Mutagenesis, Vol. 18, No. 3, 265-271,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation
Laboratory of Mutagenesis, Institute for Medical Research and Occupational Health, HR-10 000 Zagreb, Croatia
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The alkaline Comet assay was selected as a biomarker of exposure to evaluate the ongoing exposure to ionizing radiation of 50 medical workers occupationally exposed to ionizing radiation and 50 corresponding unexposed control subjects. The primary DNA damage was evaluated by measuring the extent of DNA migration in peripheral blood leukocytes. The inter-individual differences in DNA damage between exposed subjects were compared with their dosimeter readings and occupation. It was found that medical workers who were occupationally exposed to ionizing radiation for different periods of time showed highly significant increases in levels of DNA damage compared with controls. However, influences of the different occupational settings and doses absorbed on the levels of DNA damage, assessed by use of the Comet assay, might be excluded in the majority of subjects. Differences in comet parameters measured due to smoking and gender were not statistically significant in either exposed or control subjects. The results obtained have confirmed the usefulness of the alkaline Comet assay as an additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ionizing radiation is a ubiquitous environmental physical agent whose DNA-damaging effects are fairly well established. In physico-chemical interaction with cellular DNA it produces a variety of primary lesions, such as single-strand breaks (SSBs), alkali-labile sites, double-strand breaks (DSBs), DNA–DNA and DNA–protein crosslinks and damage to purine and pyrimidine bases (Natarajan, 1993
; Kruszewski et al., 1998; Chaubey et al., 2001).
The effects of low level exposure to ionizing radiation are of concern to a large number of people, including workers occupationally exposed to radiation. Medical radiation workers are employees of hospitals, clinics and private offices where radiation is used in the process of delivering health care to humans. These workers can be categorized into two groups: ‘exposed employees’ who receive at least a minimum detectable exposure during a 1 year period and ‘potentially exposed employees’ who work in the vicinity of radiation but whose exposures are below detectable limits. The exposure of patients and workers to radiation in medicine is a direct consequence of the use of radiation to improve the health of individuals. Trends in radiation exposure of both patients and workers are effected not only by developments in radiation protection, but also by the doses used in the practice of medicine (Hendee and Edwards, 1990). It is very important to estimate absorbed doses from individuals occupationally exposed to ionizing radiation in order to carry out radioprotection procedures and restrict the hazards to human health (Ramalho et al., 1998), but the extent of the health hazard is difficult to assess. Therefore, the development of procedures that can be used to precisely identify health hazards in exposed populations is important to establish effective programmes for disease prevention (Au et al., 1998).
A wide range of methods are presently used for the detection of early biological effects of DNA-damaging agents in environmental and occupational settings. Currently, unstable chromosomal aberrations in peripheral blood lymphocytes, in particular dicentrics, are the most fully developed biological indicators of ionizing radiation exposure (IAEA, 1986; Carrano and Natarajan, 1988; Bauchinger, 1995; Ramalho et al., 1998). This methodology usually complements data obtained by physical dosimetry. As a rule, it is used whenever the individual dosimeter shows an exposure to penetrating radiation above its limit of detection. One of the advantages of cytogenetic dosimetry is that this biological dosimeter can be assessed at any moment, whereas physical dosimeters are not always present on the subject (Ramalho et al., 1998).
In the last few years, the single cell gel electrophoresis (SCGE) or Comet assay has been widely used for genotoxicity testing (Singh et al., 1988; Tice, 1990; Fairbairn et al., 1995; Olive, 1999). In molecular epidemiology studies DNA damage evaluated by the Comet assay has been used as a biomarker of exposure (Betti et al., 1994; Collins et al., 1997; Garaj-Vrhovac and Kopjar, 1998; 
rám et al., 1998; Piperakis et al., 1999; Kopjar and Garaj-Vrhovac, 2001; Maluf et al., 2001; Garaj-Vrhovac et al., 2002).
The Comet assay permits the detection of primary DNA damage and the study of repair kinetics at the level of single cells (Singh et al., 1988; Olive et al., 1990; Hellman et al., 1995; Olive, 1999). A variety of possible modifications of the assay facilitate the detection of SSBs, alkali-labile sites, DSBs, incomplete excision repair sites and interstrand crosslinks. In addition to the above, DNA fragmentation associated with cell death or related to apoptosis can be evaluated with the Comet assay (Piperakis et al., 1999; Olive, 1999).
While biomonitoring studies employing cytogenetic techniques are limited to circulating lymphocytes and involve proliferating cell populations, the Comet assay can be applied to proliferating and non-proliferating cells and cells of those tissues which are the first sites of contact with genotoxic agents. Compared with other genotoxicity tests, the Comet assay is a cheap and simple method. However, it is advisable to use a computer-assisted image analysis system to measure the comet parameters. Despite the fact that the Comet assay requires isolated cells of the same type, there has been increasing interest in this test in the past years, mainly because of its advantages of sensitivity and rapidity (Cotelle and Ferard, 1999; Møller et al., 2000; Singh, 2000).
The aim of the present study was to assess and quantify the levels of DNA damage in peripheral blood leukocytes of medical workers occupationally exposed to ionizing radiation and of corresponding unexposed control subjects. As a sensitive biomarker of exposure the alkaline Comet assay was selected.
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Materials and methods |
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Subjects of study
The population studied comprised 100 volunteer blood donors: 50 of them had been occupationally exposed to ionizing radiation and 50 were unexposed control subjects. Each person completed a standardized questionnaire which included items concerning personal data (age and health status) and occupational exposure to ionizing radiation at the time of the study. The questionnaire also included items concerning non-occupational exposure to potential mutagenic hazards, such as smoking, alcohol and drug consumption, viral diseases, recent vaccinations and radiodiagnostic examinations.
The exposed group consisted of 16 female and 34 male subjects aged 22–62 years (mean age 43.3 years) working in the radiology and surgery units of six Croatian hospitals. Ten of them were nurses, 15 were physicians and 25 were radiological technicians. Their mean duration of occupational exposure was 17.8 years (range 2–37 years). During their work they all wore individual dosimeters (film badges). The range of dosimeter readings for the exposed group during the year prior to the study was 0–8548 µSv. Fifteen exposed subjects were smokers (eight female and seven male) and 35 were non-smokers (eight female and 27 male). In the year prior to the beginning of the present study exposed subjects had not been subjected to diagnostic X-ray examinations.
Control subjects were healthy students and office employees (20 female and 30 male), chosen from the general Croatian population. Twenty-nine of them were non-smokers (12 female and 17 male) and 21 of them were smokers (eight female and 13 male). The mean age of the control group was 42.1 (range 22–60 years). None of them had ever had any contact with sources of ionizing radiation or been occupationally exposed to known genotoxic agents. None of the control subjects reported alcohol consumption. During the year prior to the blood sample collection the controls had not been subjected either to diagnostic X-ray or non-ionizing examinations. None had received any therapeutic irradiation. They were also not taking any medications or oral contraceptives.
Blood sampling
Peripheral blood samples of the exposed and control subjects were collected by venipuncture into heparinized tubes during the period March 2001–March 2002. Blood sampling and processing of exposed and control donors were carried out simultaneously. All blood samples were coded, cooled and processed within a maximum 2 h period after collection. The alkaline Comet assay on whole blood samples was performed immediately after blood transportation.
The Comet assay
The Comet assay was carried out under alkaline conditions, basically as described by Singh et al.(1988). Fully frosted slides were covered with 1% normal melting point (NMP) agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. When this layer had solidified, a second layer containing the whole blood sample mixed with 0.5% low melting point (LMP) agarose (Sigma) was placed on the slides. After 10 min solidification on ice, the slides were covered with 0.5% LMP agarose. Afterwards the slides were immersed for 1 h in ice-cold freshly prepared lysis solution [2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% Na-sarcosinate (Sigma), pH 10] with 1% Triton X-100 (Sigma) and 10% dimethyl sulfoxide (Kemika) added fresh to lyse cells and allow DNA unfolding. The slides were then placed in a horizontal gel electrophoresis tank, facing the anode. The unit was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH 13.0) and the slides were set in this alkaline buffer for 20 min to allow DNA unwinding and expression of alkali-labile sites. Denaturation and electrophoresis were performed at 4°C under dim light. Electrophoresis was carried out for 20 min at 25 V (300 mA). After electrophoresis the slides were rinsed gently three times with a neutralization buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 µg/ml) and covered with a coverslip. Slides were stored at 4°C in sealed boxes until analysis.
Comet capture and analysis
A total of 100 randomly captured comets from each slide were examined at 400x magnification using an epifluorescence microscope (Zeiss) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, UK). A computerized image analysis system acquires images, computes the integrated intensity profiles for each cell, estimates the comet cell components and then evaluates the range of derived parameters. To quantify the DNA damage tail length (TL) and tail moment (TM) were evaluated. Tail length (length of DNA migration) is related directly to the DNA fragment size and presented in micrometres. It was calculated from the centre of the cell. Tail moment was calculated as the product of the tail length and the fraction of DNA in the comet tail.
Statistical analysis
Both comet parameters measured in the exposed and control groups were evaluated using the non-parametric Mann–Whitney U-test and MANOVA on log transformed data. The level of significance was set at 5%.
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Results |
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The present paper illustrates the results of an alkaline Comet assay study on peripheral blood leukocytes obtained from 50 subjects occupationally exposed to ionizing radiation and 50 control subjects. Using Comet Assay II software we evaluated parameters for measuring DNA damage. To describe the comet the global parameters tail length and tail moment are used.
The results of the alkaline Comet assay are summarized in Tables I and II. Figure 1 gives the mean values of tail length (Figure 1a) and tail moment (Figure 1b) for the exposed and control groups. Figure 2 shows the distributions of tail length and tail moment in the exposed and control groups.
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Tail length
The comet tail lengths measured in exposed subjects were in the range 15.12 ± 0.21 to 22.56 ± 0.47 µm, with a mean value 17.49 ± 0.23 µm (Table I
and Figure 2). In control subjects the values of tail length were in the range 11.92 ± 0.16 to 15.51 ± 0.21 µm, with a mean tail length of 14.05 ± 0.13 µm (Table II and Figure 2). According to the results obtained, the exposure (job) was highly significant (all exposed versus controls, P < 0.0001; Figure 1a).
Tail moment
The values of tail moment measured in the exposed group ranged between 12.59 ± 0.20 and 19.67 ± 0.44, with a mean value of 14.85 ± 0.21 (Table I and Figure 2). The observed values differed significantly (P < 0.05, Mann–Whitney U-test; Figure 1b) from the tail moment values measured in control subjects, which were in the range 8.41 ± 0.26 to 12.94 ± 0.22, with a mean value of 11.46 ± 0.15 (Table II and Figure 2).
Among the exposed group no significant effects of years of exposure, absorbed dose, gender or smoking habit were recorded. However, it has to be pointed out that within the exposed group marked inter-individual differences regarding both comet tail parameters were observed (Table I). Among radiologists tail length measures were in the order technicians > nurses > physicians (Table I), while the highest individual level of DNA migration was recorded in exposed subject no. 3 (a nurse employed in a radiology unit). Among surgery staff tail length measures were also in order technicians > nurses > physicians (Table I). Although mean values of comet parameters measured in peripheral blood leukocytes of medical personnel employed in surgery units were increased compared with those recorded in subjects employed in radiology units, the observed differences were not statistically significant (MANOVA, P > 0.05). No statistically significant differences were found between the mean values of comet tail parameters recorded in exposed smokers and non-smokers, independent of their gender and occupation (Mann–Whitney U-test, MANOVA, P > 0.05) (Table II). It was also observed that age did not significantly influence the levels of DNA damage among exposed subjects <30 and >50 years. However, in exposed subjects aged between 30 and 50 years increased DNA migration compared with both younger and older exposed age groups was observed (MANOVA, P < 0.05).
On the other hand, the distribution of comet tail parameters in control subjects was characterized by nuclei with smaller comets. Although some unexposed smokers had increased levels of DNA damage compared with non-smokers, the mean values of both comet parameters evaluated did not differ significantly. The same was observed for the differences recorded between unexposed female and male subjects (Mann–Whitney U-test and MANOVA) (Table II).
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Discussion |
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Continued development of new technologies and procedures is anticipated in medicine and these developments may continue to fuel the increased use of imaging procedures in medical diagnosis and therapy. Working in the presence of ionizing radiation is one of many managed risks within a hospital. However, over the past two decades medical occupational radiation exposure has decreased in spite of increased use of radiation in medicine (Hendee and Edwards, 1990
). Provided that the worker follows all policies and procedures set forth by the institution, all workers will have personal exposures well within the boundaries of what is considered ‘safe’. Exposure to X-rays may result in effects that are both deterministic and stochastic in nature. Radiation workers are only at increased risk of deterministic effects if they work in violation of hospital safety policies and procedures. Some stochastic effects from occupational exposure to X-rays would be cancer and genetic mutation. These are long-term effects, which do not show in the exposed population until many years after the exposure. Radiation workers are predicted to have a greater percentage risk of developing detrimental effects over the general public because of their generally greater exposure.
From the experience with radiation accidents it is evident that biological indicators are needed in order to obtain information concerning the distribution and extent of radiation exposures, as such data are lacking from physical dosimetry in many cases. These biological indicators can have a further advantage in the way that the individual radiation damage is measured, which includes variability of individual radiosensitivity. Among the many biological parameters that have been studied in this connection, chromosomal damage in lymphocytes is most promising (Streffer et al., 1998). Among individuals occupationally exposed to ionizing radiation different cytogenetic changes, for example increased frequencies of chromosome aberrations and micronuclei, are well known (IAEA, 1986; Carrano, 1986; Carrano and Natarajan, 1988; Garaj-Vrhovac et al., 1997; Maluf et al., 2001). Scoring of dicentric and ring chromosomes in metaphase preparations of peripheral lymphocytes is the method of choice for quantifying acute over-exposures to ionizing radiation (Bauchinger, 1995). One of the advantages of cytogenetic dosimetry is that this biological dosimeter can be assessed at any moment, whereas physical dosimeters are not always present on the subject. Another advantage is that subjects under study cannot intentionally modify a biological dosimeter (Ramalho et al., 1998).
Ionizing radiation is a ubiquitous environmental physical agent whose DNA-damaging effects are fairly well established. It induces DNA damage directly (as a result of deposition of energy in cells) or indirectly (as a result of free radical formation and oxidative damage). It has long been known that physico-chemical interactions between ionizing radiation and DNA produce a broad spectrum of DNA lesions, including damage to nucleotide bases, DNA–DNA and DNA–protein crosslinks and alkali-labile sites, as well as SSBs and DSBs. DSBs were originally assumed to be the critical cytotoxic lesion, whereas base damage, particularly thymine glycols, were implicated in mutagenesis. It is now accepted, however, that misrepaired DSBs are the principle lesion of importance in the induction of both chromosomal abnormalities and gene mutations (Ward, 1995; Little, 2000; Ptácek et al., 2001).
Lesions induced by ionizing radiation in DNA can be detected by the alkaline single cell gel electrophoresis or Comet assay (Møller et al., 2000; Maluf et al., 2001). The same method was evaluated in the present study on occupationally exposed medical personnel. The Comet assay is an easy, quick and accurate test that has been widely applied to measure both in vitro DNA damage and repair following exposure to various genotoxic agents and for human biomonitoring (Collins et al., 1997; rám et al., 1998; Kopjar and Garaj-Vrhovac, 2001; Maluf et al., 2001; Garaj-Vrhovac et al., 2002).
In the present study the alkaline Comet assay revealed heterogeneity in the level of DNA breakage induced in human leukocytes by occupational exposure to ionizing radiation. Although some exposed subjects had high dosimeter readings (up to 8548 µSv), we did not find a clear correlation between the DNA-damaging effects and the doses recorded by the dosimeters. This result is in agreement with studies mentioned previously (Barquinero et al., 1993; Maluf et al., 2001), in which the authors discussed the difficulty of establishing dose–effect relationships for low doses. It is also possible that certain parts of the body of the same subjects were exposed to higher radiation doses than those encompassed by the dosimeter. Another problem, discussed by other authors (Maluf et al., 2001), is that some X-ray workers have other jobs in small hospitals with the same kind of exposure. However, these phenomena were not observed in the experimental group studied here. Other reports also pointed out that the distribution of medical occupational exposures over time is poorly understood. Individuals receiving high readings in one year probably receive relatively high readings in succeeding years because specialized workers tend to perform the same tasks year after year (Hendee and Edwards, 1990).
The increased comet values measured in peripheral blood leukocytes of exposed subjects in the present study indicate highly significant levels of primary radiation-induced DNA damage compared with controls. However, the influences of the different occupational settings and doses absorbed on the levels of DNA damage assessed by use of the Comet assay in the majority of subjects might be excluded.
In the present study smoking habit did not significantly increase the levels of primary DNA damage in either control or exposed subjects. Our observations, which are in agreement with findings by Hellman et al.(1997, 1999) and Wojewódzka et al.(1999), indicate that cigarette smoking is not a very potent confounding factor on the comet parameters measured. On the other hand, some authors observed a connection between cigarette smoking and increased migration of human lymphocyte DNA during the Comet assay (Betti et al., 1994), as well as in the alkaline elution method (Fuchs et al., 1995).
Comet measurements may reflect both individual repair ability and DNA damage level. Because the measured damage level is the result of an equilibrium between damage infliction and repair, a low damage level as assessed experimentally in an individual may be the result of an actual low number of lesions or of a high efficiency of repair (Wojevodzka et al., 1999). A slightly increased level of SSBs is therefore not automatically associated with an increased incidence of gene mutations, chromosomal aberrations or other permanent genetic alterations.
The high values of DNA migration recorded in some exposed subjects are obviously the result of differential concurrent exposure. The high inter-individual variations in comet measurements obtained in the present study, independent of the doses recorded, could also be related to the ability of the Comet assay to detect primary DNA damage in different populations of leukocytes. It is well known that long-lived lymphocytes in lymphatic tissues or other organs may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of cells (Carrano and Natarajan, 1988). In those cells some types of DNA damage (especially chromosome aberrations) accumulate during the long period of occupational exposure of the exposed subjects. It is now well established that certain chromosomal rearrangements, including translocations and deletions, can, if left unrepaired, lead to tumourigenesis through mutational activation of protooncogenes and inactivation of tumour supressor genes and thus are associated with a wide variety of human cancers.
Therefore, to ensure maximum occupational safety, biomonitoring is of great value. Various methods have been established for the monitoring of long-term occupational exposure to ionizing radiation, which should be considered as internal dosimeters for the detection of increased genotoxic, and presumably also carcinogenic, risks. Our results indicate that the alkaline Comet assay might be a useful additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.
It is not acceptable under any circumstances for radiological workers to exceed their annual limit. It is well documented that modern X-ray equipment and moderately aggressive radiation protection practices can keep occupational exposures at least an order of magnitude below the annual limit for the vast majority of radiological workers. Rotation of workers through all tasks in a radiology programme would tend to distribute occupational exposures more uniformly over time.
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Acknowledgments |
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This investigation was supported in part by the Croatian Ministry of Science and Technology (grant no. 0022020).
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Notes |
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1 To whom correspondence should be addressed. Tel: +385 1 4 673 188; Fax:+385 1 4 673 303; Email: vgaraj{at}imi.hr
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References |
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Received on August 8, 2002; revised on November 21, 2002; accepted on November 25, 2002.
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