Ibxicology

letters

ELSEVIER

Toxicology

Letters 76 ( 1995) 113-I I7

Chromosomal damage in workers occupationally exposed to chronic low level ionizing radiation
Alfred0 H. Hagelstriim, Nora B. Gorla*, Irene B. Larripa
Departamento de GenPtica, Institute de Investigaciones Hematoldgicas. Academia Argentina National de Medicina, J.A Pacheco de Melo 3081, 1425 Buenos Aires.

Received 18 March 1994;revision received 2 September

1994; accepted

5 September

1994

Abstract Chromosomal aberrations were evaluated in cultures of peripheral lymphocytes from subjects working in diagnostic X-ray and nuclear medicine areas, exposed to electromagnetic ionizing radiation and particulate ionizing emissions, respectively. A 4-fold increase in the level of chromosomal aberrations was found between the exposed and control groups without qualitative or quantitative cytogenetic differences between X-rays and nuclear medicine-exposed workers. Results are discussed in view of the early damage detection from chronic exposures particularly related to biological controls, hygienic improvements and overwork in a developing country. Keywords: Biomonitoring; Occupational radiation exposure; Chromosomal damage

1. Introduction Chromosomal aberrations, as a measurement of human exposure to ionizing radiation, have become a well-established methodology during the past 2 or 3 decades. Human lymphocytes exposed to radiation in vitro account for almost the entire literature in the area. Fewer data are available on the genetic effects of human in vivo exposure to radiation, except the observations of genetic consequences after acute accidental exposures, i.e.
l Corresponding author, Genetica, Departamento de ProducGn Animal, Facultad de Agronomia y Veterinaria, Universidad National de Rio Cuarto, ruta 36, km 601, Rio

Cuarto

(Cba), Argentina. 0 1995 Elsevier Science Ireland

evaluations in survivors of the nuclear attacks on Hiroshima and Nagasaki [ 1,2] and, more recently and to a lesser extent, the Chernobyl and Goiana nuclear accidents [3,4]. Thus, the supposed effects of radiation used for diagnostic purposes in medical practice are extrapolations from the unequivocal effects observed at high doses. Evaluations of the cytogenetic impact of chronic low dose radiation exposure are scarce. The literature shows cytogenetic effects in radiological technologists exposed to low level radiation [5,6] and there are limited data linking radiation doses from diagnostic procedures in nuclear medicine with deleterious genetic effects [7]. In the present study, chromosomal aberrations were evaluated from cultures of peripheral lym-

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76 (1995) 113-117

phocytes of subjects working in diagnostic X-ray and nuclear medicine areas exposed to electromagnetic ionizing radiation and particulate ionizing emissions, respectively. 2. Materials and methods 2.1. Subjects Blood samples were obtained from 20 hospital workers exposed to low level ionizing radiation in nuclear medicine and radiodiagnostic areas at 4 different hospitals of Buenos Aires, Argentina. Of these, 10 subjects (3 physicians and 7 technicians) belonged to nuclear medicine areas and were exposed to particulate ionizing radiation (Table 1) and 10 subjects (3 physicians and 7 technicians) worked in radiodiagnostic areas and were exposed to predominantly electromagnetic ionizing radiation (Table 2). All of them gave their oral consent to participate in this study. We have also cytogenetically studied 2 groups of unexposed controls. The 20 control subjects used in this study, and the 20 exposed subjects, were selected on the basis of a questionnaire detailing habits, sex and age. Only individuals without concurrent infections and medications and no general and dental X-rays in the last 6 months were admitted. Individuals did not consume more than 3 cups of coffee per day and 10 cigarettes per day. Each exposed subject culture was set up with a control one with the same sex and range age (approximately 25-40 or 40-60 years old). Data concerning the total 40 subjects are shown in Tables 1 and 2. All the workers of nuclear medicine areas were exposed to particulate emissions from radioactive isotopes (32P, 67Ga, In 201T1, 59Fe, 57Co, SICr, 99mTc, 131, 1921r). Thk dose absorbed in exposed subjects film badges indicated a mean of 47 mrads (nuclear medicine areas workers) and 180 mrads (radiodiagnostic areas workers) over the prior 6 months. 2.2. Cytogenetics Blood samples were obtained by venipuncture and collected into heparinized tubes. Lymphocytes were cultured for 72 h at 37C according to the method of Moorhead et al. [S]. Cultures were set up with 0.8 ml whole blood, 8.5 ml FlO nutrient

mixture medium (Gibco Laboratories), 15% heatinactivated fetal bovine serum, certified grade (Gibco Laboratories) and 0.1 ml phytohemagglutinin-M (Gibco Laboratories). Colcemid solution (Gibco Laboratories), 0.1 ml, was present in the cultures for the final 1.5 h. The cells were harvested by exposure to hypotonic shock with potassium chloride 0.075 M for 20 min at 37C, and fixation in methanol and acetic acid 3: 1. Slides were prepared and conventionally stained with Giemsa. The coded slides were scored blind by 2 observers. At least 100 cells per individual were analyzed for the number and type of chromosomal aberrations. Chromosome aberrations were classified according to ISCN [9]. Cells with less than 44 chromosomes were not included. Students t-test was performed to determine the difference between exposed and control groups. The significance of differences was assessed using a two-way ANOVA model with multifactorial experiment for sex, radiation exposure and their interactions. In order to normalize the chromosomal aberrations distribution and to equalize the variances, a logarithmic transformation was applied. 3. Results and discussion Tables 1 and 2 give data on the age, sex, and results of the chromosome aberration analysis performed in blood lymphocytes of the 4 groups studied. In the control groups, the mean frequency of chromosomal aberrations1100 cells was 3.6 f 0.5 and 2.8 f 0.3 in the particulate and electromagnetic studies, respectively. In the radiationexposed groups the mean value was 13.6 f 1.5 for the nuclear medicine-, and 13.6 f 1.3 for the Xray-exposed groups, respectively. These frequencies were statistically different (P < 0.0001) between control and exposed groups. Statistically, no differences were observed between female or male exposure to either type of ionizing radiation. The 4-fold increase in the level of chromosomal aberrations due to an occupational exposure to ionizing radiation is biologically significant. We did not find differences in the level of chromosomal aberrations induced by either type of ionizing radiation, (electromagnetic or predominantly

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115

Table I Chromosomal aberrations in blood lymphocytes from subjects occupationally exposed to particulate ionizing radiation and controls Group No. Sex Age Chromosomal aberrations ctg 8 I3 3 2 5 2 0 0 0 0 csg
0

Mean aberrations/ 100 cells csb I 5 2 0 I 0 I 0 I 0 end I 0 0 0 0 0 0 0 0 0 ace I 0 0 0 0 0 0 0 0 0 qr

0 0 0 0 0 0 0 0

ctb 9 I5 5 3 IO I6 IO 8 I2 I2

Nuclear medicine I M 35 2 F 41 3 F 30 4 M 28 5 M 32 6 M 54 I F 44 8 F 46 9 M 28 IO F 60 Mean f S.D. Controls II F 28 I2 M 38 I3 M >20 I4 M >20 I5 F >20 I6 F 32 I7 M 48 I8 M 25 I9 F 32 20 F 26 Mean f S.D.

3 2 I 0 0 I 0 0 0

I 0

20 I8 I2 6 I6 I8 12 8 I4 I2 13.6 f l.5*

0 0 0

2 I I 0 I 0 0

0 0 I 0 0 0 0 0 0 0

4 0 4 I 4 3 4 2 I 3

2 0 0 0 0 0 0 0 I 0

0 3 0 0 0 0 0 0 0 0

0 0

0 0 0 0 0 0 0 0 0 0

I
0 0 0

0
0 0 0

3 3 6 3 5 4 4 3 2 3 3.6 * 0.5

ctg, chromatid gap; csg, chromosome gap; ctb, chromatid break; csb, chromosome break; end, endoreduplication; ace, acentric fragment; qr, quadriradial. *P < 0.0001 (Students t-test) for the difference between control and nuclear medicine groups.

particulate) despite their different mechanisms of action. All of the charged particles from radioactive isotopes are directly ionizing, i.e, they can directly disrupt the atomic structure of the absorber through which they pass and produce chemical and biological changes [lo]. On the other hand, electromagnetic radiations (X- and gamma rays) are indirectly ionizing. They do not produce chemical and biological damage themselves, but when absorbed in the material through which they pass they give up their energy to produce fastmoving charged particles. Indirectly ionizing radiation is usually more penetrating than directly ionizing particulate radiation [ 111. Next to be considered are the different types of aberrations found and their distribution, sum-

marized in Tables 1 and 2. Stable aberrations, principally chromatid gaps and chromatid breaks, predominate the distribution. No dicentrics or ring figures were seen. Other authors in long-term radiation exposure studies have also found a majority of stable aberrations [12]. In spite of this, it is important to discuss that dicentrics and ring figures are the unstable chromosome aberrations generally observed as a consequence of an in vitro or an acute in vivo ionizing radiation exposure [13,14]. In our population, dicentrics and rings were not found probably due to the low level of ionizing radiations chronically received. In the case of long-term chronic exposures, aberrations can be induced in Go lymphocytes and will accumulate in the long-lived lymphocyte population. The fre-

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A.H. Hagelstdm

er al. IToxicol.

Lurt. 76 (1995) 113-117

Table 2 Chromosomal aberrations in blood lymphocytes from subjects occupationally exposed to electromagnetic ionizing radiation and controls Group No.
X-rays

Chromosomal aberrations Sex Age ctg

6 4 4 3 5 3 2 2 3 0

csg
0 0
0 0

ctb
IO

csb

end
0

ace
0

Mean aberrations/ I00 cells qr

0
0 0

21 22 23 24 25 26 27 28 29 30 Mean

M 44 M 61 M 30 F 35 F 25 F 28 M 42 F 56 F 36 F 32 f S.D.

I 2 0 0 0 0

8 8 14 7 9 3 13 8 12

1 0
2

0
0 0 0

0 0 0
0 0

0
0 0 0 0 0 0

1
2 0

0
0 0 0 0

0
0 0 0

1 0 0

18 13 12 19 14 16 5 16 II 12 13.6 f 1.3;

Conlrols
31 32 33 34 35 36 37 38 39 40 Mean F M M F F M F M F F * SD. 52 41 36 28 33 28 35 58 24 21
0 0 0 0 0 0
0 0 0 0

1 0 0 0 0 1 0 0 0

2 0 3 0 4 3 3 0 2 4

0 1 0
2 0 0 0

0 0 0 0

0
0 0 0 0 0 0

0 0

0
0 0 0 0

0
0 0 0 0 0

2 2 3 2 4 3 4

1 1 0

0 0
0

0
0 0

I
3 4 2.8 f 0.3

ctg, chromatid gap; csg, chromosome gap; ctb, chromatid break; csb, chromosome break; end, endoreduplication; ace, acentric fragment; qr, quadriradial. *P < 0.0001 (Students r-test) for the difference between control and radiodiagnostic groups.

quency can therefore be used to indicate an exposure [15]. In general, chronic exposure involves breakage of 1 chromatid, whereas those that produce breakage of 2 chromatids (rings, dicentrics, translocations) require increases approximately as the square of the dose [16]. The purpose of this work was to provide data on the genetic hazards due to the occupational exposure to ionizing radiation, since the potential risks and biological consequences of nuclear medicine, namely mutagenesis, carcinogenesis and teratogenesis, have been attained through the extrapolation from acute exposures. Our results are also particularly interesting for a developing country such as ours, where biological security controls are

not so strict and extended work days are common. Biomonitoring of occupationally exposed people appears to be a sensitive way to evaluate the genotoxic effects of radiation exposures. This type of monitoring may be used as an indicator to detect early damage and to demand more controls in radiation protection. Unfortunately, it is not possible to compare the exposed radiation dose (mean 47 and 180 for the last 6 months) to response relationship with that found by other authors as many of the workers have confessed that they have not used the film badges periodically. Thus, the reported values are not an exact estimation of the exposure dose and are only taken as hypothetical minimum exposure doses.

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117

Biomonitoring for somatic mutations in human populations is also important as it is believed that somatic mutations contribute to cell lethality, loss in specific functions (171 and diseases such as cancer. There is an association between an increased rate of chromosome aberrations and cancer risks [ 181. For health surveillance, the detection of early genotoxic effects may permit the adoption of preventive biological controls such as hygienic improvements in the workplace or the reduction of hours of occupational exposure. References Ill Sasaki, MS. and Miyata, H. (1968) Biological dosimetry
in atomic bomb survivors. Nature (London) 220, 1189- 1193. PI Schull, W.L., Otake, M. and Neal, J.V. (1981) Genetic effects of the atomic bomb: a reappraisal. Science 213, 1220-1227. [31 Stephan, G. and Oestreicher, U. (1989) An increased frequency of structural chromosome aberrations in persons present in the vicinity of Chernobyl during and after the reactor accident. Is this effect caused by radiation exposure? Mutat. Res. 223, 7-12. I41 Croft, J.R. (1989) The Goiana accident. In: W.A. Crosbie and J.H. Gittus (Eds.), Medical Response to Effects of Ionising Radiation, Elsevier Applied Science, England, pp. 83-101. [51 Bigatti, P. (1988) Cytogenetic monitoring of hospital workers exposed to low-level ionizing radiation. Mutat. Res. 204, 343-347. WI Tanaka, R., Kumagai, E. and Sawada, S. (1983) Chromosome aberrations in radiological technologists. In: Report of the Study supported by Grant-in Aid (No. 00544089) for General Scientific Research, the Ministry of Education, Science and Culture, Japan, pp. 67-86. 171 Messing, K., Seifert, A.M. and Bradley, W.E.C. (1986) In vivo mutant frequency of technicians professionally exposed to ionizing radiation. In: Monitoring of Occupational Genotoxicants, Alan R. Liss., pp. 87-97.

VI Moorhead, P.S., Nowell, P., Mellman, W.J., Battips.

W.J. and Hungerford, D.A. (1960) Chromosome preparation of leukocyte cultures from human peripheral blood. Exp. Cell Res. 20, 613-616. [91 ISCN (1985) An International System for Human Cytogenetic Nomenclature. S. Karger, New York. VOI Hall, E.J. (1988) Diagnostic radiology and nuclear medicine: risk versus benefit. In: Radiobiology for the Radiologist, 3rd Edition, J.B. Lippincott Company, Philadelphia, pp. 467-503. [Ill Mettler, F.A. and Moseley, R.D. (1985) Basic radiation physics, chemistry, and biology. In: Medical Effects of Ionizing Radiation, Grune and Stratton, Inc., pp. l-30. WI Kumagai, E., Tanaka, R., Kumagai, T., Onomichi, M. and Sawada, S. (1990) Effects of long-term radiation exposure on chromosomal aberrations in radiological technologists. J. Radiat. Res. 31, 270-279. iI31 Bender, M.A., Awa, A.A., Brooks, A.L., Evans, H.J., Groer, P.G., Gayle Littlefield, L., Pereira, C., Preston, R.J. and Wachholz, B.W. (1988) Current status of cytogenetic procedures to detect and quantify previous exposures to radiation. Mutat. Res. 196, 103-159. iI41 Pohl-Ruling, J., Fisher, P., Haas, 0.. Obe, G., Natarajan, A.T., van Buul, P.P.W., Buckton, K.E., Bianchi, N.O., Larramendy, M., Kucerova, M., Polikova, Z., Leonard, A., Fabry, L., Palitti, F., Sharma, T., Binder, W., Mukeherjee, R.N. and Mukherjee, U. (1983) Effect of low-dose acute X-irradiation on the frequencies of chromosomal aberrations in human peripheral lymphocytes in vitro. Mutat. Res. 110, 71-82. 1151 Evans, H.J., Buckton, K.E., Hamilton, G.E. and Carothers, A. (1979) Radiation induced chromosome aberrations in nuclear-dockyard workers. Nature 277, 531-534. iI61 Maugh, T.H. (1982) Biological markers for chemical exposure. Science 215, 643-647. 1171 Evans, H.J. (1982) Chromosomal mutations in human populations. Cytogenet. Cell Genet. 33, 48-56. 1181 Nordenson, I., Beckman, L., Liden, S. and Stjernberg, N. (1984) Chromosomal aberrations and cancer risk. Hum. Hered. 34, 76-81.