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Induction of chromosome aberrations by 90Sr β-particles in cultured human lymphocytes
Mutation Research, 163 (1986) 277-283
277
Elsevier MTR 04269
Induction of chromosome aberrations by 9°Srr-particles in cultured human lymphocytes N. Vulpis and G. Scarpa ENEA, CRE Casaccia, Laboratory of Dosimetry and Biophysics, C.P. 2400, 00100 Rome A.D. (Italy) (Received 17 March 1986) (Revision received 10 July 1986) (Accepted 16 July 1986)
Summary Human blood was irradiated with r-particles from an external source of 9°Sr. The source was a rolled piece of silver foil, active dimensions: 100 × 12.5 mm, incorporating 3.7 × 108 Bq (10 mCi) of 9°Sr/9°Y. After culturing for 48 h, the dicentric yield in the lymphocytes at the first metaphase was measured as a function of the dose in the blood. The aberration yield fitted the linear-quadratic function well, which is consistent with the single-track and two-track model for aberration formation at low LET radiation. The curve for 9°Sr r-rays was compared with a curve for 6°Co 3'-rays. The main difference between the coefficients was in the a values. With respect to 6°Co 3,-rays, the RBE calculated from the dose-effect relationships for dicentric production was 2.8 at the dose of 0.14 Gy; it decreased with increasing doses. The distribution of dicentrics was consistent with the Poisson distribution but showed a tendency to over-dispersion in the region of higher doses. A reason for these discrepancies is discussed.
9°Sr with its daughter 90y, which is assumed to be always present in equilibrium, is a pure source of highly energetic B-particles. As a fission product of uranium, it produces a share of the global contamination in the atmosphere which results from nuclear weapons testing and as a consequence of routine and accidental releases during the nuclear fuel cycle. When discharged from a nuclear installation, it may enter terrestrial food chains (Lalit et al., 1983; Wilkins et al., 1984) and may constitute a health hazard for urban populations. Its selective localization in endosteal tissue (Spiers, 1966) and its long effective retention therein result in chronic irradiation of the bone and the contiguous bone marrow. It is, therefore, a biologically hazardous fission-product radionuclide. Radioactive luminous paints containing 9°Sr and 126Ra have been used in industry. Some cases
of radiation dermatitis and a higher incidence of cancer have been observed in luminous-dial painters (Miiller et al., 1966; Tuscany and Miiller, 1967; Wenger et al., 1966, 1968; Volf, 1971; Klener et al., 1976). In this group of workers, a significant increase in cells carrying unstable or stable chromosome aberrations has also been observed, as compared to healthy controls. In the absence of sufficient data from studies on humans, studies have been conducted with several species of laboratory animals, mainly to obtain information on the genetic risks induced by 9°Sr (Reddy et al., 1977; Brooks and McLellan, 1969). The relationship between the dose of ionizing radiation and chromosome aberrations in cultured human lymphocytes provides a useful method for evaluating the absorbed dose (cytogenetic dosimetry) (Bender and Gooch, 1962; Buckton and Pike,.
0027-5107/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
278 1964; Norman and Sasaki, 1966; Evans, 1967; Heddle, 1969; Dolphin et al., 1973; Lloyd et al., 1975; Vulpis et al., 1976). There is a large amount of published data on the dependence of chromosome aberrations on radiation quality in human lymphocytes (Lloyd and Edwards, 1983). Nevertheless, with regard to fl-rays the only calibration data available refer to results obtained from in vitro exposure of lymphocytes to tritiated water (Bocian et al., 1977; Prosser et al., 1983; Vulpis, 1984). The experiments presented here were designed to analyse the relationship between dicentric chromosome yield and dose after direct irradiation of blood with fl-particles from a 9°Sr radioactive source. Materials
and
made of vinyl plastic (PVC) and consisted of two blocks clamped together at both sides of a 9°Sr r-source. Each block had 2 recesses of rectangular shape, 40 mm in length and 19 mm in height. 1.3-ml blood samples were put into these recesses in direct contact with the source surface. As shown "in section B-B of Fig. 1, the thickness of the blood layer was 3 mm, i.e., much shorter than the range of the r-particles; its height was 13 mm, almost identical to the active height of the source (12.5 mm). Mechanical shaking was performed during irradiation to mix the sample and to increase the homogeneity of the dose distribution. The source was a rolled piece of silver foil, active dimensions: 100 × 12.5 mm, incorporating 3.7 × 108 Bq (10 mCi) of 9°Sr/9°Y (Amersham, SIC. 7). A gasket, located around the recesses, prevented any leakage of the blood. The jig was protected from dust pollution by a cover. A theoretical calculation of the dose rate (D x), at a distance x in the soft tissue from the surface of the fl source, was carried out using a formula
methods
Irradiation conditions and dosimetry The apparatus used for irradiation of the blood samples is illustrated in Fig. 1. The device .was
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0 L
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,Fig. 1. Schematicdiagram of the apparatus used to irradiate peripheral blood using a 9°Srr-source (metal foil 0.3-mm thick).
279
proposed by Loevinger et al. (1965): D x = 2.88.10
-7
dose rate on the other side. This was probably due to the fact that the active part of the source was not located in the centre of the metal foil. It was decided to irradiate the blood samples only on the side where the dose rate was higher, in order to reduce the exposure time to a minimum. The results of the dosimetry, performed on the more active side of two 9°Sr sources, are summarized in Table 1. The reproducibility of the dose rate in the same compartment was reasonable, the SD being 3-6% of the mean value. On the other hand, the dose rate varied considerably among the different compartments, probably due to a non-homogeneous distribution of the activity through each source. The overall mean value was around 7.8 Gy h -1, with an SD of 1.3 G y h -1 (17%), i.e., about 50% of the value obtained using the above-mentioned calculation. This discrepancy can be explained by both the approximation of the theoretical approach and the uncertainty of the real value of the source activity. In the present paper, only the dosimetric data obtained experimentally (7.8 Gy h-1) are shown. Irradiation was carried out at room temperature. The doses given to the blood ranged from 0.138 to 2.76 Gy with irradiation times between 1 min 6 sec and 21 rain 33 sec. Higher doses were not attempted as this would have required exposure times considerably longer than 20 rain, which is usually accepted as the limit for acute exposure (Schmid et al., 1984). According to the distance of each lymphocyte from the source, eqn. (1) gives dose rates varying from about 0.55 to 0.08 Gy min h-1 with a factor of about 7 between the maximum and minimum.
vEao
X(c[(l+lnC
vx
)
--
e 1 ~x]
+ el-vx
Gy h -1
(1) in which 1, is the apparent absorption coefficient in cm 2 g-1, ~ the mean energy of fl-particles in MeV and o the surface activity in Bq cm-2; a and c are dimensionless parameters. Assuming p = 6.06 cm 2 g-1 a = 0.333 c=l
and taking into account the decay of the source and the presence of a front window 50-mg/cm 2 thick, the dose rate, D x, averaged over a layer of 3 mm of tissue would be Dx = 14.1 G y h-1 An experimental evaluation of this dose rate was carried out using a ferrous sulphate dosimetric solution (Fricke dosimeter). The solution was prepared and used according to the technique suggested by the British N L P (Ellis, 1977). The G value was assumed to be 15.4, according to data published by Peisach and Steyn (1960). The amount of the Fricke solution used in each measurement was 1.3 ml, i.e., the same volume as the blood. The dosimetry showed a strong asymmetry of the fl-emission from the two sides of the source, the dose rate on one side being about 25% of the
TABLE 1 RESULTS OF CHEMICAL DOSIMETRY IN T H E I R R A D I A T I O N JIG (Gy h - 1 ) Compartment a No.
Experiment No. 1
2
3
4
2 4 6 8
9.037 5.617 8.939 -
9.270 6.148 7.985 7.166
8.953 6.435 7.800 7.748
9.498 5.832 8.687 7.772
2, 4, 6, 8 a See Fig. 1.
SD
9.189 6.017 8.353 7.562
0.246 0.364 0.547 0.343
7.780
1.296
280 TABLE 2 T H E Y I E L D OF U N S T A B L E C H R O M O S O M E A B E R R A T I O N S I N D U C E D IN H U M A N L Y M P H O C Y T E S BY 9°Sr r - P A R TICLES Dose (Gy)
Cells scored
Dicenttics
Acentrics
Centric tings
Dicentrics per cell _+SE
0.138 0.274 0.55 1.10 1.65 2.20 2.76
1200 425 243 131 175 188 102
14 11 20 37 70 107 90
65 20 19 16 48 45 36
3 2 10 17 10
0.012 0.026 0.082 0.282 0.400 0.569 0.882
Lymphocyte culture Samples of heparinized whole blood from two healthy male donors were used. The whole-blood microcultures and criteria used in the aberration analysis followed a procedure described elsewhere (Vulpis et al., 1976). The only difference was the addition of 10 # M of 5-bromodeoxyuridine (BrdU) to the cultures to allow subsequent combined fluorescent-plus-Giemsa (FPG) staining of the chromosome preparation (Wolff and Perry, 1974). No significant differences in aberration yields were observed between the two donors used in the experiment and the data were combined for subsequent analysis.
Statistical analysis Distributions of aberrations among the cells were determined for each dose level. To assess the data for fitting to the Poisson distribution, the u-test was performed according to the description of Papworth (1970). It has been shown that if the absolute value of u is greater than 1.96, t h e n the over- or under-dispersion is significant. Results
The doses of r-radiation given to the blood and the number of aberrations observed for each of the doses as well as the number of dicentrics per cell are shown in Table 2. The uncertainties concerning the dose were on the order of 15%, as illustrated in Table 1. Dicentric and acentric yields were fitted using the least-squares method to a linear or linear-quadratic model with a Polyfit programme (Edwards and Dennis, 1973). This
0.003 0.008 0.018 0.046 0.048 0.055 0.093
programme assumes a Poisson distribution and applies a weighting factor to aberration yields. This factor is derived from the uncertainty about the dependent variable y for each experimental point. Zero-dose data points of 0.0005 + 0.0002 dicentrics and 0.003 + 0.001 acentrics were used in the curve fitting. Referring to a second-order polynomial, Table 3 gives the linear (a) and quadratic (fl) coefficients for dicentrics, their standard error (SE), as well as the value of X2 and the probability P that a X2 value greater than the computed one could have occurred purely by chance. The P value was nearly zero using a first-degree polynomial. The a, r , X 2 and P coefficients for acentrics are not presented in view of the poor overall fits to both the first- and second-degree polynomials ( P = 1 x 10 -4 and 6 × 10 -4, respectively). The coefficients for acute y-irradiation, also shown in Table 3 for comparison, derived from our unpublished data obtained at 0.5 Gy rain -1 with 6°Co y-radiation over the dose range 0.5-5.0 Gy. Fig. 2 compares the experimental
TABLE 3 E V A L U A T I O N OF T H E P A R A M E T E R S OF T H E D O S E RESPONSE R E L A T I O N S H I P F O R T H E I N D U C T I O N OF D I C E N T R I C S BY 9°Sr r - and 6°Co y-RAYS U S I N G A SECOND-DEGREE POLYNOMIAL Radiation
a _+SE ( x 1 0 -2 G y 1)
fl + SE ( × 1 0 -2 Gy 1)
a/fl (Gy)
X2
p
9°Srfl 60 Co y
8.22+1.72 0.93 + 1.16
8.81+1.14 5.83 __.0.50
0.93 0.16
4.99 1.98
0.42 0.57
281 TABLE 4 D I S T R I B U T I O N OF D I C E N T R I C S A M O N G T H E CELLS, R E L A T I V E V A R I A N C E A N D R E S U L T S OF T H E u-TEST F O R T H E POISSON N A T U R E O F D I S T R I B U T I O N Dose (Gy)
Cells scored
Dicentrics per cell
Distribution 0 1
x2/Y _+SE 2
3
4
5
6
(Y) 0.138 0.274 0.55 1.10 1.65 2.20 2.76
1200 425 243 131 175 188 102
0.012 0.026 0.082 0.282 0.400 0.569 0.882
1186 414 223 98 126 112 46
14 11 20 29 32 56 36
4 13 11 13
points and the fitted curves obtained for 9°Sr fl-radiation and 6°Co -/-radiation, using the coefficients in Table 3. The error bars of the points refer
--] klJ C2
I
Q.. CO 0.4
4 8 3
0 2
1 1
1
0.96 ± 0.04 0.97 +_0.06 0.92 ± 0.09 0.94 + 0.12 1.32+_0.11 1.28_+0.10 1.43 + 0.14
-0.27 -0.36 -0.89 -0.48 3.02 2.70 3.10
to both dose and counting uncertainties. Table 4 shows the data for the distribution of dicentrics among the cells. The mean number of observed dicentrics per cell (Y) is given for each dose (D) along with the relative variance (o2/Y) and the unit normal deviate (u). Table 5 gives the value of RBE with respect to our 6°C0 3' data for dicentrics induction. RBE was calculated by using the best fit curve for 6°C0 ,/-rays and the experimental points for 9°Sr fl-rays. Discussion The data for acute y-irradiation shown in Table 3 and Fig. 2 were obtained prior to introduction of the F P G method. This fact could have caused an error because not only first-division cells (M1) were scored. However, there is evidence that the number of second-division cells actually observed in the cultures 48 h after initiation was consistently below 10% (Purrot et al., 1981); in fact, in
0.i
0.~
0.01
TABLE 5 0.004
0.001 O. I
RBE OF 9°Sr fl-RAYS C O M P A R E D TO 6°Co y-RAYS
O. 2
0.~.
0.5 0.8 i
2
,
6 8 10 DOSE ( G y )
Fig. 2. Frequency of dicentrics plotted as a function of graded doses of 9°Sr and 6°Co. The vertical bars indicate the standard error of the mean. The horizontal bars indicate the uncertainty of the dose for 9°Sr irradiations.
Dose (Gy)
RBE
+ SEM
0.138 0.274 0.55 1.10 1.65 2.20 2.76
2.7 2.2 2.0 1.9 1.5 1.4 1.4
0.53 0.50 0.30 0.19 0.13 0.11 0.10
282 the present experiments the average frequency of second-division metaphases was less than 3%. The comparison between the two curves in Fig. 2 therefore seems to be valid. Comparison of the a a n d / 3 coefficients for 9°Sr and 6°Co showed that the main difference lay in the a term. The linear coefficient a was higher for 9°Sr than for 6°Co by a factor of about 9, whereas the quadratic coefficient only differed by a factor of 1.5. Accordingly, the ratio a/fl decreased from 0.93 G y to 0.16 Gy. Even if the dose rates applied in the present study were not strictly comparable, it is unlikely that this contributed m u c h to the differences in the c~ and fl coefficients. In fact, it is c o m m o n l y thought that changes in dose rate or delivery time predominantly affect the/3 and not the a coefficient. A comprehensive review of this subject is given b y Lloyd et al. (1984). Although both types of radiation examined have similar energies (0.93 MeV for 9°Sr /3- and 1.25 MeV for 6°Co y-rays) and, therefore, similar L E T values, they nevertheless reveal distinct differences in their biological effectiveness. As shown in Table 5, the R B E of 9°Sr/3-rays can be estimated a r o u n d 3 at low doses and decreases with increasing doses. These differences p r o b a b l y reflect differences in the L E T spectra of the two radiation types. The same possible explanation applies to the differences observed in the a and /3 coefficients (Virsik et al., 1977). The X 2 value of 4.99 and the P value of 0.42 obtained for the dicentrics induced by 9°Sr/3-rays using a second-order polynomial are consistent with the single-track and two-track model for aberration formation at low L E T radiation. As regards the data in Table 4, it is shown that a great deal of variation exists for intercell distribution of c h r o m o s o m e aberrations induced by radiation. Frequently, the underlying probability distribution of dicentric chromosomes is f o u n d to be Poissonian and its relative variance (x2/y) is found to differ essentially from unity. This case is typical for low L E T radiation at small and moderate doses (Virsik et al., 1977). Over-dispersion, characterized b y a relative variance exceeding unity, is observed for dicentrics when high L E T radiation is applied (Edwards et al., 1979; Virsik and Harder, 1981). The values of both relative variance and the u test seemed to suggest that
lower doses lead to a distribution of dicentrics which m a y be Poissonian. Conversely, at the 3 higher doses a marked over-dispersion was shown. It is difficult to understand this difference in dispersion between lower and higher dose levels. A possible explanation could be given by the fact that during the longer irradiations the blood cells might have settled, accumulating in the lower part of the sample where the dose rate is presumably m u c h lower, because this part of the sample corresponds to the edge of the active portion of the source (see section B - B of Fig. 1). In fact, overdispersion is to be expected for dicentrics even with low L E T radiation if the cells are non-uniformly irradiated (Lloyd and Edwards, 1983).
References Bender, M.A, and P.C. Gooch (1962) Types and rates of X-ray-induced chromosome aberrations in human blood irradiated in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 48, 522-532. Bocian, E., B. Ziemba-Zak, O. Rosiek and J. Sablinski (1977) Chromosome aberrations in human lymphocytes exposed to tritiated water in vitro, Curr. Top. Radiat. Res. Q., 12, 168-181. Brooks, A.L., and R.O. McLellan (1969) Chromosome aberrations and other effects produced by 9°Sr-9°Y in Chinese hamsters, Int. J. Radiat. Biol., 16, 545-561. Buckton, K.E., and M.C. Pike (1964) Chromosome investigations on lymphocytes from irradiated patients: effect of time in culture, Nature (London), 202, 714-715. Dolphin, G.W., D.C. Lloyd and R.J. Purrott (1973) Chromosome aberration analysis as a dosimetric technique in radiological protection, Health Phys., 25, 7-15. Edwards, A.A., and J.A. Dennis (1973) Polyfit A computer program for fitting a specified degree of polynomial to data points, NRPB-Mll, U.K. National Radiological Protection Board, Harwell. Edwards, A.A., D.C. Lloyd and R.J. Purrott (1979) Radiation induced chromosome aberrations and the Poisson distribution, Radiat. Environ. Biophys., 16, 89-100. Ellis, S.C. (1977) The dissemination of absorbed dose standards by chemical dosimetry, Mechanism and use of the Fricke dosemeter, in: E. Casnati (Ed.), Ionizing Radiation Metrology, Compositori, Bologna, pp. 163-194. Evans, H.J. (1967) Dose-response relations from in vitro studies, in: H.J. Evans, W.M. Court Brown and A.S. McLean (Eds.), Human Radiation Cytogenetics, North-Holland, Amsterdam, pp. 20-36. Heddle, J.A. (1969) Radiation-induced chromosome aberrations in man: a possible biological dosimeter, Fed. Proc., 28, 1790-1793. Klener, V., R. Tuscany and V. Svoboda (1976) Occurrence of tumors and kariological findings in persons with long-term
283 226Ra and 9°Sr bone depot, Vnitr. Lek., 22, 767-772. Lalit, B.Y., T.V. Ramachandran and S. Rajan (1983) Strontium-90 and caesium-137 in Indian tea, Radiat. Environ. Biophys., 22, 75-83. Lloyd, D.C., and A.A. Edwards (1983) Chromosome aberrations in human lymphocytes: effect of radiation quality, dose, and dose rate, in: T. Ishihara and M.S. Sasaki (Eds.), Radiation-Induced Chromosome Damage in Man, Liss, New York, pp. 23-49. Lloyd, D.C., R.J. Purrott, G.W. Dolphin, D. Bolton, A.A. Edwards and M.J. Corp (1975) The relationship between chromosome aberrations and low LET radiation dose to human lymphocytes, Int. J. Radiat. Biol., 28, 75-90. Lloyd, D.C., A.A. Edwards, J.S. Prosser and M.J. Corp (1984) The dose response relationships obtained at constant irradiation times for the induction of chromosome aberrations in human lymphocytes by cobalt-60 gamma-rays, Radiat. Environ. Biophys., 23,179-189. Loevinger, R., E.M. Japha and G.L. Brownell (1956) Discrete radioisotopes sources, in: G.J. Hine and G i . Brownell (Eds.), Radiation Dosimetry, Academic Press, New York, pp. 693-799. Miiller, J., V. Klener, R. Tuscany, J. Thomas, D. Br~zikovh and M. Hou~kovh (1966) Study of internal contamination with strontium-90 and radium-226 in man in relation to chemical findings, Health Physics, 12, 993-1006. Norman, A., and M.S. Sasaki (1966) Chromosome-exchange aberrations in human lymphocytes, Int. J. Radiat. Biol., 11, 321-328. Papworth, D.G. (1970) Testing and fitting frequency distributions, Appendix to J.R.K. Savage, Sites of radiation induced chromosome exchanges, Curr. Top. Radiat. Res. Q., 6, 129-194 Peisach, M., and J. Steyn (1960) Radiolytic oxidation of ferrous solutions with standardized internal sources of phosphorus-32, Nature (London), 187, 58-59. Prosser, J.S., D.C. Lloyd, A.A. Edwards and J.W. Stather (1983) The induction of chromosome aberrations in human lymphocytes by exposure to tritiated water in vitro, Radiation Protection Dosimetry, 4, 21-26. Purrott, R.J., N. Vulpis and D.C. Lloyd (1981) Chromosome dosimetry: the influence of culture media on the proliferation of irradiated and unirradiated human lymphocytes, Radiation Protection Dosimctry, 1,203-208. Reddy, P.P., and M. Sanjeeva Rao (1977) Induction of chromosome rearrangcments in spermatogonia of mouse by Strontium-90, Indian H. Hered., 9, 45-52.
Schmid, E., M. Bauchinger, S. Streng and U. Nahrstedt (1984) The effect of 220 kVp X-rays with different spectra on the dose response of chromosome aberrations in human lymphocytes, Radiat Environ. Biophys., 23, 305-309. Spiers, F.W. (1966) Dose to bone from strontium-90: implications for the setting of the maximum permissible body burden, Radiat. Res., 28, 624-642. Tuscany, R., and J. Miiller (1967) Chromosomal study of bone marrow and peripheral blood in persons carrying body burdens 226Ra and 9°Sr, in: H.J. Evans, W.M. Court Brown and A.S. McLean (Eds.), Human Radiation Cytogenetics, North-Holland, Amsterdam, pp. 203-207. Virsik, R.P., and D. Harder (1981) Analysis of radiation-induced acentrics fragments in human Go lymphocytes, Radiat. Environ. Biophys., 19, 29-40. Virsik, R.P., D. Harder and I. Hansmann (1977) The RBE of 30 kV X-rays for the induction of dicentric chromosomes in human lymphocytes, Radiat. Environ. Biophys., 14, 109-121. Volf, V. (1971) Strontium-90 effects in man, in: M. Goldman and L.K. Bustard (Eds.), Biomedical Implications of Radiostrontium Exposure, Proceedings of a Symposium, Davis, CA (February 22-24, 1971), pp. 313-325. Vulpis, N. (1984) The induction of chromosome aberrations in human lymphocytes by in vitro irradiation with fl-particles from tritiated water, Radiat. Res., 97, 511-518. Vulpis, N., G. Panetta and L. Tognacci (1976) Radiation-induced chromosome aberrations in radiological protection, Dose-response curves at low dose-levels, Int. J. Radiat. Biol., 29, 595-600. Wenger, P., K. Soucas and Y. Annen (1966) Radium and strontium-90 toxicity in human beings, Final Report, International Atomic Energy Agency, Vienna, Research Contract No. 38/RB. Wenger, P., L.G. Bengtsson, R.A. Dudley, B.E. Godfrey, W. Karniewicz, V. Lenger and D. Newton (1968) Whole-body counting of persons containing 9°Sr and 226Ra: an interlaboratory comparison, Health Physics, 14, 209-222. Wilkins, B.T., N. Green, N.J. Dodd and D.M. Smith (1984) Concentrations of strontium-90 and caesium-137 in milk produced in the channel islands, Radiation Protection Dosimetry, 8, 253-255. Wolff, S., and P. Perry (1974) Differential Giemsa staining of sister chromatids and the study of sister-chromatid exchanges without autoradiography, Chromosoma, 48, 341-353.
277
Elsevier MTR 04269
Induction of chromosome aberrations by 9°Srr-particles in cultured human lymphocytes N. Vulpis and G. Scarpa ENEA, CRE Casaccia, Laboratory of Dosimetry and Biophysics, C.P. 2400, 00100 Rome A.D. (Italy) (Received 17 March 1986) (Revision received 10 July 1986) (Accepted 16 July 1986)
Summary Human blood was irradiated with r-particles from an external source of 9°Sr. The source was a rolled piece of silver foil, active dimensions: 100 × 12.5 mm, incorporating 3.7 × 108 Bq (10 mCi) of 9°Sr/9°Y. After culturing for 48 h, the dicentric yield in the lymphocytes at the first metaphase was measured as a function of the dose in the blood. The aberration yield fitted the linear-quadratic function well, which is consistent with the single-track and two-track model for aberration formation at low LET radiation. The curve for 9°Sr r-rays was compared with a curve for 6°Co 3'-rays. The main difference between the coefficients was in the a values. With respect to 6°Co 3,-rays, the RBE calculated from the dose-effect relationships for dicentric production was 2.8 at the dose of 0.14 Gy; it decreased with increasing doses. The distribution of dicentrics was consistent with the Poisson distribution but showed a tendency to over-dispersion in the region of higher doses. A reason for these discrepancies is discussed.
9°Sr with its daughter 90y, which is assumed to be always present in equilibrium, is a pure source of highly energetic B-particles. As a fission product of uranium, it produces a share of the global contamination in the atmosphere which results from nuclear weapons testing and as a consequence of routine and accidental releases during the nuclear fuel cycle. When discharged from a nuclear installation, it may enter terrestrial food chains (Lalit et al., 1983; Wilkins et al., 1984) and may constitute a health hazard for urban populations. Its selective localization in endosteal tissue (Spiers, 1966) and its long effective retention therein result in chronic irradiation of the bone and the contiguous bone marrow. It is, therefore, a biologically hazardous fission-product radionuclide. Radioactive luminous paints containing 9°Sr and 126Ra have been used in industry. Some cases
of radiation dermatitis and a higher incidence of cancer have been observed in luminous-dial painters (Miiller et al., 1966; Tuscany and Miiller, 1967; Wenger et al., 1966, 1968; Volf, 1971; Klener et al., 1976). In this group of workers, a significant increase in cells carrying unstable or stable chromosome aberrations has also been observed, as compared to healthy controls. In the absence of sufficient data from studies on humans, studies have been conducted with several species of laboratory animals, mainly to obtain information on the genetic risks induced by 9°Sr (Reddy et al., 1977; Brooks and McLellan, 1969). The relationship between the dose of ionizing radiation and chromosome aberrations in cultured human lymphocytes provides a useful method for evaluating the absorbed dose (cytogenetic dosimetry) (Bender and Gooch, 1962; Buckton and Pike,.
0027-5107/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
278 1964; Norman and Sasaki, 1966; Evans, 1967; Heddle, 1969; Dolphin et al., 1973; Lloyd et al., 1975; Vulpis et al., 1976). There is a large amount of published data on the dependence of chromosome aberrations on radiation quality in human lymphocytes (Lloyd and Edwards, 1983). Nevertheless, with regard to fl-rays the only calibration data available refer to results obtained from in vitro exposure of lymphocytes to tritiated water (Bocian et al., 1977; Prosser et al., 1983; Vulpis, 1984). The experiments presented here were designed to analyse the relationship between dicentric chromosome yield and dose after direct irradiation of blood with fl-particles from a 9°Sr radioactive source. Materials
and
made of vinyl plastic (PVC) and consisted of two blocks clamped together at both sides of a 9°Sr r-source. Each block had 2 recesses of rectangular shape, 40 mm in length and 19 mm in height. 1.3-ml blood samples were put into these recesses in direct contact with the source surface. As shown "in section B-B of Fig. 1, the thickness of the blood layer was 3 mm, i.e., much shorter than the range of the r-particles; its height was 13 mm, almost identical to the active height of the source (12.5 mm). Mechanical shaking was performed during irradiation to mix the sample and to increase the homogeneity of the dose distribution. The source was a rolled piece of silver foil, active dimensions: 100 × 12.5 mm, incorporating 3.7 × 108 Bq (10 mCi) of 9°Sr/9°Y (Amersham, SIC. 7). A gasket, located around the recesses, prevented any leakage of the blood. The jig was protected from dust pollution by a cover. A theoretical calculation of the dose rate (D x), at a distance x in the soft tissue from the surface of the fl source, was carried out using a formula
methods
Irradiation conditions and dosimetry The apparatus used for irradiation of the blood samples is illustrated in Fig. 1. The device .was
i
1 I I
F',
i i i
._i_~
- - -
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A-A
BLOOD
COMPARTMENTS
!~//,6"J6",¢fJ~//ffJff~
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SOURCE/ COVER
AIR BLOOD
SOURCE u
Active
part
of
90Sr s o u r c e
0 L
1
2
3
4 I
cm
,Fig. 1. Schematicdiagram of the apparatus used to irradiate peripheral blood using a 9°Srr-source (metal foil 0.3-mm thick).
279
proposed by Loevinger et al. (1965): D x = 2.88.10
-7
dose rate on the other side. This was probably due to the fact that the active part of the source was not located in the centre of the metal foil. It was decided to irradiate the blood samples only on the side where the dose rate was higher, in order to reduce the exposure time to a minimum. The results of the dosimetry, performed on the more active side of two 9°Sr sources, are summarized in Table 1. The reproducibility of the dose rate in the same compartment was reasonable, the SD being 3-6% of the mean value. On the other hand, the dose rate varied considerably among the different compartments, probably due to a non-homogeneous distribution of the activity through each source. The overall mean value was around 7.8 Gy h -1, with an SD of 1.3 G y h -1 (17%), i.e., about 50% of the value obtained using the above-mentioned calculation. This discrepancy can be explained by both the approximation of the theoretical approach and the uncertainty of the real value of the source activity. In the present paper, only the dosimetric data obtained experimentally (7.8 Gy h-1) are shown. Irradiation was carried out at room temperature. The doses given to the blood ranged from 0.138 to 2.76 Gy with irradiation times between 1 min 6 sec and 21 rain 33 sec. Higher doses were not attempted as this would have required exposure times considerably longer than 20 rain, which is usually accepted as the limit for acute exposure (Schmid et al., 1984). According to the distance of each lymphocyte from the source, eqn. (1) gives dose rates varying from about 0.55 to 0.08 Gy min h-1 with a factor of about 7 between the maximum and minimum.
vEao
X(c[(l+lnC
vx
)
--
e 1 ~x]
+ el-vx
Gy h -1
(1) in which 1, is the apparent absorption coefficient in cm 2 g-1, ~ the mean energy of fl-particles in MeV and o the surface activity in Bq cm-2; a and c are dimensionless parameters. Assuming p = 6.06 cm 2 g-1 a = 0.333 c=l
and taking into account the decay of the source and the presence of a front window 50-mg/cm 2 thick, the dose rate, D x, averaged over a layer of 3 mm of tissue would be Dx = 14.1 G y h-1 An experimental evaluation of this dose rate was carried out using a ferrous sulphate dosimetric solution (Fricke dosimeter). The solution was prepared and used according to the technique suggested by the British N L P (Ellis, 1977). The G value was assumed to be 15.4, according to data published by Peisach and Steyn (1960). The amount of the Fricke solution used in each measurement was 1.3 ml, i.e., the same volume as the blood. The dosimetry showed a strong asymmetry of the fl-emission from the two sides of the source, the dose rate on one side being about 25% of the
TABLE 1 RESULTS OF CHEMICAL DOSIMETRY IN T H E I R R A D I A T I O N JIG (Gy h - 1 ) Compartment a No.
Experiment No. 1
2
3
4
2 4 6 8
9.037 5.617 8.939 -
9.270 6.148 7.985 7.166
8.953 6.435 7.800 7.748
9.498 5.832 8.687 7.772
2, 4, 6, 8 a See Fig. 1.
SD
9.189 6.017 8.353 7.562
0.246 0.364 0.547 0.343
7.780
1.296
280 TABLE 2 T H E Y I E L D OF U N S T A B L E C H R O M O S O M E A B E R R A T I O N S I N D U C E D IN H U M A N L Y M P H O C Y T E S BY 9°Sr r - P A R TICLES Dose (Gy)
Cells scored
Dicenttics
Acentrics
Centric tings
Dicentrics per cell _+SE
0.138 0.274 0.55 1.10 1.65 2.20 2.76
1200 425 243 131 175 188 102
14 11 20 37 70 107 90
65 20 19 16 48 45 36
3 2 10 17 10
0.012 0.026 0.082 0.282 0.400 0.569 0.882
Lymphocyte culture Samples of heparinized whole blood from two healthy male donors were used. The whole-blood microcultures and criteria used in the aberration analysis followed a procedure described elsewhere (Vulpis et al., 1976). The only difference was the addition of 10 # M of 5-bromodeoxyuridine (BrdU) to the cultures to allow subsequent combined fluorescent-plus-Giemsa (FPG) staining of the chromosome preparation (Wolff and Perry, 1974). No significant differences in aberration yields were observed between the two donors used in the experiment and the data were combined for subsequent analysis.
Statistical analysis Distributions of aberrations among the cells were determined for each dose level. To assess the data for fitting to the Poisson distribution, the u-test was performed according to the description of Papworth (1970). It has been shown that if the absolute value of u is greater than 1.96, t h e n the over- or under-dispersion is significant. Results
The doses of r-radiation given to the blood and the number of aberrations observed for each of the doses as well as the number of dicentrics per cell are shown in Table 2. The uncertainties concerning the dose were on the order of 15%, as illustrated in Table 1. Dicentric and acentric yields were fitted using the least-squares method to a linear or linear-quadratic model with a Polyfit programme (Edwards and Dennis, 1973). This
0.003 0.008 0.018 0.046 0.048 0.055 0.093
programme assumes a Poisson distribution and applies a weighting factor to aberration yields. This factor is derived from the uncertainty about the dependent variable y for each experimental point. Zero-dose data points of 0.0005 + 0.0002 dicentrics and 0.003 + 0.001 acentrics were used in the curve fitting. Referring to a second-order polynomial, Table 3 gives the linear (a) and quadratic (fl) coefficients for dicentrics, their standard error (SE), as well as the value of X2 and the probability P that a X2 value greater than the computed one could have occurred purely by chance. The P value was nearly zero using a first-degree polynomial. The a, r , X 2 and P coefficients for acentrics are not presented in view of the poor overall fits to both the first- and second-degree polynomials ( P = 1 x 10 -4 and 6 × 10 -4, respectively). The coefficients for acute y-irradiation, also shown in Table 3 for comparison, derived from our unpublished data obtained at 0.5 Gy rain -1 with 6°Co y-radiation over the dose range 0.5-5.0 Gy. Fig. 2 compares the experimental
TABLE 3 E V A L U A T I O N OF T H E P A R A M E T E R S OF T H E D O S E RESPONSE R E L A T I O N S H I P F O R T H E I N D U C T I O N OF D I C E N T R I C S BY 9°Sr r - and 6°Co y-RAYS U S I N G A SECOND-DEGREE POLYNOMIAL Radiation
a _+SE ( x 1 0 -2 G y 1)
fl + SE ( × 1 0 -2 Gy 1)
a/fl (Gy)
X2
p
9°Srfl 60 Co y
8.22+1.72 0.93 + 1.16
8.81+1.14 5.83 __.0.50
0.93 0.16
4.99 1.98
0.42 0.57
281 TABLE 4 D I S T R I B U T I O N OF D I C E N T R I C S A M O N G T H E CELLS, R E L A T I V E V A R I A N C E A N D R E S U L T S OF T H E u-TEST F O R T H E POISSON N A T U R E O F D I S T R I B U T I O N Dose (Gy)
Cells scored
Dicentrics per cell
Distribution 0 1
x2/Y _+SE 2
3
4
5
6
(Y) 0.138 0.274 0.55 1.10 1.65 2.20 2.76
1200 425 243 131 175 188 102
0.012 0.026 0.082 0.282 0.400 0.569 0.882
1186 414 223 98 126 112 46
14 11 20 29 32 56 36
4 13 11 13
points and the fitted curves obtained for 9°Sr fl-radiation and 6°Co -/-radiation, using the coefficients in Table 3. The error bars of the points refer
--] klJ C2
I
Q.. CO 0.4
4 8 3
0 2
1 1
1
0.96 ± 0.04 0.97 +_0.06 0.92 ± 0.09 0.94 + 0.12 1.32+_0.11 1.28_+0.10 1.43 + 0.14
-0.27 -0.36 -0.89 -0.48 3.02 2.70 3.10
to both dose and counting uncertainties. Table 4 shows the data for the distribution of dicentrics among the cells. The mean number of observed dicentrics per cell (Y) is given for each dose (D) along with the relative variance (o2/Y) and the unit normal deviate (u). Table 5 gives the value of RBE with respect to our 6°C0 3' data for dicentrics induction. RBE was calculated by using the best fit curve for 6°C0 ,/-rays and the experimental points for 9°Sr fl-rays. Discussion The data for acute y-irradiation shown in Table 3 and Fig. 2 were obtained prior to introduction of the F P G method. This fact could have caused an error because not only first-division cells (M1) were scored. However, there is evidence that the number of second-division cells actually observed in the cultures 48 h after initiation was consistently below 10% (Purrot et al., 1981); in fact, in
0.i
0.~
0.01
TABLE 5 0.004
0.001 O. I
RBE OF 9°Sr fl-RAYS C O M P A R E D TO 6°Co y-RAYS
O. 2
0.~.
0.5 0.8 i
2
,
6 8 10 DOSE ( G y )
Fig. 2. Frequency of dicentrics plotted as a function of graded doses of 9°Sr and 6°Co. The vertical bars indicate the standard error of the mean. The horizontal bars indicate the uncertainty of the dose for 9°Sr irradiations.
Dose (Gy)
RBE
+ SEM
0.138 0.274 0.55 1.10 1.65 2.20 2.76
2.7 2.2 2.0 1.9 1.5 1.4 1.4
0.53 0.50 0.30 0.19 0.13 0.11 0.10
282 the present experiments the average frequency of second-division metaphases was less than 3%. The comparison between the two curves in Fig. 2 therefore seems to be valid. Comparison of the a a n d / 3 coefficients for 9°Sr and 6°Co showed that the main difference lay in the a term. The linear coefficient a was higher for 9°Sr than for 6°Co by a factor of about 9, whereas the quadratic coefficient only differed by a factor of 1.5. Accordingly, the ratio a/fl decreased from 0.93 G y to 0.16 Gy. Even if the dose rates applied in the present study were not strictly comparable, it is unlikely that this contributed m u c h to the differences in the c~ and fl coefficients. In fact, it is c o m m o n l y thought that changes in dose rate or delivery time predominantly affect the/3 and not the a coefficient. A comprehensive review of this subject is given b y Lloyd et al. (1984). Although both types of radiation examined have similar energies (0.93 MeV for 9°Sr /3- and 1.25 MeV for 6°Co y-rays) and, therefore, similar L E T values, they nevertheless reveal distinct differences in their biological effectiveness. As shown in Table 5, the R B E of 9°Sr/3-rays can be estimated a r o u n d 3 at low doses and decreases with increasing doses. These differences p r o b a b l y reflect differences in the L E T spectra of the two radiation types. The same possible explanation applies to the differences observed in the a and /3 coefficients (Virsik et al., 1977). The X 2 value of 4.99 and the P value of 0.42 obtained for the dicentrics induced by 9°Sr/3-rays using a second-order polynomial are consistent with the single-track and two-track model for aberration formation at low L E T radiation. As regards the data in Table 4, it is shown that a great deal of variation exists for intercell distribution of c h r o m o s o m e aberrations induced by radiation. Frequently, the underlying probability distribution of dicentric chromosomes is f o u n d to be Poissonian and its relative variance (x2/y) is found to differ essentially from unity. This case is typical for low L E T radiation at small and moderate doses (Virsik et al., 1977). Over-dispersion, characterized b y a relative variance exceeding unity, is observed for dicentrics when high L E T radiation is applied (Edwards et al., 1979; Virsik and Harder, 1981). The values of both relative variance and the u test seemed to suggest that
lower doses lead to a distribution of dicentrics which m a y be Poissonian. Conversely, at the 3 higher doses a marked over-dispersion was shown. It is difficult to understand this difference in dispersion between lower and higher dose levels. A possible explanation could be given by the fact that during the longer irradiations the blood cells might have settled, accumulating in the lower part of the sample where the dose rate is presumably m u c h lower, because this part of the sample corresponds to the edge of the active portion of the source (see section B - B of Fig. 1). In fact, overdispersion is to be expected for dicentrics even with low L E T radiation if the cells are non-uniformly irradiated (Lloyd and Edwards, 1983).
References Bender, M.A, and P.C. Gooch (1962) Types and rates of X-ray-induced chromosome aberrations in human blood irradiated in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 48, 522-532. Bocian, E., B. Ziemba-Zak, O. Rosiek and J. Sablinski (1977) Chromosome aberrations in human lymphocytes exposed to tritiated water in vitro, Curr. Top. Radiat. Res. Q., 12, 168-181. Brooks, A.L., and R.O. McLellan (1969) Chromosome aberrations and other effects produced by 9°Sr-9°Y in Chinese hamsters, Int. J. Radiat. Biol., 16, 545-561. Buckton, K.E., and M.C. Pike (1964) Chromosome investigations on lymphocytes from irradiated patients: effect of time in culture, Nature (London), 202, 714-715. Dolphin, G.W., D.C. Lloyd and R.J. Purrott (1973) Chromosome aberration analysis as a dosimetric technique in radiological protection, Health Phys., 25, 7-15. Edwards, A.A., and J.A. Dennis (1973) Polyfit A computer program for fitting a specified degree of polynomial to data points, NRPB-Mll, U.K. National Radiological Protection Board, Harwell. Edwards, A.A., D.C. Lloyd and R.J. Purrott (1979) Radiation induced chromosome aberrations and the Poisson distribution, Radiat. Environ. Biophys., 16, 89-100. Ellis, S.C. (1977) The dissemination of absorbed dose standards by chemical dosimetry, Mechanism and use of the Fricke dosemeter, in: E. Casnati (Ed.), Ionizing Radiation Metrology, Compositori, Bologna, pp. 163-194. Evans, H.J. (1967) Dose-response relations from in vitro studies, in: H.J. Evans, W.M. Court Brown and A.S. McLean (Eds.), Human Radiation Cytogenetics, North-Holland, Amsterdam, pp. 20-36. Heddle, J.A. (1969) Radiation-induced chromosome aberrations in man: a possible biological dosimeter, Fed. Proc., 28, 1790-1793. Klener, V., R. Tuscany and V. Svoboda (1976) Occurrence of tumors and kariological findings in persons with long-term
283 226Ra and 9°Sr bone depot, Vnitr. Lek., 22, 767-772. Lalit, B.Y., T.V. Ramachandran and S. Rajan (1983) Strontium-90 and caesium-137 in Indian tea, Radiat. Environ. Biophys., 22, 75-83. Lloyd, D.C., and A.A. Edwards (1983) Chromosome aberrations in human lymphocytes: effect of radiation quality, dose, and dose rate, in: T. Ishihara and M.S. Sasaki (Eds.), Radiation-Induced Chromosome Damage in Man, Liss, New York, pp. 23-49. Lloyd, D.C., R.J. Purrott, G.W. Dolphin, D. Bolton, A.A. Edwards and M.J. Corp (1975) The relationship between chromosome aberrations and low LET radiation dose to human lymphocytes, Int. J. Radiat. Biol., 28, 75-90. Lloyd, D.C., A.A. Edwards, J.S. Prosser and M.J. Corp (1984) The dose response relationships obtained at constant irradiation times for the induction of chromosome aberrations in human lymphocytes by cobalt-60 gamma-rays, Radiat. Environ. Biophys., 23,179-189. Loevinger, R., E.M. Japha and G.L. Brownell (1956) Discrete radioisotopes sources, in: G.J. Hine and G i . Brownell (Eds.), Radiation Dosimetry, Academic Press, New York, pp. 693-799. Miiller, J., V. Klener, R. Tuscany, J. Thomas, D. Br~zikovh and M. Hou~kovh (1966) Study of internal contamination with strontium-90 and radium-226 in man in relation to chemical findings, Health Physics, 12, 993-1006. Norman, A., and M.S. Sasaki (1966) Chromosome-exchange aberrations in human lymphocytes, Int. J. Radiat. Biol., 11, 321-328. Papworth, D.G. (1970) Testing and fitting frequency distributions, Appendix to J.R.K. Savage, Sites of radiation induced chromosome exchanges, Curr. Top. Radiat. Res. Q., 6, 129-194 Peisach, M., and J. Steyn (1960) Radiolytic oxidation of ferrous solutions with standardized internal sources of phosphorus-32, Nature (London), 187, 58-59. Prosser, J.S., D.C. Lloyd, A.A. Edwards and J.W. Stather (1983) The induction of chromosome aberrations in human lymphocytes by exposure to tritiated water in vitro, Radiation Protection Dosimetry, 4, 21-26. Purrott, R.J., N. Vulpis and D.C. Lloyd (1981) Chromosome dosimetry: the influence of culture media on the proliferation of irradiated and unirradiated human lymphocytes, Radiation Protection Dosimctry, 1,203-208. Reddy, P.P., and M. Sanjeeva Rao (1977) Induction of chromosome rearrangcments in spermatogonia of mouse by Strontium-90, Indian H. Hered., 9, 45-52.
Schmid, E., M. Bauchinger, S. Streng and U. Nahrstedt (1984) The effect of 220 kVp X-rays with different spectra on the dose response of chromosome aberrations in human lymphocytes, Radiat Environ. Biophys., 23, 305-309. Spiers, F.W. (1966) Dose to bone from strontium-90: implications for the setting of the maximum permissible body burden, Radiat. Res., 28, 624-642. Tuscany, R., and J. Miiller (1967) Chromosomal study of bone marrow and peripheral blood in persons carrying body burdens 226Ra and 9°Sr, in: H.J. Evans, W.M. Court Brown and A.S. McLean (Eds.), Human Radiation Cytogenetics, North-Holland, Amsterdam, pp. 203-207. Virsik, R.P., and D. Harder (1981) Analysis of radiation-induced acentrics fragments in human Go lymphocytes, Radiat. Environ. Biophys., 19, 29-40. Virsik, R.P., D. Harder and I. Hansmann (1977) The RBE of 30 kV X-rays for the induction of dicentric chromosomes in human lymphocytes, Radiat. Environ. Biophys., 14, 109-121. Volf, V. (1971) Strontium-90 effects in man, in: M. Goldman and L.K. Bustard (Eds.), Biomedical Implications of Radiostrontium Exposure, Proceedings of a Symposium, Davis, CA (February 22-24, 1971), pp. 313-325. Vulpis, N. (1984) The induction of chromosome aberrations in human lymphocytes by in vitro irradiation with fl-particles from tritiated water, Radiat. Res., 97, 511-518. Vulpis, N., G. Panetta and L. Tognacci (1976) Radiation-induced chromosome aberrations in radiological protection, Dose-response curves at low dose-levels, Int. J. Radiat. Biol., 29, 595-600. Wenger, P., K. Soucas and Y. Annen (1966) Radium and strontium-90 toxicity in human beings, Final Report, International Atomic Energy Agency, Vienna, Research Contract No. 38/RB. Wenger, P., L.G. Bengtsson, R.A. Dudley, B.E. Godfrey, W. Karniewicz, V. Lenger and D. Newton (1968) Whole-body counting of persons containing 9°Sr and 226Ra: an interlaboratory comparison, Health Physics, 14, 209-222. Wilkins, B.T., N. Green, N.J. Dodd and D.M. Smith (1984) Concentrations of strontium-90 and caesium-137 in milk produced in the channel islands, Radiation Protection Dosimetry, 8, 253-255. Wolff, S., and P. Perry (1974) Differential Giemsa staining of sister chromatids and the study of sister-chromatid exchanges without autoradiography, Chromosoma, 48, 341-353.