Mutagenesis, Vol. 17, No. 3, 223-232,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Assessment of genotoxic effects related to chronic low level exposure to ionizing radiation using biomarkers for DNA damage and repair
1 Free University of Brussels, Laboratory for Cell Genetics, Pleinlaan 2, B-1050 Brussels, Belgium, 2 Catholic University of Louvain, Unit of Industrial Toxicology and Occupational Medicine Brussels, Belgium and 3 University of Gent, Department of Biomedical Physics and Radiation Protection, Proeftuinstraat 86, B-9000 Gent, Belgium
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Abstract |
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The first objective of our study was to analyse whether biomarkers for genotoxic effects (DNA breaks and alkali-labile sites and micronucleus and non-disjunction frequencies) could be fully validated for biomonitoring workers chronically exposed to ionizing radiation (IR). Blood samples of controls and individuals chronically exposed to IR were analysed. The interindividual variation was reduced when the comet data were adjusted for interexperimental variation, but remained statistically significant. No differences were found between groups, either for smoking or for exposure status. The second objective was to determine whether the Comet assay can be used to evaluate global repair phenotype as a susceptibility biomarker for IR-induced DNA damage in nuclear workers. A pilot study was performed and blood from workers exposed or not to radiation was submitted to a challenging dose of
-rays. The repair kinetics of each individual donor were analysed by Comet assay at different time points and compared with the frequencies of biomarkers of genotoxic effects. There was a statistically significant interaction between biomarkers assessing the same damage (micronucleus and Comet assays). Multivariate analysis showed that micronucleus frequencies were positively influenced by age and the percentage of residual tail length was negatively influenced by the interaction between smoking and exposure status. The general conclusions from our study are: (i) a positive correlation exists between mechanistically related biomarkers; (ii) multivariate regression analysis confirmed that the interaction between smoking and exposure to IR negatively and statistically significantly influenced residual tail length; (iii) use of the Comet assay for the estimation of global repair phenotype with respect to IR is recommended because it is simple, fast and differences in in vitro repair capacity can be detected. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To improve cancer prevention in workers chronically exposed to low level ionizing radiation (IR) in nuclear power plants, occupational medicine applies essentially biomonitoring of exposure and effects. In the future it may also include biomonitoring of susceptibility, relating genotype and phenotype polymorphisms to differential risk for cancer.
As far as the biomonitoring of IR exposure is concerned, it is restricted to the estimation of the duration of occupational exposure, dose and type of irradiation. Several methodologies have been developed to perform the biomonitoring of effects, but their sensitivity and specificity are often too low to detect genetic effects considering the annual occupational limit of 20 mSv. Assessment of the frequencies of dicentrics and chromosome translocations by Giemsa staining (Lloyd et al., 1992
) or by chromosome-specific fluorescence in situ hybridization (FISH) painting in general is considered the most sensitive method (reviewed in IARC, 1997). However, the technique is time consuming and demands expertise. Scoring of ~1000 metaphases for translocations after chromosome painting allows the detection of doses of ~0.3 Gy (Snigiryova et al., 1997). According to Thierens et al. (1999), reaching the detection limit of 0.02 Gy after in vitro exposure to X-rays should require scoring of 10 000 metaphases with conventional Giemsa analysis of dicentrics; the detection of 0.25 Gy after in vitro exposure to -rays, using a cocktail of chromosome 2, 4 and 8 painting probes, should require a minimum of 1000 cells.
In the last decade the ex vivo/in vitro lymphocyte micronucleus (MN) test (Fenech and Morley, 1985) combined with pan-(peri-)centromeric FISH probes has provided information not only on the induction of acentric fragments (MNCen-) but also on chromosome loss (MNCen+) (Elhajouji et al., 1995; Van Hummelen et al., 1995). Scoring only the MNCen- was suggested to have higher sensitivity with a lower workload and cost (Vral et al., 1997). Based on the last observation, the scoring of MNCen- only improves the detection level from 0.25 to 0.1 Gy. Double FISH labelling of cytochalasin-blocked cells with chromosome-specific probes permits the assessment of chromosome non-disjunction (NDJ) (Kirsch-Volders et al., 1996; Elhajouji et al., 1997). Chromosome loss and NDJ were shown not to have significant consequences in human fibroblasts after in vitro exposure to X-rays (Kirsch-Volders et al., 1996) and in mouse splenocytes after in vivo exposure to the same mutagen (Hande et al., 1997). In addition, we have demonstrated a statistically significant increased rate of NDJ in human peripheral blood lymphocytes (PBLs) after in vitro exposure to 1 Gy -rays (Touil et al., 2000). Only 2000 binucleated interphase cells (BN) were required to reach this conclusion.
Another straightforward technique for the quantification of DNA damage in a cell-by-cell approach is the single cell gel electrophoresis or Comet assay (Singh et al., 1988). Its application under alkaline conditions as a sensitive technique in the regular health screening of workers who are occupationally exposed to genotoxic environmental agents to assess the possibility of different types of DNA damage [DNA strand breaks and alkali-labile sites (ALS)] is well documented (reviewed by Moller et al., 2000). For IR, the assay is relatively sensitive [the detection limit of -rays in peripheral blood lymphocytes (PBLs) and the number of cells needed for this analysis were, respectively, 0.6 cGy and 50 cells] (Malyapa et al., 1998). Recently, De Boeck et al. (2000) further validated the methodology for biomonitoring purposes with the introduction of an internal standard.
The biomonitoring of susceptibility is not yet applicable due to the lack of scientifically sound assessment of our capacity to quantify the effects of genetic polymorphisms on the cellular response to IR. Moreover, bioethical issues related to the implementation of genetic screening for IR susceptibility in occupational medicine need to be addressed in all their aspects before the implementation of methods can be considered. However, it is important to realize that the development of scientifically adequate methods to assess individual susceptibility to IR exposure may be helpful not only to prevent undesirable exposure, but also to allow a more accurate interpretation of the genotoxic effects observed during occupational follow-up. Therefore, genotyping and phenotyping studies of those polymorphisms which are relevant to exposure to IR are becoming essential. As far as IR is concerned, DNA repair, cell cycle and apoptosis genes are the most important. Information on genetic polymorphisms of repair genes is needed before implementing genotyping as a tool in biomonitoring. Polymorphisms of several related repair genes have been identified (e.g. OGG1, XRCC1 and XRCC5), some of which possibly play a role in clinical radiosensitivity (Price et al., 1997). However, development and evaluation of a global `phenotype' repair assay to assess the global repair capacity for IR-induced DNA damage might reflect the genotype status and be helpful in identifying hypersensitive individuals (Leprat et al., 1998).
This work has addressed two major questions: (i) are biomarkers of effects (DNA breaks and ALS, MN and NDJ frequencies) fully validated for biomonitoring of workers chronically exposed to ionizing radiation; (ii) can the Comet assay be used to evaluate global repair phenotype as a susceptibility biomarker for IR-induced DNA damage in nuclear workers. To achieve the second objective, a pilot study was performed and blood from workers exposed or not to a chronic dose of IR was submitted to a challenging dose of -rays. The repair kinetics of each individual donor were analysed by alkaline Comet assay and compared with the frequencies of biomarkers of effect (DNA breaks and ALS, MN and NDJ frequencies).
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Materials and methods |
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Study population
The characteristics of the study populations are summarized in Table I
. A group of workers (28 males) occupationally exposed to low doses of IR in a nuclear power plant, with 20 years average duration of employment, and a group of controls (19 men) chosen from among administrative employees were monitored. Exposure was estimated from personnel dosimeter records, the doses ranged from 19.54 to 242 mSv. Moreover, information on age, smoking habits, intake of vitamins, use of therapeutic drugs and previous exposures to diagnostic X-rays was recorded. We were unable to perform an analysis of all the biomarkers for each individual because of lack of material.
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Sampling
Blood (10 ml) drawn by venipuncture into heparinized tubes (Vacutainer; Benton Dickinson, Oxford, UK) was delivered to the laboratory by car within 4 h, protected from light. Samples from the controls and exposed subjects were handled concurrently. All assays were run on coded samples.
Biomarkers of genotoxic effect
Alkaline Comet assay
From each individual, 2.8 ml of whole blood was diluted with 3.2 ml of sterile Ca2+- and Mg2+-free phosphate-buffered saline (PBS). An aliquot of 6 ml of the diluted blood was layered on top of a 3 ml Ficoll-Paque gradient (Pharmacia-Biotech, Brussels, Belgium) and centrifuged at 400 g for 45 min at room temperature. The interphase containing the mononuclear cells was collected with a Pasteur pipette, transferred to a separate tube and washed three times with PBS. For each electrophoresis, internal Comet slides from a human K562 erythroleukaemia cell line (Lozzio and Lozzio, 1975), treated or untreated with 2 mM ethylmethane sulfonate, were included as positive and negative standards, respectively. The procedure for the preparation of these samples has been described in a previous work (De Boeck et al., 2000). The Comet assay was performed according to the method described by Singh et al. (1988), with some modifications as reported (De Boeck et al, 1998). Briefly, 1000–5000 cells/ml in 0.8% low melting point agarose was layered on top of an ordinary microscope slide that had been pre-coated with 1% normal melting point agarose. The Comet slides were left in lysis solution overnight at 4°C (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, pH 10) supplemented just before use with 10% DMSO and 1% Triton X-100. The coded slides from the human blood donors were transferred to an electrophoresis unit together with 200 µl of H2O2 (10 min).
Cytokinesis-blocked micronucleus (MN) assay
Whole blood cultures were made as follows: 1.6 ml of whole blood was placed in culture flasks at a concentration of 1x106 cells/ml and incubated in a humidified CO2 incubator at 37°C in Ham's F-10 medium supplemented with 15% fetal calf serum (FCS) (Gibco, Paisley, UK). The lymphocytes were stimulated with 2% phytohaemagglutinin (PHA 16; Murex Biotech, Belgium) and treated with 6 µg/ml cytochalasin-B (Sigma Chemical Co., Belgium) at 44 h. At 72 h cells were subjected to a cold hypotonic treatment (0.075 M KCl), immediately centrifuged at 400 g for 8 min and fixed three times with methanol/acetic acid (3:1). The fixed cells were dropped onto humidified slides and air dried. For MN analysis the slides were stained directly with 5% Giemsa for 20 min, rinsed and air dried. The remaining slides were stored at –20°C until use in FISH analysis.
FISH with chromosome-specific centromeric probes
FISH with probes for centromeric regions of chromosomes 1 (pUCl.77) and 17 (D17Z1) was applied to slides from MN preparations. The probes were labelled by nick translation according to the instructions of the supplier (Life Technologies, BRL). FISH was performed as described by Touil et al. (2000). Briefly, the slides were treated with RNase (Sigma) (0.1 mg/ml in 2x SSC) for 1 h and pepsin solution for 10 min in a water bath (37°C). Thereafter, the slides were denatured at 80°C together with the probes. Following an overnight hybridization at 37°C, the slides were washed with 50% formamide in 2x SSC at 42°C. Detection of the biotinylated probe for chromosome 17 was performed with avidin–FITC (fluorescein avidin D; Vector Laboratories, Burlingame, CA) and biotinylated goat anti-avidin antibodies (Vector Laboratories), allowing signal amplification. The digoxigenin-labelled probe (chromosome 1) was detected using a mouse anti-digoxigenin antibody (Boehringer Mannheim, Germany) followed by a Texas Red-conjugated sheep anti-mouse antibody (Amersham, Little Chalfont, UK). After dehydrating in an ethanol series (50, 75 and 98%), the cells were counterstained with 4',6-diamido-2 phenylindole (DAPI) (Boehringer Mannheim, Germany) in a phenylenediamine antifade solution.
Global repair phenotype Comet assay
For the study of repair kinetics of control and exposed workers, 2.8 ml of blood per donor was stored at 4°C in the dark until the following day. Previous experiments have shown that whole blood can be stored under these conditions for at least 24 h without affecting the level of DNA damage in lymphocytes (Vaghef et al., 1997).
Blood samples from 10 nuclear workers and 10 control individuals were used for the repair study. For each individual, two whole blood cultures of 35 ml were prepared. The blood was placed in culture flasks at a concentration of 1x106 cells/ml and incubated in a humidified CO2 incubator at 37°C in Ham's F-10 medium supplemented with 15% FCS. The lymphocytes were stimulated with 2% PHA. After 24 h, cells were exposed to 60Co -rays in the G1 phase of the cell cycle. The blood cultures were irradiated in vitro with 2 Gy at 0.1 Gy/min in a water bath at 0°C to prevent any repair. Irradiation was performed on whole blood to better mimic the in vivo situation. Dosimetry was performed at the position of the samples with a NE2571 cylindrical ionization chamber and NE2570 dosimeter (Nuclear Enterprises, Reading, UK), applying the IAEA 1987 Code of Practice. Aliquots (2x5 ml) were removed from the cultures before and immediately after exposure and the Comet slides were stored in lysis solution at 4°C with internal standards prepared on the same day. The remaining blood cultures (25 ml), having been kept on ice, were incubated at 37°C to allow acclimatization for 20 min. This time was established in preliminary experiments. Thereafter, repair assessments were performed after in vitro irradiation (0 min) at times 5, 10, 30, 60 and 120 min, aliquots of 5 ml of blood cultures being centrifuged at 400 g for 10 min. The comet slides were prepared as described above (see Alkaline Comet assay). The slides prepared at different sampling times, all originating from the same individual, were kept overnight at 4°C in lysis solution with those obtained before and immediately after irradiation and internal standards prepared on the same day. They were processed together in the same electrophoresis session the following day.
Reproducibility of the assay
Whole blood collected from one volunteer was subjected to in vitro challenge with 2 Gy -rays followed by repair, as described above for the global repair phenotype. Four electrophoreses were performed to assess the reproducibility of the assay.
Analysis
Comet assay
For the Comet assay analysis 100 cells per donor from two cultures were randomly captured on coded slides using a Leitz fluorescence microscope (x25 objective) coupled to a CCD camera and image analysis system (Komet 3.0; Kinetic Imaging, Liverpool, UK). The length of the comet tail in µm (TL), the percentage of DNA damage in the tail (TDNA) and the product of TL and TDNA in µm (TM) were recorded.
MN assay
For the MN analysis micronuclei (MNi) were scored in both BN and in non-divided lymphocytes (mononucleated cells, i.e. Monos) on a same slide prepared 72 h after mitogen stimulation. The scoring criteria followed those proposed by Thierens et al. (1999). 2000 BN per individual (1000 BN per culture) were examined for the presence of one, two or more MNi and data are presented as the frequency of micronucleated binucleate cells per thousand binucleates (
MNBN). The mononucleated lymphocytes with or without MNi were examined and data are presented as the frequency of micronucleated mononucleate cells (MNMonos).
FISH with chromosome-specific centromeric probes
For chromosomal NDJ, to restrict the scoring to the first mitosis and exclude artefacts, only BN having the diploid number of hybridization signals were analysed. The distribution of signals for both probes in BN was scored as 2/2, 1/3 and 0/4 for, respectively, two spots in each of the two nuclei, one spot in one nucleus and three spots in the second, no spots in one nucleus and four spots in the second. Both the 1/3 and 0/4 combinations were scored as a single NDJ cell. NDJ represents the number of NDJ events observed per 1000 BN. The events involving chromosome 1 were scored independently of those involving chromosome 17 but were recorded in parallel in the same cells. The preparations were examined with a Zeiss Axioscop microscope (Carl Zeiss, Oberkochen, Germany) equipped with a filter (filter block 9; Zeiss) to visualize the fluorescein/Texas red-labelled probe and DAPI counterstaining. To estimate chromosomal NDJ frequencies (%NDJ) for the total genome, assuming that NDJ occurs randomly over the chromosomes, the initial frequencies of NDJ for both chromosomes were multiplied by 23/2.
Repair kinetics
For analysis of repair kinetics, the initial DNA damage and residual DNA damage were calculated as follows (Leprat et al., 1998): initial DNA damage = DNA damage immediately after in vitro irradiation – DNA damage in control cells before in vitro irradiation; per cent residual DNA damage at time t after irradiation (%RD) = 100x(DNA damage at time t after irradiation – DNA damage in control cells before irradiation) ÷ (DNA damage immediately after irradiation – DNA damage in control cells before irradiation).
Three parameters of RD were recorded: per cent residual tail length (%RDTL), per cent residual tail DNA (%RDTDNA) and per cent residual tail moment (%RDTM).
Statistical evaluation of data
For all genotoxicity tests, the interindividual variation was analysed by a Student's t-test.
TL, TDNA, TM, MNMonos, MNBN and %NDJ showed significant departures from a normal distribution. Therefore, all data underwent log transformation and a two-way ANOVA design was performed to evaluate the possible influences of smoking and exposure on the biomarkers. A simple regression analysis was performed to verify the correlation between all genotoxicity parameters and age for each group (exposed workers and controls separately) and for all workers together.
In addition, a multiple regression analysis (stepwise multivariate analysis) was performed to determine the relative influence of the different independent variables (cumulative dose, smoking and age in the exposed group and exposure, smoking and age in all workers) on genotoxicity parameters. Statistical analyses were performed using the SPSS and SAS packages.
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Results |
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Biomarkers of effect
The comet, MN and NDJ data are summarized in Table II
. The results compare the control and exposed groups. In addition, the control and exposed groups were subdivided into control non-smokers (CNS), control smokers (CS), exposed non-smokers (ENS) and exposed smokers (ES).
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DNA damage data
The results for TL, TDNA and TM were corrected by the calibration model described in a previous work (De Boeck et al., 2000
). TL, TDNA and TM showed similar patterns (Table II
). No statistically significant effect of smoking or of exposure at the 95% confidence level was found within each group and between groups.
Micronucleus assay
A highly and statistically significant interindividual variability was found (P < 0.001) for MN frequencies in both MNMonos and MNBN using Student's t-test.
The frequency of MNMonos was not statistically different between controls and exposed workers (Table II). The frequency of MNMonos showed an increased level in CS as compared with CNS. However, this increase was not statistically significant. In ES there was a tendency for an increase in MNMonos but the difference between ENS and ES was not statistically significant.
No statistically significant increase in MNBN frequency was found in workers exposed to IR as compared with controls. In the control group the mean frequency of MNBN was higher in CS than in CNS, but the difference was not statistically significant. In the exposed group no clear effect of smoking was observed.
Chromosome NDJ
Chromosome-specific malsegregation was studied using FISH with specific centromeric probes for chromosomes 1 and 17. No statistically significant differences in NDJ frequencies between the two chromosomes were found. No significant differences in NDJ for chromosome 1 and for chromosome 17 frequencies between individuals in the control and exposed groups were detected. Frequencies of BN where NDJ was observed in the same cell for both chromosomes were extremely low. The data are shown for each group (Table II). No statistically significant differences between groups were found either for smoking or for exposure.
Global repair phenotype assay
Reliability of the assay
Reliability was determined using the intraclass correlation coefficient (ICC). There was significant ICC between the electrophoreses for the DNA repair parameters %RDTDNA and %RDTM (P = 0.0003). This is an indication that the in vitro global repair phenotype assay as performed in our laboratory is reproducible given that %RDTDNA is the most stable repair parameter.
To assess the global phenotype for repair of DNA breakage and ALS induced by IR, PHA-stimulated whole blood cultures were challenged in vitro with an acute exposure to 2 Gy 60Co -rays applied to cells from 10 nuclear workers and 10 controls. For the controls only four individuals were non-smokers, while in contrast seven individuals were non-smokers in the exposed group.
The repair capacity was estimated at different time points (5, 10, 30, 60 and 120 min) after irradiation by the alkaline Comet assay. This allows the quantification of initial damage, the assessment of repair phenotype and the calculation of residual DNA damage at each time point.
Initial DNA damage induced by in vitro radiation in control and exposed worker groups
No significant effect of smoking and occupational exposure to IR on the level of initial DNA damage was found. The background level of DNA damage determined from TL, TDNA and TM was similar in both groups (Table III).
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For all individuals a statistically significant increase in comet parameters (TL, TDNA and TM) in PHA-stimulated lymphocytes was detected immediately after in vitro exposure to 2 Gy
-rays as compared with the DNA damage before irradiation (P < 0.001). The initial DNA damage for the three comet parameters was the same in both groups.
Residual DNA damage as measured by the time-dependent reduction in comet parameters in control and exposed worker groups
The in vitro -ray-induced DNA damage was progressively repaired in PHA-stimulated lymphocytes from both control and exposed workers as measured as a time-dependent reduction in the comet parameters. The interindividual variation was high, the standard deviation (SD) being elevated in both groups. Table IV shows the repair kinetics of DNA damage in both control and exposed individuals expressed as mean values of %RDTL, %RDTDNA and %RDTM. A period of faster repair (50% of DNA damage remains after 5 min) when considering %RDTM as a measure of repair capacity was observed in occupationally exposed individuals and controls. This faster repair was more pronounced in exposed than in control workers when considering %RDTDNA (48.35 ± 26.64 and 58.20 ± 29.48, respectively). At 30 min repair time the mean %RDTDNA in exposed individuals was, however, higher than in control individuals. %RDTL followed the same pattern as that for %RDTM, remaining higher at longer repair time points in the exposed group. Despite these differences in repair between groups, there were no statistically significant differences between groups (ANOVA analysis; see the P values in Table IV). For controls (Table V) the only statistically significant effect of smoking on repair capacity was seen for %RDTL after 30 min; this residual damage was found to be higher in CS. %RDTDNA remained higher in CS than CNS at 5, 10 and 30 min repair, but reached similar values at 60 min and were lower at 120 min, however, the P values were close to one at the last repair time point (i.e. 120 min). In the exposed group (Table VI) %RDTL followed an opposite trend in ES and was much lower than in ENS.
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Global repair phenotype at the individual level
The global repair phenotype at the individual level aims at detecting individuals who have an elevated or diminished repair capacity in lymphocytes compared with an average level of repair. In addition, characterization of this capacity by defined parameters allows comparison between individuals. Therefore, the data for each individual were plotted separately for each time point on graphs giving ± 2 SD from the mean value calculated for the control non-smokers (CNS).
Figures 1–3 show %RDTL, %RDTDNA and %RDTM, respectively, measured 5, 10, 60 and 120 min after 2 Gy exposure for both control and exposed individuals.
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As seen in Figure 1
, two donors appeared to be deficient in repair capacity at the 120 min time point, one individual being CS and the other ENS. The corresponding %RDTL was closed to 90%.
Interestingly, three individuals appeared to have a high %RDTDNA after 5 min repair incubation. Two of these were CS and also showed an abnormal repair capacity at 10 min (Figure 2). One of the ENS appeared to have a high repair capacity at all time points.
%RDTM, which is in fact the product of both %RDTL and %RDTDNA, detected more individuals with less efficient repair capacity (Figure 3). Some of the donors who were CS indeed appeared to have a higher %RD damage during the faster repair period (i.e. 5 and 10 min).
Evaluation of the biomarkers
The analysis of possible mechanistic relationships between biomarkers was performed in the total population (Table VII). Significant and positive correlations were found between the different parameters (TL, TDNA and TM) assessing DNA damage (Comet assay). MNBN and MNMonos were also significantly correlated. TL correlated significantly with MNBN and TM with NDJ. For %RDTDNA, a statistically significant and positive correlation with %RDTM was found. However, a negative correlation between %RDTM and %NDJ was found.
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Interaction of confounders with genotoxicity markers
The influence of independent variables, namely cumulative dose and smoking habit, on genotoxicity parameters was examined by multiple regression analysis in all workers. Interactions between cumulative dose and smoking (I1), exposure and smoking (I2) and exposure and age (I3) were examined (Table VIII
). MNMonos frequencies were influenced positively by age and negatively by I3 (i.e. interaction between exposure and age). MNBN frequencies were influenced positively by age and %RDTL was influenced negatively by the interaction between smoking and exposure status.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This work has addressed two major questions: (i) are biomarkers of effects (DNA breaks and ALS, MN and NDJ frequencies) fully validated for biomonitoring of workers chronically exposed to ionizing radiation; (ii) can the Comet assay be used to evaluate global repair phenotype as a susceptibility biomarker for IR-induced DNA damage in nuclear workers.
The power of the present study is the use of several biomarkers assessing different types of damage after chronic low level exposure to IR. The first biomarker is the Comet assay, a biomarker of exposure and/or effect, validation of which is in progress. The other two are biomarkers of genotoxic effects. MN and non-disjunction assays were performed to analyse chromosome/genome mutations. Also, a global repair phenotype Comet assay was included in the test battery to better understand the intragroup and intergroup susceptibilities to chronic exposure to IR, taking into account smoking habits.
The controls and exposed individuals were not perfectly matched for smoking habits and age, and this could be a source of bias. Indeed, based on published data in our laboratory (Elhajouji et al., 1998), an increase in both MNi in Monos and in BN would be expected in the exposed group as compared with the controls since the exposed group was statistically significantly older. Therefore, multivariate analysis was applied to assess the effects of these confounders. Our study involved the assessment of genotoxicity markers in a rather limited number of subjects, especially for the global repair phenotype Comet assay. The latter should be considered as a pilot study to evaluate the method, by comparison with other biomarkers for genotoxicity, and to see whether it could be useful.
The strengths of this study are the application of recent standardized protocols for the Comet (De Boeck et al., 2000) and MN assays (Kirsch-Volders and Fenech, 2001).
To achieve the first objective in this work, the Comet assay, including standardization of the different comet parameters, was used. Our previous work has demonstrated that internal standards should be included with the biomonitoring samples in the electrophoreses and a calibration model should be used to minimize potential sources of bias and confounders (De Boeck et al., 2000). The interindividual variation was smaller but remained statistically significant when the data for the parameters TL, TDNA and TM were adjusted for both control and exposed individuals in our study. This indicates that the observed interindividual variation is not due to the experimental conditions. Moreover, these data are supported by the interindividual variation found for the biomarkers of effect (MN and NDJ). This variability between individuals may result from acquired or inherited susceptibility, personal exposure, differences in repair capabilities, nutritional status and/or the immune response.
In the nuclear workers TL was negatively influenced by smoking habit and TDNA by cumulative dose. Consequently, TM was influenced by the interaction of both independent variables, which is expected since TM is the product of TL and TDNA. In a recent work we clearly demonstrated that TDNA of an internal standard evaluated in 141 electrophoreses was more stable than TL (De Boeck et al., 2000). Therefore, we consider TDNA to be more reliable than TL for the evaluation of DNA damage. In addition, it measures the `real' amount of breaks in the tail. The negative slope of the independent variable cumulative dose, which influences the parameter TDNA, could be seen as a protective effect of exposure to chronic low level IR for those workers, since there was no contribution of smoking habit.
As far as MNi frequencies are concerned, the frequency of MNi in both Monos and BN was determined. Since Monos are those cells that did not enter cell division after 72 h mitogen stimulation (Fenech et al., 1997), MNMonos frequency may indicate a cumulative effect over a long period. MNBN are those cells that may also harbour MNi present before they were stimulated to divide in culture (for a recent discussion of protocols and mechanisms see Kirsch-Volders and Fenech, 2001). In our study, although the mean frequency of MNMonos was slightly elevated as compared with exposed individuals and an opposite trend for MNBN in controls was observed, the mean frequencies of MNi in both Monos and BN did not differ significantly in the exposed group as compared with controls. A multiple regression analysis was performed to determine possible confounders which may influence the frequencies of MNi in both Monos and BN in all workers. MNi frequencies were positively influenced by age and negatively influenced by the interaction of age and cumulative dose for BN. This negative slope for the interaction factor on MNBN frequency is simply because younger people had higher cumulative doses of IR.
The level of NDJ observed in the nuclear workers was similar to that seen in the control group. It was previously demonstrated by our laboratory that after in vitro exposure to -rays NDJ correlated with increasing IR dose (Touil et al., 2000). NDJ events were not detectable at -ray doses <1 Gy. In the present study no statistically significant increase in NDJ frequency with cumulative dose was observed in the exposed group. This is not surprising considering the low dose to which the workers were exposed (90.4 ± 49.3 mSv).
There are a number of factors which may prevent the detection of exposure effects by the biomarkers of effect (i.e. TDNA, MNMonos, MNBN and NDJ): (i) a low cumulative dose, which could at least explain the lack of detection of NDJ events and MNi in both Monos and BN after in vivo exposure in this study; (ii) death of damaged cells during cell division in vivo since cells, especially Monos, with chromosomal damage may be eliminated by apoptosis in vivo; (iii) chronic low level exposure could lead to the induction of an adaptive response (AR) and consequently damage leading to MNi in BN would be repaired in the in vivo situation, moreover, there was a negative influence of cumulative dose on TDNA which could also be considered as a type of AR; (iv) none of the biomarkers used actually measures a stable type of DNA damage and since the cumulative dose was relatively low, the dose received during the short period before sampling was much, much lower.
The most striking observation when using the global repair phenotype was that compared with lymphocytes from controls, lymphocytes from exposed individuals showed a higher percentage of residual TL and TM after the 120 min repair time point, suggesting better repair in the controls. No statistically significant differences in residual DNA damage between smokers and non-smoking individuals could be detected at any repair time point. The percentage of residual DNA damage for all comet parameters was found to be much lower in ES (Table VI) at later time points (120 min), while in controls %RDTL was higher in smokers at the same time points. In addition, cumulative dose and smoking habit were found to negatively influence %RDTL (Table VIII). These results suggest that exposed individuals may have shorter DNA strand breaks than controls. The active carcinogen(s) in cigarette smoke may alter the properties of DNA as a result of adduct formation or DNA crosslinks and therefore may hamper the migration of fragmented DNA during electrophoresis. The use of this biomarker as a predictor of repair capacity may be disrupted. Before drawing any conclusions, it will be necessary to reanalyse DNA damage and repair phenotype in a larger group of nuclear workers taking into consideration smoking habit.
Characterization of individual global repair capacity for acute exposure to -rays was attempted by the statistical approach proposed by Leprat et al. (1998). They analysed repair only at 60 min after irradiation, whereas our data present DNA repair for the three residual forms of damage (i.e. %RDTL, %RDTDNA and %RDTM) after several incubation times (5, 10, 30, 60 and 120 min). This aims at a better analysis of the differences in repair of single-strands breaks, which is assumed to be complete within 15 min, and repair of double-strand breaks, which generally takes >1 h.
Considering %RDTL as a measure of recovery after the in vitro challenge assay, two donors appeared to be less efficient in repair capacity at the 120 min repair time point. Interestingly, two CS individuals showed elevated DNA damage 5 min after repair incubation when considering %RDTDNA as a measure of repair capacity. These CS also showed an abnormal repair capacity at 10 min, suggesting abnormal single-strand break repair. One of the ENS individuals appeared to have a high repair capacity at all repair time intervals. %RDTDNA was shown to have a higher ICC. Therefore, %RDTDNA is more reliable for the estimation of global repair capacity. Thus, importance should be given to our observation that the level of %RDTDNA found in the exposed group was much lower than in controls (Table 4). Does this mean that people exposed to chronic low level IR have more efficient repair than the controls? If so, this suggests that these exposed individuals might not be sensitive to the challenge dose of 2 Gy -rays.
The global repair phenotype assay, although performed for only a relatively small number of workers, confirms the first study on IR by Leprat et al. (1998) and leads to the recommendation that this assay be used for the estimation of global repair phenotype with respect to IR. These conclusions are also supported by a similar work performed by Schmezer et al. (2001) using the Comet assay to study in vitro challenge with bleomycin. However, one should consider the interindividual variation in PHA stimulation as a confounding factor in the assay. The extent of activation is going to influence the extent of expression of DNA repair proteins. Moreover, we cannot conclude from our results whether an association exists between repair phenotype and repair genes. Moreover, smoking might have an impact on the susceptibility to radiation effects and might be responsible for differences among individuals in the risks of radiation-induced health effects. This confounder makes biomarker measurements difficult to interpret except on a population basis.
In conclusion, a positive correlation between mechanistically related biomarkers has confirmed the relevance of these biomarkers to assess genotoxic effects after exposure to IR. Neither exposure to chronic IR nor smoking induced a statistically significant increase in genotoxic effects in the control group versus the group of exposed workers; multivariate regression analysis confirmed that age positively influenced MNMonos and MNBN and indicated that %RDTL was negatively influenced by the interaction between smoking and exposure status. Use of the Comet assay for the estimation of global repair phenotype with respect to IR is recommended because it is simple, fast and differences in in vitro repair capacity can be detected.
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We thank Miss M.De Boeck for stimulating discussions and K.Kemp for the English language revision. The Federal Office of Scientific, Technical and Cultural Affairs in the Prime Minister's Office supported this work.
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1 To whom correspondence should be addressed. Tel: +32 2 629 34 23; Fax: + 32 2 629 27 59; Email: mkirschv{at}vub.ac.be
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De Boeck,M., Lison,D. and Kirsch-Volders,M. (1998) Evaluation of the in vitro direct and indirect genotoxic effects of cobalt compounds using the alkaline comet assay. Influence of interdonor and interexperimental variability. Carcinogenesis, 19, 2021–2029.
De Boeck,M., Touil,N., De Visscher,G., Aka Vande,P. and Kirsch-Volders,M. (2000) Validation and implementation of an internal standard in comet assay analysis. Mutat. Res., 469, 181–198.[Web of Science][Medline]
Elhajouji,A., Van Hummelen,P. and Kirsch-Volders,M. (1995) Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes. Environ. Mol. Mutagen., 26, 292–304.[Web of Science][Medline]
Elhajouji,A., Tibaldi,F. and Kirsch-Volders,M. (1997) Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes. Mutagenesis, 12, 133–140.
Elhajouji,A., Cunha,M. and Kirsch-Volders,M. (1998) Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay. Mutagenesis, 13, 193–198.
Fenech,M. and Morley,A.A. (1985) Measurement of micronuclei in lymphocytes. Mutat. Res., 147, 29–36.[Web of Science][Medline]
Fenech,M., Perepetskaya,G. and Mikhalevich,L. (1997) A more comprehensive application of the micronucleus technique for biomonitoring of genetic damage rates in human populations—experiences from the Chernobyl catastrophe. Environ. Mol. Mutagen., 30, 112–118.[Web of Science][Medline]
Hande,M.P., Boei,J.J. and Natarajan,A.T. (1997) Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridisation. III. Chromosome malsegregation/aneuploidy. Mutagenesis, 12, 125–131.
IARC (1997) Application of Biomarkers in Cancer Epidemiology, IARC Scientific Publications no. 142. IARC, Lyon.
Kirsch-Volders,M. and Fenech,M. (2001) Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes. Mutagenesis, 16, 51–58.
Kirsch-Volders,M., Tallon,I., Tanzarella,C., Sgura,A., Hermine,T., Parry,E.M. and Parry,J.M. (1996) Mitotic non-disjunction as a mechanism for in vitro aneuploidy induction by X-rays in primary human cells. Mutagenesis, 11, 307–313.
Leprat,F., Alapetite,C., Rosselli,F., Ridet,A., Schlumberger,M., Sarasin,A., Suarez,H.G. and Moustacchi,E. (1998) Impaired DNA repair as assessed by the `comet' assay in patients with thyroid tumors after a history of radiation therapy: a preliminary study. Int. J. Radiat. Oncol. Biol. Phys., 40, 1019–1026.[Web of Science][Medline]
Lloyd,D.C., Edwards,A.A., Leonard,A., Deknudt,G.L., Verschaeve,L., Natarajan,A.T., Darroudi,F., Obe,G., Palitti,F., Tanzarella,C., Tawn,E.J. (1992) Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays. Int. J. Radiat. Biol., 61, 335–343.[Web of Science][Medline]
Lozzio,C.B. and Lozzio,B.B. (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood, 45, 321–334.
Malyapa,R.S., Bi,C., Ahern,E.W. and Roti Roti,J.L. (1998) Detection of DNA damage by the alkaline comet assay after exposure to low-dose gamma radiation. Radiat. Res., 149, 396–400.[Web of Science][Medline]
Moller,P., Knudsen,L.E., Loft,S. and Wallin,H. (2000) The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiol. Biomarkers Prev., 9, 1005–1015.
Price,E.A., Bourne,S.L., Radbourne,R., Lawton,P.A., Lamerdin,J., Thompson,L.H. and Arrand,J.E. (1997) Rare microsatellite polymorphisms in the DNA repair genes XRCC1, XRCC3 and XRCC5 associated with cancer in patients of varying radiosensitivity. Somat. Cell Mol. Genet., 23, 237–247.[Web of Science][Medline]
Schmezer,P., Rajaee-Behbahani,N., Risch,A., Thiel,S., Rittgen,W., Drings,P., Dienemann,H., Kayser,K.W., Schulz,V. and Bartsch,H. (2001) Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes. Mutagenesis, 16, 25–30.
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184–191.[Web of Science][Medline]
Snigiryova,G., Braselmann,H., Salassidis,K., Shevchenko,V. and Bauchinger,M. (1997) Retrospective biodosimetry of Chernobyl clean-up workers using chromosome painting and conventional chromosome analysis. Int. J. Radiat. Biol., 71, 119–127.[Web of Science][Medline]
Thierens,H., Vral,A., Aousalah,B. and De Ridder,L. (1999) A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay. Mutat. Res., 445, 105–111.[Web of Science][Medline]
Touil,N., Elhajouji,A., Thierens,H. and Kirsch-Volders,M. (2000) Analysis of chromosome loss and chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent mistake in chromosome segregation produced by low dose exposure to ionizing radiation. Mutagenesis, 15, 1–7.
Vaghef,H., Nygren,P., Edling,C., Bergh,J. and Hellman,B. (1997) Alkaline single-cell gel electrophoresis and human biomonitoring for genotoxicity: a pilot study on breast cancer patients undergoing chemotherapy including cyclophosphamide. Mutat. Res., 395, 127–138.[Web of Science][Medline]
Van Hummelen,P., Elhajouji,A. and Kirsch-Volders,M. (1995) Clastogenic and aneugenic effects of three benzimidazole derivatives in the in vitro micronucleus test using human lymphocytes. Mutagenesis, 10, 23–29.
Vral,A., Thierens,H. and De Ridder,L. (1997) In vitro micronucleus-centromere assay to detect radiation-damage induced by low doses in human lymphocytes. Int. J. Radiat. Biol., 71, 61–68.[Web of Science][Medline]
Received on June 4, 2001; revised on January 4, 2002; accepted on January 14, 2002.
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