FA patients.
Comparison of initial induced DNA damages of FA patients and carriers with healthy volunteers
Results are shown in Figure 1. The figure presents the range of assay parameters observed in the study subjects, as well as the medians and the 10th, 25th, 75th and 90th percentiles. Non-irradiated cells of FA patients exhibited noticeably higher baseline amounts of DNA fragmentation compared to carriers and controls. As seen in Figure 1A–C, the mean values of background DNA damage were statistically different in the FA patients, heterozygotes and healthy individuals when the comets are analysed with different parameters (P < 0.05) and this difference was more pronounced when TM was considered for analysis. Induced DNA damage showed identical median and mean values for the controls, heterozygotes and the patients (P < 0.05). Net induced DNA damage was higher in FA patients than carriers and healthy controls, but this failed to reach statistical significance (P > 0.05) when either parameters is considered. In addition, a very similar range of distribution was found for the results of the controls and of the patients and is marked by the boundaries of the boxes that represent the 25th and 75th percentiles. Patients with results lying within the 25–75% range of the healthy controls were considered to show a ‘normal’ cellular reaction to gamma irradiation. In addition, the patients exhibiting less damage in non-irradiated or irradiated cells than marked by the 25–75% range were also classified as normal. Induced DNA damage (DD) in irradiated cells yield about two (20%) of the 10 controls and three (23%) of 13 heterozygotes and three (30%) of 10 FA patients have values higher than the induced cut point defined as mean + 1 SD of control values (38). Statistical analysis showed that there was no statistical difference between net induced DNA damage of FA patients, heterozygotes and healthy controls.
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Study of repair kinetics
In order to investigate the rejoining of DNA breaks, the changes in the residual DNA damage in 10 FA patients, 13 carriers and 10 healthy volunteers were investigated at 0, 2 and 4 h after exposure to gamma rays with 4 Gy under the alkaline condition and are shown in Figure 2. As seen in Figure 2A, repair kinetics of DNA damage quantified by visual analysis where the standardized DNA damage in normal and FA heterozygotes is almost similar and not significantly different with each other in either 2- or 4-h repair time. However, a marked and significant difference was observed for repair of DNA damage in cells obtained from FA patients which showed a slower repair kinetic especially at 4 h after irradiation. This difference was found significantly different with either normal or heterozygotes (P < 0.05). Figure 2B and C shows the results obtained by automatic analysis of TM and OTM, respectively, for repair kinetics of DNA damages in the study groups. The trends of DNA damage repair shown in these figures are completely compatible with those obtained by visual analysis and shown in Figure 2A. Also similar results were obtained when TM and OTM were considered for analysis. Statistical analysis showed significant difference for TM (Figure 2B) and OTM (Figure 2C) between normal individuals and FA patients for all incubation times after irradiation (P < 0.05). Results also indicated that although the repair kinetics in heterozygotes was slower than controls, it was not significantly different. The rates of residual DNA damage in carriers were less than patients in both incubation times (P < 0.05).
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The results show that the patients have higher baseline values of DNA damage than controls. Using similar technique, higher levels of baseline DNA damage in leukocytes of breast cancer patients compared to controls were also reported (38
). Persistent basal DNA damage may reflect higher exposure to carcinogens and deficient DNA repair (40). It could be argued that pre-existing DNA damage, as indicated by increased spontaneous chromosome breakage rates in FA, may be responsible for the high initial tail moment and TM values in patients’ and carriers’ cells without being irradiated (Figure 1).
After irradiation in vitro, there was a trend toward an increased induced DNA damage level (DD) in the cells from FA patients, but this failed to reach statistical significance (Figure 1). These results are similar to the study of Cassado et al. (41) who used PFGE to study DNA damage in FA lymphoblastoid cells with different complementation groups. These results as well as those shown in Figures 1 and 2 are in contrast to the results reported by Djuzenova et al. (26,27) for FA fibroblasts and FA mononuclear blood cells using the comet assay. Our observations show that the initial number of DNA breaks produced by radiation is almost similar in normal, carriers and FA cells.
A number of reports claim to have shown that FA cells are radiosensitive (14–19) and these claims are supported by clinical observations of poor outcome and increased pigmentation and desquamation of irradiated skin areas in a number of FA patients after preconditioning for bone marrow transplantation (17,42). However, there are a number of reports that fail to find evidence for increased radiosensitivity of FA cells (22,41,43,44). According to the presented data, cells from FA patients did not show radiosensitive when initial DNA damage was used as an index for radio-sensitivity. The net induced DNA damage after gamma irradiation in FA patients and controls is not significantly different (Figure 1). It is worthy to mention that observation of statistically different basal DNA damage and also similar net induced DNA damage between the study groups might not be affected by inter-individual differences. As shown in Figure 1, a very similar range of distribution was found for the results of the controls, heterozygotes and patients marked by boundaries of the boxes that represent the 25th and 75th percentiles.
What is measured by the comet assay is not radiosensitivity in the conventional sense, i.e. chromosome breakage, micronucleus formation, reduced growth and cloning survival or increased mutation frequency. Rather, the comet assay provides a measure of chromatin integrity as a function of time immediately following exposure to a clastogenic agent such as ionizing radiation. Differences in the degree of chromatin compaction may, for example, influence the results of the comet assay (45). Moreover, the recognition of DNA damage by using the comet assay is known to be influenced by a number of factors that would alter the DNA release from the nuclear protein matrix (46). Therefore, pre-existing intrinsic alterations in chromatin conformation might well determine the degree of DNA migration in response to radiation-induced damage (47,48). The failure of FA patients’ cells to respond normally to ionizing irradiation in the comet assay is consistent with the recent notion that FA might be a ‘chromatin disease’ in which a defective complex of the various FA proteins does not adequately fulfil its role in chromatin remodelling in situations of cellular stress (26,49). This fact would also suggest that the residual DNA damage measured as TM and OTM values of FA patients could not be restored to normal if given sufficient time. Our choice of sampling times following irradiation, to study repair capacity of normal and cells obtained from FA patients, was based on previous studies on cancer patients. In most of these studies, an incubation time of 
3 h was used to study the residual DNA damage (38,50,51). Previously, Djuzenova et al. (26,27) have studied on fibroblast and mononuclear blood cells of FA patients during the first 50 min, with 10 min sampling intervals after exposure to 3 Gy of X-irradiation; our results are not compatible with their report (Figure 2). In their study, the cells of both FA patients and carriers showed uniformly high initial induced DNA damage rates as assessed by the total initial tail moment. In addition, the average residual tail moment at 30–50 min and the repair half-time parameters were significantly elevated. These authors have also calculated a mean half-time repair of 4.1 ± 2.4 min for normal individuals, 9.8 ± 7.4 min for carriers and 10.4 ± 8.9 min for FA patients. Moreover, the longest repair half-time calculated for patients by these authors was 36.3 min. However, in their research, it was not clear if they exclude the basal DNA damage or not. As shown in Figure 1, the basal DNA damages in FA patients were higher than carriers and healthy donors, statistically significant (P < 0.05).
In the present study, we thought the repair time of <1 h might not differentiate between DNA damage repair of cells from normal, FA and carriers. Figure 2A–C clearly shows that there is no statistical difference between the rate of DNA damage rejoining up to 2 h, although the initial DNA damage in FA cells is higher than normal and heterozygotes. Alkaline comet assay detects SSB and DSB (33); therefore, the processes of DNA breaks rejoining study with this technique contain a fast component and a slow component (52). Therefore, in the present study, the incubation time to allow rejoining of DNA strand breaks was extended to 4 h. While the induced SSBs were rejoined with a half-life of several minutes in many cell lines, the DSB were rejoined with a half-life of 10–20 min for the fast component and 1–2 h for the slow component after irradiation (30). It is suggested that the remaining damage measured after 3 h is an excellent index of the restoration ability of DNA damage (53). It has been shown that the rejoining of radiation-induced DSBs was levelled off between 2 and 4 h in mammalian cell lines (50,54) and that the number of residual DSB did not change statistically different between 4 and 20 h using the neutral filter elution technique (55).
After 4-h incubation post-irradiation, however, the residual DNA damage in cells from FA patients was found to be at a much higher level than controls and heterozygotes (P < 0.05) (Figure 2). G2 chromosomal radiosensitivity shown for FA cells (15,18) might be due to this higher level of DNA damage that converts into chromatid type's damages when cells are irradiated at G2 phase of the cell cycle. At the molecular level, involvement of FA proteins in homologous recombination (HR) and non-homologous end joining (NHEJ) has been reported. Increasing evidence suggests that FA cells are defective in HR, although there is some inconsistency among reports; however, there seems to be at least a mild defect of HR in FA cells (see for review, Taniguchi and D'Andrea (1)). NHEJ defect in FA cells exposed to ionizing radiation for three complementation groups (FA-A, FA-C and FA-F) has been reported while the number of DNA DSBs in normal and FA cells was similar and no difference in radiosensitivity of normal and FA-A lymphoblast cells was observed (41). Moreover, there are also evidences suggesting involvement of FANCD2 in radiosensitivity of FA fibroblasts and mice (56,57).
In conclusion, in the present study, no difference was found in the initial DNA damage induced by gamma rays between FA patients, heterozygotes and healthy controls by using the comet assay. However, there was a significant difference in repair kinetics and in the amount of residual DNA damage in leukocytes of FA patients compared to other study groups. Therefore, elevated sensitivity of FA cells to intercalating and clastogenic agents might be due to inefficient and delayed repair machinery of these cells.
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Research Department of Tarbiat Modares University.
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The authors wish to express their sincere thanks to Dr P. Vosough for her advice and help for sampling, all patients and healthy individuals for blood donation and Ms Z. Tizmaghz for irradiation of samples.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +98 21 82883830; Fax: +98 21 88006544; Email: mozdarah{at}modares.ac.ir
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Received on May 13, 2008; revised on August 27, 2008; accepted on August 27, 2008.
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