Enhanced frequency of chromosome aberrations in workers occupationally exposed to diagnostic X-rays

Mutation Research, 260 (1991) 343-348 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100116D

343

MUTGEN 01678

Enhanced frequency of chromosome aberrations in workers occupationally exposed to diagnostic X-rays A.N. J h a a n d T. S h a r m a Cytogenetics Laboratory, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi-221 005 (India) (Received 29 August 1990) (Revision received 6 February 1991) (Accepted 7 February 1991)

Keywords: Peripheral blood lymphocytes; Diagnostic X-rays; Occupational exposure; Chromosome aberrations

Summary To estimate the level of radiation exposure of personnel handling diagnostic X-ray machines, the yield of chromosomal aberrations was analysed in peripheral blood lymphocyte cultures. These occupationally exposed individuals showed higher frequencies of dicentrics as well as acentrics than normal controls. Absorbed radiation doses calculated by extrapolating reference in vitro dose-response curve for dicentrics ranged between 0.13 and 0.17 Gy. This implies exposure beyond the permissible limit of 0.05 Gy/year for the whole body. However, no obvious trend of increased aberrations as a function of either duration of employment or age was noticed. The increase in the aberration yields in this personnel underscores the need of adopting measures to avoid or minimise such overexposure.

Diagnostic and therapeutic uses of ionising radiations make the largest man-made contribution to the population dose (UNSCEAR, 1982). In diagnostic radiology it is intended that the desired information is obtained with minimum exposure of the subjects and with the least risk to the technical personnel. Medical workers thus constitute the group most consistently exposed to low doses of ionising radiations (UNSCEAR, 1982). Because of the low levels of exposure, only limited Correspondence: Dr. A.N. Jha, Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).

information is available on chromosomal aberration analysis of this personnel. Needless to say, such studies have been accepted as a fairly reliable parameter for evaluating damages induced by ionising radiation and other environmental agents in humans (Bender and Gooch, 1966; Evans et al., 1979; Bauchinger et al., 1980; Ramalho et al., 1990). Stewart and Sanderson (1961) and Conen et al. (1963) demonstrated chromosomal abnormalities in individuals receiving less than 0.3 and 2.0 rad of diagnostic X-rays. Norman et al. (1964) found about 0.77% dicentrics in lymphocytes of radiation workers exposed to a cumulative dose of 10-25 rad during the period of their employment.

344 Bloom and Tjio (1964) reported 1-5% dicentrics, acentrics and rings in peripheral blood lymphocytes of 5 patients who had received 12-35 rad of diagnostic X-rays during gastrointestinal studies. However, aberrations were not found in over 3000 cells examined from the 7 individuals who had received 20-80 mrad during chest X-rays. Migeon and Mertz (1964) also reported absence of dicentrics and rings in 600 cells scored from 3 patients who had received about 20 rad during diagnostic exposure. Under in vitro conditions, Schmickel (1967) demonstrated a significantly higher number of cells with chromosomal aberrations than in controls, even at 5-6 rad. Recently, Bigatti et al. (1988) showed an increased frequency of chromosomal aberrations, including dicentrics, in 3 groups of hospital workers (physicians, nurses and technicians) who were exposed to very low levels of X- or 7-rays. In the present study yields of chromosomal aberrations in medical workers exposed to diagnostic levels of X-rays have been analysed and an attempt has been made to estimate the absorbed radiation doses.The primary purpose of such studies as advocated by UNSCEAR (1982) is to gather information for determining dose accumulation in individuals. This information is necessary to demonstrate compliance with the occupational exposure limits. Materials and methods

Peripheral blood samples were collected from 20 male individuals who had worked for 5-20 years in different clinics and establishments, including private ones. We intended to study more such individuals but could do only 20 as others did not come forward to give blood samples. For several reasons the screened workers could not provide us with records of their accumulated doses monitered by physical dosimeter. Blood samples from normal individuals who had not been exposed to X-rays served as parallel controls. Neither the radiation workers nor the controls had received chemotherapeutic or cytostatic drugs. However, because of practical limitations it was not possible to precisely match the exposed and control individuals for different factors such as social class and life style. But care was taken to be close regarding age, sex and smoking habit. Wherever

possible, blood samples were collected twice from each worker. Routine whole-blood microcultures were set up by adding 0.30 ml blood to 5 ml of Eagle's MEM supplemented with 20% heat-inactivated human AB + serum. 5-Bromodeoxyuridine (BrdU) was also added to a final concentration of 5 ~tg/ml. The cultures initiated by adding 0.15 ml phytohaemagglutinin were allowed to grow for 48 h at 37°C. The culture vials were protected from visible light. Colcemid (0.20 ~tg/ml) was added 3 h prior to harvesting. Flame-dried chromosome preparations were processed for differential staining using the BrdU-Giemsa-sunlight method of Goto et al. (1975) with slight modifications (Sharma and Das, 1981). At least 200 metaphases were scored from each individual. Chromosome aberrations, viz. dicentrics, rings and acentrics, were scored exclusively from first-division metaphases. However, it was difficult to classify acentrics as terminal deletions, interstitial deletions and isochromatid breaks. The 'Z-test' (test of equality of 2 proportions) was used for statistical comparison between control and exposed individuals. The frequency of dicentrics/cell of a worker was used for estimating the absorbed equivalent whole-body dose by linearly extrapolating the in vitro dose-response reference curve prepared in our laboratory (Jha and Sharma, unpublished) for X-ray-induced dicentrics. Linear extrapolation of the in vitro dose-response curve was considered practical on the assumption that the linearity holds good with data adequately fitting a simple linear regression (Catcheside et al., 1946; Lloyd et al., 1988). In this context, it is also known that protraction or fractionation of the dose lowers the aberration yield. Most of the available in vitro reference curves with low-LET ionising radiations use acute irradiation of the blood samples and the resultant aberration yields generally fit well to the quadratic dose-effect model. Attempts to produce 'chronic' curves involving exposure at a constant dose rate and delivering doses over different times have resulted in a poor fit of the data to the quadratic model (Lloyd and Edwards, 1990). The linear model was therefore selected in the present study as it involved exposure to chronic low doses and dose rates.

345

Results Yields of a b e r r a t i o n s in different e x p o s e d workers are s u m m a r i s e d in T a b l e 1. M e a n frequencies of a b e r r a t i o n s b e t w e e n e x p o s e d a n d control individuals f r o m the p o o l e d d a t a are comp a r e d in T a b l e 2. F r e q u e n c i e s of dicentrics a n d acentrics either i n d i v i d u a l l y or collectively were significantly higher in e x p o s e d i n d i v i d u a l s ( p < 0.05). In one case (No. 20) 1 dicentric was observed in 402 cells, b u t the frequency of acentrics was not f o u n d to b e statistically higher. A m o n g 20 i n d i v i d u a l s studied, dicentrics were n o t o b s e r v e d in 6, which could b e taken to m e a n that they h a d received p r a c t i c a l l y zero dose. However, the frequency of acentrics was f o u n d to be signific a n t l y higher in e x p o s e d i n d i v i d u a l s t h a n in controls. There was no obvious t r e n d of i n c r e a s e d a b e r r a t i o n s as a function of d u r a t i o n of e m p l o y m e n t a n d age of the i n d i v i d u a l s ( T a b l e 1). The c a l i b r a t i o n curve g e n e r a t e d in o u r l a b o r a tory for l l 0 - k V X - r a y s (dose rate: 1 G y / m i n ) was

used for the e s t i m a t i o n of a b s o r b e d doses as no d o s e - r e s p o n s e curve for l o w - d o s e - r a t e X - r a y - i n d u c e d dicentrics was available. T h e curve was constructed by taking 6 dose points ranging from 0.25 to 4 Gy. T h e yield of dicentrics gave a value of Y = - 0 . 0 2 5 + 0.152X for the linear e q u a t i o n (Y = c + a X ) when its yield at dose p o i n t 0 was taken as 0. W h e n used for e s t i m a t i o n s this equation gave values o f a b s o r b e d doses which r a n g e d f r o m 0.18 to 22 G y . H o w e v e r , this e q u a t i o n was n o t t a k e n i n t o a c c o u n t as it also e s t i m a t e d some d o s e for c o n t r o l i n d i v i d u a l s which is n o t logical. W h e n the yield of 2 dicentrics out of 7429 cells scored f r o m 45 n o r m a l i n d i v i d u a l s was i n c l u d e d in the data, the linear e q u a t i o n fitted to the values Y=-0.018+0.153X. This in vitro reference curve l o w e r e d the e s t i m a t e d dose for c o n t r o l individuals a n d was e x t r a p o l a t e d to e s t i m a t e the abs o r b e d doses a m o n g the workers. I n this case, the c a l c u l a t e d dose r a n g e d f r o m 0.13 to 0.17 G y , i n d i c a t i n g an e x p o s u r e b e y o n d the p e r m i s s i b l e level of w h o l e - b o d y e x p o s u r e of 0.05 G y / y e a r for

TABLE 1 CHROMOSOMAL ABERRATION YIELDS AND ESTIMATED ABSORBED RADIATION DOSES IN OCCUPATIONALLY EXPOSED INDIVIDUALS Subject

Work experience (years)

Age (years)

Cells scored

Dicentrics (dicentrics/cell x 10- 3)

Acentrics (acentrics/ cell x 10- 3)

Aberrant cells (aberrations/ cellX 10 -3 )

Est. dose (Gy)

95% Confidence limits (Gy)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

5 5 15 8 20 10 20 5 20 9 10 6 8 9 10 8 7 6 10 10

26 25 40 29 35 29 45 29 45 30 32 28 30 32 40 30 30 28 35 32

640 425 452 405 430 412 425 220 408 422 456 409 412 276 236 326 482 460 415 402

4 (6.3) 3 (7.0) 2 (4.4) 1 (2.5) 3 (7.0) 0 (0.0) 1 (2.4) 0 (0.0) 1 (2.5) 3 (7.1) 2 (4.4) 0 (0.0) 0 (0.0) 1 (3.6) 2 (8.5) 0 (0.0) 1 (2.1) 2 (4.3) 0 (0.0) 1 (2.5)

23 (35.9) 16 (37.6) 16 (35.4) 14 (34.6) 15 (34.9) 14 (34.0) 16 (37.6) 8 (36.4) 14 (34.3) 15 (35.6) 18 (39.5) 18 (44.0) 20 (48.5) 12 (43.5) 10 (42.4) 12 (36.8) 18 (37.3) 22 (47.8) 12 (28.9) 8 (19.9)

27 (42.2) 19 (44.7) 18 (39.8) 15 (37.0) 18 (41.9) 14 (34.0) 17 (40.0) 8 (36.4) 15 (36.8) 18 (42.7) 20 (43.9) 18 (44,0) 20 (48.5) 13 (47.1) 12 (50.8) 12 (36.8) 19 (39.4) 24 (52.2) 12 (28.9) 9 (22.3)

0.16 0.16 0.15 0.13 0.16 0.00 0.13 0.00 0.13 0.16 0.15 0.00 0.00 0.14 0.17 0.00 0.13 0.15 0.00 0.13

0.08-0.24 0.08-0.24 0.07-0.23 0.05-0.21 0.08-0.24 0.00-0.00 0.05-0.21 0.00-0.00 0.05-0.21 0.08-0.24 0.07-0.23 0.00-0.00 0.00-0.00 0.06-0.22 0.09-0.25 0.00-0.00 0.05-0.21 0.07-0.23 0.00-0.00 0.05-0.21

346 0.80 0.25 0.60

~

0.40

• 0"°0"1°0 . .AP-

0,20 "

]

InVivo o1:5 InVitro

~

1

2

3

Dose

4

(Gy)

Fig. ]. Linear extrapolation of the lower end of the in vitro dose-response reference curve for X-ray-induced dicentrics to estimate absorbed radiation dose in occupationally exposed individuals.

the workers. The observed dicentric yield was plotted against the estimated dose (Fig. 1). In many cases, the same dose was estimated for individuals with different dicentric yields (Table 1). Discussion

Increased frequencies of chromosomal aberrations are well known among occupationally exposed workers even at much below the permissible level of exposure. Qualities of radiation to which populations were exposed differed in most of the studies so a direct comparison with the present study is not logical. The workers tested in this study could not provide records of their absorbed dose monitered by physical dosimeter, therefore evaluation of physical versus biological dosimetry was limited. In the classical study on 200 nuclear dockyard workers (Evans et al., 1979), an elevated frequency of chromosomal aberrations was reported after 10 years of study. A dose-effect

relationship was also observed on the basis of their accumulated dose. Similarly, a higher frequency of dicentrics and acentrics in nuclear power plant workers was documented, but without any dose-effect relationship (Bauchinger et al., 1980). A significantly higher number of cells with chromosome-type aberrations in 3 groups of hospital workers exposed to much less than the legal limit of X- and y-rays has been reported (Bigatti et al., 1988). However, a significant difference in aberration frequencies was not found when exposed individuals were categorised with respect to the kind of work they did. In the present study, no correlation between aberration frequency and estimated absorbed dose was found. Individuals with 5-20 years' employment showed no increase in the yield as a function of duration of employment or dose-dependent response. The present observation therefore supports the findings of Bauchinger et al. (1980) and Bigatti et al. (1988). The absence of dose dependence is attributed to several factors which in combination influence the yields of aberrations (Bauchinger et al., 1980). Interindividual differences in proliferation rate of PHA-responsive lymphocytes in culture may also influence the in vivo dose-response relationship (Crossen and Morgan, 1977). However, the main reason for the absence of a dose-dependent accumulation of aberrations is thought to be the life span of PHA-responsive lymphocytes and the slow disappearance of aberration-bearing cells from the circulation. Variable half-lives of 530 (Norman et al., 1965), 1095 (Dolphin et al., 1973) and 1600 days (Buckton et al., 1967) for such circulating lymphocytes have been reported. Recently, a half-life of 130 days with a range of 95-220 days has been suggested after studying the disappearance of unstable chromosomal aberra-

TABLE 2 C O M P A R I S O N OF YIELDS OF C H R O M O S O M A L A B E R R A T I O N S IN C O N T R O L A N D O C C U P A T I O N A L L Y EXPOSED INDIVIDUALS Group

Number of donors

Mean age (years)

Cells scored

Dicentrics (dicentrics/cell × 1 0 - 3 q_ SD)

Acentrics (acentrics/cell × 1 0 - 3 ~ SD)

Control Exposed

45 20

29.00 32.75

7479 8013

2 (0.27 +_0.94) 27 (3.37 _+2.88)

121 (16.18 _+9.16) 301 (37.56 _+7.44)

347

tions from lymphocytes of individuals accidentally exposed to Cs-137 in Goiania, Brazil (Ramalho et al., 1990). The above information suggests that true dose dependence is not possible in individuals exposed for more than 5 years. In the occupationally exposed individuals in the current study an increased frequency of acentrics was frequently noticed. Most cases of increased frequency of acentrics are known to involve relatively minor exposures to low doses or dose rates of X- and T-radiations (Purrott et al., 1974). A yield of acentrics as high as 200% of the yield of dicentrics has been reported (Purrott et al., 1973). A significant increase in the frequency of acentrics has also been reported for the individuals who were present in the vicinity of Chernobyl during and after the reactor accident. It may be mentioned that in Chernobyl, chronic exposure is the expected outcome (Stephan and Oestreicher, 1989). For the increased frequency of acentrics in such cases it has been assumed, as explained by Lea (1955), that acentrics are largely 'single-hit' in origin, whereas dicentrics and rings result from '2-hit' events. If the time required for repair is short (1-2 h), very-low-dose-rate radiation could lead to an increase of 'single-hit' deletions relative to '2-hit' aberrations. This explanation is also given for the increased frequency of acentrics observed in individuals exposed to low-LET radiations at occupational levels of dose and dose rate (Purrott et al., 1973). Under in vitro conditions, in normal individuals the spontaneous yield of acentrics is higher than that of dicentrics. It is assumed that certain aberration-inducing factors, other than occupational exposure to radiation, might complicate cytogenetic dosimetry (Purrott et al., 1973). It is remarkable that an increased frequency of acentrics was also observed in individuals with no dicentrics. Purrott et al. (1974) reported on an individual who worked for many years in a chronic low dose environment and had accumulated 28 rad. This individual had an accident in which a cobalt-60 source fell into his pocket and on estimation a whole-body dose of 3 rad was calculated. Analysis of 500 cells of this individual yielded no dicentrics but 27 acentrics. The large number of acentrics was attributed to the prolonged chronic low-dose working environment. As in the above situation, it may be reasonable to assume that in

the present observation continuous exposure to a low dose and dose rate for long periods might have induced higher frequencies of acentrics. One of the practical limitations of cytogenetic dosimetry is its imprecision for dose estimation, particularly for lower doses. On the basis of in vitro studies, it has been suggested that the absence of dicentrics in 500 cells scored estimates a zero dose, but there is a 1 in 40 chance that the dose is above 15 rad (Purrott et al., 1974). On the other hand, 1 dicentric in 500 cells indicates a dose of approximately 15 tad of X- or v-rays. This dose could be accumulated within permissible limits in 15-36 months when the formula for the estimation of maximum permissible dose 5 ( N 18) is used. In this formula, N denotes the age of the worker assuming that he or she started work at the age of 18 years. As the occurrence of a single damaged cell is based on chance, it carries 95% confidence limits extending from the normal background up to about 35 rad. The technique, therefore, cannot distinguish between closely similar doses (Purrott et al., 1974). A similar situation of imprecise dose estimation is found in the present study. This is evident when different yields of dicentrics produce the same estimates for absorbed dose. In the contrary situation, the same dose is extrapolated when the dicentric yield is not the same. It has been argued that there are no precise criteria for deciding on the accuracy that biological dose estimates need to achieve. It has, however, been taken that a dose estimate within 30% of the real dose is a good accuracy to attain (Lloyd et al., 1987). It would nevertheless have been useful to have more blood samples from the workers showing higher dicentric yields in the present study. Chromosomal aberrations only indicate gross genetic damage but their absence does not exclude other possible damage to DNA. However, the presence of chromosomal aberrations will indicate a possible link with exposure to radiations. In such cases steps should be taken to avoid overexposure.

Acknowledgements The work was supported by IAEA, Vienna (Research Contracts 3171/RB and 3 1 7 1 / R I / R B ) ,

348 DAE, Bombay and UGC, New Delhi. We are t h a n k f u l to the v o l u n t e e r s w h o d o n a t e d b l o o d to m a k e the s t u d y p o s s i b l e . W e are also t h a n k f u l to Dr A.A. Edwards (National Radiological Protect i o n B o a r d , C h i l t o n , U . K , ) for critical c o m m e n t s a n d D r A . H . Z w i n d e r m a n ( D e p a r t m e n t of M e d i cal Statistics, U n i v e r s i t y o f L e i d e n , T h e N e t h e r l a n d s ) for h e l p in the statistical t r e a t m e n t o f t h e d a t a . W e are i n d e b t e d to P r o f e s s o r D r A . T . N a t a r a j a h ( U n i v e r s i t y o f L e i d e n , T h e N e t h e r l a n d s ) for encouraging criticism and valuable suggestions.

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