Abstract
Background/Aim: Biomonitoring is currently applied in the estimation of health risks after overexposure to ionizing radiation (IR). The aim of this study was to compare the association of dicentric chromosomes and acentric fragments (AF) with cancer risk in subjects exposed to IR, as well as in control subjects. Materials and Methods: The study was performed on 3,574 subjects (2,030 subjects exposed to IR and 1,544 control subjects). The mean follow-up period was 8 years. Results: In subjects reporting exposure to IR, the presence of AFs and dicentric chromosomes was associated with a significant increase in cancer risk, hazard ratio (HR)=1.78 (95% confidence interval (CI)=1.01-3.13) and HR=1.73 (95% CI=1.03-2.90), respectively. Conclusion: AFs are associated with cancer risk and have a similar sensitivity to dicentric chromosomes in subjects exposed to IR. Because automated AF scoring can be easily introduced using fast flow cytometry combined with the pan-centromere staining, this biomarker may hold promise as a potential sensitive biomarker of exposure to IR and cancer risk.
- Ionizing radiation
- cancer risk
- acentric fragments
- chromosomal aberrations
- carcinogenesis
- chromosome damage
- acentric fragments
In nuclear accidents like Chernobyl and Fukushima, the majority of irradiated subjects received doses that do not cause acute radiological disease, but prolonged health effects with possible long-term latency period. Estimation of received doses by physical personal dosime-ters of large populations has significant limitations due to logistic problems with their distribution, stockpile size and calibration.
Biodosimetry represents a much more reliable approach in the estimation of health risks after accidental overexposure to ionizing radiation (IR) than physical dosimetry as it measures final biological effects that are age- and gender-related. Chromosomal aberrations (CA) in peripheral blood lymphocytes have been the most reliable biomarker of exposure to IR during the last several decades.
The frequency of dicentric chromosomes is the “gold standard” of the IR biodosimetry. The main strengths of the dicentric chromosome as a biomarker are available from regulatory compliance standards (1, 2) and a technical manual with standardized methodology (3). Although it is not specific to IR, the advantages of the dicentric chromosome as a biomarker are its low frequency in the general population and good sensitivity as the lower limit of dose detection for low linear energy transfer (LET) radiation is 0.1-0.2 Gy (3).
The first epidemiological evidence of CA as biomarkers of increased cancer risk was published in the 1990s in Nordic countries and in Italy (4-7). In a large European population study, Bonassi et al. provided evidence confirming chromosome aberrations as a reliable biomarker that could predict cancer risk in healthy subjects (8). This study included 22,358 subjects from 11 countries exposed to different chemical agents and IR, as well as unexposed controls. It showed that the relative risk (RR) of cancer was increased for subjects in the medium RR=1.31, 95% confidence interval (CI)=1.07-1.60) and in the high (RR=1.41; 95% CI=1.16-1.72) tertiles of chromosome-type aberrations when compared with the low tertile. This study demonstrated the association between cancer risk and the frequency of unstable chromosomal aberrations in subjects exposed to IR.
A significant disadvantage of biodosimetry, based on genome damage, is the long period between sampling and results. Densely populated regions may demand risk assessment of several thousand subjects in a short time; a capacity that currently available biodosimetry laboratories do not have (3, 9, 10).
Currently, there is no suitable available automated system using neither dicentric chromosome nor other frequently used biomarkers for exposure to IR, such as micronucleus or translocation frequency, due to still not sufficiently developed image analysis programs.
Although micronucleus assay and CA (not dicentric chromo-somes) are shown to be predictive for increased cancer risk (8), there is still no data on correlation between acentric fragments (AFs) and cancer risk.
As AFs arise from double strand breaks of DNA via illegitimate repair processes or during formation of dicentric and ring chromosome, as well as incomplete translocation, they might be suggested as an additional potential sensitive biomarker as their number usually exceeds the frequency of dicentric chromosomes. The spontaneous incidence of dicentric chromosomes in the general population varies between studies; however, the International Atomic Energy Agency (IAEA) reached a consensus on 1 to 2 in 1,000 cells (3, 11, 12), while AF frequency varies from 2 to 6 per 1,000 cells, and no consensus was reached on their frequency by IAEA (11-15). The sensitivity of AFs as biomarker for IR is known from premature chromosome condensation (PCC) studies, which show reliability at doses of 0.06 Gy (16).
The ratio between dicentric chromosomes and AFs in occupationally exposed populations varies. Thus, workers exposed to uranyl compounds in industry show higher or lower frequency of dicentric chromosomes than AFs (14, 17-19). In hospital workers occupationally exposed to low doses of ionizing radiation, acentric frequency was also reported to be higher than of dicentrics (0.77% versus 0.095%, respectively) (20, 21).
According to experimental data at low LET, the frequency of excess AFs is about 60% of the number of dicentric chromosomes and this relationship is not dose- and rate-dependent, suggesting the same kinetics of AFs and dicentric chromosomes (22).
Until now, the induction of chromosome-type AFs, as a biomarker, has been neglected and their potential to predict cancer risk has never been properly evaluated. The advantage of AF is in a potentially elegant future approach to the development of an automated system that would perform rapid analyses of large cohorts in cases of emergencies caused by nuclear accidents. Such system may be based on flow cytometry and fluorescent in situ hybridization (FISH).
The aim of this study was to compare the association of dicentric chromosomes and chromosome AFs with cancer risk in a sub-cohort of subjects exposed to IR and control sub-jects from the European cohort evaluated by Bonassi et al. (8).
Materials and Methods
Control subjects were selected for this study if they (i) had never been exposed to IR (except for natural background ionizing radiation and routine medical x-rays 6 months before sam-pling), (ii) never were diagnosed with cancer or leukemia, (iii) never had radio- or chemotherapy, (iv) never had been diagnostically evaluated by radio-imaging (v) had viral infection and antibiotic therapy 6 months before sampling.
The study included 1,432 subjects continuously occupationally exposed to IR in radiological departments of hospitals in Croatia, Hungary, Slovakia and Poland, as well as 598 Lithuanian citizens who worked as liquidators in Chernobyl. Both groups were extracted from the pooled database of the European study group on cytogenetic biomarkers and human cancer risk (8). The mean age of subjects exposed to IR at the end of the follow-up was 47 years (69% males) and of control subjects 47 years (58% males). Subjects from Chernobyl were analyzed within 6 years after exposure, thus within the half-life span of naive T lympho-cytes (23). The mean received dose was 0.13 Gy (range=0.001-0.47 Gy). In occupationally exposed subjects (hospital workers), exposure was less than 20 mSv annually. In subjects from Chernobyl no data on doses were available. Subjects were screened for chromosome aberration frequency during the period 1981-2002. The mean follow-up period was 8.7±4.5 years. The mean age of the group at the time of testing was 38.7±10.5 years (64.2% males, 46.5% smokers). The 1,544 control subjects worked in administration and were never occupationally or accidentally exposed to IR. For each individual a detailed questionnaire was used. Individuals who underwent exposure to IR for medical purposes during the last 6 month were not included in the study. Information on cancer incidence was collected by linking with national and local cancer registries, except for Hungary. In this case, an active follow-up system was set up for the period 1978-1998, linking Individual Identification Numbers with the morbidity database of local hospitals and family doctors. After 1998, the National Cancer registry was used to assess the presence of cancer diagnoses. Cancer risk associated with AF frequency was evaluated and compared to that for dicentric frequency. The range of years of cancer diagnosis was 1982-2004. A full description of methods can be found in Bonassi et al. (8).
The study was approved for each cohort by the National Ethics Committees and was conducted following the principles of the Helsinki Declaration. All subjects were informed about the aim of the study and gave their written consent. The results of the study were reported to the subjects.
Heparinized venous blood samples were used for lymphocyte cultures (48 h). Half milliliter of blood was added to 5 ml culture mixture consisted of culture medium available ac-cording to the country: RPMI 1640 or F10 (Sigma-Aldrich Chemie Gmbh Munich, Germany), supplemented with 20% fetal calf serum (Sigma-Aldrich), 1% phytogaemaaglutinin P (Difco or Sigma-Aldrich), glutamine 2 mM and antibiotic. Bromodeoxyuridine (Sigma-Aldrich) was added in cell culture at final concentration of 15 μM. The cells were harvested 48 h following stimulation. Colchicine was added 3 h before harvest at a concentration of 0.004% (Sigma-Aldrich). Chromosome preparations were performed according to standard procedures and stained with Giemsa. One hundred metaphases per individual were counted. Acentric fragments, which were not associated with any dicentric chromosome or ring, were registered.
Comparison between exposure groups of patients/healthy subjects using the healthy unexposed subjects as reference level.
To link the frequency of chromosome type AF and dicentric chromosomes with cancer incidence, the Cox proportional hazards model (24) was applied. These biomarkers have a very low frequency with a number of 0 values that exceed 50% in the case of AF and even 84% in the case of dicentric chromosomes. Therefore, in the Cox model both markers were recorded into two categories depending on whether the count was 0 or more than 0. This method avoids the influence of the extreme values and reduces the heterogeneity between laboratories. Thus, through the Cox model, we estimated the hazard ratio (HR), i.e. the ratio between hazard rates of subjects with at least one dicentric chromosome or AF to those with-out dicentric chromosome or AF. The hazard rate is the number of cancer cases divided by the number of individuals at risk per unit time, as the time interval approaches zero.
Moreover, the mean frequencies of dicentric chromosomes or AFs in healthy controls and cancer cases were compared in subjects exposed, or not, to IR. Dicentric chromosome or AF frequency is a discrete variable created by a count; hence, negative binomial (NB) regres-sion is the most suitable statistical model to analyze these markers (25). This model is particularly effective given its property to take into account “overdispersion” (25), a phenomenon that frequently occurs with counts. The regression parameters estimated from this model are interpretable as ratios between the mean frequencies of biomarkers in the exposed to the mean frequencies in controls mean ratio (MR). To adjust the HRs and MRs by the effect of con-founding factors, gender, age (continuous), smoking status (yes/no) and a variable identifying the laboratories, were included in the statistical models. All statistical analyses were performed using STATA statistical software (26).
Results
The comparison between the frequencies of dicentric chromosomes and acentric fragments within the analyzed groups are shown in Table I. Results show that in exposed subjects, both healthy or diagnosed with cancer, the frequency of acentric fragments was significantly higher than in unexposed controls with MR of 1.44 (95% CI=1.14-1.81; p=0.002) and 1.87 (95% CI=1.36-2.56; p<0.001), respectively. The dicentric chromosomes show quite similar behavior being in excess in healthy individuals compared to cancer patients exposed to IR (MR=1.59; 95% CI=1.13-2.22; p=0.007 and mean ratio=2.18; 95% CI=1.27-3.76; p=0.005, respectively). Both the AFs and dicentric chromosomes appear to be able to highlight the occurrence of genotoxic damage, with the second having a greater sensitivity.
The association of AF and dicentric chromosomes with cancer incidence in subjects exposed to IR and in unexposed controls is presented in Figure 1. This multi-panel figure shows cancer-free survival by follow-up time. Cancer-free survival refers the time period from the chromosome aberration test to the first cancer diagnosis. In the subjects exposed to IR, both for AFs (60 cancer cases; 14,795 person-years) and for dicentric chromosmes (71 cancer cases; 17,916 person-years), the presence of 1 or more of these chromosome aberrations is associated to a significant increase in cancer risk: HR=1.78 (95% CI=1.01-3.13; p=0.046) and HR=1.73 (95% CI=1.03-2.90; p=0.039), respectively. In the unexposed controls the hazard ratios (HR) were 1.12 (95% CI=0.61-2.07; p=0.717) for the AF (46 cancer cases; 6,334 person-years) and 1.29 (95% CI=0.57-2.92; p=0.535) for dicentric chromosomes (59 cancer cases; 13,151 person-years), both not significant. The small number of cases prevents reliable inference regarding specific cancers; however, in people exposed to IR, lung cancer seems to be more related to the AF than dicentric chromosomes, while in the control population breast cancer presented high hazard ratios both for AF and dicentric chromosomes (data not shown). Subjects with at least one AF in the control population added up to 1890 person-years. In such a small group of unexposed people, the statistical power to evaluate the link with less frequent tumors is low.
Discussion
Large nuclear accidents like Chernobyl and Fukushima, despite having occured 30 years apart, show that it is still not possible to perform biodosimetry for large populations. This is true for several reasons, such as logistic problems in the field, troubled communication, lack of laboratory capacities, etc. The dynamics of nuclear accident management demands response within hours to days. Current biodosimetry does not have reliable methods to be included in triage decisions on evacuation within a short time period or decisions on distribution of radioprotective drugs. Additionally, sampling, sample storage and transport to the nearest biodosimetry centre, which can be expected to be difficult in conditions immediately after a nuclear accident, as well as time consuming analysis of genome damage, question the role of biodosimetry in the decision making process of health risk prevention. The possible solution may be a portable biodosimetric automatic system, that can be located at the site of a nuclear accident that sends raw data by analog or digital signals so they can be interpreted by experts located anywhere on Earth. However, there is a need for selection of such a biomarker that (a) can be analyzed automatically (b) is used as prognostic factor of certain health risk after exposure to doses of IR that do not cause radiation disease and (c) requires a small sample so it can be used for all age groups.
Survival curves, estimated from Cox proportional hazard model for total cancer incidence (International Classification of Diseases IX 140-208), of acentric fragments and dicentric chromosomes based on pooled data from 5 countries. Cancer-free probability refers to time from chromosome aberration test to the first cancer diagnosis. HR, Hazard ratio; CI, confidence interval.
Over the last few decades several new biomarkers have been introduced but dicentric chromosome measurement is still considered to be the gold-standard in biodosimetry (3). Biodosimetry has focused on dicentric chromosomes for decades, as it was believed that they are preferentially specific for IR. Although AFs were also repeatedly applied as a biomarker of exposure to ionizing radiation (27-29), the informativeness of this biomarker was anecdotally investigated. With the development of whole chromosome painting (30), translocations are now as easy to measure as dicentric chromosomes (31), although a fully automated translocation or dicentric chromosome analysis system is still not commercially available. However, AFs, due to their structure, enable application of combined fluorescent in situ hybridization in suspension and fast flow cytometry (32, 33). For the short term (six months to a year post exposure), such an approach offers significant contribution in high throughput biodosimetry.
Thus far, except for a limited number of papers describing the mechanism of the origin of AFs, usually on in vitro models during 1980s, evaluation of applicability of AFs in biodo-simetry has not been done. Therefore, this study investigated potency and predictivity of this biomarker for cancer risk in a large cohort of healthy unexposed subjects and subjects occupationally exposed to IR as a preparative step for development of automated AF scoring. Our results, for the first time, confirm that AFs can be used instead of dicentric chromosomes in biodosimetry of subjects exposed to IR. The correlation between cancer risk, dicentric and acentric frequency shows that both dicentric chromosomes and AFs are good predictors of risk in an exposed population to IR with significant increase in cancer risk of 1.78 for AFs and 1.73 for dicentric chromosomes. However, such predictivity was not possible to be applied in control populations.
The sensitivity of AFs as a biomarker can be seen in papers on populations exposed to IR after the Chernobyl nuclear accident. The level of soil contamination by radionuclides and frequency of dicentric chromosomes and AFs correlate but AFs, at higher doses, show higher frequencies than dicentric chromosomes (34). Similarly, in a study of children after the Cher-nobyl nuclear accident, it is shown that the frequency of AFs is higher than the frequency of dicentrics and that there is a better dose response for AFs than for dicentric chromosomes (34). This is also in concordance with results in Chernobyl liquidators sampled up to two years after exposure in those whose AF frequency was reported to be higher than that of dicentric chromosomes (35). In children exposed to 137Cs and 90Sr living in the South Ural region, due to radioactive contamination, the frequency of AFs is higher than of dicentric and ring chromosomes together (0.49% versus 0.07%, respectively) (36). Similarly, in the adult population of the Dolon region close to Semiplatinsk nuclear weapon test site, there is a higher frequency of AFs than of dicentric and ring chromosomes (0.83% vs. 0.26%, respectively) (37). In testicular cancer patients after radiotherapy (38, 39), it has also been shown that the number of AFs are increasingly steeper than dicentric chromosomes in relation to dose and their rate of decline is slower, which is an advantage for biodosimetry.
As both dicentirc chromosomes and AFs are unstable biomarkers, the rate of their decline is also a very important parameter. It is suggested that the rate of decline for dicentric chromosomes may be different from that of AFs and that decline depends on the fraction of irradiation and location, as well as volume of irradiated organs of the body (40). However, decline in in vitro conditions after exposure to X rays of 1.5 Gy and 3 Gy is 60% per cell generation from initial frequency (41), which is the same for both dicentric chromosomes and AFs.
An elegant approach for automatic scoring of AFs may be achieved using fluorescent in situ hybridizitaion in suspension (S-FISH). This method was initially introduced for clinical cytogenetics when it was important to recognize bone marrow cells in the case of subjects who received a sex-mismatched allogeneic bone marrow transplantation (42-44). Using an avidin-biotin system, where avidin is the probe label, e.g. pan-centromere probe (45), and biotin is attached to a solid support (32), AF can be separated from all centromere-bearing chromosomes. The efficiency of such an approach has already been shown in an experimental model (46). The solution of separated fragments can then be easily processed with a single laser flow cytometer, thus allowing for a quick and sensitive analysis of chromosomal damage in large populations. Additionally, by employing unstipulated-peripheral blood lymphocytes (PBLs), obtained by premature chromosome condensation, which can be accomplished within 3-4 h, cell culture time can be eliminated (47). This method also eliminates the risk of underestimation of genome damage due to examinee's immunological stress and, consequently, cell culture quality.
In conclusion, the results of this study support the use of AFs as a cancer risk biomarker in subjects exposed to IR, with similar performance as dicentric chromosomes. This means that AFs can be measured as a proxy for all aberrations. Quantities associated with the same quantity are associated with each other. The advantage of AFs is the possibility of their scoring by a combination of S-FISH and flow cytometry, which would eliminate possible bias of overlooked small acentric fragment and dramatically increase the number of analyzed subjects in a short period of time.
Acknowledgements
The Authors thank technical writers, Jennifer Collins and Makso Herman, for editing this manuscript. This study was supported by Croatian Ministry for the Science, Education and Sport.
- Received February 19, 2016.
- Revision received April 1, 2016.
- Accepted April 4, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.