1 Aberrations of Genetic Material as Biomarkers of Ionizing Radiation Effects S. Milačić University of Belgrade, Faculty of Medicine, Institute of Occupational Medicine and Radiological Protection, Deligradska 29, Belgrade, Serbia and Montenegro Abstract. Ionizing radiation is the most powerful mutagen in environmental and working conditions. The result of genotoxic effect of radiation is the development of chromosome aberrations. The structural chromosome aberrations in peripheral blood lymphocytes are dicentric, ring, acentric fragment. The observation of chromosome aberration frequency in lymphocyte karyotype is the conclusive method to assess the absorbed dose of ionizing radiation. Our study compared the incidence of chromosome aberrations in occupationally exposed healthy medical workers and in nonexposed healthy population. We analyzed the effect of working place, dose by thermo luminescence personal dosimeter (TLD), duration of occupational exposure (DOE) and age to the sum of aberrant cells and aberrations. Four-year study included 462 subjects, mean-aged 42.3 years, who were occupational exposed to ionizing radiation and 95 subjects, mean-aged 35,2 years, who were not exposed to ionizing radiation, during the same time period and from the same territory. All of them possess thermo luminescence personal dosimeter (TLD) which is read by scanner for thermo luminescence dosimeters. Modified Moorhead s micro method for peripheral blood lymphocytes and conventional cytogenetic technique of chromosome aberration analysis were used for analysis of chromosome aberrations. Stained preparations (Giemsa) are observed in immersion by light microscope. The karyotype of 200 lymphocytes in metaphase is analyzed the most characteristic aberration: dicentric, then the ring and acentric fragments. The increased incidence of chromosome aberrations was found to be 21.6% in the exposed group and 2.1% in the controls, while the findings within the limits (non-specific chromosome lesions gaps, breaks, elongations, and exchanges) were equal in both groups (22%). Among occupationally exposed medical workers, the highest incidence was found in nuclear medicine workers (42.6%), then in orthopedists (27.08%). There is highly significant difference of the number of aberrant cells and the sum of chromosome aberrations between the exposed and control groups (t-test; p<0.001). The sum of chromosome aberrations and the number of aberrant cells was in positive correlation with the duration of exposure (p< 0.001), and to a less degree of probability with the age (p< 0.05) in the exposed workers. In the controls, this correlation was negative and insignificant. The group of subjects with the duration of occupational exposure up to 15 years had significantly less number of aberrant cells and chromosome aberrations in comparison to the subjects with longer duration of occupational exposure, over 15 years. Long-term occupational exposure to low doses had the effect to the development and frequency of chromosome aberrations, especially unstable ones (dicentric), but it varied in relation to different working places in public health system. The majority of subjects had no genetic modifications affected by low doses, arguing for the significance of individual variability s in radio sensitivity and genetic predisposition. 1. Introduction Deposition of ionizing radiation energy in genetic material induces the development of stable and unstable structural chromosome aberrations in equal proportion (1, 2). The unstable ones are dicentric, polycentric, ring and terminal deletion, followed by acentric fragment. The aberrant cell may survive 10 divisions most (2, 3 and 4). However, acentric fragments do not follow the inversions and translocations and they may survive in tissue for a long time, representing the constant imbalance of karyotype (5, 6). Reparative processes misrepaire or misreplications have a great role for the development of chromosome aberrations, transforming the primary lesions of DNA chains into the secondary ones with characteristic forms of aberrant chromosomes (7, 8). The structural aberrations may occur in G1 phase of cell cycle (chromosome) and in S phase (chromatic) (9). The analysis of chromosome aberrations is extremely important since it represents the reliable biomarker of radiation effect (10, 11, 12, 13, and 14).
2 One of the sequels of deposition of energy during the ionizing radiation of different LET (Lynear Energy Transpher) is the distribution of chromosome aberrations in peripheral blood lymphocytes (3, 4, 5, and 6). The absorbed dose of ionizing radiation is determined on the basis of the frequency of lymphocyte aberrations (10, 11). In occupational exposure, that is, the exposure to low doses of ionizing radiation, the measurement of absorbed dose is based on biological effects to genetic material (12, 13 and 14). The presence of at least one dicentric in 200 tested metaphases is the evidence of higher absorbed dose of ionizing radiation, namely, the evidence of MTD exceeding - maximal tolerated dose which is 20mSv for occupationally exposed persons. Calibration curves are used for accurate estimation of high doses (10). 2. Subjects and methods Four-year study included 462 subjects, mean-aged 42.3 years, who were exposed to low doses of ionizing radiation and 95 subjects, mean-age 35.2 years, who were not exposed to ionizing radiation, during the same time period and from the same territory. The exposed subjects comprised the exposed group (E) including the medical workers of various professions from different working places within the area of ionizing radiation in the public health system: Radiologists 93 X-ray units radiological technicians 274 X-ray units physicians and laboratory technicians in NM 47 isotopic laboratories orthopedists, cardiologists, anesthetists (others) 48 X-ray units exposed to direct beam, contrast imaging, interventional radiology All of them possess thermo luminescence personal dosimeter (TLD) which is read by scanner for thermo luminescence dosimeters. Dosimeter absorbs the radiation falling on the body (and dosimeter) during the operation with the radiation sources. The average annual absorbed doses for the exposure period indicate the dynamics of the absorbed dose at the working place in the controlled professional conditions, when it must meet the defined maximal tolerated doses (MTD: 20 msv). In distinction from the occupationally exposed subjects, the subjects not occupationally exposed to ionizing radiation are the controls (C) and have no TLD. Modified Moorhead s micro method for peripheral blood lymphocytes and conventional cytogenetic technique of chromosome aberration analysis were used for analysis of chromosome aberrations. The total blood for human lymphocyte culturing is taken by venous puncture from the subject and poured into the sterile test-tube containing heparin. Heparinized blood may be stored for 24 hours, at the temperature of 4 o C. Cultivation has been performed in RPMI medium with the addition of 0.1% phytohemaglutinin, inducing the transformation of lymphocytes into blastoid cells, where the intensive synthetic activity is started followed by morphological changes (the increase of cell volume over three-times of normal), resulting in successive mitotic divisions to provide the largest possible number of cells in metaphase when the cellular chromosome complement will be the most appropriate for cytogenetic analysis; 2-3 hours before the time of cultivation is up, the cultures are added with 0.2 ml of colcemide, enabling the transformation of cell from metaphase to anaphase of mitosis, in the way that it interferes with the function of mitotic spindle (9). Stained preparations (Giemsa) are observed in immersion by light microscope. The karyotype of 200 lymphocytes in metaphase has been analyzed. The most characteristic aberration that
3 is observed is dicentric, then the ring, acentric fragment, pericentric inversion and translocation. Chromosome lesions (gaps), e.g., breaks, modifications, and elongations are not characteristic for the ionizing radiation effect only, but they also occur as a result of toxic, infectious, pharmacological and other factors from the working and living environment (12-14), and therefore, being non-specific, they have been designated as the findings within the limits. 3. Results and discussion The average annual absorbed dose measured by TLD is 10.5 msv in occupationally exposed subjects. The controls were exposed to natural phone; namely, the average annual absorbed dose for the population of this region was 2.8 msv. On the basis of the history and general clinical examination defined by the Law on protection from ionizing radiation in our country, complying with the ICRP recommendation, all subjects were healthy and were not exposed to genotoxic agents at the time of chromosome aberration test (13, 14). The increased incidence of chromosome aberrations (c.a.) was found to be 21.6% in the exposed group and 2.1% in the controls, while the findings within the limits (non-specific chromosome lesions) were equal in both groups (22%). Accordingly, non-specific changes of chromosomes cannot be the indicator of the absorbed dose because they were found in general population in the same proportion, while the changes occurring much easily and affected by less toxic elements and not so intense carcinogens as the ionizing radiation were fewer (12). Dicentric form was the most prevailing one (12.53%), while stable structural aberrations (translocations and inversions) were found in 6.26%. Among all aberrations, the unstable ones accounted for two-thirds or 74.6%, out of which 57% were dicentric, only 5.27% were ring. Dicentric, occurring due to double break of DNA chain in two different chromosomes, is too big lesion rarely found in healthy population (only one case was reported in our controls). It is used to determine the absorbed dose of the ionizing radiation and it is the indicator of the desorbed dose under the occupational exposition conditions. Among occupationally exposed medical workers, the highest incidence was found in nuclear medicine workers (Table I), then in orthopedists, cardiologists and anesthetists (others), who are generally exposed to direct X-ray beam during their activities. The nuclear medicine workers work in laboratories and perform diagnostic procedures with radioisotopes, open sources, and, therefore, there is always the risk of internal contamination (8). Unfortunately, the regular measurement of urine radioactivity is not possible in our conditions, what will be the actual evidence of internal contamination considering the use of short-lived radionuclides having the short-acting time of half-excretion (e.g., hippuran-j131 - only 2.5 days). The annual monitoring of 24-hour urine radioactivity revealed significantly lower values in relation to ALI (annual limit of intake), ranging from 0.1 to 10 Bq/l. However, the taken radionuclide becomes the internal source of radiation, especially if organ depot is present, while in case of its equal distribution and short retaining in the body, it also has great possibility to act directly to the lymphocyte nucleus causing the chromatin ionization and producing the radiation recompose forms in chromosomes. In the external radiation, the indirect effect is higher, through free radicals diffusing slowly from the extra cellular fluid into the nucleus (3). Table I. The incidence of chromosome aberrations (c.a.) in relation to professions Profession No. of Higher Percentage No higher Percentage examinations incidence incidence Radiologists , ,8 Radiographers , ,4 Nuclear Medicine workers , ,4 Other , ,92 Total , ,6
4 Radiologists and radiographers are only occasionally in close proximity to the source of radiation, in contrast medium radiography and imaging, since they are behind the protective shield or in another room. Moreover, they know best the principles of radiological protection by nature of their job. Medical workers of other specialties (other than radiological) should not operate with X-ray machines, particularly with the direct X-ray beam in the interventional radiology. There is highly significant difference of the number of aberrant cells and the sum of chromosome aberrations between the exposed and control groups, what could be expected considering the exposure to ionizing radiation as the cause of genetic material transformations (Table II). Table II. The difference of the aberrant cell number and the sum of chromosome aberrations (c.a.) between the exposed and control groups Group No. of subjects No. of aberrant cells Sum of c.a. Exposed group 462 0,52+-0,03 0,71+-0,05 Control group 95 0,24+-0,04 0,24+-0,05 T-test 3,83; p=0,0001 4,27; p=0,0001 The effects of accumulation of low doses were manifested. Nevertheless, over a half (56.8%) of the exposed workers had no chromosome aberrations. The differences of genetic material modifications under the conditions of exposure may be interpreted by individual features of occupationally exposed subjects that were considered in this study: immunological status, deficiency of DNA repair, genetic instability (predisposition, sensitivity), and lack of foliates in diet, fatigue, sex, age, etc. (13). It indicates the need for professional selection relating to the work in the area of the ionizing radiation through the preliminary tests to radio sensitivity (10) and their implementation in the regular practice and legislation. Table III. The difference of the aberrant cell number and the sum of chromosome aberrations (c.a.) between two exposed groups (E) of workers E DOE year Number of subjects No. of aberrant cells Sum of c.a. E I ,46 +/- 0,03 0,63 +/- 0,05 E II P 135 0,66 +/- 0,07 0,002 0,90 +/- 0,1 0,01 The group of subjects with the duration of occupational exposure (DOE) up to 15 years (E1=327) had significantly less number of aberrant cells and chromosome aberrations in comparison to the subjects (E2=135) with longer duration of occupational exposure, over 15 years (Table III). Although radio sensitivity decreases with the age, long-term exposure led to accumulation, and, therefore, radiobiological effect of the absorbed doses was higher in older subjects with longer period of exposure. The sum of chromosome aberrations and the number of aberrant cells was in positive correlation with the duration of exposure p< (Table IV), and with the age (p< 0.05) in the exposed workers (Table V). In the controls, this correlation was negative and insignificant (Table IV). Radio sensitivity tends to decrease with the age (2, 3). Conspicuously less number of aberrant cells was found in the subjects over 40 years of age (Table VI). Table IV. Results of correlation between the sum of chromosome aberrations (c.a.) and the aberrant cell number, and the duration of occupational exposure (DOE) DOE; sum of c.a. Correlation DOE; number of aberrant cells - Correlation r = 0,128 r = 0,121 p = 0,006 p = 0,009
5 Table V. Results of correlation of the age, and sum of chromosome aberrations (c.a.) and the aberrant cell number in the exposed group Age; sum of c.a. Correlation Age; number of aberrant cells - Correlation r = 0,110 r = 0,097 p = 0,018 p = 0,038 Table VI. Results of correlation of the age, and sum of chromosome aberrations (c.a.) and the aberrant cell number in the control group Age; sum of c.a. Correlation Age; number of aberrant cell - Correlation r=-0,001 P=0,993 R=-0,060 P=0,565 There is no correlation between the incidence of chromosome aberrations and the absorbed doses measured by TLD. TLD doses ranged diversely, from 1.5mSv to 19.2 msv (10.5mSv in average), but this distribution within low doses, below 20mSv, is not significant. The significance of personal dosimeters for radiation determination would be higher in case of measurement of the exposure dose and effective equivalent and collective doses, involving, besides TL dosimeters, some other parameters such as the source distance, and length of time in any individual case, what is actually unfeasible in our conditions. Therefore, the correlation of aberration frequency and the duration of exposure have higher significance, due to cumulative effects of low doses that may not be avoided in the continuous occupational exposure. According to literature, under the conditions of exposure to low doses of the ionizing radiation, as well as in our study, noticeable lesions of chromosomes in blood cells may be noted (12, 13, and 14). The radiation workers employed in the hospitals, exposed to X-rays, to doses lower to maximal tolerated limits, estimated by the international standards, have manifested chromosome lesions. Higher risk of carcinogens in healthy subjects with elevated proportion of chromosome aberrations in lymphocytes has been described in epidemiological studies. Regardless the role of exposure to carcinogens, the increase of risk of cancer in people with higher incidence of aberrant cells is significant (3, 13). This suggests that the chromosome changes in lymphocytes should be recognized as biomarkers of higher probability of carcinogens (3). 4. Conclusions It is apparent that the unstable aberrations are more prevalent, that are not fixed in the cell division, and manifest higher absorbed dose at the working place. Since the absorbed doses in the exposed workers are within limits and differ rather slightly (according to measurements at working places and TLD), the distinctions of results are interpreted, before all, by significant deviation of the individual sensitivity, and especially the efficiency of DNA reparative processes as well as possible effect of genotoxic environmental agents. Healthy subjects, occupationally exposed to the ionizing radiation, may have structural chromosome aberrations, regardless the low, tolerated dose and the correctness of the apparatus and the source at working places. Most frequently, they are unstable and decline in cell division, so they have no high clinical significance. Nevertheless, some of them may be present in tissues for a longer period of time and be responsible for late sequel.
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