Mutagenesis, Vol. 14, No. 4, 427-432,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Change in centromeric and acentromeric micronucleus frequencies in human populations after chronic radiation exposure
1 Institute of Environmental Health Sciences and Institute of Public Health, School of Medicine and 2 Department of Medical Engineering and Technology, National Yang Ming University, Taipei, 3 Department of Family Medicine, Taipei Municipal Jan-Ai Hospital, Taipei, 4 Institute of Statistical Sciences, Academia Sinica, Taipei, Taiwan and 5 Department of Epidemiology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD, USA
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Abstract |
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Acute radiation exposure of humans was observed to induce various forms of cytogenetic damage, including increased frequencies of micronuclei and chromosomal aberrations. However, the cytogenetic effects of chronic low dose radiation exposure in vivo needs further characterization. Sixteen subjects with chronic low dose rates of
-radiation exposure from 60Co-contaminated steel in radioactive buildings were compared with seven non-exposed reference subjects for micronucleus frequencies after they relocated. By in situ hybridization using a digoxigenin-labeled anti-
all human centromere probe, the exposed subjects were shown to have a significant increase in cytochalasin B-modulated micronucleus (CBMN) frequencies, as well as a significant increase in centromere-positive (C+) CBMN, centromere-negative (C–) CBMN, total C+signals, single C+ MN signals and multiple C+ signals/1000 binucleated cells (BN). However, decreases in the ratios C+MN/C– MN and C+MN/total CBMN (%) were also noted in the exposed subjects. By mixed effects analysis, considering individuals from the same families, the C– MN and single C+ MN/1000 BN were both positively and moderately associated with previous cumulative exposure. When the time period of relocation post-exposure (relocation time or RT) was considered, total C+MN and multiple C+MN/1000 BN were negatively and significantly associated with RT. Moreover, the C+MN, C– MN, C+MN/C– MN ratio and single C+MN/1000 BN were all negatively and moderately associated with RT, but not with exposure dose. This suggested that acentromeric and single or multiple centromeric CBMN cytogenetic damage seems to disappear differentially in human subjects post chronic low dose radiation exposure. |
Introduction |
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More than 180 residential buildings were constructed with 60Co-contaminated steel rods in Taiwan around 1983–1984 (Chang and Kau, 1993
; Chang 1993; Lubenau and Yusko, 1995; Chang et al., 1997a). It was not until August 1992 that some of them started to be identified. The rates of exposure (0.5–270 µSv/h) in these buildings, measured in 1994, have been estimated to be several to >1000 times the background radioactivity (0.08–0.1 µSv/h) in general Taiwanese constructions. Due to great concern about adverse health effects potentially incurred, an epidemiological study based on these exposed residents was initiated at the end of 1995.
In a previous study, 73 residents in one of the contaminated buildings, the Ming-shan Villa (MSV), were identified as carrying a significant increase in micronucleus (MN) frequencies in their peripheral T lymphocytes via the cytokinesis block MN assay (CBMN assay), as compared with age- and sex-matched relatives or community controls (Chang et al., 1997b). In order to further evaluate the kinetics of MN remaining in these exposed residents, MN frequencies and included centromeres were evaluated during a follow-up.
MN frequencies have been demonstrated to be valuable biomarkers in evaluating various environmental mutagenic exposures (Heddle and Carrano, 1977). The introduction of cytochalasin B (CBMN), which inhibits microfilament assembly, made evaluation of MN more efficient (Fenech and Morley, 1989). Chromatin which forms MN may consist of either acentromeric chromosomal fragments (Countryman and Heddle, 1976), single chromosomes (Frackowiak et al., 1986; Vig and Swearngin, 1986; Sterns and Vig, 1989) or multicentric chromosomes (Krepinsky and Heddle, 1983). After exposure to aneuploidogenic agents like colchicine, vincristine and diethylstilbestrol, kinetochore-positive MN were more likely to be formed than those without (Eastmond and Tucker, 1989). In contrast, fewer kinetochore-containing MN were identified after clastogenic exposures such as ionizing radiation (Heddle and Carrano, 1977; Eastmond and Tucker, 1989). This latter observation has been reproduced in vitro as a dose–response effect decrease in kinetochore-positive MN in X-irradiated human melanoma cells (Weissenborn and Streffer, 1991) and -irradiated human fibroblasts (Cornforth and Goodwin, 1991). However, the correlation between chronic low dose ionizing radiation exposure in vivo and induced MN and the probability of forming acentric fragments or lagging chromosomes remains to be determined. Moreover, the physiological half-lives of this chromosomal damage should be further examined for its significance in biodosimetry and correlation with cancer risk. Previously, the half-lives of MN in some of those exposed were estimated to be >30 months and were affected significantly by the duration between exposure and evaluation (W.P.Chang et al., submitted for publication). The kinetics of decline in binucleates with single MN and multiple MN were further shown to be significantly different (Chang et al., 1999). We have therefore employed several analyses to determine changes in MN in this unique cohort population after they relocated from radioactive buildings.
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Materials and methods |
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Blood sampling
Sixteen residents, including nine females and seven males in seven families, who lived in radioactive apartments for 1–11 years and received 14 to 1493 mSv exposure cumulatively (Cardarelli et al., 1997
) were invited for this study after they had moved out of the radioactive buildings for various periods during 1995–1997 (Chang et al., 1997a). After giving their written consent, each individual provided detailed personal information, including a personal medical history and time and age when they moved in and relocated from the radioactive apartments. The cumulative doses for each individual were reconstructed by the Taipei Cumulative Dose Model (TCD), incorporating information on detailed duration of exposure and environmental radioactivity in each individuals' living space (Cardarelli et al., 1997; Hwang et al., 1998). Simultaneously, seven non-exposed reference subjects were invited to participate in this study voluntarily. A detailed questionnaire was obtained from each participant after they had provided informed consent. None of the participants had a history of severe medical illness, genetic disorders or had received radio- or chemotherapy. They all denied a history of significant occupational mutagenic exposure.
Cell culture and fluorescence in situ hybridization
Peripheral blood was collected from these residents and processed as described previously (Chang et al., 1996, 1997b). In brief, lymphocytes collected from fresh venous blood were isolated by Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) sedimentation, washed twice with phosphate-buffered saline (PBS) and stimulated with 5 µg/ml phytohemaglutinin-P (PHA) (Gibco BRL, Penrose, Auckland, New Zealand) in RPMI 1640 (Gibco BRL) medium, containing 20% HL-1 (BioWhitaker, Walkersville, MD), 10% heat-inactivated fetal bovine serum (JRH Inc., Lenexa, KS) and the antibiotics penicillin and streptomycin. These stimulated cells were then returned to the incubator in 37°C and 5% CO2. For the CBMN, 6.0 µg/ml cytochalasin B (Sigma, St Louis, MO) was added to the culture 44 h post PHA stimulation until 72 h and cells were fixed with 3:1 methanol:glacial acetic acid. At least 1000 binucleated cells (BN) were randomly scored under a light microscope by one investigator, who was blind to the codes for each sample.
In situ hybridization was performed as described previously (Miller et al., 1992; Miller and Nusse, 1993). Cells harvested on slides were pretreated with 2x SSC (0.3 M NaCl, 0.1 M sodium citrate, pH 7.0) for 30 min, sequentially dehydrated with 70, 80 and 95% ethanol, then denatured with 70% formamide/2x SSC (70°C, 5 min). They were then dipped into 70% ethanol (–20°C) and dehydrated with 80 and then 95% ethanol (–20°C). A digoxigenin-labeled anti- all human centromere probe (Oncor, Gaithersburg, MD), a 171 bp tandem random repeat considered to be a selection of alphoid sequences which hybridized to the centromere of all human chromosomes (Titenko-Holland et al., 1994), was preheated at 70°C for 5 min, then mixed with harvested cells on the slides at 37°C for 8 h. The slide was then dipped into 50% formamide/2x SSC to wash away unbound probes. Anti-digoxigenin antibody labeled with fluorescein isothiocyanate (FITC; Oncor) was then mixed with the above slide preparation, dipped in 1x phosphate buffer detergent (PBD; Oncor) three times for 2 min each, to wash out unbound antibody. Finally, cells were counterstained with 0.3 µg/ml propidium iodide (Oncor) and observed under a fluorescence microscope for further evaluation.
Scoring of MN and C + MN
Binucleated cells with single, double, triple or quadruple MN were scored by previous criteria (Chang et al., 1996, 1997b) (Table I). In the centromere labeling for binucleated or micronucleated cells, only those with >20 very bright centromeric spots or signals within both red main nuclei were scored. Frequencies (
) of binucleates with one or more than one MN and with any centromere-positive (C +) signals were designated C +MN (item [C]), while those without any centromeric signals were designated C– MN (item [D] of Table I). Therefore, total C + MN and C– MN equals MN/1000 BN (item [B]) in one individual. The ratio of C + MN frequencies over that of C– MN in the same individual or the C + MN/C– MN ratio was designated item [E]. C + MN composed of a single C + signal were included in item [G] and more than one C + signal in item [H]. Total numbers of C + signals/1000 BN are shown as item [F], which was composed of a single to five C + signals in one MN. C + MN frequency is shown as C + MN over MN/1000 BN or [C]/[B] (item [I]).
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Statistical analysis
Comparison of various parameters between the exposed and non-exposed reference population was first conducted by the Wilcoxon rank sum test, because these parameters were not normally distributed. To further explore possible associations between the explanatory variables and these biological markers, we fitted the following linear models to the data:
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has a dependent structure, i.e. the covariance of i and j will be 
2 if i = j or 
2 if individuals i and j belong to the same family, and 0 otherwise. It is believed that a family effect may contribute to the response. Therefore, if the data for each family is fitted to the above linear models, different estimates of the intercept and slope coefficients may be obtained. The family factor in the study is assumed to be a random sample from a larger population of families. The variation in estimated coefficients among the families are usually modeled by random components, i.e. the two models are finalized as
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a2 and b2, respectively.
These models are termed general linear mixed effects models and are expanded from the general linear model by including random effects factors especially to study correlated data (Chen,J.J. et al., 1995). The computing procedure MIXED is also available in a SAS statistical package to fit a variety of data to linear mixed effect models (Little et al., 1996), which provides us with a more accurate modeling of the uncertainty.
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Results |
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The distribution of age, sex, dose of exposure, relocation time, CBMN frequencies and several patterns of centromere-containing CBMN in the 16 exposed and seven non-exposed subjects are shown in detail in Table I
. The mean age of the 16 exposed subjects (item [A], 20.6 ±12.5 mean ± SD) was slightly but not significantly lower than that of the seven non-exposed subjects (25.0 ± 6.4, Wilcoxon rank sum test, P = 0.72; Table I). The average excess exposure, based on the dose reconstruction program TCD, was 632 ± 531 mSv (max. 1493 mSv, min. 14 mSv).
The mean CBMN frequency of the exposed subjects (item [B], 25.4 ± 14.6 per 1000 BN) was significantly higher than that of the non-exposed reference subjects (9.7 ± 5.4 per 1000 BN, Wilcoxon rank sum test, P = 0.02) or that of another non-exposed population evaluated in our laboratory (9.0 ± 4.0 or 11.0 ± 8.0; Chang et al., 1996, 1997b). The frequencies of C + MN and C– MN in the exposed subjects (items [C] and [D], 10.4 ± 5.6 and 15.0 ± 9.3 per 1000 BN) were both significantly higher than those of the reference subjects (5.6 ± 3.4 and 4.2 ± 2.2, Wilcoxon rank sum tests, P = 0.03 and 0.008, respectively), whereas the ratio C + MN/C– MN in the exposed subjects (item [E], 0.75 ± 0.15) was shown to be significantly lower than in the non-exposed subjects (1.6 ± 0.9, P = 0.005). The exposed subjects were also shown to have a significant increase in the total C + signals (item [F], 17.9 ± 10.4), single C + signals (item [G], 5.9 ± 3.2) and multiple C + signals (item [H], 3.4 ± 2.9) per 1000 BN than those of the non-exposed subjects (8.7 ± 3.7, 3.4 ± 2.9 and 2.2 ± 1.0, respectively, Wilcoxon rank sum tests, P = 0.035, 0.007 and 0.048, respectively). Finally, the exposed subjects were shown to have lower C + MN fractions (item [I], 42.8 ± 5.2%) than the non-exposed subjects (58.3 ± 10.9, Wilcoxon rank sum test, P = 0.0006).
In order to understand the kinetics of decline in acentric or centromeric MN in the exposed subjects after they had relocated from the radioactive environments, the mixed effects model was employed to correlate these changes with factors of individual exposure and relocation duration (RT). In order to stabilize the variances in the proportions of acentric and centromeric MN for each parameter, y was transformed as sin–1 [y]1/2 for the following analysis, except for the ratio C + MN/C– MN. For the simple correlation in Model 1, the frequencies of C– MN and single C + MN were both moderately associated with cumulative exposure (items [D] and [G], 1 estimate ± SE, 0.0022 ± 0.0012 and 0.0013 ± 0.0006, P = 0.097 and 0.066, respectively; Table II). The other variables were not significantly associated with exposure dose.
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Model 2 was used to further assess the correlation with RT (Table III
). RT was found to be significantly and negatively associated with the parameters C + MN, C– MN, total C + signal, single C + and multi C + frequencies (items [C], [D], [F], [G] and [H], 2 estimate ± SE, –0.066 ± 0.025, –0.091 ± 0.035, –0.101 ± 0.035, –0.043 ± 0.019 and –0.057 ± 0.018, P = 0.023, 0.022, 0.013, 0.045 and 0.007, respectively). The ratio C + MN/C– MN (item [E]) was moderately but positively associated with RT (estimate ± SE, 0.0049 ± 0.007, P = 0.091).
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Discussion |
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Increased frequencies of MN have been demonstrated in a group of people with chronic low dose, but cumulative high dose,
-radiation (Chang et al., 1997b). In this study, the frequencies of cytogenetic damage apparent as MN frequencies were significantly higher in the 16 exposed subjects. Surprisingly, the frequencies of C +MN (item [C]), C– MN (item [D]), C +/C– ratio (item [E]), total C + MN signals (item [F]), single C + MN (item [G]), multiple C + MN (item [H]) and C + MN/total MN (item [I]) (Table I) were all significantly increased more in the exposed than the non-exposed subjects. These changes were unlikely to be caused by the differences in age and gender, as the exposed subjects were relatively younger than the non-exposed subjects and both groups contained similar numbers of males and females. The exposed subjects were also questioned as to previous cigarette smoking and exposure to occupational mutagens and indicated no significant exposure. The frequencies of C– MN and single C + MN were further demonstrated to have moderate association with exposure by mixed effects analysis with family factors considered (Table II). This was comparable with observations made in -irradiated (Eastmond and Tucker 1989) and X-irradiated healthy human lymphocytes (Fenech and Morley, 1989). However, single C + MN frequencies representing at least one single lagging chromosome inside the MN were also associated with exposure dose.
MN are assumed to arise primarily from acentric fragments (Countryman and Heddle, 1976) and less from single chromosomes (Frackowiak et al., 1986; Vig and Swearngin, 1986; Sterns and Vig, 1989) or multicentric chromosomes (Krepinsky and Heddle, 1983). Many fewer kinetochore-containing MN were identified after X-irradiation of human melanoma cells (Weissenborn and Streffer, 1991) or -irradiation of human lymphocytes (Littlefield et al., 1989) or fibroblasts (Cornforth and Goodwin, 1991). However, the rate of exposure was associated with the probability of acentric fragments being included in the MN. With acute high doses of radiation exposure, acentric fragments would be less likely to be incorporated into single MN separately and there would be a tendency to form MN with multiple acentric fragments (Littlefield et al., 1989). If this could be applied to low dose rate exposure of the subjects in this study, acentric fragments could be fully expressed as independent MN. In our analysis, C– MN frequencies, as well as single C + MN frequencies, were observed to be moderately correlated with exposure dose (P = 0.097 and 0.066, respectively; Table II). Previously, low doses of ionizing radiation were shown to induce aneuploidy (Uchida et al., 1975) and a fraction of radiation-induced MN contained whole chromosomes, demonstrated by kinetochore staining inside the MN (Degrassi and Tanzarella, 1988; Thomson and Perry, 1988; Fenech and Morley, 1989). Recently, NIH 3T3 fibroblasts were shown with 17% containing one centromeric hybridization signal and about four telomeres, suggesting their origin from whole chromosomes (Miller et al., 1992). Protracted low dose radiation exposure seemed to generate ill-defined complex molecular DNA damage in G0 lymphocytes which was probably caused by asymmetrical inter- and intra-strand exchanges in the peripheral circulation.
Low dose ionizing radiation has been shown to alter gene expression and differential mRNA expression (Wolff, 1996), induce apoptosis in thymocytes (Liu et al., 1996), induce G2 delay and S phase prolongation and alterations in cell cycle progression (Salone et al., 1996) and/or is accompanied by changes in mitotic indices (Skladowski et al., 1993). These effects could also be modified by interactions between sub-lethal lesions, further altering cell survival probability (Zaider and Wuu, 1995), and enhanced genetic mutations such as MN formation (Zetterberg and Grawe, 1993). Although it was at a low dose rate, continual ionizing radiation exposure might have produced significant genetic damage in stem cells, shown as chromosomal instability (Chang and Little, 1992; Kadhim et al., 1995). We have recently shown potential genomic instability in 10–30% of the 26 exposed subjects of this cohort population evaluated for CBMN frequencies 3–4 years post-relocation from radioactive environments (Chang et al., 1999); some of these centromeric and acentromeric MN lesions might arise from genomic instability processes in the hematopoietic stem cells in vivo.
When RT was included in the mixed effect analysis, it became the most significant determinant for all the MN-related outcomes (Table III) for the exposed subjects. The cytochalasin B anti-cytokinesis approach for MN analysis in this study enabled enumeration of chromosomal damage in those T lymphocytes which had divided only once. Lymphocytes in G0 phase, which might have carried high levels of DNA damage, may be unlikely to enter S phase and could be eliminated by apoptosis (Abend et al., 1995). An under-estimation of the spectrum and scale of DNA or chromosomal damage may, therefore, occur in CBMN analysis (Abend et al., 1995; Fenech, 1998). As the biological half-lives of lymphocytes carrying various DNA damage was estimated to be <12 months (Buckton, 1983; Fenech et al., 1990) or longer (Braselmann et al., 1994), RT should always be considered for all cytogenetic assays on human lymphocytes. This was also reflected in a non-significant difference in the frequencies of various chromosomal aberrations in a limited number of samples with similar exposure (Chen et al., 1997).
To our knowledge, this is the first study employing centromere-specific FISH in CBMN analysis of human subjects in a longitudinal follow-up study. The results should provide a more in-depth understanding of the mechanisms of MN formation in human subjects and application of the CBMN assay in biodosimetry and in determining radiation signature in the future.
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Acknowledgments |
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Drs Shyh-Dye Lee of the National Taiwan University Hospital and Pai-tsan Hwang of the Chan-hwa Christian Hospital helped with sample collection and medical care for the exposed during the follow-up periods. Special thanks are due to Dr Teresa Yang of Yale University, Dr Peter Keng of the University of Rochester and Dr H.W. Chen of the University of California Riverside for technical support and Dr John B. Little of Harvard University for help in writing the manuscript. This study was supported partly by the NSC Taiwan and the NHRI Taiwan (DD01-86IX-RA501P).
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Notes |
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6 To whom correspondence should be addressed at: Institute of Environmental Health Sciences, National Yang Ming University Medical School, 155 Section 2 Lih-long Street, Taipei, Taiwan 12211. Tel: +886 2 8267053; Fax: +886 2 8202837; Email: wpc94{at}mailsrv.ym.edu.tw
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References |
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Received on January 4, 1999; accepted on March 18, 1999.
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F. Maffei, C. Fimognari, E. Castelli, G. F. Stefanini, G. C. Forti, and P. Hrelia Increased cytogenetic damage detected by FISH analysis on micronuclei in peripheral lymphocytes from alcoholics Mutagenesis, November 1, 2000; 15(6): 517 - 523. [Abstract] [Full Text] [PDF]
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C. Andrianopoulos, G. Stephanou, E. Politi, and N.A. Demopoulos Evaluation and characterization of micronuclei induced by the antitumour agent ASE [3{beta}-hydroxy-13{alpha}-amino-13,17-seco-5{alpha}-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate] in human lymphocyte cultures Mutagenesis, May 1, 2000; 15(3): 215 - 221. [Abstract] [Full Text] [PDF]
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