Mutagenesis, Vol. 16, No. 3, 251-255,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Dynamics of changes in micronucleus frequencies in subjects post cessation of chronic low-dose radiation exposure
1 Institute of Environmental Health Sciences, National Yang Ming University, Taipei, Taiwan, 2 Institute of Statistical Sciences, Academia Sinica, Taipei, Taiwan and 3 Department of Family Medicine, Taipei Municipal Jan-Ai Hospital, Taipei, Tawain
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Abstract |
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To assess DNA damage remaining in peripheral lymphocytes, 48 individuals were evaluated twice for lymphocyte micronucleus frequencies by the cytokinesis-blocking cytochalasin B (CBMN) analysis post relocation from radio-contaminated apartments after various periods of time. The frequencies of CBMN at the first evaluation were significantly higher than those at the second examination (Chang et al., 1999c
). These individuals were categorized into three groups: those with cumulative exposure of >300 mSv (defined as high exposure, HDose), those with 100–300 mSv (MDose) and those with <100 mSv (LDose). Using the Poisson mixed-effect model (Little et al., 1996), the estimated mean CBMN frequencies (
) for individuals in HDose, MDose and LDose exposure categories when they had only recently relocated were 21.8, 17.6 and 15.4, respectively. The estimated mean duration post relocation for the CBMN frequencies of these individuals to reduce to 10.2, the second CBMN frequency, on average, was 47.5, 37.2 and 28.3 months in the three exposure groups, respectively. The rates of change in CBMN frequencies were shown to be significantly higher in the HDose group than in the MDose and LDose groups. The results suggested a characteristic dose-dependent decline in the CBMN frequencies in the exposed population post cessation of chronic low-dose ionizing radiation exposure. |
Introduction |
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By the end of 1998, more than 180 civilian building complexes in Taiwan had been identified as having, since early 1983, steel rods contaminated with excessive levels of 60Co (Chang et al., 1997a
). As these contaminated buildings—a total of more than 1800 contaminated apartments and school classrooms—were not disclosed until mid 1992, over 10 000 people were assumed to have been exposed to low-dose rate 
-radiation on a daily basis for 2 to >15 years (Chang, 1993; Chang and Kau, 1993; Chang et al., 1997a; Chang, 1999c). The exposure dose-rate was estimated to range from 0.7 to 150 mSv per year (Cardarelli et al., 1997) and on average much higher than the 1 mSv per year recommended by the ICRP-60 (ICRP, 1997).
Previously, the cytokinesis-blocking cytochalasin B (CBMN) frequencies for 71 exposed individuals who once lived in contaminated apartments in Ming-Shan Villa were significantly greater than those sex- and age-matched non-exposed reference populations (Chang et al., 1997b). The micronucleus assay has been employed to evaluate cytogenetic or mutagenic loading post specific environmental mutagenic exposure in human populations (Fenech and Morely, 1986; Fenech and Neville, 1992), and was shown to be a reliable biomarker for evaluation following acute (da Cruz et al., 1994; Wuttke et al., 1996) or chronic radiation exposure (Chang et al., 1997b). However, little has been reported on the life span of lymphocytes with micronuclei. In individuals who had received acute high-dose radiotherapy, CBMN frequencies were observed to decline significantly, i.e. they decreased to 57 ± 10% in 12 months in 11 patients (Fenech et al., 1990). This was similar to a separate observation in which unstable chromosomal aberrations disappeared from the peripheral blood in <3 years, or decreased by 50% in 12 months (Buckton, 1983), or by 90% in 4 years in patients with X-irradiation for ankylosing spondilitis (Buckton et al., 1978). The unstable chromosomal aberrations were further observed following a two-term exponential function (Ramalho et al., 1995) to show a rapid decline before 470 days and a slower decline beyond 470 days post exposure, respectively. This bi-phasic curve of decrease was also observed in dicentrics of T cell subsets and was suggested to be caused by a differential rate of disappearance in CD45RO (memory cells) and CD45RA cells (naive cells; Michie et al., 1992; McLean and Michie, 1995).
Previously, we repeatedly evaluated 48 subjects with chronic low-dose -radiation exposure for their CBMN frequencies after they relocated from radio-contaminated buildings. They were shown to have a significant decrease in CBMN frequency after 26.2 months (Chang et al., 1999c). Moreover, the decrease in the proportions of binucleates (BNs) with multiple micronuclei was significantly faster than those with a single micronucleus. In order to understand the physiological changes of lymphocytes with various cytogenetic damage in vivo, we further evaluated factors relating to the decreased CBMN frequencies in the same individuals in this study. More importantly, the correlation between the decline in the CBMN frequencies and those of previous cumulative exposure were further evaluated.
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Materials and methods |
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Sampling the exposed population
Forty-eight residents in 18 families with various doses of excessive exposure were enrolled into this prospective cohort study (Chang et al., 1999c
). Soon after they were informed of the contamination (during 1993 and 1994) they were advised to move from their apartments. Around 9.0 ± 6.0 months after they relocated from the radio-contaminated apartments, they received the first CBMN assay. Between 1996 and 1997, 26.2 ± 8.4 months after the first evaluation, they were recalled and re-evaluated for the second CBMN assay. After giving informed consent, we gathered each individual's detailed personal information, including medical and smoking history, time and age when they moved in and relocated from the radio-contaminated apartments and the relocation time (RT), i.e. the duration between moving out of the radio-contaminated apartments and the CBMN analysis. The cumulative doses of exposure for each individual were reconstructed using the Taiwan Cumulative Dose (TCD) Program, which incorporates information on time of exposure and environmental radioactivity in living spaces for each exposed individual (Cardarelli et al., 1997; Hwang et al., 1998).
Lymphocyte culture and CBMN scoring
Peripheral blood lymphocytes were processed immediately after collection and separated by Ficoll-Paque (Pharmacia Biotech) sedimentation. The collected lymphocytes were cultivated in a RPMI 1640 medium containing 10% completely defined, serum-free culture medium HL-1 (BioWhittaker), 10% fetal bovine serum (Hyclone), 10 µg/ml phytohaemagglutinin-p (PHA-P), 1% penicillin and streptomycin. The stimulated T lymphocytes were incubated with cytochalasin-B (6 µg/ml; Sigma) from 44 to 72 h after cellular cultivation, fixed with methanol and acetic acid (10:1), stained with 6% Giemsa and scored under the criteria as described previously (Chang et al., 1996, 1997b, 1999c). The total numbers of micronuclei included in single or multiple micronucleated cells per 1000 randomly examined BNs were designated as the CBMN frequencies and were scored by one well-trained investigator under double blindness throughout the study. The micronuclei read by the investigator were double-checked by another senior investigator and the quality was comparable with the micronuclei read during the first CBMN evaluation.
Statistical analysis
Since the CBMN assay produces frequency data, it is reasonable to assume the response variable follows a Poisson distribution. The Poisson regression model is a generalized linear model that assumes a Poisson distribution for response data and uses the log transformation for the expected value of response. The explanatory variables we collected include sex, smoking history, age, relocation time (RT) and cumulative exposure. According to results from an extensive exploratory data analysis, the subjects can be classified by exposure into three groups: low, medium and high cumulative exposure.
Note that each individual was evaluated twice and some of the subjects are from the same family. Due to the reasons of inheritance, circumstance, lifestyle etc., we believe that the correlation of the two evaluations between the same individual is greater than the evaluations between different individuals, and the correlation of the evaluations between individuals coming from the same family is greater than those coming from different families. So it is reasonable to assume the response has a dependent covariance structure. We assume that the covariance structure has a compound symmetry. Furthermore, the households in the study are assumed to be a random sample from a larger population of households. Therefore, the coefficients of intercept and relocation time among the families could be modeled as random. Hence, the Poisson mixed-effect model (Little et al., 1996) was considered a better model to fit the data.
Specifically, the proposed Poisson mixed-effect model can be presented as follows:
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; Chang et al., 1999a). The coefficients, 
0, 1, 2, ß0, ß1, ß2, 1, 2, 3 and 4 are fixed effects with the intercept of three exposure groups and relocation time of three exposure groups, and covariates, SEX, SMOKE, AGE, and the interaction of RT and AGE. The coefficients, 0, 1, 2, ß0, ß1 and ß2 are random effects with the intercept of three exposure groups and covariates, RT of three exposure groups. Estimates of these coefficients can be obtained using the computing procedure GLIMIX macro available in the SAS statistical package (Little et al., 1996). |
Results |
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Distribution of sex, smoking habits, age, dose of cumulative exposure, RT, duration between each individual's two evaluations and the CBMN frequencies in these two evaluations are illustrated in Tables I and II
. The 48 individuals were categorized into three groups to better evaluate dose effect. The first set of CBMN frequencies in three exposure groups were 17.9 ± 8.1, 21.1 ± 8.8 and 21.6 ± 12.2 per 1000 binucleated cells, respectively. The second set of CBMN frequencies in three exposure groups were 9.2 ± 3.4, 11.4 ± 6.3 and 9.7 ± 5.1 per 1000 binucleated cells, respectively. The mean CBMN frequencies of these 48 individuals in the first and the second evaluation were 20.3 ± 9.7 and 10.2 ± 5.2 per 1000 binucleated cells, respectively. Using the Wilcoxon signed rank test the CBMN frequencies at the second evaluation were significantly lower than those of the first evaluation in the same individual (P < 0.0001).
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The estimates for the fixed effects in the Poisson mixed-effects model are shown in Table III
. The variables for LDose, MDose and HDose are to estimate the effect of cumulative exposure and the ß variables of RTxLDose, RTxMDose and RTxHDose are to estimate the effect of relocation time among the different exposure groups. The P values of the coefficients of LDose, MDose, HDose, RTxHDose, RTxMDose and RTxLDose are <0.05. This means that we have strong evidence that these variables are significantly related to the CBMN evaluated results. After adjusting for factors of sex, smoking habits, age, RT and interaction of age and RT, the estimated intercepts [s], which are the estimated CBMN frequencies when the subjects only recently relocated from radio-contaminated buildings, ± standard error (SE), are 2.7 ± 0.5, 2.9 ± 0.5 and 3.1±0.4 for the low, medium and high exposure groups, respectively. The estimated intercepts ß0, ß1 and ß2 represent the estimated decline rate of CBMN frequencies in low, medium and high cumulative exposure with the same sex, smoking habits, age and interaction of age and RT. ß2 is smaller than ß0, ß1, which means the decrease in CBMN frequencies in the high exposure group is faster than in the other two exposure groups.
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The estimated mean CBMN for the three exposure groups, shown in Figure 1
, can be obtained by the equation CBMN (T) = exp[ + ßxRT] adjusting for sex, smoking habits, age, interaction of age and RT. When RT equals 0 i.e., when these individuals only recently relocated and excessive exposure ceased, the estimated mean CBMN frequencies were 15.4 [(9.5, 24.8) an approximated 95% confidence interval] for the low exposure group, whereas for the medium and high exposure groups they were 17.6 (11.2, 27.7) and 21.8 (14.3, 33.2), respectively. When relocation time increased by 1 month, the CBMN frequency decreased by 1 – e–0.0363*1 (~3.6%) per month in the high exposure group, 1 – e–0.0261*1 (~2.6%) and 1 – e–0.0291*1 (~2.9%) per month in the medium and low exposure groups, respectively. The estimated mean durations for the mean CBMN frequencies of the exposed individuals to decline to 10.2—the average CBMN frequency of all individuals at the second CBMN evaluation—were 47.5, 37.2 and 28.3 months in the three exposure groups, respectively. The duration for the CBMN frequency to decline to 10.2 was significantly shorter for the HDose group than for the LDose group (P value < 0.05).
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Discussion |
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The CBMN frequencies in the 48 exposed subjects at the first CBMN evaluation were significantly higher than at the second CBMN evaluation in these same individuals (Table II
). This suggests that chronic protracted low-dose radiation exposure incurred increased genetic damage in these individuals and the genetic loading shown as CBMN frequencies in the same individual apparently decreased once they relocated. Four individuals exhibited slight increases in CBMN frequencies during this period, including three male subjects who had smoked more than two packets of cigarettes per day for >10 years and did not stop smoking post relocation. Individuals who smoked a high number of cigarettes per day were shown to have significantly elevated CBMN frequencies compared with those of low-level smokers (Fenech, 1993). Other laboratories have also reported significant increments in CBMN frequency related to smoking (Tomanin et al., 1991; Duffaud et al., 1999). Smoking habit may partly explain the increase in the CBMN frequencies observed in these three male subjects.
Age is an important factor associated with CBMN frequency. Micronuclei have been shown to increase in number with age (Huber et al., 1989; Fenech, 1993). However, our data indicated that sex, age at the second CBMN assay and smoking were not significantly associated with the decline rate in CBMN frequencies. This might be caused by several factors. The CBMN frequencies were shown to increase by 3.4% per year by conventional assay and by 2.2% via CB assay (Huber et al., 1989). In a similar approach, the relationship between age and the CBMN frequency was suggested to be: Y = 0.715xAGE (in years) – 1.411 (Fenech, 1993). The coefficient of the age response in this model (Table III) was negative and suggested that older subjects carried a minor change in CBMN frequencies compared with younger subjects. However, the decrease in CBMN frequencies post relocation from a radioactive environment in these subjects was significantly more rapid than the age-dependent increase in CBMN frequencies. It is likely that the age-dependent decline was masked by other factors. Moreover, the range of age distribution in this study was not wide enough to affect the decline in CBMN frequencies. This might explain why age was not more significantly associated with the change in CBMN frequencies than cumulative exposure.
Different decline slopes in these CBMN frequencies in the three exposure subgroups were further observed (Table III and Figure 1). Interestingly, those with the highest exposure were shown to have the fastest decline, followed by the medium and the low exposure groups. This was similar to that seen in patients with high or moderate doses of radiotherapy (>0.2 Gy) in which the frequencies of aberrations fell to ~5% of the initial frequencies 7.5 years after exposure (Ramalho et al., 1998). But the individuals whose absorbed doses were <0.2 Gy were shown to have a much slower decrease in aberration frequency. Based on the model approach, individuals with higher exposure were shown to have higher CBMN frequencies when they had only recently relocated, and also to have the fastest decline in the CBMN frequencies from then on. We have previously observed that BNs with multiple micronuclei were demonstrated to decline from peripheral circulation significantly faster than those BNs with a single micronucleus (Chang et al., 1999c). We assume that subjects with higher exposure carried higher CBMN frequencies and, seemingly, carried more multi-micronuclei BN just before they relocated (Figure 1). The rapid decline in these multi-micronuclei BNs from the peripheral blood would possibly lead to a faster decline in their total CBMN frequencies.
Nonetheless, all the exposed subjects in this study were shown to have a much slower decline in CBMN frequencies than those reported in studies on acute radiation exposure. Several reasons might explain this difference. The micronuclei carried by the lymphocytes of individuals in this study could be mostly at their late plateau phase of decline as each individual received protracted chronic exposure for up to several years. It was shown that the chromosomal aberrations or micronuclei had less of a sharp decline at certain months post irradiation (Fenech et al., 1990; Buckton et al., 1978; Ramalho et al., 1995). Moreover, the exposed individuals in this study were evaluated for their first CBMN frequencies on average 9 ± 6 months (mean ± SD) after they relocated from the radio-contaminated environment. It has been demonstrated that the half-life for the disappearance of circulating lymphocytes with dicentrics was longer at a later post-exposure time than at an earlier post-exposure time (Bauchinger et al., 1989). Due to the long half-life of lymphocytes with micronuclei-like changes, the slower decline in CBMN frequencies in these exposed subjects could have been in an even later or plateau phase.
Furthermore, the annual average exposure in these subjects was 32.8 ± 28.1 mSv, which is much lower than individuals receiving radiotherapy. It may be unlikely for T lymphocytes with high levels of DNA damage to enter the S phase, and they could be eliminated by apoptosis (Abend et al., 1995). Cells with such high levels of DNA damage would be less compatible with further cellular proliferation than cells with less DNA damage, which tend to form micronuclei containing whole chromosomes. We have recently observed relatively higher proportions of centromeric signals in the micronuclei in these exposed subjects (Chang et al., 1999b). Similar results were also shown in medical workers with occupational exposure to X- and -rays (Thierens et al., 2000). However, acute and relatively high radiation exposure tended to generate higher proportions of acentric fragments than whole lagging chromosomes included in micronuclei (Fenech and Morley, 1989; Littlefield et al., 1989; Cornforth and Goodwin, 1991; Weissenborn and Streffer, 1991; Miller et al., 1992). This suggests there are significant proportions of micronuclei containing whole chromosomes in subjects with previous exposure to chronic low-dose -rays and could partly explain the longer circulation duration observed in this study.
This is the first study modeling change in CBMN frequencies in human populations with chronic low-dose radiation exposure. The different observations made from those with acute high-dose radiation exposure should provide further perspectives on biological monitoring for human population studies.
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Acknowledgments |
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We appreciate the assistance of Dr S.D.Lee of the National Taiwan University (NTU) Hospital for providing clinical health care, Dr J.D.Wang of the College of Public Health of NTU for consultation in epidemiological design, Dr Y.H.Li of Tzu-chi Medical University and Ms C.S.Hsu for providing statistical assistance and financial support from the National Health Research Institute (DD0186IX-RA510P), Taiwan, for this program study since 1995.
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Notes |
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4 To whom correspondence should be addressed at: Institute of Environmental Health Sciences and Institute of Public Health, National Yang Ming University, 155, Section 2, Lih-non Street, Shih-pai, Taipei, 112 Taiwan. Tel: +886 2 28267053; Fax: +886 2 28207883; Email: wpc94{at}mailsrv.ym.edu.tw
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Received on June 15, 2000; accepted on December 28, 2000.
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