BIODOSIMETRY FOR A RADIATION WORKER USING MULTIPLE ASSAYS T. Straume, J. N. Lucas, J. D. Tucker, W. L. Bigbee and R. G. Langlois* interval was underestimated by at least a factor of 5 . Because there are no biodosimetry methods that have been fully validated for the types of exposures received by the worker (ie., low levels, long-term exposures), it was clear to us at the outset that definitive conclusions would not be possible. However, it was considered of scientific interest to explore the utility of state-of-theart biodosimetry methods in the evaluation of such a large discrepancy (Le., 0.56 Sv vs. >2.5 Sv). The worker agreed to be evaluated using four biodosimetric methods-glycophorin-A (GPA) somatic mutations, chromosome translocations, micronuclei, and dicentrics. Two of these biodosimeters (GPA and translocations)are stable with time postexposure (Buckton et al. 1978; Langlois et al. 1987; Straume et al. 1991) and are therefore expected to integrate radiation damage under chronic-exposure conditions. The other two are unstable (micronuclei and dicentrics) and can only detect relatively recent exposures (Buckton et al. 1978; Krepinsky and Heddle 1983). Two different approaches were used to detect chromosome translocations in peripheral blood lymphocytes-fluorescence in situ hybridization (FISH) and G-banding. GPA and FISH were developed at the Lawrence Livermore National Laboratory (LLNL) and have previously been applied to human populations exposed to radiation (Langlois et al. 1987; Lucas et al. 1991; Straume et al. 1991) and chemicals (Bigbee et al. 1990). The GPA assay is an indirect but quantitative method of identifying somatic allele-loss mutations in the GPA gene of bone marrow erythroid precursor cells detected as variant peripheral blood erythrocytes that fail to express an allelic form of GPA (Langlois et al. 1986, 1990). The FISH assay was developed to detect chromosome translocations using in situ hybridization of chromosome-specific DNA probes (Pinkel et al. 1986; Lucas et al. 1989a, 1989b, 1991). Micronuclei in human lymphocytes have also been used to measure radiation exposure (e.g., Krepinsky and Heddle 1983; Littlefield et al. 1989). The easy and rapid scoring of micronuclei makes this biodosimeter particularly useful for recent, acute exposures; however, it is not useful for chronic exposures (Krepinsky and Heddle 1983). Chromosome dicentric aberrations in human peripheral blood lymphocytes have been the mainstay in radiation biodosimetry (e.g., Bender et al. 1988; Ra-
Abstract-Four state-of-the-art biodosimeters-GPA mutations, chromosome translocations, micronuclei, and dicentrics-were used to evaluate a radiation worker who believed that the official dosimetry records substantially underestimated his actual dose. Dosimetry records indicated that the worker received 0.56 Sv during a 36-y employment history, always within the dose limits. In contrast, the worker believed that his dose equivalent may have been more than 2.5 Sv because much of the exposure was received during the early days of health physics when dosimetry capabilities and practices were not as good as they are today. Because there are no biodosimetric assays that have been fully validated for the long-term low-level exposures received by the worker, we did not expect to obtain particularly useful point-estimates of dose. However, because the discrepancy between the dosimetry records and the worker’s belief was so large, we believed that biodosimetry using multiple assays together with probabilistic assessment of the uncertainties would provide useful insight. Results showed that the frequencies of chromosome translocations and GPA mutations (stable biodosimeters) were significantly elevated when compared with those for unexposed controls. Our analysis suggests that dose-equivalent estimates in the -0.4 to -2 S v range (which include the value in the dosimetry records) cannot be confidently excluded at this time based on biodosimetry; however, a value greater than 2.5 S v appears unlikely. Important new information on the temporal stability of chromosome translocations is also presented. Health Phys. 62(2):122-130; 1992 Key words: biodosimetry; translocation; exposure, occupational; nuclear workers
INTRODUCTION A RADIATIONworker who disagreed with official exposure records was evaluated biodosimetrically. According to dosimetry records, the worker received 0.56 Sv during more than three decades of exposure, always within the occupational dose limits. However, because of dosimetry capabilities and practices during his initial employment (1953 to 1965, the early days of health physics), the worker believed his dose during that time * University of California, Lawrence Livermore National Laboratory, P.O.Box 5507, Livermore, CA 94550. (Manuscript received 22 August 1991; revised manuscript received 19 September 1991, accepted 15 October 1991) 0017-9078/92/$3.00/0 Copyright 0 1992 Health Physics Society 122
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
malho et al. 1988; Littlefield et al. 1990). These and numerous other studies demonstrate the utility of dicentrics for dose assessments within a few weeks after acute or subacute exposure. However, it is well established that dicentrics are not particularly useful for quantifying exposures that occurred a long time ago or those protracted over months or years (e.g., Buckton et al. 1978; Littlefield et al. 1990). Somatic mutations and chromosome aberrations have previously been detected in recently exposed radiation workers, e.g., elevated HPRT mutations (Messing et al. 1989) and unstable chromosome aberrations (Evans et al. 1979; Lloyd et al. 1980). However, the assays used in those studies present problematical biodosimetric interpretations for long-term exposures or for those exposed a long time ago because of the temporal instability of those assays (Hakoda et al. 1988; Littlefield et al. 1990).
MATERIALS AND METHODS Case history The dosimetry records for this 59-y-old white 'radiation worker indicated that his occupational exposure to radiation began in 1953. At the time of blood sampling for this biodosimetry study in 1989, dosimetry records indicated that he had received a whole-body dose equivalent of 0.56 Sv from all work-related sources. Gamma rays contributed 0.54 Sv and neutrons 0.02 Sv (i,e., 0.002 Gy). About 90% of the dose equivalent was received from 1953 to 1967; the remainder was received between 1968 and 1985. Since 1985, records indicate that he has received only 0.00048 Sv. Internal exposures were limited to a combined total of 0.0018 Sv from 241Am,239Pu,and 238U. For comparison, the lifetime dose equivalent to the bone marrow from natural background radiation was estimated to be 0.066 Sv (NCRP 1987), only a fraction of that received occupationally. An interview with the radiation worker indicated that he was a nonsmoker and did not appear to have a medical or other non-occupational exposure history that could be a potential source of confounding factors. Blood sample preparation Peripheral blood was obtained with informed consent in January 1989 and again in June 1989 by the Medical Department at LLNL. Each of the two samples consisted of 15 mL of blood drawn into a heparinized vacutainer. The samples were coded with an identification number, and the donor was determined to be a MN heterozygote by serotyping with commercial antiM and anti-N sera (Ortho Diagnostics, Raritan, NJ). The blood samples were then divided into aliquots for use in each of the assays. The blood used for the GPA assay was fixed, and erythrocytes were labeled with monoclonal antibodies and analyzed on a flow cytometer (Langlois et al. 1990). The blood for aberration analysis was cultured with
123
phytohemagglutinin (PHA) to stimulate growth of T lymphocytes and 25 pM bromodeoxyuridine (BrdUrd) to discriminate between first- and later-division cells. Colcemid was added at 48 h to arrest the lymphocytes in metaphase. At 52 h the cells were swollen with 0.075 M KCl and fixed with 3:l methanol and acetic acid, and microscope slides were prepared for cytogenetic analysis. Cells for FISH were cultured in the same manner except that BrdUrd was not included in the culture medium. For micronuclei, lymphocytes were isolated and cultured as described previously (Eastmond and Tucker 1989). Cytochalasin B was added at 44 h, and at 72 h the cells were centrifuged onto clean dry slides, quickly dried, and fixed in 100% methanol for 15 min. The slides were stored in NZatmosphere at -20°C until immediately before staining. GPA somatic mutation assay The GPA assay has been described previously (Langlois et al. 1986, 1990). The assay uses immunofluorescence labeling and flow cytometry to enumerate the frequency of red blood cells lacking expression of one allelic form of the cell-surface sialoglycoprotein GPA, presumably as a result of allele-loss mutations at the GPA locus in erythroid precursor cells. The GPA gene is located on human chromosome 4 and is present in the population as two common alleles at approximately equal frequencies, so that -50% of the population is MN heterozygous. The proteins coded by these two alleles carry the M and N blood group antigens and differ by only two amino acids out of 131. The GPA assay was originally developed using a dual-laser flow sorter (Langlois et al. 1986); the data in this paper were obtained with a modified method (designated the BR6 assay) performed on a simpler flow cytometer (Langlois et al. 1990). This assay enumerates the frequency of NQ)variant cells, i.e., cells that have lost expression of the M allele but express the N allele normally. Chromosome aberration analysis using FISH Translocation detection using FISH is illustrated in Fig. 1. The FISH method, described previously, can be used to detect both translocations and dicentrics (Pinkel et al. 1986; Lucas et al. 1989a, 1989b, 1991; Straume et al. 1991). FISH was performed using two cocktails of composite DNA probes, one specific for chromosome 4 only and the other specific for chromosomes 1,3, and 4 combined. The use of two different cocktails had no special significance other than the availability of DNA probes. For the January 1989 sample, the hybridization target was chromosome 4 only, for whichf, (the fraction of the genome hybridized) is 0.0668 (Mendelsohn et al. 1973). For the July 1989 sample, the hybridization target was chromosomes 1, 2, and 4 combined, for whichJ; is 0.238 (Mendelsohn et al. 1973). The hybridization target for the FISH controls was chromosomes 1, 3, and 4 combined, for whichf, is 0.223 (Mendelsohn et al. 1973), or chromosome 4. To compare results using this approach with
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February 1992, Volume 62, Number 2
Fig. 1. Translocation detection in human lymphocytes using FISH. Left: Metaphase chromosomes from a normal cell showing two homologous #4 chromosomes that have been selectively stained yellow using FISH. The other chromosomes have been counterstained red. Right: Metaphase chromosomes from an irradiated cell showing one normal #4 chromosome and another that has broken into two pieces and translocated to the correspondingpieces from a broken red chromosome (arrows).
results from conventional cytogenetic methods, the data obtained from the smaller target are scaled up to equal the full genome. The method used to scale to the full genome has been described previously (Lucas et al. 1989~). Conventional aberration analysis and G-banding Established methods were used to stain metaphase chromosomes on slides and to score for dicentrics and translocations. For unbanded chromosomes, slides were stained using the fluorescence plus Giemsa method (Ferry and Wolff 1974; Minkler et al. 1978) to ensure that only first-division cells were scored. For banded chromosomes, slides were treated with trypsin and stained with Giemsa according to established techniques (Evans et al. 1971). For each type of analysis, only cells with 45 or more centromeres were scored. Micronucleus analysis The chromosomes were stained as described previously (Eastmond and Tucker 1989) with an antikinetochore antibody' and a fluoresceinated rabbit antihuman second antibody. Counterstaining was achieved with 0.25 pg/mL DAPI, and slides were scored with simultaneous phase contrast and DAPI excitation. The presence or absence of a kinetochore label within each micronucleus was determined by viewing the cells under fluorescence excitation. Antibodies, Inc., Davis, CA.
RESULTS Results for both integrating and non-integrating biodosimeters are listed in Table 1. Frequencies for GPA, chromosome translocations, micronuclei, and dicentrics are listed for the radiation worker as well as for unexposed controls. The data for the radiation worker are mean values from two separate blood samplings, one in January 1989 and another in July 1989. For GPA, a total of 5 X lo6 cells were measured for each assay run. Duplicate assay runs were made for each of the two blood samplings. The N 0 frequency of (23 3) X is the mean kSD for the radiation worker. The control value of (8 f 3.5) X is the mean +SD for six unexposed individuals matched to the radiation worker by age, sex, non-smoking history, and time period of assay. The GPA variant frequency from the radiation worker is significantly higher than control values, as indicated in Table 1 ( p = 0.006). This is illustrated further in Fig. 2, where the N 0 frequencies measured in the six matched controls are compared with the frequencies measured in the two samples obtained from the radiation worker. Additional information on control frequencies is presented in the Uncertainties section. The values for translocations in Table 1 include combined results from both FISH and G-banding. A total of eight translocations were detected in 482 full genome-equivalent cells from the radiation worker, which is significantly higher than the control frequency ( p = 0.0004). Results for FISH and G-banding were also statistically significant when evaluated separately:
+
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
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Table 1. Measurement results for integrating and non-integratingbiodosimeters. Biodosimeter
Cells
Radiation worker Events Frequency
Cells
Unexposed controls" Events Frequency
Statisticsb ~
Integrating GPA Translocations Nonintegrating Micronuclei Dicentrics
20 X lo6 482d
455' 8
(23 f 3) >: (17 f 6) X
30 X lo6 2278'
240 6
(8 & 3.5) X p=0.006 (2.6 f 1) X low3 p=0.0004
(1 1 f 3) x 10-3 1000 9 (9 2 3) x lo-) p = o x (1.5 1.5) x 10-3 21,000 26 (1.2 f 0.3) X p=0.50 a Laboratory controls were obtained by the same methods and scorers and during the same general time frame as the results for the radiation worker being evaluated here. GPA based on six controls; translocations based on four controls; micronuclei based on one control; dicentrics based on 1 17 controls. The p-value for GPA is based on comparing the radiation worker mean with means and standard deviations from six matched controls using t-statistic with 5 degrees of freedom. For translocations, micronuclei, and dicentrics,p-values are based on a test for equal binomial proportions. N o variant cells detected in four independent measurements, two each from two separate blood samplings. Includes 332 full genome-equivalentcells scored using FISH (four events) and 150 cells scored using G-banding (four events). FISH results were scaled to full genome using method in Lucas et al. (1989~).The translocation frequency obtained for the radiation worker using FISH is not significantly different from that obtained using G-banding; p = 0.12. Full genome-equivalent cells scored using FISH. Conventional staining, 350 cells; FISH, 332 full genome-equivalent cells.
1000 682'
11
I
'
FISH, p = 0.005, and G-banding, p = 4 x To combine translocation results obtained using these two different methods, it was necessary to 1) scale the FISH results to full genome equivalents and 2) verify that FISH and banding produce equivalent dose-response
"
C-1 C-2 C-3 C-4 C-5 C-6 RW RW Aug 88 Mar 90 Mar 90 Mar 90 June 90 July 90 Jan 89 July 89
Fig. 2. Comparison of GPA N 0 variant cell frequencies measured in six controls (white bars) and in the two samplings from the radiation worker (gray bars). The group mean + I SD for the six controls is indicated as are the dates when the blood samples were obtained. The controls were matched to the worker by sex, age (within 10 y), and non-smoking history, as well as the general time interval during which the samples
were obtained and analyzed.
results. Scaling to full genome-equivalents was accomplished using the method of Lucas et al. (1989~)and thefs values listed in the Materials and Methods section. A large study comparing FISH and G-banding in Hiroshima atomic-bomb survivors has recently been completed in this laboratory, and results clearly demonstrate that these two methods produce equivalent translocation frequencies (Lucas et al. 1991). Four unexposed donors from our laboratory were used as controls for translocations. Controls were matched to the radiation worker for non-smoking history, sex, and general time period of sample acquisition and analysis. Results are listed in Table 1. The controls for translocations were 40 to 45 y of age, 14 to 19 y younger than the radiation worker. However, the frequencies of stable chromosome aberrations do not appear to increase significantly between 40 and 60 y of age (Tonomura et al. 1983; Bender et al. 1988). Results for non-integrating biodosimeters (micronuclei and dicentrics) are also listed in Table 1. Nonintegrating biodosimeters will only detect relatively recent radiation exposures (within the past few years). As expected from the worker's exposure history, the results for micronuclei and dicentrics were not significantly different from unexposed controls. Eleven micronuclei per 1000 cells were observed in the radiation worker and 9 per 1000 cells in the control ( p = 0.33). For dicentrics, one event was observed in 682 full genomeequivalent cells scored, which is also not significantly different from the frequency in controls ( p = 0.50).
BIODOSIMETRY AND DISCUSSION For GPA, dose-response curves from previous studies (Langlois et al. 1987; Straume et al. 1991) were used as standards to convert the observed mean N 0 variant cell frequency into dose. The dose equivalent
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( H ) in Sv was estimated from the GPA data using eqn (1): H
=
[(NQ),, - NQ)h)/slopeof standard curve]
X
Q,
(1) where N 0 , is the frequency of NQ) variants measured (Table 1); Nab is in the radiation worker, 23 X the background frequency measured in six matched (Table 1); and Q is the unexposed controls, 8 x radiation quality factor. The slope ( N 0 per Gy) of the “standard” GPA dose-response curve was estimated from two different sources of data, Hiroshima atomicbomb survivors and radiation accident victims in Goiinia, Brazil. In the case of Hiroshima survivors, the GPA dose response was obtained from previously published data (Langlois et al. 1987) that were reanalyzed here using DS86 marrow doses instead of the old T65DR tissue kerma values used in Langlois et al. According to DS86, the radiation from the Hiroshima bomb was almost pure gamma rays, with only -0.5 to 1% of the dose from neutrons (Roesch 1987). This small neutron component in DS86 is estimated to have contributed only -10% of the effect in Hiroshima (Brenner 1991) and is therefore ignored in the estimation of the dose equivalent (i.e., the Q for Hiroshima radiation is assumed to equal 1). The slope for the Hiroshima GPA curve was obtained from a subset of the Hiroshima GPA data (Langlois et al. 1987) that contained 58 individuals for whom DS86 dose information was available. This analysis included 18 distally exposed survivors who received DS86 marrow doses less than 0.005 Gy and 40 proximally exposed survivors who received DS86 marrow doses ranging from 0.1 to 5 Gy (mean, 1.2 Gy). The resulting linear fit parameters for GPA allele-loss and a slope of variants were a y-intercept of 11 x 48 x per Gy ( r = 0.46). Independent GPA measurements by Japanese investigators (Kyoizumi et al. 1989) also provided N 0 values for Hiroshima atomicbomb survivors using DS86 doses to the marrow; their Gy-’, similar to the value slope estimate was 40 x obtained here. Because previous results suggest that GPA mutations are stable with time postexposure (Langlois et al. 1987; Kyoizumi et al. 1989; Straume et al. 1991), the use of results for Hiroshima atomic-bomb survivors (exposed -40 y previously) as a standard curve to convert results from a radiation worker who received most of his dose -30 y ago would appear reasonable as long as the differences in dose rates are considered. The radiation exposure received by the worker was not acute like that received by atomic-bomb survivors but rather was highly protracted and therefore is expected to be less effective. The BEIR V Committee (NAS 1990) recently reviewed the experimental data on genetic effects and determined that low-dose-rate gamma rays are -5 times less effective than high-doserate gamma rays (this is the dose-rate effectiveness
February 1992, Volume 62, Number 2
factor [DREF]). Thus, the slope for GPA from Hiroshima survivors was divided by 5 to account for the difference between the acute exposure from the bomb and the chronic exposure in this individual. The pointestimate of dose equivalent obtained from the GPA data is 1.5 Sv when using 1/5 times the slope for Hiroshima in eqn (1) (uncertainties are discussed in a subsequent section). GPA results from the radiation accident victims in Goifnia, Brazil were also used as a “standard” for dose estimation (Straume et al. 1991). In this case, 13’Cs gamma-ray exposure was received during a period of about 2 wk, approximating fractionated or chronic exposure conditions. Based on dose-rate studies (e.g., Lloyd and Edwards 1983), the dose-response relationship for the Goiinia accident victims is expected to be nearly independent of dose rate, as is the case with the radiation worker. Thus, at first approximation,a DREF is not required. A linear regression was fitted to the Goilnia GPA data using dose estimates from Ramalho Gy-l et al. (1988). The resultant slope is 12.8 X and, when used in eqn (1) with a Q of 1.O, the point estimate of the dose equivalent for the radiation worker is 1.2 Sv. Dose is also estimated from the translocation data. To obtain these estimates, we relied on (1) our previous in vitro results for translocations obtained using FISH (Lucas et al. 1989b, 1989c, 1991), (2) information demonstrating that the yields of aberrations induced in lymphocytes after in vitro exposures are identical to those observed after in vivo exposures (Brewen and Gengozian 1971; Buckton et al. 1971; Schmid et al. 1974; Bajerska and Liniecki 1975; Littlefield et al. 1990), and (3) the observation that translocations are stable with time postexposure (Buckton et al. 1978; Lucas et al. 1991; Straume et al. 1991). In this connection, it is indeed significant that the translocation frequencies measured using FISH in atomic-bomb survivors some 45 y after exposure compare well with predictions based on in vitro measurements evaluated in first-division cells. This is illustrated in Fig. 3, where the plotted ratios are the translocation frequencies measured in lymphocytes of 19 Hiroshima survivors divided by the frequencies obtained in vitro from first-division cells. If translocation frequencies in individual survivors have remained unchanged since 1945, they should be identical to those obtained in vitro and the ratios should be unity. This is essentially what is observed in Fig. 3, where 16 out of the 19 survivors exhibit no significant departure from a ratio of 1.O. The error bars in Fig. 3 represent the confidence intervals from 5% to 95% and were obtained by propagating the Poisson uncertainties associated with the in vivo and in vitro translocation frequencies, as well as the 30% SD on the Hiroshima dose (Roesch 1987), using a Monte Carlo computer code.$ It is noteworthy that of the
*
Crystal Ball (Version 2), A Forecasting and Risk Management Program for the Macintosh, Comtech Services, Inc., Denver, CO.
Biodosimetry for a radiation worker 0 T. STRAUME ET AL. 100
I
I
127
Q. The point estimate of the dose equivalent obtained from the chromosome translocation results is 0.6 Sv.
UNCERTAINTIES
1 I
.01 0
I
I
1
2
I
.3
Dose (W
Fig. 3. In vivolin vitro ratios are translocation frequencies measured in blood lymphocytes of 19 Hiroshima survivors (Lucas et al. 1991) divided by the translocation frequencies expected from identical doses in vitro at the first postirradiation division. The fit parameters for the in vitro dose-response curve for acute gamma rays are a = 0.023 Gy-’ and p == 0.053 Gy-* (Lucas et al. 1991). All translocations were measured using FISH. Because Hiroshima survivors were sampled 45 y after exposure, this ratio is a measure of the combined stability and interpersonal variability of the translocation frequency in individuals a long time after whole-body exposure. If the translocation frequency for an individual is totally stable and interpersonal variations are minimal, then the ratios should not be very different from unity. It is observed that the ratios for 16 of 19 individuals are not significantly different from 1.0. Furthermore, the three that do differ significantly from unity have error bars that extend to within a factor of 2 of 1.O.
almost 10,000 cells scored in the 19 Hiroshima survivors using FISH, not a single clone has been detected. It has been shown that as gamma-ray dose rate is decreased, the quadratic coefficient (p) of the linearquadratic dose-response curve for exchange aberrations diminishes and appears to become zero at the very chronic exposure rates considered in this paper (Bauchinger et al. 1979; Lloyd and Edwards 1983; Lloyd et al. 1984). Therefore, for the radiation worker, only the linear coefficient ( a ) would have contributed to the translocation frequency. The dose equivalent in Sv was estimated from the translocation data using eqn (2):
H = K Y - k)/al X Q, (2) where Y is the number of translocations per cell measured in blood lymphocytes of the radiation worker, k is the spontaneous background frequency for translocations, and a is the linear coefficient for translocations obtained from the linear-quadratic dose-response relationship for gamma-ray-induced translocations in vitro using FISH (obtained from data in Lucas et al. 1989b, 1989~). The dose-equivalent estimate for translocations was obtained using a of 0.023 0.016 (SD) translocations per cell per Gy, and Y and k were obtained from Table 1. Because both the radiation worker and the in vitro data are for gamma rays, a value of 1.O is used for
Firm conclusions about dose must be tempered by knowledge that the long-term low-level exposures received by the radiation worker have not been fully validated biodosimetrically. However, because both translocations and GPA mutations were significantly elevated in the radiation worker (i.e., involving two different cell lineages), it appears unlikely that the result is spurious. Also, because the dicentric and micronuclei frequencies are not elevated, it is unlikely that the radiation worker has an unusually high background frequency, such as is seen in individuals with various chromosomal fragility or DNA repair disorders (Taylor 1983; Bigbee et al. 1989). For the two integrating biodosimeters (translocations and GPA), the principal uncertainties in this evaluation are related to (1) possible time-dependent instabilities, (2) interindividual and intersample variabilities, and (3) relevancy and precision of “standard” reference curves used to estimate dose. Information is available for each of these uncertainties. For translocations, it has been known for some time that the mean aberration frequency in blood lymphocytes of a radiation-exposed population appears to be stable with time postexposure (e.g., see Buckton et al. 1978). From recent data (Fig. 3 and Lucas et al. 199I), it now appears that the translocation frequency in an individual may also be stable with time postexposure. Of the 19 Hiroshima survivors evaluated in Fig. 3, the variations can be fully explained in 16 on the basis of known uncertainties in the measured parameters, and the remaining three have 90% confidence intervals that extend to within a factor of 2 of the expected value for perfect stability. The variation of the translocation frequency between individuals or between small populations has also been studied. The upper bound on the interindividual variability in translocation frequency for persons exposed several decades ago can be inferred from Fig. 3. Accounting for known variabilities, such as Poisson statistics and dose uncertainties, the upper limit on the between-person variation would appear to be less than a factor of about 2. A summary of the data available for translocations in “unexposed” human populations (Bender et al. 1988) shows that frequencies tend to range from 0.5 to -4 translocations per 1000 cells. In Hiroshima controls, the translocation frequencies are somewhat higher than those seen generally, i.e., 5.6 to 7.9 translocations per 1000 cells (Bender et al. 1988). This is consistent with our results using FISH to measure translocation frequencies in 4000 full-genomeequivalent cells from five “control” Hiroshima survivors (those that received less than 0.01 Gy from the bomb). We obtained 7.3 (SD 1.4) translocations per 1000
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cells, very similar to the results for Hiroshima cited above. The unusually high translocation frequencies observed in Hiroshima may be a result of the frequent medical x-ray exams received during routine follow-up of atomic-bomb survivors (Yamamoto et al. 1988). The frequency of 17 translocations per 1000 cells for the radiation worker is significantly higher than the frequencies measured in any of the unexposed populations discussed above, including those in Hiroshima survivors ( p < 0.005). A number of high-quality in vitro dose-response curves are available for exchange aberrations that can be used as “standards” for dose estimation from the translocation results. These include curves obtained using FISH where the reproducibility of translocation detection is within Poisson statistics (Lucas et al. 1989b, 1991). However, the uncertainties in the take-off slopes of these curves (required for biodosimetry following low-level exposures) are substantial, i.e., SDs are 50% to 100% of mean (70% for the in vitro curve used here) and contribute significantly to the overall uncertainty in the biodosimetric estimates for translocations. Information is also available on the temporal stability of GPA. For example, the dose-response relation measured in Hiroshima survivors some 45 y postexposure is consistent with the response measured in GoiHnia accident victims -1 y after the accident, as long as the differences in dose rates are taken into account. Also, the slopes (mutation frequency per Gy) of the GPA curves for Hiroshima and GoiHnia are reasonably consistent with mutation frequencies measured immediately after radiation exposure in various in vitro mutation assays (e.g., Sanderson et al. 1984; Akiyama et al. 1991). The interpersonal variability for GPA has been addressed to some extent in Fig. 1 for matched controls, and the intersample variability is illustrated by repeat measurements of the July 1989 sample from the radiation worker. Ten repeat measurements consisting of l million cells each detected 20, 18, 16, 13, 18, 20, 32, 18, 22, and 27 N 0 variant cells (mean, 20.4; SE, 1.7). In addition, we now have GPA results from studies of the general population totalling 358 unexposed controls (Bigbee et al., unpublished results). These results, which include both sexes, smokers and non-smokers, and ages from 8 to 80 y, are consistent with the range of variant cell frequencies observed in the matched controls, and both are significantly lower than the frequency measured in the radiation worker. For example, of the 358 persons evaluated, only 9 (or 2.5%) had N 0 frequencies equal to or higher than that of the radiation worker. Furthermore, when we limit the population to those (65 y of age, only 5 out of 326 persons (or 1.5%) had N 0 frequencies higher than that of the radiation worker. This clearly shows that the radiation worker has a N 0 frequency that is exceptionally high compared with the frequencies observed in the general population. The analytical reproducibility of GPA is well established through repeat measurements of samples and is
February 1992, Volume 62, Number 2
reflected in the SD for the radiation worker listed in Table 1. Very similar GPA frequencies are obtained from repeat blood samplings of the same individual as long as additional exposures have not been received. As with translocations, the uncertainty in the slope of the “standard” curve for GPA is substantial. An SD of -25% for the slope of the dose-response curve for GPA is estimated from a combination of atomic-bomb survivor data (Langlois et al. 1987; Kyoizumi et al. 1989) and data for victims of the radiation accident in Goiinia, Brazil (Straume et al. 1991). Also, DREFs for genetic effects tend to fall within the 3 to 7 range (NAS 1990). If we assume this range to represent a 90% confidence interval, an SD of 1.3 (or 26% of mean) is estimated for the DREF for GPA. 1 .oo
-
GPA (Hiroshima)
.75.50.25 -
0
I
I
I
.25L!----A 0 0
0.75
1.50
2.25
3.00
Dose equivalent (Sv)
Fig. 4. Cumulative probability distributions of dose estimates for the radiation worker obtained using GPA and translocation results. Distributions were generated using a Monte Carlo computer code that randomly samples from the uncertainty distributions estimated to be associated with each individual parameter in eqns (1) and (2) (see text). The distribution in the upper panel was obtained using the slope ofthe Hiroshima GPA curve as a standard while the center panel employed the slope of the Goidnia curve as standard. The most probable ( p = 0.50) values from the distributions are 1.5 Sv for GPA (Hiroshima), 1.2 Sv for GPA (Goiinia), and 0.7 Sv for translocations.
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
To calculate probability distributions for the estimated dose equivalents, the distributions associated with each parameter in eqns (1) and (2) were estimated and propagated using a Monte Carlo computer code (identified above) developed for such probabilistic analyses. We assigned Poisson distributions to the number of GPA and translocation events, and lognormal distributions to the slopes of the “standard” curves and the DREF. Using the SDs given above and in Table 1, and the assumption of full temporal stability for translocations and GPA, the resultant cumulative probability distributions associated with the three biodosimetrically derived doses are shown in Fig. 4.The 90% confidence intervals are 0.6 to 3.2 Sv for GPA (Hiroshima standard), 0.5 t o 2.1 Sv for GPA (Goiinia standard), and 0.2 to 2.4 Sv for translocations. We also used the Monte Carlo approach t o deter-, mine the effect on overall uncertainty of increasing the precision of certain individual parameters. For exam-, ple, if we would have scored 6000 cells for translocations in the radiation worker instead ofjust 482, the 90% confidence interval would have improved only slightly, i.e., 0.3 t o 2.2 Sv. To achieve substantial reductions in uncertainties, the precision in the low-dose linear coefficient ( a )of the translocation dose-response curve would have to be increased. This could be accomplished by an in vitro experiment using FISH and should be of high priority. For GPA, substantial reduction in uncertainties would likely be achieved if the assay could be developed in vitro, thus permitting experimental measurement of various radiobiological variables. It is also likely that helpful information will become available from GPA studies now underway on persons exposed at Chernobyl. It is recommended that parallel GPA and FISH studies be performed on a group of 20 to 30 upper-level exposed radiation workers, with matched controls. CONCLUSION Dose-equivalent estimates for the radiation worker in the -0.4 to -2 Sv range, which include the value in the worker’s dosimetry records, cannot be confidently excluded at this time based on state-of-the-art biodosimetry. Values larger than 2.5 Sv (as believed by the worker) appear to be unlikely.
Acknowledgement(s)-This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract W-7405-Eng-48 with support from the Defense Nuclear Agency, Projects 89-870 and 90-819.
REFERENCES Akiyama, M.; Kusunoki, Y.; Shigeko, U.; Yuko, H.; Nakamura, N.; Kyoizumi, S. Evaluation of four somatic mutation assays as biological dosimeters in humans. In: Radiation research: A twentieth-century perspective. San Diego’: Academic Press, Inc. (in press).
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Bajerska, A.; Liniecki, J. The yield of chromosomal aberrations in rabbit leukocytes after irradiation in vitro and in vivo. Mutat. Res. 27:271-281; 1975. Bauchinger, M.; Schmid, E.; Dresp, J. Calculation of the doserate dependence of the dicentric yield after ‘j0Co-y-irradiation of human lymphocytes. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 35:229-233; 1979. Bender, M. A.; Awa, A. A.; Brooks, A. L.; Evans, H. J.; Groer, P. G.; Littlefield, L. G.; Pereira, C.; Preston, R. J.; Wachholtz, B. W. Current status of cytogenetic procedures to detect and quantify previous exposuresto radiation. Mutat. Res. 196:103-159; 1988. Bigbee, W. L.; Langlois, R. G.; Swift, M.; Jensen, R. H. Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia. Am. J. Hum. Genet. 44:402-408; 1989. Bigbee, W. L.; Wyrobek, A. J.; Langlois, R. G.; Jensen, R. H.; Everson, R. B. The effect of chemotherapy on the in vivo frequency of glycophorin A “null” variant erythrocytes. Mutat. Res. 240:165-175; 1990. Brenner, D. J. Significance of neutrons from the atomic bomb at Hiroshima for revised radiation risk estimates. Health Phys. 60:439-442; 199 1. Brewen, J. G.; Gengozian, N. Radiation-induced human chromosome aberrations. 11. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes. Mutat. Res. 13:383-39 1; 197 1. Buckton, K. E.; Hamilton, G. E.; Paton, L.; Langlands, A. 0. Chromosome aberrations in irradiated ankylosing spondylitis. In: Mutagen-induced chromosome damage in man. London: University Press: 1978:142- 150. Buckton, K. E.; Langlands, A. 0.;Smith, P. G.;Woodcock, G. E.; Looby, P. C. Further studies on chromosome aberrations: Production after whole-body irradiation in man. Int. J. Radiat. Biol. 19:369-378; 1971. Eastmond, D. A.; Tucker, J. D. Identification of aneuploidyinducing agents using cytokinesis-blockedhuman lymphocytes and an antikinetochore antibody. Environ. Molec. Mutagen. 13:34-43; 1989. Evans, H. C.; Buckton, K. E.; Hamilton, G. E.; Carothers, A. Radiation-induced chromosome aberrations in nucleardockyard workers. Nature 27753 1-534; 1979. Evans, H. C.; Buckton, K. E.; Sumner, A. T. Cytological mapping of human chromosomes: Results obtained with quinacrine fluorescence and the acetic-saline-Giemsatechniques. Chromosoma 35:310-325; 1971. Hakoda, M.; Akiyama, M.; Kyoizumi, S.; Awa, A. A.; Yamakido, M.; Otake, M. Increased somatic cell mutation frequency in atomic bomb survivors. Mutat. Res. 201:3948; 1988. Krepinsky, A. B.; Heddle, J. A. Micronuclei as a rapid and inexpensive measure of radiation-induced chromosomal aberrations. In: Ishihara, T.; Sasaki, M. S., eds. Radiationinduced chromosome damage in man. New York Alan R. Liss, Inc.; 1983:93-109. Kyoizumi, S.; Nakamura, N.; Hakoda, M.; Awa, A. A.; Bean, M. A.; Jensen, R. H.; Akiyama, M. Detection of somatic mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors using a single beam flow sorter. Cancer Res. 49581-588: 1989. Langlois, R. G.; Bigbee, W. L.; Jensen, R. H. Measurements of the frequency of human erythrocytes with gene expression loss phenotypes at the glycophorin A locus. Hum. Genet. 74:353-362; 1986. Langlois, R. G.; Bigbee, W. L.; Kyoizumi, S.; Nakamura, N.;
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Bean, M. A.; Akiyama, M.; Jensen, R. H. Evidence for increased somatic cell mutations at the glycophorin A locus in atomic bomb survivors. Science 236:445-448; 1987. Langlois, R. G.; Nisbet, B.; Bigbee, W. L.; Ridinger, D. L.; Jensen, R. H. An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans. Cytometry 11513-521; 1990. Littlefield, L. G.; Joiner, E. E.; Hubner, K. F. Cytogenetic techniques in biological dosimetry: Overview and example of dose estimation in 10 persons exposed to gamma radiation in the 1984 Mexican 6oCoaccident. In: Mettler, F. A., Jr.; Kelsey, C. A.; Ricks, R. C., eds. Medical management of radiation accidents. Boca Raton, FL: CRC Press, Inc.; 1990:109- 126. Littlefield, L. G.; Sayer, A. M.; Frome, E. L. Comparisons of dose-response parameters for radiation-induced acentric fragments and micronuclei observed in cytokinesis-arrested lymphocytes. Mutagenesis 4:265-270; 1989. Lloyd, D. C.; Edwards, A. A. Chromosome aberrations in human lymphocytes: Effect of radiation quality, dose, and dose rate. In: Ishihara, T.; Sasaki, M. S., eds. Radiationinduced chromosome damage in man. New York: Alan R. Liss, Inc.; 1983:23-49. Lloyd, D. C.; Edwards, A. A.; Prosser, J. S.; Corp, M. J. The dose-response relationship obtained at constant irradiation times for the induction of chromosome aberrations in human lymphocytes by 6oCogamma rays. Radiat. Env. Biophys. 23: 179- 189; 1984. Lloyd, D. C.; Purrott, R. J.; Reeder, E. J. The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people. Mutat. Res. 72523-532; 1980. Lucas, J.; Awa, A.; Kodama, Y.; Nakano, M.; Ohtaki, K.; Pinkel, D.; Poggensee, M.; Straume, T.; Weier, U.; Gray, J. Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. Livermore, CA: Lawrence Livermore National Laboratory; Report UCRL107165; 1991. Lucas, J.; Tenjin, T.; Straume, T.; Pinkel, D.; Gray, J. Rapid detection of human chromosome aberrations using fluorescence in situ hybridization. In: Proceedings of the 27th Hanford Life Sciences Symposium on Health and the Environment. Richland, WA: Battelle Press; 1989a:149155. Lucas, J.; Tenjin, T.; Straume, T.; Pinkel, D.; Moore, D.; Litt, M.; Gray, J. Rapid determination of human chromosome translocation frequency using a pair of chromosome-specific DNA probes. Int. J. Radiat. Biol. 56:35-44; 1989b. Lucas, J.; Tenjin, T.; Straume, T.; Pinkel, D.; Moore, D.; Litt, M.; Gray, J. Letter to the editor. Int. J. Radiat. Biol. 56:20 1; 1989c. Mendelsohn, M. L.; Mayall, B. H.; Bogart, E.; Moore, D. H.; Perry, B. H. DNA content and DNA-based centromeric
February 1992, Volume 62, Number 2 index of the 24 human chromosomes. Science 179: i 1261129; 1973. Messing, K.; Ferraris, J.; Bradley, W. E. C.; Swatz, J.; Seifert, A. M. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation. Health Phys. 57537-544; 1989. Minkler, J.; Stetka, D., Jr.; Carrano, A. V. An ultraviolet light source for consistent differential staining of sister chromatids. Stain Technol. 53: 359-360; 1978. National Academy of Sciences. Health effects of exposure to low levels of ionizing radiation (BEIR V). Washington, DC: U.S. National Academy of Sciences, National Academy Press; 1990. National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States. Bethesda, MD: NCRP; NCRP Report No. 93; 1987. Perry, P.; Wolff, S. New Giemsa method for the differential staining of sister chromatids. Nature 25 1:156- 158; 1974. Pinkel, D.; Straume, T.; Gray, J. W. Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. U.S.A. 83:2934-2938; 1986. Ramalho, A. T.; Nascimento, A. C. H.; Natarajan, A. T. Dose assessments by cytogenetic analysis in the Goidnia (Brazil) radiation accident. Radiat. Prot. Dosim. 23:97-100; 1988. Roesch, W. C. (ed.). Final report on US.-Japan reassessment of atomic bomb radiation dosimetry in Hiroshima and Nagasaki. Hiroshima: Radiation Effects Research Foundation; 1987. Sanderson, B. J. S.; Dempsey, J. L.; Morley, A. A. Mutations in human lymphocytes: Effect of x- and UV-irradiation. Mutat. Res. 140:223-227; 1984. Schmid, E.; Bauchinger, M.; Bunde, E.; Ferbert, H. F.; Lieven, H. V. Comparison of the chromosome damage and its dose response after medical whole-body exposures to 6oCo y-rays and irradiation of blood in vitro. Int. J. Radiat. Biol. 26:31-37; 1974. Straume, T.; Langlois, R. G.; Lucas, J.; Jensen, R. H.; Bigbee, W. L.; Ramalho, A. T.; Brandso-Mello, C. E. Novel biodosimetry methods applied to the victims of the Goilnia accident. Health Phys. 60:7 1-76; 1991. Taylor, A. M. R. The effect of radiation on the chromosomes of patients with an unusual cancer susceptibility. In: Radiation-induced chromosome damage in man. New York: Alan R. Liss, Inc.; 1983:167-199. Tonomura, A.; Kishi, K.; Saito, F. Types and frequencies of chromosome aberrations in peripheral lymphocytes of general populations. In: Radiation-induced chromosome damage in man. New York Alan R. Liss, Inc.; 1983:605616. Yamamoto, 0.; Shigetoshi, A.; Russell, W. J.; Fujita, S.; Sawada, S. Medical x-ray exposure doses as contaminants of atomic bomb doses. Health Phys. 54:257-269; 1988. M M
Abstract-Four state-of-the-art biodosimeters-GPA mutations, chromosome translocations, micronuclei, and dicentrics-were used to evaluate a radiation worker who believed that the official dosimetry records substantially underestimated his actual dose. Dosimetry records indicated that the worker received 0.56 Sv during a 36-y employment history, always within the dose limits. In contrast, the worker believed that his dose equivalent may have been more than 2.5 Sv because much of the exposure was received during the early days of health physics when dosimetry capabilities and practices were not as good as they are today. Because there are no biodosimetric assays that have been fully validated for the long-term low-level exposures received by the worker, we did not expect to obtain particularly useful point-estimates of dose. However, because the discrepancy between the dosimetry records and the worker’s belief was so large, we believed that biodosimetry using multiple assays together with probabilistic assessment of the uncertainties would provide useful insight. Results showed that the frequencies of chromosome translocations and GPA mutations (stable biodosimeters) were significantly elevated when compared with those for unexposed controls. Our analysis suggests that dose-equivalent estimates in the -0.4 to -2 S v range (which include the value in the dosimetry records) cannot be confidently excluded at this time based on biodosimetry; however, a value greater than 2.5 S v appears unlikely. Important new information on the temporal stability of chromosome translocations is also presented. Health Phys. 62(2):122-130; 1992 Key words: biodosimetry; translocation; exposure, occupational; nuclear workers
INTRODUCTION A RADIATIONworker who disagreed with official exposure records was evaluated biodosimetrically. According to dosimetry records, the worker received 0.56 Sv during more than three decades of exposure, always within the occupational dose limits. However, because of dosimetry capabilities and practices during his initial employment (1953 to 1965, the early days of health physics), the worker believed his dose during that time * University of California, Lawrence Livermore National Laboratory, P.O.Box 5507, Livermore, CA 94550. (Manuscript received 22 August 1991; revised manuscript received 19 September 1991, accepted 15 October 1991) 0017-9078/92/$3.00/0 Copyright 0 1992 Health Physics Society 122
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
malho et al. 1988; Littlefield et al. 1990). These and numerous other studies demonstrate the utility of dicentrics for dose assessments within a few weeks after acute or subacute exposure. However, it is well established that dicentrics are not particularly useful for quantifying exposures that occurred a long time ago or those protracted over months or years (e.g., Buckton et al. 1978; Littlefield et al. 1990). Somatic mutations and chromosome aberrations have previously been detected in recently exposed radiation workers, e.g., elevated HPRT mutations (Messing et al. 1989) and unstable chromosome aberrations (Evans et al. 1979; Lloyd et al. 1980). However, the assays used in those studies present problematical biodosimetric interpretations for long-term exposures or for those exposed a long time ago because of the temporal instability of those assays (Hakoda et al. 1988; Littlefield et al. 1990).
MATERIALS AND METHODS Case history The dosimetry records for this 59-y-old white 'radiation worker indicated that his occupational exposure to radiation began in 1953. At the time of blood sampling for this biodosimetry study in 1989, dosimetry records indicated that he had received a whole-body dose equivalent of 0.56 Sv from all work-related sources. Gamma rays contributed 0.54 Sv and neutrons 0.02 Sv (i,e., 0.002 Gy). About 90% of the dose equivalent was received from 1953 to 1967; the remainder was received between 1968 and 1985. Since 1985, records indicate that he has received only 0.00048 Sv. Internal exposures were limited to a combined total of 0.0018 Sv from 241Am,239Pu,and 238U. For comparison, the lifetime dose equivalent to the bone marrow from natural background radiation was estimated to be 0.066 Sv (NCRP 1987), only a fraction of that received occupationally. An interview with the radiation worker indicated that he was a nonsmoker and did not appear to have a medical or other non-occupational exposure history that could be a potential source of confounding factors. Blood sample preparation Peripheral blood was obtained with informed consent in January 1989 and again in June 1989 by the Medical Department at LLNL. Each of the two samples consisted of 15 mL of blood drawn into a heparinized vacutainer. The samples were coded with an identification number, and the donor was determined to be a MN heterozygote by serotyping with commercial antiM and anti-N sera (Ortho Diagnostics, Raritan, NJ). The blood samples were then divided into aliquots for use in each of the assays. The blood used for the GPA assay was fixed, and erythrocytes were labeled with monoclonal antibodies and analyzed on a flow cytometer (Langlois et al. 1990). The blood for aberration analysis was cultured with
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phytohemagglutinin (PHA) to stimulate growth of T lymphocytes and 25 pM bromodeoxyuridine (BrdUrd) to discriminate between first- and later-division cells. Colcemid was added at 48 h to arrest the lymphocytes in metaphase. At 52 h the cells were swollen with 0.075 M KCl and fixed with 3:l methanol and acetic acid, and microscope slides were prepared for cytogenetic analysis. Cells for FISH were cultured in the same manner except that BrdUrd was not included in the culture medium. For micronuclei, lymphocytes were isolated and cultured as described previously (Eastmond and Tucker 1989). Cytochalasin B was added at 44 h, and at 72 h the cells were centrifuged onto clean dry slides, quickly dried, and fixed in 100% methanol for 15 min. The slides were stored in NZatmosphere at -20°C until immediately before staining. GPA somatic mutation assay The GPA assay has been described previously (Langlois et al. 1986, 1990). The assay uses immunofluorescence labeling and flow cytometry to enumerate the frequency of red blood cells lacking expression of one allelic form of the cell-surface sialoglycoprotein GPA, presumably as a result of allele-loss mutations at the GPA locus in erythroid precursor cells. The GPA gene is located on human chromosome 4 and is present in the population as two common alleles at approximately equal frequencies, so that -50% of the population is MN heterozygous. The proteins coded by these two alleles carry the M and N blood group antigens and differ by only two amino acids out of 131. The GPA assay was originally developed using a dual-laser flow sorter (Langlois et al. 1986); the data in this paper were obtained with a modified method (designated the BR6 assay) performed on a simpler flow cytometer (Langlois et al. 1990). This assay enumerates the frequency of NQ)variant cells, i.e., cells that have lost expression of the M allele but express the N allele normally. Chromosome aberration analysis using FISH Translocation detection using FISH is illustrated in Fig. 1. The FISH method, described previously, can be used to detect both translocations and dicentrics (Pinkel et al. 1986; Lucas et al. 1989a, 1989b, 1991; Straume et al. 1991). FISH was performed using two cocktails of composite DNA probes, one specific for chromosome 4 only and the other specific for chromosomes 1,3, and 4 combined. The use of two different cocktails had no special significance other than the availability of DNA probes. For the January 1989 sample, the hybridization target was chromosome 4 only, for whichf, (the fraction of the genome hybridized) is 0.0668 (Mendelsohn et al. 1973). For the July 1989 sample, the hybridization target was chromosomes 1, 2, and 4 combined, for whichJ; is 0.238 (Mendelsohn et al. 1973). The hybridization target for the FISH controls was chromosomes 1, 3, and 4 combined, for whichf, is 0.223 (Mendelsohn et al. 1973), or chromosome 4. To compare results using this approach with
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February 1992, Volume 62, Number 2
Fig. 1. Translocation detection in human lymphocytes using FISH. Left: Metaphase chromosomes from a normal cell showing two homologous #4 chromosomes that have been selectively stained yellow using FISH. The other chromosomes have been counterstained red. Right: Metaphase chromosomes from an irradiated cell showing one normal #4 chromosome and another that has broken into two pieces and translocated to the correspondingpieces from a broken red chromosome (arrows).
results from conventional cytogenetic methods, the data obtained from the smaller target are scaled up to equal the full genome. The method used to scale to the full genome has been described previously (Lucas et al. 1989~). Conventional aberration analysis and G-banding Established methods were used to stain metaphase chromosomes on slides and to score for dicentrics and translocations. For unbanded chromosomes, slides were stained using the fluorescence plus Giemsa method (Ferry and Wolff 1974; Minkler et al. 1978) to ensure that only first-division cells were scored. For banded chromosomes, slides were treated with trypsin and stained with Giemsa according to established techniques (Evans et al. 1971). For each type of analysis, only cells with 45 or more centromeres were scored. Micronucleus analysis The chromosomes were stained as described previously (Eastmond and Tucker 1989) with an antikinetochore antibody' and a fluoresceinated rabbit antihuman second antibody. Counterstaining was achieved with 0.25 pg/mL DAPI, and slides were scored with simultaneous phase contrast and DAPI excitation. The presence or absence of a kinetochore label within each micronucleus was determined by viewing the cells under fluorescence excitation. Antibodies, Inc., Davis, CA.
RESULTS Results for both integrating and non-integrating biodosimeters are listed in Table 1. Frequencies for GPA, chromosome translocations, micronuclei, and dicentrics are listed for the radiation worker as well as for unexposed controls. The data for the radiation worker are mean values from two separate blood samplings, one in January 1989 and another in July 1989. For GPA, a total of 5 X lo6 cells were measured for each assay run. Duplicate assay runs were made for each of the two blood samplings. The N 0 frequency of (23 3) X is the mean kSD for the radiation worker. The control value of (8 f 3.5) X is the mean +SD for six unexposed individuals matched to the radiation worker by age, sex, non-smoking history, and time period of assay. The GPA variant frequency from the radiation worker is significantly higher than control values, as indicated in Table 1 ( p = 0.006). This is illustrated further in Fig. 2, where the N 0 frequencies measured in the six matched controls are compared with the frequencies measured in the two samples obtained from the radiation worker. Additional information on control frequencies is presented in the Uncertainties section. The values for translocations in Table 1 include combined results from both FISH and G-banding. A total of eight translocations were detected in 482 full genome-equivalent cells from the radiation worker, which is significantly higher than the control frequency ( p = 0.0004). Results for FISH and G-banding were also statistically significant when evaluated separately:
+
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
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Table 1. Measurement results for integrating and non-integratingbiodosimeters. Biodosimeter
Cells
Radiation worker Events Frequency
Cells
Unexposed controls" Events Frequency
Statisticsb ~
Integrating GPA Translocations Nonintegrating Micronuclei Dicentrics
20 X lo6 482d
455' 8
(23 f 3) >: (17 f 6) X
30 X lo6 2278'
240 6
(8 & 3.5) X p=0.006 (2.6 f 1) X low3 p=0.0004
(1 1 f 3) x 10-3 1000 9 (9 2 3) x lo-) p = o x (1.5 1.5) x 10-3 21,000 26 (1.2 f 0.3) X p=0.50 a Laboratory controls were obtained by the same methods and scorers and during the same general time frame as the results for the radiation worker being evaluated here. GPA based on six controls; translocations based on four controls; micronuclei based on one control; dicentrics based on 1 17 controls. The p-value for GPA is based on comparing the radiation worker mean with means and standard deviations from six matched controls using t-statistic with 5 degrees of freedom. For translocations, micronuclei, and dicentrics,p-values are based on a test for equal binomial proportions. N o variant cells detected in four independent measurements, two each from two separate blood samplings. Includes 332 full genome-equivalentcells scored using FISH (four events) and 150 cells scored using G-banding (four events). FISH results were scaled to full genome using method in Lucas et al. (1989~).The translocation frequency obtained for the radiation worker using FISH is not significantly different from that obtained using G-banding; p = 0.12. Full genome-equivalent cells scored using FISH. Conventional staining, 350 cells; FISH, 332 full genome-equivalent cells.
1000 682'
11
I
'
FISH, p = 0.005, and G-banding, p = 4 x To combine translocation results obtained using these two different methods, it was necessary to 1) scale the FISH results to full genome equivalents and 2) verify that FISH and banding produce equivalent dose-response
"
C-1 C-2 C-3 C-4 C-5 C-6 RW RW Aug 88 Mar 90 Mar 90 Mar 90 June 90 July 90 Jan 89 July 89
Fig. 2. Comparison of GPA N 0 variant cell frequencies measured in six controls (white bars) and in the two samplings from the radiation worker (gray bars). The group mean + I SD for the six controls is indicated as are the dates when the blood samples were obtained. The controls were matched to the worker by sex, age (within 10 y), and non-smoking history, as well as the general time interval during which the samples
were obtained and analyzed.
results. Scaling to full genome-equivalents was accomplished using the method of Lucas et al. (1989~)and thefs values listed in the Materials and Methods section. A large study comparing FISH and G-banding in Hiroshima atomic-bomb survivors has recently been completed in this laboratory, and results clearly demonstrate that these two methods produce equivalent translocation frequencies (Lucas et al. 1991). Four unexposed donors from our laboratory were used as controls for translocations. Controls were matched to the radiation worker for non-smoking history, sex, and general time period of sample acquisition and analysis. Results are listed in Table 1. The controls for translocations were 40 to 45 y of age, 14 to 19 y younger than the radiation worker. However, the frequencies of stable chromosome aberrations do not appear to increase significantly between 40 and 60 y of age (Tonomura et al. 1983; Bender et al. 1988). Results for non-integrating biodosimeters (micronuclei and dicentrics) are also listed in Table 1. Nonintegrating biodosimeters will only detect relatively recent radiation exposures (within the past few years). As expected from the worker's exposure history, the results for micronuclei and dicentrics were not significantly different from unexposed controls. Eleven micronuclei per 1000 cells were observed in the radiation worker and 9 per 1000 cells in the control ( p = 0.33). For dicentrics, one event was observed in 682 full genomeequivalent cells scored, which is also not significantly different from the frequency in controls ( p = 0.50).
BIODOSIMETRY AND DISCUSSION For GPA, dose-response curves from previous studies (Langlois et al. 1987; Straume et al. 1991) were used as standards to convert the observed mean N 0 variant cell frequency into dose. The dose equivalent
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( H ) in Sv was estimated from the GPA data using eqn (1): H
=
[(NQ),, - NQ)h)/slopeof standard curve]
X
Q,
(1) where N 0 , is the frequency of NQ) variants measured (Table 1); Nab is in the radiation worker, 23 X the background frequency measured in six matched (Table 1); and Q is the unexposed controls, 8 x radiation quality factor. The slope ( N 0 per Gy) of the “standard” GPA dose-response curve was estimated from two different sources of data, Hiroshima atomicbomb survivors and radiation accident victims in Goiinia, Brazil. In the case of Hiroshima survivors, the GPA dose response was obtained from previously published data (Langlois et al. 1987) that were reanalyzed here using DS86 marrow doses instead of the old T65DR tissue kerma values used in Langlois et al. According to DS86, the radiation from the Hiroshima bomb was almost pure gamma rays, with only -0.5 to 1% of the dose from neutrons (Roesch 1987). This small neutron component in DS86 is estimated to have contributed only -10% of the effect in Hiroshima (Brenner 1991) and is therefore ignored in the estimation of the dose equivalent (i.e., the Q for Hiroshima radiation is assumed to equal 1). The slope for the Hiroshima GPA curve was obtained from a subset of the Hiroshima GPA data (Langlois et al. 1987) that contained 58 individuals for whom DS86 dose information was available. This analysis included 18 distally exposed survivors who received DS86 marrow doses less than 0.005 Gy and 40 proximally exposed survivors who received DS86 marrow doses ranging from 0.1 to 5 Gy (mean, 1.2 Gy). The resulting linear fit parameters for GPA allele-loss and a slope of variants were a y-intercept of 11 x 48 x per Gy ( r = 0.46). Independent GPA measurements by Japanese investigators (Kyoizumi et al. 1989) also provided N 0 values for Hiroshima atomicbomb survivors using DS86 doses to the marrow; their Gy-’, similar to the value slope estimate was 40 x obtained here. Because previous results suggest that GPA mutations are stable with time postexposure (Langlois et al. 1987; Kyoizumi et al. 1989; Straume et al. 1991), the use of results for Hiroshima atomic-bomb survivors (exposed -40 y previously) as a standard curve to convert results from a radiation worker who received most of his dose -30 y ago would appear reasonable as long as the differences in dose rates are considered. The radiation exposure received by the worker was not acute like that received by atomic-bomb survivors but rather was highly protracted and therefore is expected to be less effective. The BEIR V Committee (NAS 1990) recently reviewed the experimental data on genetic effects and determined that low-dose-rate gamma rays are -5 times less effective than high-doserate gamma rays (this is the dose-rate effectiveness
February 1992, Volume 62, Number 2
factor [DREF]). Thus, the slope for GPA from Hiroshima survivors was divided by 5 to account for the difference between the acute exposure from the bomb and the chronic exposure in this individual. The pointestimate of dose equivalent obtained from the GPA data is 1.5 Sv when using 1/5 times the slope for Hiroshima in eqn (1) (uncertainties are discussed in a subsequent section). GPA results from the radiation accident victims in Goifnia, Brazil were also used as a “standard” for dose estimation (Straume et al. 1991). In this case, 13’Cs gamma-ray exposure was received during a period of about 2 wk, approximating fractionated or chronic exposure conditions. Based on dose-rate studies (e.g., Lloyd and Edwards 1983), the dose-response relationship for the Goiinia accident victims is expected to be nearly independent of dose rate, as is the case with the radiation worker. Thus, at first approximation,a DREF is not required. A linear regression was fitted to the Goilnia GPA data using dose estimates from Ramalho Gy-l et al. (1988). The resultant slope is 12.8 X and, when used in eqn (1) with a Q of 1.O, the point estimate of the dose equivalent for the radiation worker is 1.2 Sv. Dose is also estimated from the translocation data. To obtain these estimates, we relied on (1) our previous in vitro results for translocations obtained using FISH (Lucas et al. 1989b, 1989c, 1991), (2) information demonstrating that the yields of aberrations induced in lymphocytes after in vitro exposures are identical to those observed after in vivo exposures (Brewen and Gengozian 1971; Buckton et al. 1971; Schmid et al. 1974; Bajerska and Liniecki 1975; Littlefield et al. 1990), and (3) the observation that translocations are stable with time postexposure (Buckton et al. 1978; Lucas et al. 1991; Straume et al. 1991). In this connection, it is indeed significant that the translocation frequencies measured using FISH in atomic-bomb survivors some 45 y after exposure compare well with predictions based on in vitro measurements evaluated in first-division cells. This is illustrated in Fig. 3, where the plotted ratios are the translocation frequencies measured in lymphocytes of 19 Hiroshima survivors divided by the frequencies obtained in vitro from first-division cells. If translocation frequencies in individual survivors have remained unchanged since 1945, they should be identical to those obtained in vitro and the ratios should be unity. This is essentially what is observed in Fig. 3, where 16 out of the 19 survivors exhibit no significant departure from a ratio of 1.O. The error bars in Fig. 3 represent the confidence intervals from 5% to 95% and were obtained by propagating the Poisson uncertainties associated with the in vivo and in vitro translocation frequencies, as well as the 30% SD on the Hiroshima dose (Roesch 1987), using a Monte Carlo computer code.$ It is noteworthy that of the
*
Crystal Ball (Version 2), A Forecasting and Risk Management Program for the Macintosh, Comtech Services, Inc., Denver, CO.
Biodosimetry for a radiation worker 0 T. STRAUME ET AL. 100
I
I
127
Q. The point estimate of the dose equivalent obtained from the chromosome translocation results is 0.6 Sv.
UNCERTAINTIES
1 I
.01 0
I
I
1
2
I
.3
Dose (W
Fig. 3. In vivolin vitro ratios are translocation frequencies measured in blood lymphocytes of 19 Hiroshima survivors (Lucas et al. 1991) divided by the translocation frequencies expected from identical doses in vitro at the first postirradiation division. The fit parameters for the in vitro dose-response curve for acute gamma rays are a = 0.023 Gy-’ and p == 0.053 Gy-* (Lucas et al. 1991). All translocations were measured using FISH. Because Hiroshima survivors were sampled 45 y after exposure, this ratio is a measure of the combined stability and interpersonal variability of the translocation frequency in individuals a long time after whole-body exposure. If the translocation frequency for an individual is totally stable and interpersonal variations are minimal, then the ratios should not be very different from unity. It is observed that the ratios for 16 of 19 individuals are not significantly different from 1.0. Furthermore, the three that do differ significantly from unity have error bars that extend to within a factor of 2 of 1.O.
almost 10,000 cells scored in the 19 Hiroshima survivors using FISH, not a single clone has been detected. It has been shown that as gamma-ray dose rate is decreased, the quadratic coefficient (p) of the linearquadratic dose-response curve for exchange aberrations diminishes and appears to become zero at the very chronic exposure rates considered in this paper (Bauchinger et al. 1979; Lloyd and Edwards 1983; Lloyd et al. 1984). Therefore, for the radiation worker, only the linear coefficient ( a ) would have contributed to the translocation frequency. The dose equivalent in Sv was estimated from the translocation data using eqn (2):
H = K Y - k)/al X Q, (2) where Y is the number of translocations per cell measured in blood lymphocytes of the radiation worker, k is the spontaneous background frequency for translocations, and a is the linear coefficient for translocations obtained from the linear-quadratic dose-response relationship for gamma-ray-induced translocations in vitro using FISH (obtained from data in Lucas et al. 1989b, 1989~). The dose-equivalent estimate for translocations was obtained using a of 0.023 0.016 (SD) translocations per cell per Gy, and Y and k were obtained from Table 1. Because both the radiation worker and the in vitro data are for gamma rays, a value of 1.O is used for
Firm conclusions about dose must be tempered by knowledge that the long-term low-level exposures received by the radiation worker have not been fully validated biodosimetrically. However, because both translocations and GPA mutations were significantly elevated in the radiation worker (i.e., involving two different cell lineages), it appears unlikely that the result is spurious. Also, because the dicentric and micronuclei frequencies are not elevated, it is unlikely that the radiation worker has an unusually high background frequency, such as is seen in individuals with various chromosomal fragility or DNA repair disorders (Taylor 1983; Bigbee et al. 1989). For the two integrating biodosimeters (translocations and GPA), the principal uncertainties in this evaluation are related to (1) possible time-dependent instabilities, (2) interindividual and intersample variabilities, and (3) relevancy and precision of “standard” reference curves used to estimate dose. Information is available for each of these uncertainties. For translocations, it has been known for some time that the mean aberration frequency in blood lymphocytes of a radiation-exposed population appears to be stable with time postexposure (e.g., see Buckton et al. 1978). From recent data (Fig. 3 and Lucas et al. 199I), it now appears that the translocation frequency in an individual may also be stable with time postexposure. Of the 19 Hiroshima survivors evaluated in Fig. 3, the variations can be fully explained in 16 on the basis of known uncertainties in the measured parameters, and the remaining three have 90% confidence intervals that extend to within a factor of 2 of the expected value for perfect stability. The variation of the translocation frequency between individuals or between small populations has also been studied. The upper bound on the interindividual variability in translocation frequency for persons exposed several decades ago can be inferred from Fig. 3. Accounting for known variabilities, such as Poisson statistics and dose uncertainties, the upper limit on the between-person variation would appear to be less than a factor of about 2. A summary of the data available for translocations in “unexposed” human populations (Bender et al. 1988) shows that frequencies tend to range from 0.5 to -4 translocations per 1000 cells. In Hiroshima controls, the translocation frequencies are somewhat higher than those seen generally, i.e., 5.6 to 7.9 translocations per 1000 cells (Bender et al. 1988). This is consistent with our results using FISH to measure translocation frequencies in 4000 full-genomeequivalent cells from five “control” Hiroshima survivors (those that received less than 0.01 Gy from the bomb). We obtained 7.3 (SD 1.4) translocations per 1000
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cells, very similar to the results for Hiroshima cited above. The unusually high translocation frequencies observed in Hiroshima may be a result of the frequent medical x-ray exams received during routine follow-up of atomic-bomb survivors (Yamamoto et al. 1988). The frequency of 17 translocations per 1000 cells for the radiation worker is significantly higher than the frequencies measured in any of the unexposed populations discussed above, including those in Hiroshima survivors ( p < 0.005). A number of high-quality in vitro dose-response curves are available for exchange aberrations that can be used as “standards” for dose estimation from the translocation results. These include curves obtained using FISH where the reproducibility of translocation detection is within Poisson statistics (Lucas et al. 1989b, 1991). However, the uncertainties in the take-off slopes of these curves (required for biodosimetry following low-level exposures) are substantial, i.e., SDs are 50% to 100% of mean (70% for the in vitro curve used here) and contribute significantly to the overall uncertainty in the biodosimetric estimates for translocations. Information is also available on the temporal stability of GPA. For example, the dose-response relation measured in Hiroshima survivors some 45 y postexposure is consistent with the response measured in GoiHnia accident victims -1 y after the accident, as long as the differences in dose rates are taken into account. Also, the slopes (mutation frequency per Gy) of the GPA curves for Hiroshima and GoiHnia are reasonably consistent with mutation frequencies measured immediately after radiation exposure in various in vitro mutation assays (e.g., Sanderson et al. 1984; Akiyama et al. 1991). The interpersonal variability for GPA has been addressed to some extent in Fig. 1 for matched controls, and the intersample variability is illustrated by repeat measurements of the July 1989 sample from the radiation worker. Ten repeat measurements consisting of l million cells each detected 20, 18, 16, 13, 18, 20, 32, 18, 22, and 27 N 0 variant cells (mean, 20.4; SE, 1.7). In addition, we now have GPA results from studies of the general population totalling 358 unexposed controls (Bigbee et al., unpublished results). These results, which include both sexes, smokers and non-smokers, and ages from 8 to 80 y, are consistent with the range of variant cell frequencies observed in the matched controls, and both are significantly lower than the frequency measured in the radiation worker. For example, of the 358 persons evaluated, only 9 (or 2.5%) had N 0 frequencies equal to or higher than that of the radiation worker. Furthermore, when we limit the population to those (65 y of age, only 5 out of 326 persons (or 1.5%) had N 0 frequencies higher than that of the radiation worker. This clearly shows that the radiation worker has a N 0 frequency that is exceptionally high compared with the frequencies observed in the general population. The analytical reproducibility of GPA is well established through repeat measurements of samples and is
February 1992, Volume 62, Number 2
reflected in the SD for the radiation worker listed in Table 1. Very similar GPA frequencies are obtained from repeat blood samplings of the same individual as long as additional exposures have not been received. As with translocations, the uncertainty in the slope of the “standard” curve for GPA is substantial. An SD of -25% for the slope of the dose-response curve for GPA is estimated from a combination of atomic-bomb survivor data (Langlois et al. 1987; Kyoizumi et al. 1989) and data for victims of the radiation accident in Goiinia, Brazil (Straume et al. 1991). Also, DREFs for genetic effects tend to fall within the 3 to 7 range (NAS 1990). If we assume this range to represent a 90% confidence interval, an SD of 1.3 (or 26% of mean) is estimated for the DREF for GPA. 1 .oo
-
GPA (Hiroshima)
.75.50.25 -
0
I
I
I
.25L!----A 0 0
0.75
1.50
2.25
3.00
Dose equivalent (Sv)
Fig. 4. Cumulative probability distributions of dose estimates for the radiation worker obtained using GPA and translocation results. Distributions were generated using a Monte Carlo computer code that randomly samples from the uncertainty distributions estimated to be associated with each individual parameter in eqns (1) and (2) (see text). The distribution in the upper panel was obtained using the slope ofthe Hiroshima GPA curve as a standard while the center panel employed the slope of the Goidnia curve as standard. The most probable ( p = 0.50) values from the distributions are 1.5 Sv for GPA (Hiroshima), 1.2 Sv for GPA (Goiinia), and 0.7 Sv for translocations.
Biodosimetry for a radiation worker 0 T. STRAUME ET AL.
To calculate probability distributions for the estimated dose equivalents, the distributions associated with each parameter in eqns (1) and (2) were estimated and propagated using a Monte Carlo computer code (identified above) developed for such probabilistic analyses. We assigned Poisson distributions to the number of GPA and translocation events, and lognormal distributions to the slopes of the “standard” curves and the DREF. Using the SDs given above and in Table 1, and the assumption of full temporal stability for translocations and GPA, the resultant cumulative probability distributions associated with the three biodosimetrically derived doses are shown in Fig. 4.The 90% confidence intervals are 0.6 to 3.2 Sv for GPA (Hiroshima standard), 0.5 t o 2.1 Sv for GPA (Goiinia standard), and 0.2 to 2.4 Sv for translocations. We also used the Monte Carlo approach t o deter-, mine the effect on overall uncertainty of increasing the precision of certain individual parameters. For exam-, ple, if we would have scored 6000 cells for translocations in the radiation worker instead ofjust 482, the 90% confidence interval would have improved only slightly, i.e., 0.3 t o 2.2 Sv. To achieve substantial reductions in uncertainties, the precision in the low-dose linear coefficient ( a )of the translocation dose-response curve would have to be increased. This could be accomplished by an in vitro experiment using FISH and should be of high priority. For GPA, substantial reduction in uncertainties would likely be achieved if the assay could be developed in vitro, thus permitting experimental measurement of various radiobiological variables. It is also likely that helpful information will become available from GPA studies now underway on persons exposed at Chernobyl. It is recommended that parallel GPA and FISH studies be performed on a group of 20 to 30 upper-level exposed radiation workers, with matched controls. CONCLUSION Dose-equivalent estimates for the radiation worker in the -0.4 to -2 Sv range, which include the value in the worker’s dosimetry records, cannot be confidently excluded at this time based on state-of-the-art biodosimetry. Values larger than 2.5 Sv (as believed by the worker) appear to be unlikely.
Acknowledgement(s)-This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract W-7405-Eng-48 with support from the Defense Nuclear Agency, Projects 89-870 and 90-819.
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Bajerska, A.; Liniecki, J. The yield of chromosomal aberrations in rabbit leukocytes after irradiation in vitro and in vivo. Mutat. Res. 27:271-281; 1975. Bauchinger, M.; Schmid, E.; Dresp, J. Calculation of the doserate dependence of the dicentric yield after ‘j0Co-y-irradiation of human lymphocytes. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 35:229-233; 1979. Bender, M. A.; Awa, A. A.; Brooks, A. L.; Evans, H. J.; Groer, P. G.; Littlefield, L. G.; Pereira, C.; Preston, R. J.; Wachholtz, B. W. Current status of cytogenetic procedures to detect and quantify previous exposuresto radiation. Mutat. Res. 196:103-159; 1988. Bigbee, W. L.; Langlois, R. G.; Swift, M.; Jensen, R. H. Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia. Am. J. Hum. Genet. 44:402-408; 1989. Bigbee, W. L.; Wyrobek, A. J.; Langlois, R. G.; Jensen, R. H.; Everson, R. B. The effect of chemotherapy on the in vivo frequency of glycophorin A “null” variant erythrocytes. Mutat. Res. 240:165-175; 1990. Brenner, D. J. Significance of neutrons from the atomic bomb at Hiroshima for revised radiation risk estimates. Health Phys. 60:439-442; 199 1. Brewen, J. G.; Gengozian, N. Radiation-induced human chromosome aberrations. 11. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes. Mutat. Res. 13:383-39 1; 197 1. Buckton, K. E.; Hamilton, G. E.; Paton, L.; Langlands, A. 0. Chromosome aberrations in irradiated ankylosing spondylitis. In: Mutagen-induced chromosome damage in man. London: University Press: 1978:142- 150. Buckton, K. E.; Langlands, A. 0.;Smith, P. G.;Woodcock, G. E.; Looby, P. C. Further studies on chromosome aberrations: Production after whole-body irradiation in man. Int. J. Radiat. Biol. 19:369-378; 1971. Eastmond, D. A.; Tucker, J. D. Identification of aneuploidyinducing agents using cytokinesis-blockedhuman lymphocytes and an antikinetochore antibody. Environ. Molec. Mutagen. 13:34-43; 1989. Evans, H. C.; Buckton, K. E.; Hamilton, G. E.; Carothers, A. Radiation-induced chromosome aberrations in nucleardockyard workers. Nature 27753 1-534; 1979. Evans, H. C.; Buckton, K. E.; Sumner, A. T. Cytological mapping of human chromosomes: Results obtained with quinacrine fluorescence and the acetic-saline-Giemsatechniques. Chromosoma 35:310-325; 1971. Hakoda, M.; Akiyama, M.; Kyoizumi, S.; Awa, A. A.; Yamakido, M.; Otake, M. Increased somatic cell mutation frequency in atomic bomb survivors. Mutat. Res. 201:3948; 1988. Krepinsky, A. B.; Heddle, J. A. Micronuclei as a rapid and inexpensive measure of radiation-induced chromosomal aberrations. In: Ishihara, T.; Sasaki, M. S., eds. Radiationinduced chromosome damage in man. New York Alan R. Liss, Inc.; 1983:93-109. Kyoizumi, S.; Nakamura, N.; Hakoda, M.; Awa, A. A.; Bean, M. A.; Jensen, R. H.; Akiyama, M. Detection of somatic mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors using a single beam flow sorter. Cancer Res. 49581-588: 1989. Langlois, R. G.; Bigbee, W. L.; Jensen, R. H. Measurements of the frequency of human erythrocytes with gene expression loss phenotypes at the glycophorin A locus. Hum. Genet. 74:353-362; 1986. Langlois, R. G.; Bigbee, W. L.; Kyoizumi, S.; Nakamura, N.;
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February 1992, Volume 62, Number 2 index of the 24 human chromosomes. Science 179: i 1261129; 1973. Messing, K.; Ferraris, J.; Bradley, W. E. C.; Swatz, J.; Seifert, A. M. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation. Health Phys. 57537-544; 1989. Minkler, J.; Stetka, D., Jr.; Carrano, A. V. An ultraviolet light source for consistent differential staining of sister chromatids. Stain Technol. 53: 359-360; 1978. National Academy of Sciences. Health effects of exposure to low levels of ionizing radiation (BEIR V). Washington, DC: U.S. National Academy of Sciences, National Academy Press; 1990. National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States. Bethesda, MD: NCRP; NCRP Report No. 93; 1987. Perry, P.; Wolff, S. New Giemsa method for the differential staining of sister chromatids. Nature 25 1:156- 158; 1974. Pinkel, D.; Straume, T.; Gray, J. W. Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. U.S.A. 83:2934-2938; 1986. Ramalho, A. T.; Nascimento, A. C. H.; Natarajan, A. T. Dose assessments by cytogenetic analysis in the Goidnia (Brazil) radiation accident. Radiat. Prot. Dosim. 23:97-100; 1988. Roesch, W. C. (ed.). Final report on US.-Japan reassessment of atomic bomb radiation dosimetry in Hiroshima and Nagasaki. Hiroshima: Radiation Effects Research Foundation; 1987. Sanderson, B. J. S.; Dempsey, J. L.; Morley, A. A. Mutations in human lymphocytes: Effect of x- and UV-irradiation. Mutat. Res. 140:223-227; 1984. Schmid, E.; Bauchinger, M.; Bunde, E.; Ferbert, H. F.; Lieven, H. V. Comparison of the chromosome damage and its dose response after medical whole-body exposures to 6oCo y-rays and irradiation of blood in vitro. Int. J. Radiat. Biol. 26:31-37; 1974. Straume, T.; Langlois, R. G.; Lucas, J.; Jensen, R. H.; Bigbee, W. L.; Ramalho, A. T.; Brandso-Mello, C. E. Novel biodosimetry methods applied to the victims of the Goilnia accident. Health Phys. 60:7 1-76; 1991. Taylor, A. M. R. The effect of radiation on the chromosomes of patients with an unusual cancer susceptibility. In: Radiation-induced chromosome damage in man. New York: Alan R. Liss, Inc.; 1983:167-199. Tonomura, A.; Kishi, K.; Saito, F. Types and frequencies of chromosome aberrations in peripheral lymphocytes of general populations. In: Radiation-induced chromosome damage in man. New York Alan R. Liss, Inc.; 1983:605616. Yamamoto, 0.; Shigetoshi, A.; Russell, W. J.; Fujita, S.; Sawada, S. Medical x-ray exposure doses as contaminants of atomic bomb doses. Health Phys. 54:257-269; 1988. M M
