1975, British Journal of Radiology, 48, 487-493
JUNE
1975
Radiation-induced chromosomal aberrations in human lymphocytes after partial-body exposure to 60Co gamma-irradiation and in vitro exposure to 230 kV X-irradiation By Gwyneth E. Watson, B.Sc, Ph.D.,* and N. E. Gillies, B.Sc, Ph.D. Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London W1P 6DB {Received July 1974) ABSTRACT
This paper reports results for the yield of chromosome aberrations in first division cells in blood samples after a single partial-body therapeutic exposure to different anatomical sites. Comparisons were made with a standard dose response to 230 kV X-irradiation in vitro.
Small yields of chromosome aberrations were present in lymphocytes from 12 patients observed at first metaphase in culture. Blood samples were obtained at various times, up to three days, after single partial-body therapeutic exposure ranging from 75 to 400 rads of 60Co y-irradiation. When all patients were considered there was no correlation between treatment dose and aberration frequency, but on subdivision into two groups on the basis of whether the reticuloendothelial system was involved in the cancer, linear regression analysis could be fitted to the data for each group. An in vitro dose response curve for dicentrics induced by 230 kV X rays at a dose rate of 23-3 rads per minute was constructed for use as a standard calibration curve for 48 hour cultures. The yield of dicentric aberrations was best fitted by a4 power law model, Y=kDn in which & = (l-59± 0-66) lO- and n = 1-49±0-08, (P=0-96).
The induction of chromosome aberrations in peripheral blood lymphocytes has been utilized (Brewen, Preston and Littlefield, 1972) as a biological dosimeter after accidental whole-body radiation exposure. It has been shown, in animals (Clemenger and Scott, 1971, 1973; Brewen and Gengozian, 1971; Preston, Brewen and Jones, 1972), that the production of aberrations in leucocytes is the same whether they are irradiated in vivo or in vitro. In man, there are only a few examples of whole-body irradiation but from the available data it appears that there is reasonably good agreement between the production of aberrations in lymphocytes after exposure in vivo or in vitro (Buckton et al., 1969; Langlands et al, 1968; Dolphin et al, 1970; Brewen, et al, 1972). Information on the yield of chromosome aberrations in lymphocytes can be derived from studies on patients who have received radiotherapy. Most of these involve partial-body irradiation and, in practice, accidental exposure is usually also partial-body. Most of the available data (Warren and Meisner, 1965; Millard, 1965) have been obtained after exposure of patients to large fractionated doses of radiation, but Buckton, Langlands and Smith (1967) studied aberrations after exposure of patients to single doses to the spinal region only. *Present address: Medical Research Council, Radiobiology Unit, Harwell, Didcot, Berks OX11 0RD.
MATERIALS AND METHODS
Selection of patients The patients selected for study had received no previous radiation therapy, nor had been exposed to known chromosome breaking agents including both recent viral infections and ionizing radiations during employment. The nature of the study was explained to the patients who agreed to participate in it. Leucocyte culture methods Macrocultures of leucocytes irradiated in vivo were prepared, using a method based on the technique of Moorhead et al. (1960). The red cells in 10 ml. of venous blood samples were sedimented by the addition of 2 ml. of 6 per cent dextran in 0-9 per cent sodium chloride (Benger Lab. Ltd.). The leucocytes, suspended in plasma, were added to culture medium TC 199 (Glaxo) so that the final cell count was approximately 1,000 cells per mm3. 0-15 ml. of phytohaemagglutinin (PHA) (Wellcombe Lab. Ltd.) was added to 7 ml. of culture. Whole blood microcultures (Arakaki and Sparkes, 1963) were irradiated in vitro. These consisted of 0-2 ml. of whole blood, 6 ml. of TC 199 culture medium and 1 ml. of human AB serum. 0-15 ml. of PHA were added to each culture immediately following irradiation. Both micro- and macrocultures were incubated at 37°C for 46-5 hours, when 0-15 ml. of Colcemid (lxlO~ 6 g/ml.) were added to each culture and incubation continued for 1 -5 hours. At 48 hours, the leucocyte cultures were treated with warm 0-8 per cent trisodium citrate solution for 25 minutes prior to fixation in one part acetic acid: three parts methanol. Air-dried chromosome preparations, stained with 2 per cent lacto-acetic orcein, were coded and
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VOL. 48, No. 570
Gwyneth E. Watson, and N. E. Gillies TABLE I DETAILS OF PATIENTS AND FREQUENCIES OF CHROMOSOME-TYPE ABERRATIONS
D
atient
No. and sex
Age in
years
1 (F)
40
2(M)
45
3(M)
42
4(M)
37
5(M)
21
6(M)
30
7(F)
25
8(F)
60
Diagnosis
9(F)
45
10 (M)
35
Testicular seminoma
11 (M) 31
Testicular seminoma Teratoma
12(M)
37
13 (F)
78
Region
Neck, mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Neck, Hodgkin's disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum (granulomatous) and both axillae Reticulum cell Groin sarcoma (localized) Neck and Cancer of mediastinum thyroid (inoperable)
Hodgkin's disease
Cancer of larynx
Lower abdomen and groin Lower abdomen Central abdomen and both groins Anterior neck
Irradiated field size (cm)
Dose SampDirate First ling (rad/ dose time Cells cenmin.) (rad) (h) scored trics
Estimated peripheral blood y-dose Frag(rad) Rings ments (EPBD)
35x35
50
300
24
105
3
0
3
43
33x33
50
200
4 24 48
105 110 110
5 4 8
1 1 0
2 0 4
51
41 x45 17-4
200
4 24
100 100
2 5
0 2
2 1
63
41x34
200
60 100
0 8
1
2 1
87
2
—
—
—
—•
121 95
5 6
0 3
1 3
74
2* 6
50 50
1 4
3 0
2 4
87
\
1 2 4 1 1 3 2 1 2 5 2 3 1 1
0 0 0 0 0 0 0 1 0 0 1 0 2
0 2 2 3 1 1 4 0 1 2 0 1 0 2
87
z 4* 29 45 4 24 72 4 24 4 26
100 50 50 100 100 60 113 100 100 100 100 100 105 80
24 48
100 100
2 4
0 0
6 2
30x40
17-4
22-2
24 150
i
4\ 24/
No
mitotic
36x38
20-5
150
34x35
10
300
10x15
25
300
cells
4 24
6 24
12x20
60
300
20x21
20-5
75
18x28
40
18x21
55
200
12x7
60
400
250
2
63
34 45 25
34
Cases 1-7. Case 1 was generalized and in Cases 2-7 the enlarged lymph nodes were confined to the upper trunk. (3ases1 and 9 were the only bedfast patients and Case 1 died within three weeks of blood examination. analysed for all types of chromosomal damage by one observer. The scores were made only on cells containing 46 centromeres. Only dicentric and centric ring aberrations accompanied by their associated fragments were scored. Interstitial and terminal deletions were combined in one category as fragments. Approximately 100 cells were scored for each blood sample.
Radiation conditions In vivo. Patients receiving radiotherapy were irradiated with 60Co y-rays from a Mobaltron unit (TEM Ltd.) at dose rates ranging from 17 to 60 rads per minute. Large field sizes were used. 10 ml. venous blood samples were obtained immediately prior to the first therapeutic exposure and at various times up to three days following treatment.
488
JUNE 1975
Radiation-induced chromosomal aberrations in human lymphocytes In vitro. Non-PHA stimulated whole blood mi- size (cm2) did not fall on one linear regression line crocultures of leucocytes from a healthy female (Fig. 1). Total chromosome breakage (calculated by were irradiated in glass bottles with 230 kVp counting exchange aberrations as two breaks and X rays (5 mA, with no nitration) at a dose rate of all other aberrations as one break) also did not lie on 23-4 rads per minute. The irradiations were per- a single line. formed at room temperature. Three dose response The patients were then divided into two groups, experiments were performed, using the same blood group one containing patients Nos. 1-8 with indonor on each occasion. Microcultures of control volvement of the reticuloendotheJial system and non-irradiated blood from patients 5 and 8 (Table I) group two containing patients Nos. 9-13 with canwere also irradiated in vitro under the same condi- cer arising elsewhere. For group one the yield of tions. dicentrics versus dose was well fitted to a linear regression line (Fig. 1), the slope of which was (2-27^ RESULTS 0-33)10-4 (P=0-27), but the response for the freIn vivo. Details of the 13 patients from whom quency of dicentrics versus dose Xfieldsize did not blood samples were collected are given in Table I. fit a linear relationship (P=v. small). When the Lymphocytes were successfully cultured from all response for total chromosomal breakage was conpatients with the exception of one (No. 5) who sidered, the linear regression worsened with both had Hodgkin's disease. Fewer PHA-transformed dose (P=0-0027) and dose X field size (P=small). lymphocytes were present in cultures of cells from For group two the yield of dicentrics versus dose patients with Hodgkin's disease than from other was well fitted to a linear regression (Fig. 1) with a patients, agreeing with the findings of Trubowitz, slope of (0-84±0-21)10-4 (P=0-50), and also for the Masek and del Rosario (1966) and Lawler, Penty- yield against dose X field size with a slope of (2-65 ± cross and Reeve (1968). The karyotypes of all pa- 0-50)10"7 (P=0-41). The total chromosomal breaktients were found to be either normal 46, XX, or 46, age was fitted as a function of dose to a linear reXY, and thus changes in radiosensitivity due to ab- gression line with a slope of (2-95±0-39)10"4 normal chromosome complements need not be considered (Sasaki, Tonomura and Matsubara, 1970). One hundred cells per patient were examined from cultures of control, unirradiated lymphocytes. 0.08 © © None of these cells contained chromosome-type 0.06 ® aberrations. Chromosome-type aberrations were observed in © 0.04 lymphocytes following a single partial-body dose m ® 0.02 in the range of 75 to 400 rads at times up to three days following treatment (Table I). 0 2 4 6 8 K) 20 30 It was necessary to determine whether or not the Dosex Field Size (»10 ) time at which blood was obtained from patients 0.10 after radiotherapy had any effect on aberration yield. 0.08 The frequency of dicentrics was 0-028 per cent (831 cells scored) at four to six hours after irradia0.06 tion (ten patients), 0-041 per cent (1,000 cells scored) 0.04 at 24 to 29 hours (12 patients) and 0-034 per cent (232 cells scored) at 45 to 48 hours (three patients). 0.02 Although the observed frequency is lower at four to six hours than at later times after radiation treatment, 300 400 the overall differences between the three frequencies Treatment Dose (Rads) 2 is not statistically significant (x =62-76; P=0-25). FIG. 1. The results were not sub-divided by treatment dose Dicentrics per cell as a function of treatment dose Xfieldsize because of the low frequency of aberrations. (upper figure). Dicentrics per cell as a function of treatTherefore the yields of aberrations observed at 4 to ment dose in rads (lower figure) fitted to linear regression 29 hours after irradiation were combined and con- Y=(2-27±0-33)10-4D for lines: patient group 1 represented by sidered with respect to treatment dose. When all r '• y = (0-84±0-21)10 4£> f patient group 2, represented by 12 patients were considered, the frequency of dior • — •_ centrics per cell versus dose, or versus dose X field
1"
4
The patient number is shown within the group symbols. 489
VOL.
48, No. 570 Gwyneth E. Watson and N. E. Gillies T A B L E II NUMBER OF CHROMOSOME ABERRATIONS AND FREQUENCY PER CELL (IN PARENTHESIS) INDUCED IN VITRO BY 230 KV X RAYS IN HUMAN LYMPHOCYTES CULTURED FOR 48 HOURS
Dose (rads)
Cells scored
Normal female
0 75 110 150 185 225 300 375
Normal female
Lymphocyte donor
Dicentrics
Rings
Fragments
100 201 234 245 79 66 49 61
0 25(0-12) 39(0-17) 62(0-25) 36 (0-46) 35(0-53) 43 (0-88) 59(0-97)
0 2(0-01) 9 (0-04) 20(0-08) 6(0-03) 7(0-11) 9(0-18) 11(0-18)
0 17(0-08) 29(0-12) 50(0-26) 29(0-37) 24(0-36) 22(0-45) 51(0-84)
0 75 150 225 300
100 354 210 120 200
0 35(0-10) 52(0-25) 56(0-47) 162(0-81)
0 3(0-01) 10(0-05) 8(0-07) 26(0-13)
0 25 (0-07) 42(0-20) 50(0-42) 143 (0-72)
Normal female
0 23-4 58-5 117 140-4 175-5 234
100 100 100 100 100 100 100
0 2(0-02) 5 (0-05) 19(0-19) 24(0-24) 38(0-38) 51 (0-51)
0 1 (0-01) 4(0-04) 5(0-05) 4(0-04) 9(0-09) 21(0-21)
1 (0-01) 9(0-09) 6(0-06) 19(0-19) 11(0-11) 20 (0-20) 50(0-50)
Female with reticulum cell sarcoma
0 58-5 117 234
100 100 100 90
0 3(0-03) 20 (0-20) 34(0-38)
0 3(0-03) 3 (0-03) 10(0-11)
0 8(0-08) 22(0-22) 45(0-50)
(P=O16). However, the total chromosomal breakage as a function of dose X field size did not fit a linear regression (P=O00067). The linear coefficient for dicentrics was 2-6 times greater for group one than for group two but the field size was also about three times larger on average. A representative proportion of the cells from the irradiated patients was analysed for the presence of chromatid aberrations. Only 14 chromatid breaks and one isolocus break were observed in 1,970 cells. Fourteen gaps and 16 isogaps were also recorded. The low yields of chromatid aberrations were similar to those reported for a random unirradiated sample of the population of Edinburgh (Court Brown et al., 1966). In vitro. The frequencies of dicentric, centric ring and fragment aberrations induced in three separate experiments using blood from the same donor are shown in Table II. There was good agreement between experiments for the yields of dicentric chromosomes (P=0-75) as shown in Fig. 2. The data for each aberration type from the three experiments were combined and fitted to three dose response models assuming a Poisson distribution of aberrations among cells. For dicentric aberrations, the response was equally well fitted to the power law
model Y=kD", [&=(l-59±0-66)10- 4 and n = l-49± 0-08, (P=0-69)], and to the quadratic relationship Y=aD+/3Z)2, [a=(9-52±l-66)10- 4 and £=(5-33 ±0-85)10- 6 , (P=0-95)]. The data could not be fitted to a linear model (P=0-0006). The yield of fragment aberrations was poorly fitted to all three models. Whole blood microcultures from patient 8, irradiated in vitro, yielded frequencies of aberrations at the three doses studied which agreed well with the response of lymphocytes from the normal healthy donor (Fig. 2). Comparison of aberration yields induced in vivo and
in vitro Using the standard X-ray in vitro dose response curve (Fig. 2), estimates of the X-ray dose received by the peripheral blood in vivo can be made. The estimated doses were multiplied by 0-75, to allow for the lesser efficiency of y rays with respect to X rays (Scott et al, 1970). This estimated y-ray dose is defined as'the "estimated peripheral blood dose" (EPBD) (Table I). This is sometimes referred to as the equivalent whole body dose (Dolphin, 1969; Purrot et al., 1972) but, in the absence of a reliable in vivo calibration curve for uniform
490
JUNE 1975
Radiation-induced chromosomal aberrations in human lymphocytes
DISCUSSION
calibration curve in assessing radiation dose in vivo. Low yields of chromosome-type aberrations were present in lymphocytes of all patients within a day of partial-body irradiation with doses up to 400 rads. As the lymphocytes were irradiated in metaphase at the Go stage of the DNA synthesis cycle (Howard and Pelc, 1951), chromatid-type aberrations would not be produced by irradiation, and, as expected, the frequency of chromatid-type aberrations was unaffected by radiation treatment. For all doses combined, the yield of dicentrics was not statistically lower at four hours than at 24 hours after treatment. Previously Buckton et al. (1967) reported lower frequencies of cells with unstable aberrations (Cu cells) in blood samples taken immediately than after 24 hours after partial-body irradiation to a spinal field for ankylosing spondylitis, but the differences between yields were significant for only two of the five doses studied. In the present study the yield of dicentrics was a linear function of dose but values for the slopes of the lines differed for each group of patients. The results from the two groups could not be combined to fit a single regression line. In general, group two patients receive treatment to smaller areas than group one patients and in addition the anatomical regions treated did not include so much lymphoid tissues. Group one patients received doses to the cervical mediastinal and axillary lymph nodes. As the lymphocytes in the peripheral blood constitute a very small proportion of the total lymphocyte population (Osgood, 1954), the greater part of the cytogenetic damage observed was probably induced in lymphocytes in the lymph nodes at the time of irradiation; if these lymphocytes were then released into circulation, this would explain the observation that the EPBD ranged from 9-49 per cent of the treatment dose. Only about 1 per cent of the total lymphocyte population would be irradiated in the peripheral blood.
A repeatable in vitro dose response calibration curve was obtained for the production of dicentric aberrations in human lymphocytes examined at first post-irradiation metaphase. The dose exponent n in the power law model was 1-49^0-08 for dicentrics and this dose response clearly differs from the almost linear dose response measured by Evans (1967) and Bajerska and Liniecki (1969) and the dose squared response found by Bender and Barcinski (1969), Norman and Sasaki (1966), but is in agreement with the findings of Sasaki (1971). Because of the differences in the dose response for dicentrics observed in different laboratories, it is necessary for each worker to construct an in vivo
Comparison of aberration yield in vivo and in vitro The in vitro dose response for dicentrics was curvilinear and well fitted to the quadratic response model while the in vivo treatment dose response for dicentrics was linear. As the level of dicentrics observed following in vivo irradiation was at the lower end of the in vitro dose response curve where the linear term of the quadratic expression predominates the in vitro and in vivo dose response curves can therefore be compared. The lower yields of chromosome-type aberrations in the peripheral blood of patients receiving partialbody irradiation as compared with the yields
FIG. 2. In vitro dose response curve for dicentric chromosomes induced in human leukocytes by 230 kV X rays and fitted to a power law response model, F=(l-59)10-4Z> i-49±o-O8. o, • and A experiments 1, 2 and 3 respectively. • , lymphocytes from patient (No. 8) with reticulum cell sarcoma. The in vitro dose calibration curve was constructed at Harwell.
whole body irradiation, equivalence cannot be assumed. For all patients the EPBD ranged from 9 to 49 per cent of the treatment dose and for most patients was in the range of 14 to 32 per cent. For group two patients, with smaller sizes of treatment areas, the EPBD was generally a smaller percentage of the treatment dose than was the case for group one patients.
491
VOL. 48, No. 570
Gwyneth E. Watson and N. E. Gillies induced by similar in vitro doses of radiation may be attributed to a combination of factors. One is the dilution of irradiated lymphocytes by non-irradiated cells during lymphocyte recirculation (Gowans and Knight, 1964). This occurs rapidly since the mean time of residence of the small lymphocyte in the peripheral blood has been estimated to be 4-7-5 minutes (Sharpe et ah, 1968). A second factor is the greater likelihood of the division of unirradiated than irradiated cells, thereby reducing the observed yield of aberrations in a mixture (Sharpe, 1969; Lloyd, Purrot and Dolphin, 1973). Thus the standard in vitro dose response curve is unlikely to represent the in vivo situation. Biological dosimetry From the results obtained from lymphocytes irradiated in vitro it might be concluded that the induction of dicentrics might be used as a reliable indication of radiation dose in vivo. However, it is obvious from the data obtained after partial body irradiation that this is not so. A major problem is the lack of knowledge concerning the distribution of lymphocytes in the body at the time of irradiation and their subsequent rate of release into the peripheral blood. Certainly, in the present study, the diseases for which the patients were being treated might affect the release of lymphocytes from the lymph nodes in a manner which may not occur in healthy individuals. At present, there is no way of ascertaining unambiguously from chromosome aberrations alone whether accidental exposure was received by the whole or only part of the body: the presence of complete chromosome-type exchange aberrations in first division human peripheral blood leucocytes only confirms that a radiation dose was received. Cells with aberrant chromosomes have been unambiguously identified in cultures of dermal fibroblasts irradiated in vivo (Bigger, Savage and Watson, 1972; Visfeldt, 1966). Therefore, a skin biopsy from the probable site of irradiation may be a more useful guide to the magnitude of partial-body exposure.
of chromosomal aberrations in the peripheral blood lymphocytes. International Journal of Radiation Biology, 16, 467-482. BENDER, M. A., and BARCINSKI, M. A., 1969. Kinetics of
two-break aberration production by X-rays in human leucocytes. Cytogenetics, 8, 241-246. BIGGER, T. B. L., SAVAGE, J. R. K., and WATSON, G. E.,
1972. A scheme for characterising ASG banding and an illustration of its use in identifying complex chromosomal rearrangements in irradiated human skin. Chromosoma (Berlin), 39, 297-309. BREWEN, J. G., and GENGOZIAN, N., 1971. Radiation-in-
duced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes. Mutation Research, 13, 383— 391. BREWEN, J. G., PRESTON, R. J., and LITTLEFIELD, L. G., 1972
Radiation-induced human chromosome aberration yields following an accidental whole-body exposure to 60Co yrays. Radiation Research, 49, 647-656. BUCKTON, K. E., LANGLANDS, A. D., SMITH, P. G., WOOD-
COCK, G. E., and LOOBY, P. G., 1971. Further studies on
chromosome aberration production after whole-body irradiation in man. International Journal of Radiation Biology, 19, 369-378. BUCKTON, K. E., LANGLANDS, A. O., SMITH, P. G., LOOBY, P. C , WOODCOCK, G. E., and MCLELLAND, J., 1969.
Chromosome aberrations induced in human peripheral blood by 2-MeV X-irradiation to the whole-body and in vitro. Radiation induced cancer. International Atomic Energy Agency, Vienna, 135. BUCKTON, K. E., LANGLANDS, A. O., and SMITH, P. G.,
1967. Chromosome aberrations following partial- and whole-body X-irradiation in man. Dose response relationships. In Human Radiation Cytogenetics, eds. H. J. Evans, W. M. Court-Brown and A. S. McLean pp. 122135. (Amsterdam, North Holland Publishing Co.). CLEMENGER, J. F., and SCOTT, D., 1971. In vitro and in vivo
sensitivity of cultured blood lymphocytes to radiation induction of chromosome aberrations. Nature, New Biology, 234,154. 1973. A comparison of aberration yields in rabbit blood lymphocytes irradiated in vitro and in vivo. International Journal of Radiation Biology, 24, 487-496. COURT-BROWN, W. M., JACOBS, P. A., BUCKTON, K. E., TOUGH, I. M., KUENSSBERG, E. V., and KNOX, J. D. E.,
1966. Chromosome studies on adults. Eugenics Laboratory Memoirs XLII, p. 80 (Cambridge University Press). DOLPHIN, G. W., 1969. Biological dosimetry with particular reference to chromosome aberration analysis. Symposium on the Handling of Radiation Accidents, Vienna, May 1969 (SM-119) 215-224 (I.A.E.A., Vienna). DOLPHIN, G. W., BOLTON, D., HUMPHREYS, D. L. O., SPEIGHT, D. A., and STRADLING, G. N., 1970. Biological
and physical dosimetry after a radiation accident. Nature {London), 227, 165. EVANS, H. J., 1967. Dose response relations from in vitro studies. In Human Radiation Cytogenetics, eds. H. J. Evans, W. M. Court-Brown, A. S. McLean, pp. 20-36 (North Holland Publishing Co.). GOWANS, J. L., and KNIGHT, E. J., 1964. The route of
ACKNOWLEDGMENTS
re-circulation of lymphocytes in the rat. Proceedings of We would like to thank Dr. A. M. Jellife, of the Radiothe Royal Society 159 B, 257-282. therapy Department, The Middlesex Hospital, for perA., and PELC, S. R., 1951. Nuclear incorporation mission to study his patients and Mr. D. G. Papworth for HOWARD, of P 32 as demonstrated by autoradiographs. Experimental the statistical analysis of our data. We are most grateful Cell Research, 2, 178-187. to Dr. R. H. Mole for his helpful advice and suggestions in LANGLANDS, A. D., SMITH, P. G., BUCKTON, K. E., WOODthe preparation of this manuscript. COCK, G. E., and MCLELLAND, J., 1968. Chromosome,
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B., and ELTHAM, E., 1972. The study of chromosome aberration yield in human lymphocytes as an indicator of radiation dose. II. A review of cases investigated 1970-71. National Radiological Protection Board -R5. SASAKI, M. S., 1971. Radiation-induced chromosome aber- WARREN, S., and MEISNER, L., 1965. Chromosomal changes rations in lymphocytes. Possible biological dosimetry in in leukocytes of patients receiving irradiation therapy man. In Biological Aspects of Radiation Protection, eds. Journal of American Medical Association, 193, 101-108'
493
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1975
Radiation-induced chromosomal aberrations in human lymphocytes after partial-body exposure to 60Co gamma-irradiation and in vitro exposure to 230 kV X-irradiation By Gwyneth E. Watson, B.Sc, Ph.D.,* and N. E. Gillies, B.Sc, Ph.D. Department of Biology as Applied to Medicine, The Middlesex Hospital Medical School, London W1P 6DB {Received July 1974) ABSTRACT
This paper reports results for the yield of chromosome aberrations in first division cells in blood samples after a single partial-body therapeutic exposure to different anatomical sites. Comparisons were made with a standard dose response to 230 kV X-irradiation in vitro.
Small yields of chromosome aberrations were present in lymphocytes from 12 patients observed at first metaphase in culture. Blood samples were obtained at various times, up to three days, after single partial-body therapeutic exposure ranging from 75 to 400 rads of 60Co y-irradiation. When all patients were considered there was no correlation between treatment dose and aberration frequency, but on subdivision into two groups on the basis of whether the reticuloendothelial system was involved in the cancer, linear regression analysis could be fitted to the data for each group. An in vitro dose response curve for dicentrics induced by 230 kV X rays at a dose rate of 23-3 rads per minute was constructed for use as a standard calibration curve for 48 hour cultures. The yield of dicentric aberrations was best fitted by a4 power law model, Y=kDn in which & = (l-59± 0-66) lO- and n = 1-49±0-08, (P=0-96).
The induction of chromosome aberrations in peripheral blood lymphocytes has been utilized (Brewen, Preston and Littlefield, 1972) as a biological dosimeter after accidental whole-body radiation exposure. It has been shown, in animals (Clemenger and Scott, 1971, 1973; Brewen and Gengozian, 1971; Preston, Brewen and Jones, 1972), that the production of aberrations in leucocytes is the same whether they are irradiated in vivo or in vitro. In man, there are only a few examples of whole-body irradiation but from the available data it appears that there is reasonably good agreement between the production of aberrations in lymphocytes after exposure in vivo or in vitro (Buckton et al., 1969; Langlands et al, 1968; Dolphin et al, 1970; Brewen, et al, 1972). Information on the yield of chromosome aberrations in lymphocytes can be derived from studies on patients who have received radiotherapy. Most of these involve partial-body irradiation and, in practice, accidental exposure is usually also partial-body. Most of the available data (Warren and Meisner, 1965; Millard, 1965) have been obtained after exposure of patients to large fractionated doses of radiation, but Buckton, Langlands and Smith (1967) studied aberrations after exposure of patients to single doses to the spinal region only. *Present address: Medical Research Council, Radiobiology Unit, Harwell, Didcot, Berks OX11 0RD.
MATERIALS AND METHODS
Selection of patients The patients selected for study had received no previous radiation therapy, nor had been exposed to known chromosome breaking agents including both recent viral infections and ionizing radiations during employment. The nature of the study was explained to the patients who agreed to participate in it. Leucocyte culture methods Macrocultures of leucocytes irradiated in vivo were prepared, using a method based on the technique of Moorhead et al. (1960). The red cells in 10 ml. of venous blood samples were sedimented by the addition of 2 ml. of 6 per cent dextran in 0-9 per cent sodium chloride (Benger Lab. Ltd.). The leucocytes, suspended in plasma, were added to culture medium TC 199 (Glaxo) so that the final cell count was approximately 1,000 cells per mm3. 0-15 ml. of phytohaemagglutinin (PHA) (Wellcombe Lab. Ltd.) was added to 7 ml. of culture. Whole blood microcultures (Arakaki and Sparkes, 1963) were irradiated in vitro. These consisted of 0-2 ml. of whole blood, 6 ml. of TC 199 culture medium and 1 ml. of human AB serum. 0-15 ml. of PHA were added to each culture immediately following irradiation. Both micro- and macrocultures were incubated at 37°C for 46-5 hours, when 0-15 ml. of Colcemid (lxlO~ 6 g/ml.) were added to each culture and incubation continued for 1 -5 hours. At 48 hours, the leucocyte cultures were treated with warm 0-8 per cent trisodium citrate solution for 25 minutes prior to fixation in one part acetic acid: three parts methanol. Air-dried chromosome preparations, stained with 2 per cent lacto-acetic orcein, were coded and
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Gwyneth E. Watson, and N. E. Gillies TABLE I DETAILS OF PATIENTS AND FREQUENCIES OF CHROMOSOME-TYPE ABERRATIONS
D
atient
No. and sex
Age in
years
1 (F)
40
2(M)
45
3(M)
42
4(M)
37
5(M)
21
6(M)
30
7(F)
25
8(F)
60
Diagnosis
9(F)
45
10 (M)
35
Testicular seminoma
11 (M) 31
Testicular seminoma Teratoma
12(M)
37
13 (F)
78
Region
Neck, mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum and both axillae Neck, Hodgkin's disease mediastinum and both axillae Hodgkin's Neck, disease mediastinum (granulomatous) and both axillae Reticulum cell Groin sarcoma (localized) Neck and Cancer of mediastinum thyroid (inoperable)
Hodgkin's disease
Cancer of larynx
Lower abdomen and groin Lower abdomen Central abdomen and both groins Anterior neck
Irradiated field size (cm)
Dose SampDirate First ling (rad/ dose time Cells cenmin.) (rad) (h) scored trics
Estimated peripheral blood y-dose Frag(rad) Rings ments (EPBD)
35x35
50
300
24
105
3
0
3
43
33x33
50
200
4 24 48
105 110 110
5 4 8
1 1 0
2 0 4
51
41 x45 17-4
200
4 24
100 100
2 5
0 2
2 1
63
41x34
200
60 100
0 8
1
2 1
87
2
—
—
—
—•
121 95
5 6
0 3
1 3
74
2* 6
50 50
1 4
3 0
2 4
87
\
1 2 4 1 1 3 2 1 2 5 2 3 1 1
0 0 0 0 0 0 0 1 0 0 1 0 2
0 2 2 3 1 1 4 0 1 2 0 1 0 2
87
z 4* 29 45 4 24 72 4 24 4 26
100 50 50 100 100 60 113 100 100 100 100 100 105 80
24 48
100 100
2 4
0 0
6 2
30x40
17-4
22-2
24 150
i
4\ 24/
No
mitotic
36x38
20-5
150
34x35
10
300
10x15
25
300
cells
4 24
6 24
12x20
60
300
20x21
20-5
75
18x28
40
18x21
55
200
12x7
60
400
250
2
63
34 45 25
34
Cases 1-7. Case 1 was generalized and in Cases 2-7 the enlarged lymph nodes were confined to the upper trunk. (3ases1 and 9 were the only bedfast patients and Case 1 died within three weeks of blood examination. analysed for all types of chromosomal damage by one observer. The scores were made only on cells containing 46 centromeres. Only dicentric and centric ring aberrations accompanied by their associated fragments were scored. Interstitial and terminal deletions were combined in one category as fragments. Approximately 100 cells were scored for each blood sample.
Radiation conditions In vivo. Patients receiving radiotherapy were irradiated with 60Co y-rays from a Mobaltron unit (TEM Ltd.) at dose rates ranging from 17 to 60 rads per minute. Large field sizes were used. 10 ml. venous blood samples were obtained immediately prior to the first therapeutic exposure and at various times up to three days following treatment.
488
JUNE 1975
Radiation-induced chromosomal aberrations in human lymphocytes In vitro. Non-PHA stimulated whole blood mi- size (cm2) did not fall on one linear regression line crocultures of leucocytes from a healthy female (Fig. 1). Total chromosome breakage (calculated by were irradiated in glass bottles with 230 kVp counting exchange aberrations as two breaks and X rays (5 mA, with no nitration) at a dose rate of all other aberrations as one break) also did not lie on 23-4 rads per minute. The irradiations were per- a single line. formed at room temperature. Three dose response The patients were then divided into two groups, experiments were performed, using the same blood group one containing patients Nos. 1-8 with indonor on each occasion. Microcultures of control volvement of the reticuloendotheJial system and non-irradiated blood from patients 5 and 8 (Table I) group two containing patients Nos. 9-13 with canwere also irradiated in vitro under the same condi- cer arising elsewhere. For group one the yield of tions. dicentrics versus dose was well fitted to a linear regression line (Fig. 1), the slope of which was (2-27^ RESULTS 0-33)10-4 (P=0-27), but the response for the freIn vivo. Details of the 13 patients from whom quency of dicentrics versus dose Xfieldsize did not blood samples were collected are given in Table I. fit a linear relationship (P=v. small). When the Lymphocytes were successfully cultured from all response for total chromosomal breakage was conpatients with the exception of one (No. 5) who sidered, the linear regression worsened with both had Hodgkin's disease. Fewer PHA-transformed dose (P=0-0027) and dose X field size (P=small). lymphocytes were present in cultures of cells from For group two the yield of dicentrics versus dose patients with Hodgkin's disease than from other was well fitted to a linear regression (Fig. 1) with a patients, agreeing with the findings of Trubowitz, slope of (0-84±0-21)10-4 (P=0-50), and also for the Masek and del Rosario (1966) and Lawler, Penty- yield against dose X field size with a slope of (2-65 ± cross and Reeve (1968). The karyotypes of all pa- 0-50)10"7 (P=0-41). The total chromosomal breaktients were found to be either normal 46, XX, or 46, age was fitted as a function of dose to a linear reXY, and thus changes in radiosensitivity due to ab- gression line with a slope of (2-95±0-39)10"4 normal chromosome complements need not be considered (Sasaki, Tonomura and Matsubara, 1970). One hundred cells per patient were examined from cultures of control, unirradiated lymphocytes. 0.08 © © None of these cells contained chromosome-type 0.06 ® aberrations. Chromosome-type aberrations were observed in © 0.04 lymphocytes following a single partial-body dose m ® 0.02 in the range of 75 to 400 rads at times up to three days following treatment (Table I). 0 2 4 6 8 K) 20 30 It was necessary to determine whether or not the Dosex Field Size (»10 ) time at which blood was obtained from patients 0.10 after radiotherapy had any effect on aberration yield. 0.08 The frequency of dicentrics was 0-028 per cent (831 cells scored) at four to six hours after irradia0.06 tion (ten patients), 0-041 per cent (1,000 cells scored) 0.04 at 24 to 29 hours (12 patients) and 0-034 per cent (232 cells scored) at 45 to 48 hours (three patients). 0.02 Although the observed frequency is lower at four to six hours than at later times after radiation treatment, 300 400 the overall differences between the three frequencies Treatment Dose (Rads) 2 is not statistically significant (x =62-76; P=0-25). FIG. 1. The results were not sub-divided by treatment dose Dicentrics per cell as a function of treatment dose Xfieldsize because of the low frequency of aberrations. (upper figure). Dicentrics per cell as a function of treatTherefore the yields of aberrations observed at 4 to ment dose in rads (lower figure) fitted to linear regression 29 hours after irradiation were combined and con- Y=(2-27±0-33)10-4D for lines: patient group 1 represented by sidered with respect to treatment dose. When all r '• y = (0-84±0-21)10 4£> f patient group 2, represented by 12 patients were considered, the frequency of dior • — •_ centrics per cell versus dose, or versus dose X field
1"
4
The patient number is shown within the group symbols. 489
VOL.
48, No. 570 Gwyneth E. Watson and N. E. Gillies T A B L E II NUMBER OF CHROMOSOME ABERRATIONS AND FREQUENCY PER CELL (IN PARENTHESIS) INDUCED IN VITRO BY 230 KV X RAYS IN HUMAN LYMPHOCYTES CULTURED FOR 48 HOURS
Dose (rads)
Cells scored
Normal female
0 75 110 150 185 225 300 375
Normal female
Lymphocyte donor
Dicentrics
Rings
Fragments
100 201 234 245 79 66 49 61
0 25(0-12) 39(0-17) 62(0-25) 36 (0-46) 35(0-53) 43 (0-88) 59(0-97)
0 2(0-01) 9 (0-04) 20(0-08) 6(0-03) 7(0-11) 9(0-18) 11(0-18)
0 17(0-08) 29(0-12) 50(0-26) 29(0-37) 24(0-36) 22(0-45) 51(0-84)
0 75 150 225 300
100 354 210 120 200
0 35(0-10) 52(0-25) 56(0-47) 162(0-81)
0 3(0-01) 10(0-05) 8(0-07) 26(0-13)
0 25 (0-07) 42(0-20) 50(0-42) 143 (0-72)
Normal female
0 23-4 58-5 117 140-4 175-5 234
100 100 100 100 100 100 100
0 2(0-02) 5 (0-05) 19(0-19) 24(0-24) 38(0-38) 51 (0-51)
0 1 (0-01) 4(0-04) 5(0-05) 4(0-04) 9(0-09) 21(0-21)
1 (0-01) 9(0-09) 6(0-06) 19(0-19) 11(0-11) 20 (0-20) 50(0-50)
Female with reticulum cell sarcoma
0 58-5 117 234
100 100 100 90
0 3(0-03) 20 (0-20) 34(0-38)
0 3(0-03) 3 (0-03) 10(0-11)
0 8(0-08) 22(0-22) 45(0-50)
(P=O16). However, the total chromosomal breakage as a function of dose X field size did not fit a linear regression (P=O00067). The linear coefficient for dicentrics was 2-6 times greater for group one than for group two but the field size was also about three times larger on average. A representative proportion of the cells from the irradiated patients was analysed for the presence of chromatid aberrations. Only 14 chromatid breaks and one isolocus break were observed in 1,970 cells. Fourteen gaps and 16 isogaps were also recorded. The low yields of chromatid aberrations were similar to those reported for a random unirradiated sample of the population of Edinburgh (Court Brown et al., 1966). In vitro. The frequencies of dicentric, centric ring and fragment aberrations induced in three separate experiments using blood from the same donor are shown in Table II. There was good agreement between experiments for the yields of dicentric chromosomes (P=0-75) as shown in Fig. 2. The data for each aberration type from the three experiments were combined and fitted to three dose response models assuming a Poisson distribution of aberrations among cells. For dicentric aberrations, the response was equally well fitted to the power law
model Y=kD", [&=(l-59±0-66)10- 4 and n = l-49± 0-08, (P=0-69)], and to the quadratic relationship Y=aD+/3Z)2, [a=(9-52±l-66)10- 4 and £=(5-33 ±0-85)10- 6 , (P=0-95)]. The data could not be fitted to a linear model (P=0-0006). The yield of fragment aberrations was poorly fitted to all three models. Whole blood microcultures from patient 8, irradiated in vitro, yielded frequencies of aberrations at the three doses studied which agreed well with the response of lymphocytes from the normal healthy donor (Fig. 2). Comparison of aberration yields induced in vivo and
in vitro Using the standard X-ray in vitro dose response curve (Fig. 2), estimates of the X-ray dose received by the peripheral blood in vivo can be made. The estimated doses were multiplied by 0-75, to allow for the lesser efficiency of y rays with respect to X rays (Scott et al, 1970). This estimated y-ray dose is defined as'the "estimated peripheral blood dose" (EPBD) (Table I). This is sometimes referred to as the equivalent whole body dose (Dolphin, 1969; Purrot et al., 1972) but, in the absence of a reliable in vivo calibration curve for uniform
490
JUNE 1975
Radiation-induced chromosomal aberrations in human lymphocytes
DISCUSSION
calibration curve in assessing radiation dose in vivo. Low yields of chromosome-type aberrations were present in lymphocytes of all patients within a day of partial-body irradiation with doses up to 400 rads. As the lymphocytes were irradiated in metaphase at the Go stage of the DNA synthesis cycle (Howard and Pelc, 1951), chromatid-type aberrations would not be produced by irradiation, and, as expected, the frequency of chromatid-type aberrations was unaffected by radiation treatment. For all doses combined, the yield of dicentrics was not statistically lower at four hours than at 24 hours after treatment. Previously Buckton et al. (1967) reported lower frequencies of cells with unstable aberrations (Cu cells) in blood samples taken immediately than after 24 hours after partial-body irradiation to a spinal field for ankylosing spondylitis, but the differences between yields were significant for only two of the five doses studied. In the present study the yield of dicentrics was a linear function of dose but values for the slopes of the lines differed for each group of patients. The results from the two groups could not be combined to fit a single regression line. In general, group two patients receive treatment to smaller areas than group one patients and in addition the anatomical regions treated did not include so much lymphoid tissues. Group one patients received doses to the cervical mediastinal and axillary lymph nodes. As the lymphocytes in the peripheral blood constitute a very small proportion of the total lymphocyte population (Osgood, 1954), the greater part of the cytogenetic damage observed was probably induced in lymphocytes in the lymph nodes at the time of irradiation; if these lymphocytes were then released into circulation, this would explain the observation that the EPBD ranged from 9-49 per cent of the treatment dose. Only about 1 per cent of the total lymphocyte population would be irradiated in the peripheral blood.
A repeatable in vitro dose response calibration curve was obtained for the production of dicentric aberrations in human lymphocytes examined at first post-irradiation metaphase. The dose exponent n in the power law model was 1-49^0-08 for dicentrics and this dose response clearly differs from the almost linear dose response measured by Evans (1967) and Bajerska and Liniecki (1969) and the dose squared response found by Bender and Barcinski (1969), Norman and Sasaki (1966), but is in agreement with the findings of Sasaki (1971). Because of the differences in the dose response for dicentrics observed in different laboratories, it is necessary for each worker to construct an in vivo
Comparison of aberration yield in vivo and in vitro The in vitro dose response for dicentrics was curvilinear and well fitted to the quadratic response model while the in vivo treatment dose response for dicentrics was linear. As the level of dicentrics observed following in vivo irradiation was at the lower end of the in vitro dose response curve where the linear term of the quadratic expression predominates the in vitro and in vivo dose response curves can therefore be compared. The lower yields of chromosome-type aberrations in the peripheral blood of patients receiving partialbody irradiation as compared with the yields
FIG. 2. In vitro dose response curve for dicentric chromosomes induced in human leukocytes by 230 kV X rays and fitted to a power law response model, F=(l-59)10-4Z> i-49±o-O8. o, • and A experiments 1, 2 and 3 respectively. • , lymphocytes from patient (No. 8) with reticulum cell sarcoma. The in vitro dose calibration curve was constructed at Harwell.
whole body irradiation, equivalence cannot be assumed. For all patients the EPBD ranged from 9 to 49 per cent of the treatment dose and for most patients was in the range of 14 to 32 per cent. For group two patients, with smaller sizes of treatment areas, the EPBD was generally a smaller percentage of the treatment dose than was the case for group one patients.
491
VOL. 48, No. 570
Gwyneth E. Watson and N. E. Gillies induced by similar in vitro doses of radiation may be attributed to a combination of factors. One is the dilution of irradiated lymphocytes by non-irradiated cells during lymphocyte recirculation (Gowans and Knight, 1964). This occurs rapidly since the mean time of residence of the small lymphocyte in the peripheral blood has been estimated to be 4-7-5 minutes (Sharpe et ah, 1968). A second factor is the greater likelihood of the division of unirradiated than irradiated cells, thereby reducing the observed yield of aberrations in a mixture (Sharpe, 1969; Lloyd, Purrot and Dolphin, 1973). Thus the standard in vitro dose response curve is unlikely to represent the in vivo situation. Biological dosimetry From the results obtained from lymphocytes irradiated in vitro it might be concluded that the induction of dicentrics might be used as a reliable indication of radiation dose in vivo. However, it is obvious from the data obtained after partial body irradiation that this is not so. A major problem is the lack of knowledge concerning the distribution of lymphocytes in the body at the time of irradiation and their subsequent rate of release into the peripheral blood. Certainly, in the present study, the diseases for which the patients were being treated might affect the release of lymphocytes from the lymph nodes in a manner which may not occur in healthy individuals. At present, there is no way of ascertaining unambiguously from chromosome aberrations alone whether accidental exposure was received by the whole or only part of the body: the presence of complete chromosome-type exchange aberrations in first division human peripheral blood leucocytes only confirms that a radiation dose was received. Cells with aberrant chromosomes have been unambiguously identified in cultures of dermal fibroblasts irradiated in vivo (Bigger, Savage and Watson, 1972; Visfeldt, 1966). Therefore, a skin biopsy from the probable site of irradiation may be a more useful guide to the magnitude of partial-body exposure.
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ACKNOWLEDGMENTS
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