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Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes
Mutation Research 701 (2010) 86–91
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes b ˜ Pasquale Mosesso a,∗ , Fabrizio Palitti a , Gaetano Pepe a , Joaquin Pinero , a c,1 a Raffaella Bellacima , Gunnar Ahnstrom , Adayapalam T. Natarajan a b c
Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis s.n.c., 01100 Viterbo, Italy University of Seville, Department of Cell Biology, C/Reina Mercedes sn, 41012 Seville, Spain Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 5 March 2010 Accepted 9 March 2010 Available online 16 March 2010 Keywords: FISH–Comet assay DNA repair Heterochromatin CpG islands DMSO Radical scavengers Exchange aberrations and chromatin architecture
a b s t r a c t Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced • OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET–FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ionizing radiation (IR) is an efficient inducer of chromosome aberrations (CA), which represent the outcome of very complex events, involving the conversion of DNA double strand breaks (DSBs) and other lesions through various DNA repair pathways into microscopically detectable events. Though IR is expected to induce DNA lesions in a random manner in the genome, some studies using FISH technique with chromosome specific DNA probes, have reported that the frequency of radiation-induced chromosome exchanges was proportional to the length or to the DNA content of target chromosomes [1–3], whereas other studies have reported that the induction of chromosome exchanges is nonrandom among the chromosomes studied [4–10]. The possible causes for this reported non-random distribution of exchanges
∗ Corresponding author. Tel.: +39 0761 357257; fax: +39 0761 357257. E-mail address: [email protected] (P. Mosesso). 1 Deceased. 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.03.005
have been attributed to various factors such as, differential primary damage of DNA and its repair which could be influenced by transcriptional activity [11–14], chromatin structure [4,5,15,16] and gene density [17,18] of the studied chromosomes. This implies that CA we observe, which are the consequences of mis-repair of IR induced DSBs, may not reflect the initial distribution of DNA damage in the genome. Earlier studies using the technique of premature chromosome condensation (PCC) by fusion with mitotic cells, have shown that in human lymphocytes, exchange type of aberrations are formed within a few minutes, following low doses (<2 Gy) of Xrays where the two lesions required to produce the exchange are significantly generated by one ionization track, whereas at higher doses where the probability of the two lesions being produced by two independent tracks is higher, these aberrations increase with the duration of recovery [19,20]. This reflects the relative roles of slow and fast repairing components of induced DNA double strand breaks (DSBs) leading to CA. The underlying basis for the slow and fast repairing components of the DNA breaks may be related to the differential localization of the damage in the genome, i.e., between relaxed and condensed regions. The genome is highly het-
P. Mosesso et al. / Mutation Research 701 (2010) 86–91
erogeneous and it has been shown that genes are clustered among chromosomes in a sub-set of R bands, most of which are known as T bands containing the highest concentration of CpG islands typical of relaxed regions. While G bands are late replicating, relatively ATrich, highly condensed and transcriptionally silent, R and T bands are early replicating, less condensed with unmethylated DNA and accessible HpaII sensitive sites on chromosomes [21]. We have tried to elucidate the relative roles of slow and fast repairing components of induced DSBs in relaxed and condensed regions by means of cytogenetic analysis in prematurely condensed chromosomes (PCC) and alkaline Comet assay in unstimulated (G0) human lymphocytes irradiated with X-rays in the absence and presence of dimethyl sulfoxide (DMSO). DMSO, an efficient and selective scavenger of radiation-induced • OH radicals, can be used as a probe for events taking place in regions of different chromatin compactness, since DNA damage induced by • OH radicals is known to be high in more opened chromatin [22–25]. Furthermore, we probed the damage and repair in transcriptionally silent condensed regions of chromosomes or from relaxed regions with very high gene density by COMET–FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques [26] in human chromosomes, 18 and 19, which possess ideal properties in respect to their structural organization to be considered as models of condensed and relaxed chromatin respectively. Both chromosomes are similar in size, but exhibit very different chromatin organisation and banding patterns. While chromosome 19 has a very high gene density with strong hybridization signals for T bands, chromosome 18 exhibits very few active genes and very little hybridization signals for T bands [27,28]. 2. Materials and methods 2.1. Cell preparation and treatment conditions Lymphocytes were isolated from bags of “buffy coat” generated from approximately 500 ml of heparinised fresh venous whole blood drawn from three healthy male donors, supplied by a local hospital. “Buffy coats” were diluted 1:1 in phosphate-buffered saline (PBS) and lymphocytes separated using Ficoll–Histopaque 1077 (Sigma). Briefly, 15–20 ml diluted “buffy coat” was layered over 12.5 ml Ficoll–Histopaque in 50 ml polystyrene conical centrifuge tubes (Falcon) and centrifuged for 35 min at 450 × g at room temperature. During centrifugation, mononuclear cells and platelets were concentrated in a fluffy white layer below the plasma. The cell layers were collected with Pasteur pipettes and washed twice with fresh culture medium. Then the cell pellets were resuspended by gentle vortexing, and cell suspensions with a final concentration of 1 × 107 cells/ml in PBS were prepared. Greiner® 14 ml conical cell culture tubes contained 0.5 ml of cell suspension (5 × 106 lymphocytes) and 4.5 ml medium (Ham’s F-10, supplemented with 20% foetal calf serum heat-inactivated at 56 ◦ C, 100 IU/ml penicillin, 100 g/ml streptomycin, 0.4 mM l-glutamine and 2% Hepes buffer). All the chemicals used for lymphocyte cultures were supplied by Gibco® . Cultures were irradiated with 3 Gy X-rays at 300-kV and 6 mA at a rate of 1.0 Gy/min directly prior to stimulation with 2% phytohaemagglutinin (PHA 15 Murex, Italy) when lymphocytes are in a G0 phase (unstimulated cultures), or 12 h after stimulation with PHA when lymphocytes are in the G1 phase of the cell cycle. For treatments in the presence of dimethyl sulfoxide (Sigma–Aldrich), CAS registry number: 67-68-5, cell suspensions were inoculated in 4.5 ml PBS containing DMSO (2 M final concentration). Human lymphocytes from treated cultures were processed to prepare Comet slides, immediately after irradiation (0 h recovery time) and always on ice or placed in an incubator at 37 ◦ C for the appropriate recovery time (0.5, 1, 2, 4 and 24 h), or fused with mitotic CHO cells to obtain PCC immediately after irradiation (0 h recovery time) and at 2 and 4 h recovery times. Mitotic CHO cells collected by mitotic shake off and frozen at −80 ◦ C in complete growth medium containing DMSO 10% and 0.2 g/ml colcemid, were thawed immediately before use. Control cultures both in the absence and presence of DMSO were not irradiated. 2.1.1. Cytogenetic analysis in prematurely condensed (PCC) human chromosomes Cell fusion between mitotic CHO cells and G0 lymphocytes was mediated by polyethylene glycol (PEG) according to Pantelias and Maillie [29]. Briefly, mitotic cells and appropriately treated human lymphocytes (see Section 2.1) were mixed in a ratio of 1:3 in round-bottom culture tubes. After centrifugation, the cells were suspended in 2 ml of Ham’s F-10 medium without serum and centrifuged again. The supernatant was discarded without disturbing the pellet and 0.25 ml of PEG (MW 1450 from Sigma–Aldrich) was added and left for 1 min. For
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the next 3 min, 2 ml of phosphate-buffered saline (PBS) were added drop wise; the tube was gently shaken after each drop. The cell suspensions were centrifuged and resuspended in 0.5 ml culture medium containing colcemid (0.2 g/ml) and incubated at 37 ◦ C for 1 h [30,31]. Finally cells were treated with hypotonic KCl solution (75 mM) for 10 min and fixed in methanol–acetic acid (3:1). Cell suspension was dropped onto pre-cleaned wet slides, air-dried and aged for at least 1 week. In order to improve detection of dicentric chromosomes in PCC, we employed fluorescence in situ hybridization (FISH) using centromeric DNA probes directly labelled with rhodamine (TRITC) which identify the centromeric region of each human chromosome (Appligene-Oncor). To apply pan-centromeric probes, slides were denatured for 4 min at +80 ◦ C with 70% formamide/2× SSC and dehydrated by serial ethanol washing (70, 90 and 100%). The pan-centromeric probe was pre-warmed for 20 min at 37 ◦ C, 6 l mixed with 14 l hybridization buffer (50% formamide, 10% dextran sulfate, 2× SSC), denatured at +80 ◦ C for 10 min and chilled on ice. Twenty microliters of the probe were put onto each slide, which were then sealed and hybridized at 37 ◦ C overnight in a moist-chamber. Then, slides were preincubated twice with 50% formamide in 2× SSC at 42 ◦ C for 5 min, the coverslips were removed and washed once with 0.005% Tween 20 in PBS. Finally slides were dehydrated by serial ethanol washing (70, 90 and 100%), and embedded in 25 l Vectashield (Vector Laboratories) with DAPI at a final concentration of 0.15 g/ml. 2.1.2. “Conventional” alkaline Comet and FISH/alkaline Comet assays Comet slides were prepared following published protocols for the alkaline “Comet assay” [32] with modifications for FISH with whole chromosome painting probes, as previously described [33,34]. Briefly, 10 l of cell suspension was mixed with 65 l of 0.7% (w/v) low-melting point agarose (Bio-Rad Lab.) and sandwiched between a lower layer of 1% (w/v) normal-melting point agarose (Bio-Rad Lab.) and an upper layer of 0.7% (w/v) low-melting point agarose on microscope slides (Carlo Erba, Milan, Italy). Triplicate slides were prepared from each treatment. Slides were immersed in lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10) containing 10% DMSO and 1% Triton x-100 (ICN Biomedicals Inc.) at 4 ◦ C overnight. Following lysis, slides were placed in a horizontal gel electrophoresis tank with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA, pH ≥ 13) and incubated for 25 min at 4 ◦ C to allow the DNA to unwind and to express the alkali-labile sites. Electrophoresis was carried out at 4 ◦ C for 15 min at 30 V (1 V/cm) and 300 mA. After electrophoresis, slides were immersed in 0.3 M sodium acetate in ethanol for 30 min. One set of slides from each treatment series was dehydrated by passing them through an alcohol series (2 min at 70, 85 and 100%), air-dried and stained with 20 g/ml ethidium bromide immediately before analysis of DNA breakage by conventional Comet assay (tail moment), as described in Section 2.2.2. The remaining two sets of slides from each treatment series were dehydrated by passing them through an alcohol series (2 min at each 70, 85 and 100%), dried thoroughly at 37 ◦ C before detection of probes and counterstained by COMET–FISH analysis. This procedure was adopted to improve stabilization of the gel, which otherwise disintegrates during hybridization. Dual-colour FISH was performed as follows: flow-sorted human chromosomes 18 and 19 (kindly supplied by Prof. M. Rocchi, University of Bari, Italy) were amplified and directly labelled with digoxigenin-11-dUTP or with biotin-16-dUTP (Boehringer, Mannheim, Germany), respectively, by degenerative oligonucleotide primer-polymerase chain reaction (DOP-PCR). 400 ng of each labelled probe, mixed with 100 l of 70% deionised formamide in 2× standard sodium citrate solution (SSC), was applied to cells on the air-dried slides. Cells and probes were denatured at +74 ◦ C for 5 min before cover slips (24 mm × 50 mm) were sealed with rubber cement over the gels. Hybridization was performed overnight at +37 ◦ C in a humid chamber. Slides were then washed in 50% formamide, 2× SSC (pH 7.0) for 5 min at +42 ◦ C followed by three washes in 0.01× SSC for 5 min at +60 ◦ C and a final wash with 4× SSC, 0.05% Tween 20 (pH 7.0) for 5 min. Biotinylated probes were detected with avidin–FITC, biotin–avidin and avidin–FITC (Vector Laboratories, Burlingame, CA, USA). The probes labelled with digoxigenin were detected with sequential mouse anti-digoxigenin, sheep anti-mouse–digoxigenin and sheep anti-digoxigenin–TRITC (Vector Laboratories, Burlingame, CA, USA). Finally slides were dehydrated, dried and mounted in Vectashield (Vector Laboratories) containing 0.15 g/ml 4 ,6diamidino-2-phenylindole (DAPI) and antifade. 2.2. Analysis 2.2.1. PCC analysis PCC slides were analyzed with a Leitz Ergolux microscope equipped with single band pass filters for DAPI, FITC, TRITC and a fully automated metaphase finder (MetaSystems GmbH, Altlussheim, Germany). An Isis digital imaging system (MetaSystems GmbH, Altlussheim, Germany) was used to capture, digitalise, annotate and print fluorescence images. A minimum of 100 PCC plates were scored for each test point per experiment. Chromosomes with two or more centromeric signals were scored as dicentric or polycentric chromosomes (which were converted into dicentrics for the purpose of calculation) while chromosomes without centromeric signal were classified as fragments. A typical example of PCC is shown in Fig. 1. 2.2.2. Conventional alkaline Comet assay Slides stained with DAPI were examined at 40× magnification using an automated image analysis system specific for Comet assays (Comet Assay III; Perceptive
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P. Mosesso et al. / Mutation Research 701 (2010) 86–91 Table 1 Effect of DMSO on exchange aberrations and fragments obtained at different recovery times in PCC of human G0 lymphocytes irradiated with 3 Gy X-rays. Recovery time (h)
Mean frequencies per 100 cells ± SE Exchange aberrations
0 2 4
Fig. 1. G0 PCC stained by fluorescence in situ hybridization (FISH) using DNA probes which identify the centromeric region of each human chromosome. For details see Section 2.1.1.
Instruments, UK) connected to a fluorescence microscope (Zeiss Axioskop 2). Tail moment and percentage of migrated DNA was determined to quantify DNA damage. One hundred cells from each slide were analysed for each experimental point. 2.2.3. Dual-colour FISH alkaline Comet assay Fluorescence microscopy was performed using a Leitz Ergolux microscope equipped with single band pass filters for detection of DAPI, FITC, TRITC signals. Images were captured with a CCD camera mounted onto the microscope. An Isis digital imaging system (MetaSystems GmbH, Altlussheim, Germany) was used for analysis. After FISH analysis using whole chromosome painting probes specific for human chromosomes 18 and 19, undamaged nuclei (Fig. 2a) showed two labelled areas: green for FITC-labelled chromosome 19 and red for TRITC-labelled chromosome 18, following merging of individual fluorescent images obtained with single band pass filters tuned to detect DAPI, FITC, TRITC (Fig. 2b–d). Blue signal
Fragments
−DMSO
+DMSO
−DMSO
+DMSO
101 ± 20 126 ± 17 161 ± 25
21 ± 9 30 ± 13 27 ± 10
949 ± 61 701 ± 53 532 ± 46
756 ± 55 576 ± 48 494 ± 44
(DAPI) refers to the whole genome DNA. Damaged cells showed instead a spread of hybridization signals (green spots for chromosome 19 and red spots for chromosome 18) in the tail of comets (Fig. 3a) after merging of individual fluorescent images obtained with single band pass filters for detection of DAPI, FITC and TRITC, respectively (Fig. 3b–d). Similarly to undamaged cells, blue signal (DAPI) refers to the whole genome DNA. To quantify DNA breakage in chromosomes 18 and 19, at least 100 damaged cells per independent experiment (cells showing a visible tail) with hybridization signals were analyzed for each treatment. Differential DNA fragmentation in chromosomes 18 and 19 was evaluated by measuring the distance of the different hybridization spots in the tail from the main nucleus. On this basis we were able to analyze differences of DNA damage in chromosomes 18 and 19 (Fig. 3 c and d).
3. Results 3.1. Cytogenetic analysis of PCCs Results obtained from cytogenetic analysis of at least 300 PCC per test point from three different blood donors (100 from each of the three experiments performed) are shown in Table 1 as mean incidences of exchange aberrations per 100 cells scored. The standard error of the mean (SE) is also shown. In the absence of DMSO the majority of exchange aberrations is formed during the fusion process (0 h recovery time). At later sampling times (2 and 4 h)
Fig. 2. (a) Agarose-embedded untreated G0 human lymphocyte stained by fluorescence in situ hybridization (FISH) using DNA probes specific for human chromosomes 18 and 19. Image acquisition was performed with a fluorescence microscope equipped with single band pass filters for DAPI, FITC, TRITC. (b–d) Fluorescent images of the same cell generated with single band pass filters for DAPI, FITC and TRITC respectively. a: Merge of images b–d.
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Table 2 Percent cells with Comets (tails ≥ 15 m). Lymphocytes from one donor were irradiated before G0 or after stimulation with PHA (G1) with 3 Gy X-rays. 100 cells each were analyzed per entry following hybridization with painting probes for chromosomes 18 or 19. Recovery time (h)
0 0.5 1 2 4 24
Chromosome 18
Chromosome 19
G0
G1
G0
G1
75 88 92 93 98 99
84 90 90 95 100 100
25 12 8 7 2 1
16 10 10 5 0 0
The frequencies of Comets with DNA fragmentation of chromosome 18 are significantly higher than those of chromosome 19 (p < 0.001; Chi-squared test).
tion spots) at the different recovery times analyzed (0, 0.5, 1, 2, 4 and 24 h). Red hybridization spots (chromosome 18) were generally more displaced at the edge of the Comet tails than the green hybridization spots (chromosome 19) in the majority of damaged cells analyzed for both unstimulated G0 and stimulated G1 lymphocytes, ranging from a minimum of 75 and 84% at the 0 h recovery time (G0 and G1, respectively) to 99 and 100% at the 24 h recovery time (G0 and G1, respectively) as shown in Table 2. This indicates that following X-irradiation of lymphocytes, DNA from chromosome 18 (red hybridization spots) is more extensively fragmented or alternatively rejoined more slowly than DNA from chromosome 19 (green hybridization spots) both in G0 and G1 phases. 3.3. Conventional alkaline Comet assay
Fig. 3. Agarose-embedded G0 human lymphocyte irradiated with 3 Gy X-rays and stained by fluorescence in situ hybridization (FISH) using DNA probes specific for human chromosomes 18 and 19. Processed in the dual-colour Comet/FISH protocol in the presence of two whole chromosome painting probes specific for human chromosomes 18 and 19. Image acquisition was performed with a fluorescence microscope equipped with single band pass filters for DAPI (b), FITC (c) and TRITC (d). a: Merge of images b–d. White bars displayed in Images a–d indicate the length in m of the tail of damaged cell from the main nucleus.
mean incidences increased by 25 and 59%, respectively. The incidence of fragments which is more than nine times higher than exchange aberrations at 0 h recovery time, decreases drastically to 74 and 56% of initial yield at 2 and 4 h recovery times, respectively. In the presence of DMSO the mean incidence of both exchange aberrations and fragments is significantly reduced to approximately 20 and 80%, respectively, of the corresponding values observed in treatments performed in the absence of DMSO at 0 h recovery time. At later recovery times (2 and 4 h) the incidence of exchange aberrations is not significantly modified compared to the initial yield while fragments decrease to 76 and 65% at 2 and 4 h recovery times, respectively.
Fig. 4 shows the mean tail moment (MTM) observed in unstimulated lymphocytes following X-ray irradiation (3 Gy) in the absence and presence of DMSO at 0, 0.5, 1, 2, 4 and 24 h recovery times obtained from three experiments. At 0 h recovery time the mean tail moment observed was markedly higher in the absence of DMSO (9.3) than in its presence (5.2) (ANOVA test). At 0.5 h recovery time, the initial MTM observed in the absence of DMSO dropped to approximately the same value observed in its presence, indicating a very fast rejoining of DNA breaks. Beyond 0.5 h recovery time, both curves showed a similar trend with respect to the kinetics of rejoining of DNA breaks with MTM values obtained without DMSO being slightly higher than those measured with DMSO, though not statistically different (ANOVA test). At 4 h recovery time MTM values in the absence and presence of DMSO were very similar and dropped to 4.12 and 2.99, respectively. MTM values returned to control values after 24 h recovery time.
3.2. Dual-colour FISH alkaline Comet assay Table 2 presents the frequencies of damaged cells (cells exhibiting a visible tail) showing greater fragmentation of DNA from chromosomes 18 (red hybridization spots) or 19 (green hybridiza-
Fig. 4. Mean tail moments of G0 human lymphocytes irradiated with 3 Gy X-rays in the absence and presence of 2 M DMSO. *: Statistically significant at p < 0.01 (ANOVA test).
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4. Discussion Cytogenetic analyses by premature chromosome condensation (PCC) in human lymphocytes have shown that exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the time of recovery [19,20]. This seems to rule out that slow and fast repairing components of induced DNA double strand breaks (DSBs) leading to CA may be related to the differential localization of the damage in the genome, i.e., between relaxed and condensed chromatin. We attempted to get some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced • OH radicals, acting mostly in the more accessible relaxed regions, followed by PCC and (b) probing the damage and repair in condensed and relaxed regions by COMET–FISH techniques employing two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes, i.e., representing condensed and relaxed regions, respectively. Results obtained from the analysis of PCC (Table 1) indicate that both fast appearing and slowly formed chromosome exchanges occur in more opened chromatin regions since their formation is affected to a similar degree by DMSO. The majority of exchange aberrations (63%) are formed early during the fusion process of PCC which takes approximately 1 h. This confirms previous findings which indicate that irradiation of cells in the presence of the • OH radical scavenger DMSO reduces DNA damage in chromatin regions where • OH radicals can access more easily [24,25]. A more direct proof for these conclusions comes from results obtained in the alkaline Comet/FISH with chromosomes 18 and 19. The results presented in Table 2 clearly show a highly differential distribution of the X-ray induced DNA breakage in the human chromosomes 18 and 19 in both G0 and G1 phases of cell cycle. Immediately after treatment (virtually at 0 h recovery time, because fast repair activities can still proceed, even at low temperature [35] due to the procedure of the Comet assay which takes at least 15–20 min until lysis of cells and complete block of metabolism) the number of damaged cells showing DNA breakage of chromosome 18 was significantly higher than the number of damaged cells showing a higher breakage of chromosome 19 both in the G0 and G1 phases of cell cycle. One possible explanation for this higher fragmentation of condensed chromatin (chromosome 18) may be the lower enzymatic repair activity observed in these regions [14]. In contrast, this could also imply that the higher enzymatic repair activity present in the relaxed regions [14] may induce rapidly both correct and incorrect DNA rejoining, resulting in an overall reduced DNA fragmentation in the chromatin. A support for this conclusion can be also drawn from findings on DNA rejoining with conventional Comet assay obtained at global genome level following irradiation of unstimulated G0 human lymphocytes in the absence and presence of DMSO. As shown in Fig. 4, G0 irradiated cells in the absence of DMSO show a statistically significant greater level of DNA breakage compared to those irradiated in its presence at 0 h recovery time. A significant contribution to this higher DNA breakage comes likely from DNA lesions caused by • OH radicals essentially in the relaxed chromatin as a result of directly induced DNA single strand breaks (SSBs) or incomplete DNA repair clusters (e.g. base repair and/or excision repair) generated by oxidised DNA bases. However, the damage is repaired or mis-repaired faster than in more compact chromatin since DNA breakage in the cells X-irradiated in the absence of DMSO drops very close to the DNA breakage level of the parallel treatment performed in its presence at 0.5 h recovery time. At later recovery times the rejoining curve obtained in the absence of DMSO shows similar profiles to those obtained in its presence, though slightly higher in absolute terms but not significantly different. On the basis of the results obtained it can be concluded that, relaxed chromatin appears to be less fragmented than heterochro-
matin under the reported experimental conditions because the radiation-induced damage is repaired or mis-repaired faster than in more compact chromatin [17,36] indicating that accessibility of DNA repair enzymes to damaged chromatin regions appears to be an important factor for the rate of repair. This is also in agreement with the reported distribution of X-ray induced CA which occurs preferentially in the gene rich regions (i.e., relaxed regions) of chromatin of Chinese hamster chromosomes following irradiation in G1 or G2 stages of cell cycle [4,16,37,38]. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement This work was partially supported by University of Tuscia. References [1] J.D. Tucker, M.J. Ramsey, D.A. Lee, J.L. Minkler, Validation of chromosome painting as a bio-dosimeter in human peripheral lymphocytes following acute exposure to ionizing radiation in vivo, Int. J. Radiat. Biol. 64 (1993) 27–37. [2] A. Matsuoka, J.D. Tucker, M. Hayashi, N. Yamazaki, T. Sofuni, Chromosome painting analysis of X-ray-induced aberrations in human lymphocytes in vitro, Mutagenesis 9 (1994) 151–155. [3] K.L. Johnson, J.D. Tucker, J. Nath, Frequency, distribution and clonality of chromosome damage in human lymphocytes by multi-colour FISH, Mutagenesis 13 (1998) 217–227. [4] P. Slijepcevic, A.T. Natarajan, Distribution of radiation-induced G1 exchange and terminal deletion breakpoints in Chinese hamster chromosomes as detected by G banding, Int. J. Radiat. Biol. 66 (1994) 747–755. [5] M.C. Muhlmann-Diaz, J.S. Bedford, Breakage of human chromosomes 4, 19 and Y in G0 cells immediately after exposure to gamma-rays, Int. J. Radiat. Biol. 65 (1994) 165–173. [6] T.K. Pandita, V. Gregoire, K. Dhingra, W.N. Hittelman, Effect of chromosome size on aberration levels caused by gamma radiation as detected by fluorescence in situ hybridization, Cytogenet. Cell Genet. 67 (1994) 94–101. [7] J.D. Tucker, J.R. Senft, Analysis of naturally occurring and radiation-induced breakpoint locations in human chromosomes 1, 2 and 4, Radiat. Res. 140 (1994) 31–36. [8] S. Knehr, S. Zitzelsberger, H. Braselmann, U. Nahrstedt, M. Bauchinger, Chromosome analysis by fluorescence in situ hybridization: further indications for a non-DNA-proportional involvement of single chromosomes in radiationinduced structural aberrations, Int. J. Radiat. Biol. 70 (1996) 385–392. [9] J.J. Boei, S. Vermeulen, A.T. Natarajan, Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation, Int. J. Radiat. Biol. 72 (1997) 139–145. [10] J.F. Barquinero, S. Knehr, H. Braselmann, M. Figel, M. Bauchinger, DNAproportional distribution of radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization painting of all chromosomes of a human female karyotype, Int. J. Radiat. Biol. 74 (1998) 315–323. [11] V.A. Bohr, C.A. Smith, D.S. Okumoto, P.C. Hanawalt, DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall, Cell 40 (1985) 359–369. [12] V.A. Bohr, D.H. Phillips, P.C. Hanawalt, Heterogeneous DNA damage and repair in the mammalian genome, Cancer Res. 47 (1987) 6426–6436. [13] L.H. Mullenders, H. Vrieling, J. Venema, A.A. van Zeeland, Hierarchies of DNA repair in mammalian cells: biological consequences, Mutat. Res. 250 (1991) 223–228. [14] E.C. Friedberg, Relationships between DNA repair and transcription, Annu. Rev. Biochem. 65 (1996) 15–42. [15] G.A. Folle, W. Martinez-López, E. Boccardo, G. Obe, Localization of chromosome breakpoints: implication of the chromatin structure and nuclear architecture, Mutat. Res. 404 (1998) 17–26. [16] P. Slijepcevic, A.T. Natarajan, Distribution of X-ray-induced G2 chromatid damage among Chinese hamster chromosomes: influence of chromatin conformation, Mutat. Res. 323 (1994) 113–119. [17] J. Surralles, S. Sebastián, A.T. Natarajan, Chromosomes with high gene density are preferentially repair in human cells, Mutagenesis 12 (1997) 437–442. [18] J. Surralles, S. Puerto, M.J. Ramirez, A. Créus, R. Marcos, L.H. Mullenders, A.T. Natarajan, Links between chromatin structure, DNA repair and chromosome fragility, Mutat. Res. 404 (1998) 39–44. [19] F. Darroudi, M. Fomina, M. Meijers, A.T. Natarajan, Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes using PCC and FISH, Mutat. Res. 404 (1998) 55–65. [20] R. Greinert, E. Detzler, B. Volkmer, D. Harder, Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocyte: analysis by premature chromosome condensation with delayed fusion, Radiat. Res. 144 (1995) 190–197.
P. Mosesso et al. / Mutation Research 701 (2010) 86–91 [21] J.M. Craig, W.A. Bickmore, Chromosome bands—flavours to savour, Bioessays 15 (1993) 349–354. [22] S.M. Chiu, L.R. Friedman, L.X. Xue, N.L. Oleinick, Modification of DNA damage in trascriptionally active vs. bulk chromatin, Int. J. Radiat. Oncol. Biol. Phys. 12 (1986) 1529–1532. [23] L.G. Littlefield, E.E. Joiner, S.P. Colyer, A.M. Sayer, E.L. Frome, Modulation of radiation-induced chromosome aberrations by DMSO, an • OH radical scavenger. I. Dose–response studies in human lymphocytes exposed to 220 kV X-rays, Int. J. Radiat. Biol. 53 (1988) 875–890. [24] C.M. De Lara, T.J. Jenner, K.M.S. Townsend, S.J. Marsden, P. O’Neill, The effect of dimethyl sulfoxide on the induction of DNA double strand breaks in V79-4 mammalian cells by alpha particles, Radiat. Res. 144 (1995) 43–49. [25] G. Ahnström, J. Nygren, S. Eriksson, The effect of dimethyl sulphoxide on the induction and repair of double strand breaks in human cells after irradiation with gamma rays and accelerated ions: rapid or slow repair may depend on accessibility of breaks in chromatin of different compactness, Int. J. Radiat. Biol. 76 (2000) 533–538. [26] G. Spivak, R.A. Cox, P.C. Hanawalt, New applications of the Comet assay: Comet–FISH and transcription-coupled DNA repair, Mutat. Res. 681 (2009) 44–50. [27] S. Saccone, C. Federico, I. Solovei, M.F. Croquette, G. Della Valle, G. Bernardi, Identification of the gene-richest bands in human prometaphase chromosomes, Chromosome Res. 7 (1999) 379–386. [28] J.M. Craig, W.A. Bickmore, The distribution of CpG islands in mammalian chromosomes, Nat. Genet. 7 (1994) 376–382. [29] G.E. Pantelias, H.D. Maillie, A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol, Somatic Cell Genet. 9 (1983) 533–547.
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[30] G.E. Pantelias, H.D. Maillie, The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry, Radiat. Res. 99 (1984) 140–150. [31] F. Darroudi, A.T. Natarajan, Cytogenetic characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique, Mutat. Res. 213 (1989) 249–255. [32] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191. [33] S.J. Santos, N.P. Singh, A.T. Natarajan, Fluorescence in situ hybridization with comets, Exp. Cell Res. 232 (1997) 407–411. [34] A. Rapp, C. Bock, H. Dittmar, K.O. Greulich, UV-A breakage sensitivity of human chromosomes as measured by COMET–FISH depends on gene density and not on the chromosome size, J. Photochem. Photobiol. B 56 (2000) 109–117. [35] C. Bock, H. Dittmar, H. Gemeinhardt, E. Bauer, K.O. Greulich, Comet assay detects cold repair of UV-A damages in a human B-lymphoblast cell line, Mutat. Res. 408 (1998) 111–120. [36] D.M. López-Larraza, N.O. Bianchi, DNA response to bleomycin in mammalian cells with variable degrees of chromatin condensation, Environ. Mol. Mutagen. 21 (1993) 258–264. [37] G.A. Folle, W. Martinez-Lopez, E. Boccardo, G. Obe, Localization of chromosome breakpoints: implication of the chromatin structure and nuclear architecture, Mutat. Res. 404 (1998) 17–26. [38] W. Martinez-Lopez, G.A. Folle, G. Obe, P. Jeppesen, Chromosome regions enriched in hyperacetylated hystone H4 are preferred sites for endonucleaseand radiation-induced breakpoints, Chromosome Res. 9 (2001) 69–75.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes b ˜ Pasquale Mosesso a,∗ , Fabrizio Palitti a , Gaetano Pepe a , Joaquin Pinero , a c,1 a Raffaella Bellacima , Gunnar Ahnstrom , Adayapalam T. Natarajan a b c
Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis s.n.c., 01100 Viterbo, Italy University of Seville, Department of Cell Biology, C/Reina Mercedes sn, 41012 Seville, Spain Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 5 March 2010 Accepted 9 March 2010 Available online 16 March 2010 Keywords: FISH–Comet assay DNA repair Heterochromatin CpG islands DMSO Radical scavengers Exchange aberrations and chromatin architecture
a b s t r a c t Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced • OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET–FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ionizing radiation (IR) is an efficient inducer of chromosome aberrations (CA), which represent the outcome of very complex events, involving the conversion of DNA double strand breaks (DSBs) and other lesions through various DNA repair pathways into microscopically detectable events. Though IR is expected to induce DNA lesions in a random manner in the genome, some studies using FISH technique with chromosome specific DNA probes, have reported that the frequency of radiation-induced chromosome exchanges was proportional to the length or to the DNA content of target chromosomes [1–3], whereas other studies have reported that the induction of chromosome exchanges is nonrandom among the chromosomes studied [4–10]. The possible causes for this reported non-random distribution of exchanges
∗ Corresponding author. Tel.: +39 0761 357257; fax: +39 0761 357257. E-mail address: [email protected] (P. Mosesso). 1 Deceased. 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.03.005
have been attributed to various factors such as, differential primary damage of DNA and its repair which could be influenced by transcriptional activity [11–14], chromatin structure [4,5,15,16] and gene density [17,18] of the studied chromosomes. This implies that CA we observe, which are the consequences of mis-repair of IR induced DSBs, may not reflect the initial distribution of DNA damage in the genome. Earlier studies using the technique of premature chromosome condensation (PCC) by fusion with mitotic cells, have shown that in human lymphocytes, exchange type of aberrations are formed within a few minutes, following low doses (<2 Gy) of Xrays where the two lesions required to produce the exchange are significantly generated by one ionization track, whereas at higher doses where the probability of the two lesions being produced by two independent tracks is higher, these aberrations increase with the duration of recovery [19,20]. This reflects the relative roles of slow and fast repairing components of induced DNA double strand breaks (DSBs) leading to CA. The underlying basis for the slow and fast repairing components of the DNA breaks may be related to the differential localization of the damage in the genome, i.e., between relaxed and condensed regions. The genome is highly het-
P. Mosesso et al. / Mutation Research 701 (2010) 86–91
erogeneous and it has been shown that genes are clustered among chromosomes in a sub-set of R bands, most of which are known as T bands containing the highest concentration of CpG islands typical of relaxed regions. While G bands are late replicating, relatively ATrich, highly condensed and transcriptionally silent, R and T bands are early replicating, less condensed with unmethylated DNA and accessible HpaII sensitive sites on chromosomes [21]. We have tried to elucidate the relative roles of slow and fast repairing components of induced DSBs in relaxed and condensed regions by means of cytogenetic analysis in prematurely condensed chromosomes (PCC) and alkaline Comet assay in unstimulated (G0) human lymphocytes irradiated with X-rays in the absence and presence of dimethyl sulfoxide (DMSO). DMSO, an efficient and selective scavenger of radiation-induced • OH radicals, can be used as a probe for events taking place in regions of different chromatin compactness, since DNA damage induced by • OH radicals is known to be high in more opened chromatin [22–25]. Furthermore, we probed the damage and repair in transcriptionally silent condensed regions of chromosomes or from relaxed regions with very high gene density by COMET–FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques [26] in human chromosomes, 18 and 19, which possess ideal properties in respect to their structural organization to be considered as models of condensed and relaxed chromatin respectively. Both chromosomes are similar in size, but exhibit very different chromatin organisation and banding patterns. While chromosome 19 has a very high gene density with strong hybridization signals for T bands, chromosome 18 exhibits very few active genes and very little hybridization signals for T bands [27,28]. 2. Materials and methods 2.1. Cell preparation and treatment conditions Lymphocytes were isolated from bags of “buffy coat” generated from approximately 500 ml of heparinised fresh venous whole blood drawn from three healthy male donors, supplied by a local hospital. “Buffy coats” were diluted 1:1 in phosphate-buffered saline (PBS) and lymphocytes separated using Ficoll–Histopaque 1077 (Sigma). Briefly, 15–20 ml diluted “buffy coat” was layered over 12.5 ml Ficoll–Histopaque in 50 ml polystyrene conical centrifuge tubes (Falcon) and centrifuged for 35 min at 450 × g at room temperature. During centrifugation, mononuclear cells and platelets were concentrated in a fluffy white layer below the plasma. The cell layers were collected with Pasteur pipettes and washed twice with fresh culture medium. Then the cell pellets were resuspended by gentle vortexing, and cell suspensions with a final concentration of 1 × 107 cells/ml in PBS were prepared. Greiner® 14 ml conical cell culture tubes contained 0.5 ml of cell suspension (5 × 106 lymphocytes) and 4.5 ml medium (Ham’s F-10, supplemented with 20% foetal calf serum heat-inactivated at 56 ◦ C, 100 IU/ml penicillin, 100 g/ml streptomycin, 0.4 mM l-glutamine and 2% Hepes buffer). All the chemicals used for lymphocyte cultures were supplied by Gibco® . Cultures were irradiated with 3 Gy X-rays at 300-kV and 6 mA at a rate of 1.0 Gy/min directly prior to stimulation with 2% phytohaemagglutinin (PHA 15 Murex, Italy) when lymphocytes are in a G0 phase (unstimulated cultures), or 12 h after stimulation with PHA when lymphocytes are in the G1 phase of the cell cycle. For treatments in the presence of dimethyl sulfoxide (Sigma–Aldrich), CAS registry number: 67-68-5, cell suspensions were inoculated in 4.5 ml PBS containing DMSO (2 M final concentration). Human lymphocytes from treated cultures were processed to prepare Comet slides, immediately after irradiation (0 h recovery time) and always on ice or placed in an incubator at 37 ◦ C for the appropriate recovery time (0.5, 1, 2, 4 and 24 h), or fused with mitotic CHO cells to obtain PCC immediately after irradiation (0 h recovery time) and at 2 and 4 h recovery times. Mitotic CHO cells collected by mitotic shake off and frozen at −80 ◦ C in complete growth medium containing DMSO 10% and 0.2 g/ml colcemid, were thawed immediately before use. Control cultures both in the absence and presence of DMSO were not irradiated. 2.1.1. Cytogenetic analysis in prematurely condensed (PCC) human chromosomes Cell fusion between mitotic CHO cells and G0 lymphocytes was mediated by polyethylene glycol (PEG) according to Pantelias and Maillie [29]. Briefly, mitotic cells and appropriately treated human lymphocytes (see Section 2.1) were mixed in a ratio of 1:3 in round-bottom culture tubes. After centrifugation, the cells were suspended in 2 ml of Ham’s F-10 medium without serum and centrifuged again. The supernatant was discarded without disturbing the pellet and 0.25 ml of PEG (MW 1450 from Sigma–Aldrich) was added and left for 1 min. For
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the next 3 min, 2 ml of phosphate-buffered saline (PBS) were added drop wise; the tube was gently shaken after each drop. The cell suspensions were centrifuged and resuspended in 0.5 ml culture medium containing colcemid (0.2 g/ml) and incubated at 37 ◦ C for 1 h [30,31]. Finally cells were treated with hypotonic KCl solution (75 mM) for 10 min and fixed in methanol–acetic acid (3:1). Cell suspension was dropped onto pre-cleaned wet slides, air-dried and aged for at least 1 week. In order to improve detection of dicentric chromosomes in PCC, we employed fluorescence in situ hybridization (FISH) using centromeric DNA probes directly labelled with rhodamine (TRITC) which identify the centromeric region of each human chromosome (Appligene-Oncor). To apply pan-centromeric probes, slides were denatured for 4 min at +80 ◦ C with 70% formamide/2× SSC and dehydrated by serial ethanol washing (70, 90 and 100%). The pan-centromeric probe was pre-warmed for 20 min at 37 ◦ C, 6 l mixed with 14 l hybridization buffer (50% formamide, 10% dextran sulfate, 2× SSC), denatured at +80 ◦ C for 10 min and chilled on ice. Twenty microliters of the probe were put onto each slide, which were then sealed and hybridized at 37 ◦ C overnight in a moist-chamber. Then, slides were preincubated twice with 50% formamide in 2× SSC at 42 ◦ C for 5 min, the coverslips were removed and washed once with 0.005% Tween 20 in PBS. Finally slides were dehydrated by serial ethanol washing (70, 90 and 100%), and embedded in 25 l Vectashield (Vector Laboratories) with DAPI at a final concentration of 0.15 g/ml. 2.1.2. “Conventional” alkaline Comet and FISH/alkaline Comet assays Comet slides were prepared following published protocols for the alkaline “Comet assay” [32] with modifications for FISH with whole chromosome painting probes, as previously described [33,34]. Briefly, 10 l of cell suspension was mixed with 65 l of 0.7% (w/v) low-melting point agarose (Bio-Rad Lab.) and sandwiched between a lower layer of 1% (w/v) normal-melting point agarose (Bio-Rad Lab.) and an upper layer of 0.7% (w/v) low-melting point agarose on microscope slides (Carlo Erba, Milan, Italy). Triplicate slides were prepared from each treatment. Slides were immersed in lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10) containing 10% DMSO and 1% Triton x-100 (ICN Biomedicals Inc.) at 4 ◦ C overnight. Following lysis, slides were placed in a horizontal gel electrophoresis tank with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA, pH ≥ 13) and incubated for 25 min at 4 ◦ C to allow the DNA to unwind and to express the alkali-labile sites. Electrophoresis was carried out at 4 ◦ C for 15 min at 30 V (1 V/cm) and 300 mA. After electrophoresis, slides were immersed in 0.3 M sodium acetate in ethanol for 30 min. One set of slides from each treatment series was dehydrated by passing them through an alcohol series (2 min at 70, 85 and 100%), air-dried and stained with 20 g/ml ethidium bromide immediately before analysis of DNA breakage by conventional Comet assay (tail moment), as described in Section 2.2.2. The remaining two sets of slides from each treatment series were dehydrated by passing them through an alcohol series (2 min at each 70, 85 and 100%), dried thoroughly at 37 ◦ C before detection of probes and counterstained by COMET–FISH analysis. This procedure was adopted to improve stabilization of the gel, which otherwise disintegrates during hybridization. Dual-colour FISH was performed as follows: flow-sorted human chromosomes 18 and 19 (kindly supplied by Prof. M. Rocchi, University of Bari, Italy) were amplified and directly labelled with digoxigenin-11-dUTP or with biotin-16-dUTP (Boehringer, Mannheim, Germany), respectively, by degenerative oligonucleotide primer-polymerase chain reaction (DOP-PCR). 400 ng of each labelled probe, mixed with 100 l of 70% deionised formamide in 2× standard sodium citrate solution (SSC), was applied to cells on the air-dried slides. Cells and probes were denatured at +74 ◦ C for 5 min before cover slips (24 mm × 50 mm) were sealed with rubber cement over the gels. Hybridization was performed overnight at +37 ◦ C in a humid chamber. Slides were then washed in 50% formamide, 2× SSC (pH 7.0) for 5 min at +42 ◦ C followed by three washes in 0.01× SSC for 5 min at +60 ◦ C and a final wash with 4× SSC, 0.05% Tween 20 (pH 7.0) for 5 min. Biotinylated probes were detected with avidin–FITC, biotin–avidin and avidin–FITC (Vector Laboratories, Burlingame, CA, USA). The probes labelled with digoxigenin were detected with sequential mouse anti-digoxigenin, sheep anti-mouse–digoxigenin and sheep anti-digoxigenin–TRITC (Vector Laboratories, Burlingame, CA, USA). Finally slides were dehydrated, dried and mounted in Vectashield (Vector Laboratories) containing 0.15 g/ml 4 ,6diamidino-2-phenylindole (DAPI) and antifade. 2.2. Analysis 2.2.1. PCC analysis PCC slides were analyzed with a Leitz Ergolux microscope equipped with single band pass filters for DAPI, FITC, TRITC and a fully automated metaphase finder (MetaSystems GmbH, Altlussheim, Germany). An Isis digital imaging system (MetaSystems GmbH, Altlussheim, Germany) was used to capture, digitalise, annotate and print fluorescence images. A minimum of 100 PCC plates were scored for each test point per experiment. Chromosomes with two or more centromeric signals were scored as dicentric or polycentric chromosomes (which were converted into dicentrics for the purpose of calculation) while chromosomes without centromeric signal were classified as fragments. A typical example of PCC is shown in Fig. 1. 2.2.2. Conventional alkaline Comet assay Slides stained with DAPI were examined at 40× magnification using an automated image analysis system specific for Comet assays (Comet Assay III; Perceptive
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P. Mosesso et al. / Mutation Research 701 (2010) 86–91 Table 1 Effect of DMSO on exchange aberrations and fragments obtained at different recovery times in PCC of human G0 lymphocytes irradiated with 3 Gy X-rays. Recovery time (h)
Mean frequencies per 100 cells ± SE Exchange aberrations
0 2 4
Fig. 1. G0 PCC stained by fluorescence in situ hybridization (FISH) using DNA probes which identify the centromeric region of each human chromosome. For details see Section 2.1.1.
Instruments, UK) connected to a fluorescence microscope (Zeiss Axioskop 2). Tail moment and percentage of migrated DNA was determined to quantify DNA damage. One hundred cells from each slide were analysed for each experimental point. 2.2.3. Dual-colour FISH alkaline Comet assay Fluorescence microscopy was performed using a Leitz Ergolux microscope equipped with single band pass filters for detection of DAPI, FITC, TRITC signals. Images were captured with a CCD camera mounted onto the microscope. An Isis digital imaging system (MetaSystems GmbH, Altlussheim, Germany) was used for analysis. After FISH analysis using whole chromosome painting probes specific for human chromosomes 18 and 19, undamaged nuclei (Fig. 2a) showed two labelled areas: green for FITC-labelled chromosome 19 and red for TRITC-labelled chromosome 18, following merging of individual fluorescent images obtained with single band pass filters tuned to detect DAPI, FITC, TRITC (Fig. 2b–d). Blue signal
Fragments
−DMSO
+DMSO
−DMSO
+DMSO
101 ± 20 126 ± 17 161 ± 25
21 ± 9 30 ± 13 27 ± 10
949 ± 61 701 ± 53 532 ± 46
756 ± 55 576 ± 48 494 ± 44
(DAPI) refers to the whole genome DNA. Damaged cells showed instead a spread of hybridization signals (green spots for chromosome 19 and red spots for chromosome 18) in the tail of comets (Fig. 3a) after merging of individual fluorescent images obtained with single band pass filters for detection of DAPI, FITC and TRITC, respectively (Fig. 3b–d). Similarly to undamaged cells, blue signal (DAPI) refers to the whole genome DNA. To quantify DNA breakage in chromosomes 18 and 19, at least 100 damaged cells per independent experiment (cells showing a visible tail) with hybridization signals were analyzed for each treatment. Differential DNA fragmentation in chromosomes 18 and 19 was evaluated by measuring the distance of the different hybridization spots in the tail from the main nucleus. On this basis we were able to analyze differences of DNA damage in chromosomes 18 and 19 (Fig. 3 c and d).
3. Results 3.1. Cytogenetic analysis of PCCs Results obtained from cytogenetic analysis of at least 300 PCC per test point from three different blood donors (100 from each of the three experiments performed) are shown in Table 1 as mean incidences of exchange aberrations per 100 cells scored. The standard error of the mean (SE) is also shown. In the absence of DMSO the majority of exchange aberrations is formed during the fusion process (0 h recovery time). At later sampling times (2 and 4 h)
Fig. 2. (a) Agarose-embedded untreated G0 human lymphocyte stained by fluorescence in situ hybridization (FISH) using DNA probes specific for human chromosomes 18 and 19. Image acquisition was performed with a fluorescence microscope equipped with single band pass filters for DAPI, FITC, TRITC. (b–d) Fluorescent images of the same cell generated with single band pass filters for DAPI, FITC and TRITC respectively. a: Merge of images b–d.
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Table 2 Percent cells with Comets (tails ≥ 15 m). Lymphocytes from one donor were irradiated before G0 or after stimulation with PHA (G1) with 3 Gy X-rays. 100 cells each were analyzed per entry following hybridization with painting probes for chromosomes 18 or 19. Recovery time (h)
0 0.5 1 2 4 24
Chromosome 18
Chromosome 19
G0
G1
G0
G1
75 88 92 93 98 99
84 90 90 95 100 100
25 12 8 7 2 1
16 10 10 5 0 0
The frequencies of Comets with DNA fragmentation of chromosome 18 are significantly higher than those of chromosome 19 (p < 0.001; Chi-squared test).
tion spots) at the different recovery times analyzed (0, 0.5, 1, 2, 4 and 24 h). Red hybridization spots (chromosome 18) were generally more displaced at the edge of the Comet tails than the green hybridization spots (chromosome 19) in the majority of damaged cells analyzed for both unstimulated G0 and stimulated G1 lymphocytes, ranging from a minimum of 75 and 84% at the 0 h recovery time (G0 and G1, respectively) to 99 and 100% at the 24 h recovery time (G0 and G1, respectively) as shown in Table 2. This indicates that following X-irradiation of lymphocytes, DNA from chromosome 18 (red hybridization spots) is more extensively fragmented or alternatively rejoined more slowly than DNA from chromosome 19 (green hybridization spots) both in G0 and G1 phases. 3.3. Conventional alkaline Comet assay
Fig. 3. Agarose-embedded G0 human lymphocyte irradiated with 3 Gy X-rays and stained by fluorescence in situ hybridization (FISH) using DNA probes specific for human chromosomes 18 and 19. Processed in the dual-colour Comet/FISH protocol in the presence of two whole chromosome painting probes specific for human chromosomes 18 and 19. Image acquisition was performed with a fluorescence microscope equipped with single band pass filters for DAPI (b), FITC (c) and TRITC (d). a: Merge of images b–d. White bars displayed in Images a–d indicate the length in m of the tail of damaged cell from the main nucleus.
mean incidences increased by 25 and 59%, respectively. The incidence of fragments which is more than nine times higher than exchange aberrations at 0 h recovery time, decreases drastically to 74 and 56% of initial yield at 2 and 4 h recovery times, respectively. In the presence of DMSO the mean incidence of both exchange aberrations and fragments is significantly reduced to approximately 20 and 80%, respectively, of the corresponding values observed in treatments performed in the absence of DMSO at 0 h recovery time. At later recovery times (2 and 4 h) the incidence of exchange aberrations is not significantly modified compared to the initial yield while fragments decrease to 76 and 65% at 2 and 4 h recovery times, respectively.
Fig. 4 shows the mean tail moment (MTM) observed in unstimulated lymphocytes following X-ray irradiation (3 Gy) in the absence and presence of DMSO at 0, 0.5, 1, 2, 4 and 24 h recovery times obtained from three experiments. At 0 h recovery time the mean tail moment observed was markedly higher in the absence of DMSO (9.3) than in its presence (5.2) (ANOVA test). At 0.5 h recovery time, the initial MTM observed in the absence of DMSO dropped to approximately the same value observed in its presence, indicating a very fast rejoining of DNA breaks. Beyond 0.5 h recovery time, both curves showed a similar trend with respect to the kinetics of rejoining of DNA breaks with MTM values obtained without DMSO being slightly higher than those measured with DMSO, though not statistically different (ANOVA test). At 4 h recovery time MTM values in the absence and presence of DMSO were very similar and dropped to 4.12 and 2.99, respectively. MTM values returned to control values after 24 h recovery time.
3.2. Dual-colour FISH alkaline Comet assay Table 2 presents the frequencies of damaged cells (cells exhibiting a visible tail) showing greater fragmentation of DNA from chromosomes 18 (red hybridization spots) or 19 (green hybridiza-
Fig. 4. Mean tail moments of G0 human lymphocytes irradiated with 3 Gy X-rays in the absence and presence of 2 M DMSO. *: Statistically significant at p < 0.01 (ANOVA test).
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4. Discussion Cytogenetic analyses by premature chromosome condensation (PCC) in human lymphocytes have shown that exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the time of recovery [19,20]. This seems to rule out that slow and fast repairing components of induced DNA double strand breaks (DSBs) leading to CA may be related to the differential localization of the damage in the genome, i.e., between relaxed and condensed chromatin. We attempted to get some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced • OH radicals, acting mostly in the more accessible relaxed regions, followed by PCC and (b) probing the damage and repair in condensed and relaxed regions by COMET–FISH techniques employing two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes, i.e., representing condensed and relaxed regions, respectively. Results obtained from the analysis of PCC (Table 1) indicate that both fast appearing and slowly formed chromosome exchanges occur in more opened chromatin regions since their formation is affected to a similar degree by DMSO. The majority of exchange aberrations (63%) are formed early during the fusion process of PCC which takes approximately 1 h. This confirms previous findings which indicate that irradiation of cells in the presence of the • OH radical scavenger DMSO reduces DNA damage in chromatin regions where • OH radicals can access more easily [24,25]. A more direct proof for these conclusions comes from results obtained in the alkaline Comet/FISH with chromosomes 18 and 19. The results presented in Table 2 clearly show a highly differential distribution of the X-ray induced DNA breakage in the human chromosomes 18 and 19 in both G0 and G1 phases of cell cycle. Immediately after treatment (virtually at 0 h recovery time, because fast repair activities can still proceed, even at low temperature [35] due to the procedure of the Comet assay which takes at least 15–20 min until lysis of cells and complete block of metabolism) the number of damaged cells showing DNA breakage of chromosome 18 was significantly higher than the number of damaged cells showing a higher breakage of chromosome 19 both in the G0 and G1 phases of cell cycle. One possible explanation for this higher fragmentation of condensed chromatin (chromosome 18) may be the lower enzymatic repair activity observed in these regions [14]. In contrast, this could also imply that the higher enzymatic repair activity present in the relaxed regions [14] may induce rapidly both correct and incorrect DNA rejoining, resulting in an overall reduced DNA fragmentation in the chromatin. A support for this conclusion can be also drawn from findings on DNA rejoining with conventional Comet assay obtained at global genome level following irradiation of unstimulated G0 human lymphocytes in the absence and presence of DMSO. As shown in Fig. 4, G0 irradiated cells in the absence of DMSO show a statistically significant greater level of DNA breakage compared to those irradiated in its presence at 0 h recovery time. A significant contribution to this higher DNA breakage comes likely from DNA lesions caused by • OH radicals essentially in the relaxed chromatin as a result of directly induced DNA single strand breaks (SSBs) or incomplete DNA repair clusters (e.g. base repair and/or excision repair) generated by oxidised DNA bases. However, the damage is repaired or mis-repaired faster than in more compact chromatin since DNA breakage in the cells X-irradiated in the absence of DMSO drops very close to the DNA breakage level of the parallel treatment performed in its presence at 0.5 h recovery time. At later recovery times the rejoining curve obtained in the absence of DMSO shows similar profiles to those obtained in its presence, though slightly higher in absolute terms but not significantly different. On the basis of the results obtained it can be concluded that, relaxed chromatin appears to be less fragmented than heterochro-
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