Mutagenesis Advance Access published online on May 22, 2008
Mutagenesis, doi:10.1093/mutage/gen027
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Characterization of chromosomes and chromosomal fragments in human lymphocyte micronuclei by telomeric and centromeric FISH
New Technologies and Risks, Work Environment Development, Finnish Institute of Occupational Health, Topeliuksenkatu 41aA, FI-00250 Helsinki, Finland
Micronuclei (MN), used as a biomarker of effect in exposure to genotoxic carcinogens, derive from chromosomes and chromosomal fragments lagging behind in anaphase. The two types of MN are usually distinguished from each other by centromeric fluorescence in situ hybridization (FISH), centromere-positive (C+) MN representing entire chromosomes and centromere-negative (C–) MN chromosomal fragments. The incorporation of various types of chromosomal fragments and chromosomes and chromatids to MN is still poorly understood. We used directly labelled pancentromeric and pantelomeric DNA probes to examine the contents of MN in cultured binucleate lymphocytes of four unexposed, healthy subjects (two men and two women) 35–56 years of age. The presence and number of telomeric and centromeric signals was evaluated in 200 MN (50 MN per subject). These data were used to estimate the proportion of MN harbouring terminal/interstitial fragments, acentric/centric fragments, chromatid-type/chromosome-type fragments and entire chromatids/chromosomes. The majority of the C+ MN (96% in men and 86% in women) found contained telomeric (T+) sequences. Most of the C+ T+ MN had one centromere and two or one telomere signals, suggesting that single chromatids were more frequently involved in MN than both sister chromatids. Among the C– MN, telomere signals were found in 91% (men) and 79% (women), showing that fragments in MN were mostly terminal. Most C– T+ MN had one telomere signal, indicating higher prevalence for chromatid-type than chromosome-type terminal fragments. Combined centromeric and telomeric FISH is expected to increase the sensitivity of detecting exposure-related effects, when the exposure induces specific types of MN and its effect is low. This approach could particularly have use in discriminating between MN harbouring chromatid- and chromosome-type fragments in studies of human exposure to chemical clastogens and ionizing radiation.
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Introduction |
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Micronuclei (MN) are commonly used as indicators of genotoxic effects in genotoxicity testing and in studies of human exposure to genotoxic agents. MN can be formed from whole chromosomes or chromosomal fragments that lag behind in anaphase. The two types of MN are usually distinguished from each other by fluorescence in situ hybridization (FISH) using centromeric DNA probes, centromere-positive (C+) MN representing entire chromosomes and centromere-negative (C–) MN representing chromosomal fragments. Numerous in vitro studies indicate that clastogens preferentially produce C– MN while aneuploidogens induce C+ MN (1
). Many genotoxic agents may increase both types of MN (2), even though one type is usually clearly more prevalent than the other.
In human lymphocytes, age and gender have a large impact on the ratio of the two types of MN. Age increases C+ MN, and the higher micronucleus frequency of women, as compared with men, is also due to an increase in MN that contain whole chromosomes. Much of the age and gender effects on micronucleus frequency result from excess micronucleation of the sex chromosomes (1,3).
As the constitution of MN is influenced by the type of exposure (clastogenic or aneugenic) as well as biological factors (age and gender), the characterization of the contents of MN should be particularly important when the micronucleus assay is used for biomonitoring of human genotoxic effects. This is because human exposures, and thereby the induced genotoxic effects, are usually relatively small. If the analysis can be targeted on those MN that the exposure specifically induces, the effects are probably more easily detected. For instance, exposure to a chemical clastogen which induces chromatid-type chromosomal aberrations in peripheral lymphocytes may be better discovered, if MN harbouring chromatid fragments could be identified. This can, in principle, be accomplished by using a pantelomeric DNA probe together with a pancentromeric probe. MN with terminal chromatid fragments should contain one telomere but no centromere.
Telomeres, composed of tandem repeats of DNA and associated proteins at the end of eukaryotic chromosomes, have several important functions, including preservation of chromosome integrity and regulation of the replicative span of somatic cells (4). There are only a few earlier studies on the occurrence of telomeres in MN, despite the fact that the presence of centromeres in MN has extensively been examined previously (1). Yankiwski et al. (5) studied the nuclear structure of Bloom syndrome fibroblasts and observed, using centromeric and telomeric FISH, that MN apparently budding out from the cells in S phase could contain both telomeric and centromeric sequences. MN induced by 
-ray irradiation in two-cell human–hamster embryos (6) contained mainly acentric fragments with only telomeric signals (77%), in accordance with the well-known clastogenic effect of ionizing radiation. In unirradiated human–hamster embryos, 
67% of MN harboured only a telomere signal and 26% showed both a centromeric and a telomeric signal, in agreement with the fact that both structural chromosomal aberrations and numerical changes can lead to formation of spontaneous MN (6).
In a few studies, combined telomere and centromere FISH were used to characterize MN in mouse NIH 3T3 fibroblasts in vitro. Almost 60% of radiation-induced MN had only telomeric signals and, therefore, probably had their origin in terminal acentric fragments (7). Approximately 17% of the MN in the irradiated NIH 3T3 cells showed one centromeric and about four telomeric signals, suggesting they originated from whole chromosomes. Twenty-two per cent of the induced MN did not show any signals (7). When NIH 3T3 cells were treated with 2-chlorobenzylidene malonitrile (8), 63–73% of induced MN contained centromeric and telomeric signals, suggesting they harboured whole chromosomes. Almost half of these MN showed one centromeric signal and were assumed to have one single chromosome. Acentric (terminal) fragments, showing telomeric signals only, constituted 23–28% of the MN, while 4.5% of the MN showed no signal at all (8).
Jie and Jia (9) observed that mitomycin C (a clastogen) primarily induced MN lacking centromeric signals (78.6%) in NIH 3T3 cells, while 74.5% of MN induced by colchicine (an aneugen) contained both centromeric and telomeric signals, indicating the presence of whole chromosomes. Acryl amide and the Chinese medicine Tripterygium hypoglaucum Hutch induced MN containing both centromeric and telomeric signals as well as MN harbouring only telomeric signals, which suggested that these agents had both aneugenic and clastogenic potential (9).
Primed in situ DNA synthesis (PRINS) is another method that has been used to detect telomeric and centromeric sequences in MN. In one study (10), mouse splenocyte cultures were treated with mitomycin C or colcemid, and PRINS was performed with centromere- and telomere-specific primers. A higher number of MN with less than four telomeres was found in cultures treated with mitomycin C than colcemid. All MN with a single telomere lacked centromere label; the authors considered that these MN represent ‘true chromosome acentric fragments’. MN with four telomere signals always carried a centromere sequence (10). In a murine liver epithelial cell line (11), centromeric and telomeric PRINS revealed that colcemid induced a high frequency of C+ MN many of which also carried three or four telomeres. This suggested that the MN contained a duplicated chromosome with two chromatids. Diepoxybutane-induced MN lacked centromere sequences, and they did not carry more than two telomeres with a few exceptions (11).
We recently assessed the centromeric and telomeric contents of MN in 9-day cultures of interleukin-2-treated human lymphocytes at normal and deprived level of folate (12). In those conditions, MN contained more often terminal acentric fragments (telomere label only) than whole chromosomes (both centromere and telomere label), tentative acentric (no label) or centric (centromeric label only) interstitial fragments being less frequent.
As the contribution of various types of chromosomal fragments and chromosomes and chromatids to MN is poorly understood, especially in normal human cells, we used telomeric and centromeric FISH to examine the contents of MN in usual 3-day cultures of untreated human lymphocytes. On the basis of the number of centromere and telomere signals in MN, we estimated the proportion of MN harbouring terminal/interstitial fragments, acentric/centric fragments, chromatid-type/chromosome-type fragments and entire chromatids/chromosomes.
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Lymphocyte cultures and slide preparation
Four unexposed, healthy subjects (two men and two women), 35–56 years of age, donated a blood sample (20 ml in heparinized vacuettes) for the study. Mononuclear cells were isolated in Leucosep tubes (Greiner, Frickenhausen, Germany) using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden). The number of cells was determined using a Coulter counter (Coulter AcT Diff; Beckman Coulter, Miami, FL, USA). Lymphocyte cultures were prepared at a concentration of 2 x 106 cells/ml in 5 ml of RPMI 1640 medium (Gibco, Paisley, Scotland, UK) containing 1% L-glutamine (Gibco), 1% penicillin/streptomycin (10 000 IU/ml penicillin, 10 000 µg/ml streptomycin; Gibco), 1% phytohaemagglutinin (Murex, Kent, UK) and 15% foetal calf serum (Gibco). The cultures were incubated at 37°C and 5% CO2 in a humidified incubator. Cytochalasin B (6 µg/ml) (Sigma C-6762; St Louis, MO, USA) was added at 44 h to inhibit cytokinesis. At 72 h, the cells were harvested onto microscope slides using a cytocentrifuge (Shandon, Cytospin 3, Cheshire, UK). The slides were then air dried, fixed in absolute methanol and kept at –20°C until FISH.
FISH
FISH was performed with directly labelled human pancentromeric and pantelomeric probes. The slides were treated with pepsin (Sigma P-7012; 5 µg/ml, in 0.01 N HCl, pH 3.0) at 37°C for 15 min, washed in distilled water and phosphate-buffered saline (PBS; Cambrex, Verviers, Belgium), fixed in 1% formaldehyde at 4°C for 5 min, washed in distilled water and PBS, dehydrated, allowed to dry, denatured in 70% formamide at 65°C for 2 min, dehydrated and allowed to dry. The pancentromeric (STAR*FISH 1141-F; fluorescein isothiocyanate label; Cambio, Cambridge, UK) and pantelomeric (STAR*FISH 1271-Cy3; N,N'-(dipropyl)-tetramethyl-indocarbocyanine label; Cambio) DNA probes were prewarmed at 37°C for 5 min. In all, 2.5 µl of both probes were mixed in a tube, and the probe mixture was denatured for 10 min at 85°C and chilled on ice. Five microlitres of the probe mixture was applied onto each slide, and the slides were then coated with coverslips, sealed and hybridized overnight at 37°C in a moist chamber. On the following day, the slides were washed in 2x SSC (20x SSC: 173.3 g NaCl, 88.2 g sodium citrate and 1 l distilled water, pH 7.0) at 37°C for 5 min, 50% formamide/2x SSC at 37°C twice for 5 min and 2x SSC at 37°C for 5 min. Finally, the slides were counterstained in the dark for 5 min in 4x SSC, 0.05% Tween-20 solution containing 5 µg/ml 4',6-diamidino-2-phenylindole (DAPI), washed in tap water, air dried and mounted in antifade solution (Vectashield; Vector, Burlingame, CA, USA).
Slide analysis
FISH analysis was performed by one microscopist on coded slides under a Zeiss Axioplan 2E Universal fluorescence microscope (Zeiss, Jena, Germany). Binucleate cells were identified by DAPI staining. Cells with a clear pattern of centromere and telomere label in the nuclei were chosen for further analysis. Telomere (red) and centromere (green) label in MN was identified with the help of a x100 objective using red, green and double green/red filters (Chroma, Rockingham, VT, USA). Altogether 200 MN (50 MN per subject) in binucleate lymphocytes were characterized for the presence and number of telomeric (T) and centromeric (C) signals.
Metaphase examination showed that the efficiency of centromere detection was near 100%, while a FISH signal was seen in 92% of telomeres. Therefore, it was assumed that 8% of telomeres escaped our detection. When interpreting the results, the telomere data were also corrected for the labelling efficiency. Figure 1 shows microscopic images of a metaphase (A) and binucleate cells with different types of MN (B–D) after FISH identification of centromeres and telomeres.
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The distributions of MN in various categories of C and T labels among the subjects were compared statistically using Pearson's chi-square test and Fisher's exact test was used in comparison of men and women. The tests were two sided and were performed using StatXact-6 (Cytel Studio 6.3.0, Cytel Software Corp., Cambridge, MA, USA).
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The frequency and prevalence of different types of MN in the subjects studied are presented in Table I. The two women showed a higher frequency of MN per 1000 binucleate cells than the men (P = 0.03, Fisher's exact test, two sided). Considering only the presence of C and T label, the MN could be divided in apparent whole chromosomes (C+ T+), terminal fragments (C– T+), centric interstitial fragments (C+ T–) and acentric interstitial fragments (C– T–). The distribution of MN in these categories differed statistically significantly among the subjects (P = 0.001, Pearson's chi-square test, two sided), but there was no difference between the two men and between the two women. Therefore, the results were viewed separately for men and women. In general, correction for labelling efficiency had little effect on the results, except for rare types of MN. Therefore, we mostly used the detected percentages and frequencies in our evaluation, although also the corrected values are shown in Tables I and II.
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Most C+ MN (96% in men and 86% in women) contained telomeres, indicating that they harboured intact chromosomes. Also the majority of C– MN (91% in men and 79% in women) showed telomere label, suggesting that they contained terminal fragments. The difference between men and women was mainly due to the fact that women had more C+ T+ MN (61% of all MN; 12.5 C+ T+ MN per 1000 binucleate cells) than men (43%, 4.9 MN per 1000 binucleate cells) (P = 0.02, Fisher's exact test, two sided). The frequencies of C– T+ MN per 1000 binucleate cells were similar in both sexes, but the proportion of C– T+ MN of all MN was higher (P = 0.001, Fisher's exact test, two sided) in men (50%) than in women (23%).
MN harbouring apparent centric (C+ T–) and acentric (C– T–) interstitial fragments were rare, the former type constituting 2% of all MN in men and 10% in women and the latter 5% in men and 6% in women. After correction for labelling efficiency, these values decreased to 0.7, 8.4, 1.9 and 4.7%, respectively. The percentage of MN with centric interstitial fragments was significantly lower in men than in women (P = 0.03, Fisher's exact test, two sided), although this finding was based on a low number of MN.
A more detailed view on MN contents could be obtained when also the number of C and T signals was taken into account (Table II). Examples of different forms of MN based on the number of C and T signals are also schematically presented in Figure 2. MN with one centromere and two telomere signals, probably containing single chromatids, represented more than one-third of C+ T+ MN in both men (35%; 15% of all MN) and women (39%; 24% of all MN). In men, the second largest group of C+ T+ MN contained one centromere but only one telomere (14% of all MN). If it is assumed that all telomeres are separately visible, these MN would harbour chromosomes of which one telomere is missing. Correction for telomere labelling efficiency slightly reduced the contribution of 1C+ 1T+ MN. At any rate, the majority of the C+ T+ MN (67% in men and 59% in women) had one centromeric and two or one telomeric signals, suggesting that MN with a complete or incomplete single chromatid was the commonest group of MN containing whole chromosomes.
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If all centromeres and telomeres were separately detected, a whole chromosome with two chromatids in a micronucleus should have one or two centromere signals (depending on the separation of the chromatids) and four telomere signals. Such MN constituted 5% of all MN in both men and women; however, MN harbouring apparent incomplete chromosomes, with one or two centromere signals and three or one to three telomeres, respectively, were more common in men (8% of all MN) and formed the second most prevalent type of C+ T+ MN in women (16% of all MN). A small portion of all MN (1% in men and 4% in women) contained more than two centromere signals or more than four telomere signals. These MN probably included more than two chromatids. The higher overall prevalence of C+ T+ MN in women than in men was due to MN containing one chromatid, two incomplete chromatids or more than two chromatids, but the differences between the sexes concerning these MN subgroups were not statistically significant.
The majority of C– T+ MN (70% in men and 65% in women; 35 and 15% of all MN, respectively) had one telomere signal, indicating the presence of a chromatid-type terminal fragment. Apparent chromosome-type terminal fragments with two telomere signals were present in 18% of the C– T+ MN (9% of all MN) in men and 35% in women (8% of all MN). The rest of the C– T+ MN in men (6% of all MN; none in women) had three or four telomeres, presumably representing MN with a composite terminal fragment or more than one terminal fragment. MN with a chromatid-type fragment or more than one fragment were statistically significantly more common (P = 0.001 and P = 0.03, respectively) among all MN in men than women.
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Discussion |
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The contribution of different types of chromosomal fragments and chromatids or chromosomes to human MN is poorly understood. The aim of the present study was to shed some light on this question by investigating the centromere and telomere composition of MN in cytokinesis-blocked human lymphocytes. Despite the widespread use of the cytokinesis-block assay in genetic toxicology, no previous information is available on the simultaneous detection of centromeres and telomeres in MN of binucleate human lymphocytes in usual 3-day cultures.
About 96% (in men) and 86% (in women) of C+ MN also contained a telomeric signal, indicating that centromere labelling alone gives a fairly accurate estimate of the presence of whole chromosomes (if defined as C+ T+) in MN. Among C– MN, telomere signals were found in 91% (men) and 79% (women), suggesting that most fragments in MN are terminal. Chromosomes and terminal fragments were together responsible for 93% of all MN in men and 84% in women. The rest of the MN had no detectable telomeres and appeared to contain either centric (C+) or acentric (C–) interstitial fragments. It is unclear if such fragments can be formed by only one initial lesion or if two independent lesions are required for their formation. If the latter is true, the proportion of both types of interstitial fragments seems to be higher than expected, even if corrected for telomere labelling efficiency, since the probability of two independent lesions in the same chromosome is low (13).
There is presently very little information about the contribution of interstitial fragments to MN in normal human cells. In tumour cells with genomic instability, centric and acentric interstitial fragments can be frequent. For instance, Majone and Jeang (14) discovered that the expression of human T cell leukaemia virus type I Tax oncoprotein in HeLa cells increased the proportion of C+ MN without telomeres. However, in non-cancerous cells, true centric fragments with a complete centromere are expected to be rare. In our study, two-thirds of the C+ T– MN with centric interstitial fragments were actually seen in one female donor, and it is possible that these MN represented micronucleated X chromosomes since the inactive X of women has been observed to show age-dependent shortening of telomeres (15). As regards C– T– MN, in vitro metaphase studies have suggested that many chromosome-type acentric fragments induced by ionizing radiation in human lymphocytes and NIH 3T3 cells and by bleomycin in Chinese hamster cells are interstitial (7,13,16,17).
Our data on the number of telomeric signals in C– MN suggested that chromatids (1C+ 2T+) are more commonly found in MN than duplicated chromosomes with both sister chromatids (1C+ or 2C+ and 4T+). MN harbouring a chromatid (1C+ 1T+) or a chromosome (1 or 2C+ 3T+ and 2C+ 1–3T+) with incomplete number of telomeres, suggestive of telomere loss or non-optimal detection of telomeres, were also rather frequent, especially in women. They might represent micronucleated X chromosomes.
Our results suggest that most terminal fragments in MN are of the chromatid type, chromatid fragments being more common in MN than chromosome fragments. This agrees with most metaphase studies. In men, a few MN with three to four telomeric signals were found, suggesting the existence of more than one terminal fragment or a composite fragment.
We have previously applied the present FISH method to examine the centromere and telomere contents of MN in interleukin-2-mediated 9-day lymphocyte cultures of women (aged 40–48 years) at 12–120 nM concentrations of folic acid or 5-methyltetrahydrofolate (12). Such long-term cultures were used because they showed a relatively high frequency of nuclear buds which were also characterized in the study. Although MN harbouring terminal (mostly chromatid) fragments and chromosomes formed the main categories also in the 9-day cultures, MN with terminal fragments were clearly more prevalent (68%) than MN with chromosomes (19%) already at the ‘adequate’ level of 120 nM folic acid, probably reflecting the culture conditions. The frequency of MN with terminal fragments increased when the concentration of folic acid was decreased. In comparison with the women of the present study, contribution to all MN (at 120 nM folic acid) was higher (12.5 versus 6%) for acentric interstitial fragments but lower (0.5 versus 10%) for centric interstitial fragments.
In an early study on mouse bone marrow cells, telomere and centromere signals were brighter in metaphase chromosomes than in MN, suggesting that FISH can have a lower efficiency in interphase cells (18). FISH techniques have much improved in recent years, and in the present study we were not able to detect marked differences in signal intensity between interphase and metaphase cells based on visual examination. Metaphase analysis indicated an excellent labelling efficiency for centromeres and an acceptable 92% efficiency for telomeres. A rough upper estimate for the underappreciation of telomeric signals (due to FISH inefficiency or overlapping) in MN can be obtained using the data in Tables I and II, by assuming that all C+ T– MN, C– T– MN and C+ MN with lower than expected number of telomere signals were incorrectly classified (which cannot be entirely true). This approach suggests that up to 25% of telomeres could escape detection, in agreement with previous studies on lymphocyte metaphases where 75–82% of telomeres could be detected (4,19,20). As indicated above for the X chromosome, some chromosomes probably do lose their telomeres, and this phenomenon may contribute to their micronucleation. Another possible explanation for the incomplete chromosomes in MN is overlapping of signals. The chromosomal material in a micronucleus is packed into a small space, and it is possible that telomeric signals near to each other are seen as one signal. In nuclei of non-cycling G1-phase human cells, the p- and q-telomeres of a chromosome were described to pair frequently (21), but this phenomenon did not seem to concern cycling cells (22) (which cultured lymphocytes are), and it is not known if it could also occur in MN. Nevertheless, at present, true telomere loss cannot be distinguished from overlapping and detection failure.
In conclusion, we showed, by using centromeric and telomeric FISH, that most MN in cultured human lymphocytes harbour either whole chromosomes or terminal acentric fragments. Among the former group of MN, those containing a single chromatid are the most prevalent, while the latter group is dominated by MN harbouring chromatid-type terminal fragments. Combined centromeric and telomeric FISH appears to be a practical method which is expected to enhance the specificity of the MN assay. This approach may improve assay sensitivity in human biomonitoring studies at low exposure levels when exposure effects are expected to be small. It may particularly have use in distinguishing chromatid-type fragments from chromosome-type fragments in studies of human exposure to chemical clastogens or ionizing radiation. For instance, if the contribution of chromosome-type fragments to all MN is <10%, as in the present study, a doubling of these MN by ionizing radiation would increase the total MN frequency only by <10%, which is very difficult to detect without centromere and telomere identification. The specificity of the assay should further be assessed in vitro by using model mutagens, e.g. inducers of chromatid breaks and exchanges, chromosome breaks and aneugens. Although metaphase data suggest acceptable labelling efficiency for the present telomere probe, the detection of telomeres could possibly be improved by using a peptide nucleic acid probe or PRINS.
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This research was partly supported by EC Contract No. QLK4-2000-00628 (CancerRiskBiomarkers) and Finnish Work Environment Fund.
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We thank the volunteers who donated blood for these experiments.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +358 30 4742622; Fax: +358 30 4742110; Email: hanna.lindberg{at}ttl.fi
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Received on February 27, 2008; revised on April 15, 2008; accepted on April 17, 2008.
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