INT . J. RADIAT. BIOL ., 1980, VOL . 37, NO . 2, 23 1-236

Chromosome aberrations induced by low doses of X-rays inhuman lymphocytes in vitro

B. ZIEMBA-ZOLTOWSKA, E . BOCIAN, O. ROSIEKand J . SABLINSKIDepartment of Radiobiology and Health Protection,Institute of Nuclear Research, Warszawa, Poland

(Received 29 May 1979 ; accepted 5 September 1979)

1 . IntroductionInvestigations of the relationship between the yield of aberrations in human

lymphocytes and dose of ionizing radiation are usually performed at doses above50 rad. The curves derived from the data obtained in these experiments are bestrepresented by the quadratic equation y=ao + a1D+a2D2 , where y is the aberrationyield, D is the radiation dose, a o is the spontaneous aberration yield, and a1 and a2 areconstants defining the `one track and two track' components respectively .

Data on the yields of aberrations after irradiation with low doses are relativelyscanty, although such data are of interest from the point of view of biologicaldosimetry as well as for elucidation of the mechanism of formation of radiation-induced chromosome aberrations . Experiments with low doses of radiation are verytime-consuming because analysis of a large number of mitotic cells is necessary andthis is probably the main reason for few data being available .

Investigations at the low dose range have been performed by Kucerova,Anderson, Buckton and Evans (1972), Lloyd, Purrott, Dolphin, Bolton, Edwardsand Corp (1975), Vulpis, Panetta and Tognacci (1976) and Luchnik and Sevankaev(1976) . The data obtained by these authors suggest that the curves described by thequadratic equation are inadequate at the low dose range . This is caused by the effectof the quadratic component decreasing with decreasing radiation dose so that below afew tens of rad of low LET radiation the linear component begins to dominate .Therefore for biological dosimetry purposes it is necessary to prepare an adequatecalibration curve .

In this paper we present the calibration curve obtained in our experiments at alow dose range of ionizing radiation (115-57 . 5 rad) . In addition, a dose-responsecurve for dicentrics plus centric rings covering the range of 5-60 rad from the pooleddata of Kucerova et al . (1972), Vulpis et al . (1976) and present studies was derived .

2 . Materials and methods2.1 . Culture conditions

Blood from three healthy female donors (about 23 years old) was used in theseexperiments. Whole blood was cultured by the modified Evans method (Evans1965). Samples (0 . 3 ml) of freshly drawn heparinized venous blood were added to aculture solution consisting of TC 199 medium (4 ml), calf serum (1 ml) and PHA M,Difco Laboratory (0 . 1 ml) . After 47 hours 0 .4 yg/ml Colcemid (Ciba) was added andthe cells were harvested 3 h later (i .e . after 50 hours). Thereafter the cells were

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treated with 0 .075 M KC1 for 8 minutes at 37°C, fixed with absolute methyl alcohol-acetic acid (3 : 1) and spread on microscope slides . The slides were stained withGiemsa and analysed for dicentrics, centric rings and acentric fragments . Dicentricsand centric rings were counted only when an accompanying fragment could be seen .For 151 dicentrics plus centric rings counted we have observed only 3 withoutaccompanying fragments . Those dicentrics were rejected from our calculations .Acentric rings, interstitial deletions (minutes) and terminal deletions were pooled tobe classified as acentrics .

2.2. Irradiation conditionsVenous blood was drawn into a sterile heparinized plastic syringe and exposed to

various doses of X-rays in the range 11 .5-57 . 5 rad . The samples were irradiated witha 180 kV Siemens Stabilipan machine at 18 mA with a 1 mm Cu filter, at an exposure-rate of 12 .12 R/min. The X-ray exposure was measured using an ion chamber andTLD-100 thermoluminescent dosemeters (Harshaw Chemical Company, USA) .Exposures were converted to doses in rad by applying the factor 0 . 95 . Irradiationswere performed at room temperature .

2.3 . Data analysisStandard errors for aberration yields were calculated on the assumption of the

Poisson distribution of aberrations between the cells .The yields of dicentrics plus centric rings and acentrics have been submitted to

linear, quadratic and power regressions . The data were evaluated by the general leastsquares program DATFIT for a Cyber 73 computer, produced by the Control DataCorporation (Van Der Voort and Halleux 1973) . Together with the parameterscalculated, 95 per cent confidence limits bounded by an ellipsoid in parameter spacehave been established . An interpretation of confidence limits for parametersexpressed this way are also discussed in the work of Van Der Voort and Halleux(1973) . The extreme values of the ellipsoid were assumed as upper and lower valuesof parameters at the 95 per cent confidence interval .

To test the goodness of fit, the x 2 test was used to compare theoretical andexperimental distributions of aberration yields .

3 . Results and discussionThe results obtained for a given class of aberration in three separate experiments

did not significantly differ, so the data for each radiation dose were pooled . Theresults are presented in the table . The data were analysed using linear, quadratic andpower law models . The three equations obtained for dicentrics plus centric rings arerespectively :

y=(0 . 148-V0 .205)10-2 +(0 .463+0.059)10 -3D

y=(4 . 75 T-2 .45)10 -2 +(0 .595+0 .070)10 -3D+(-0 .228+0 . 145)10 -5D2

y=(0.918+0,103)10 3 Do .841+0 .03

The frequency of dicentrics plus centric rings is better fitted by the linear dose-response model (x2 =2-77, p>0-25) than the quadratic (x2 =2-04, p>0-1) . Thenumber of degrees of freedom was assumed to be k = n - c - 1, where c is the numberof parameters in the model, n is the number of the data points .

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The dose relationship for acentrics is also better fitted to a linear model (x2 = 2 . 93,p > 0 .15) than the quadratic model (x 2= 321, p > 0 . 1) . The linear regression gave thevalues y=(0.746--0.435)10 -2 +(0679+0 . 125)10 -3D.

Both for dicentrics plus centric rings and acentrics the data gave the worst fit to apower response (x 2 = 2 . 88, p > 01 and x 2 = 3

p > 0- 1, respectively) .Figures 1 and 2 show the yields of dicentrics plus centric rings and acentrics per

cell, respectively, and the curves represent the 95 per cent confidence limits ofregression lines . These figures show that both dicentrics plus centric rings andacentrics increase linearly with dose. The curve for acentrics has an intercept of00074 because of acentrics occurring in unirradiated cells (figure 2) . For dicentricsplus centric rings the curve starts practically at zero (figure 1) .

The best fit to linear equation indicates that asymmetrical chromosomeexchanges at low doses of radiation are produced predominantly by a single-trackmechanism .

The linear relationship for dicentrics plus centric rings obtained in ourexperiments confirms results published by Lloyd et al . (1975), Kucerova et al. (1972)and Vulpis et al. (1976) for low doses of 250 kV X-rays. However, Kucerova et al .(1972) found no difference in the number of dicentrics in comparison with thecontrol for doses up to 15 rad. Luchnik and Sevankaev (1976) found more dicentricsthan expected from extrapolation of the dose-response curve from higher doses of y-radiation. However a plateau was observed between 10 and 30 rad. Our results are ingood agreement with the data of Vulpis et al . (1976) . The regression coefficients arevery similar to the linear equation for dicentrics (0 .483 x 10-3) in the report of Vulpiset al. (1976) and for dicentrics plus centric rings (0.463 x 10

_3 )in our study .

Comparable experimental conditions allow us to pool the data of Kucerova et al .(1972), Vulpis et al . (1976) and those from our experiments and to establish therelationship between the number of dicentrics plus centric rings and radiation dosefor greater cell numbers . The pooled data were analysed using linear and quadraticdose-response models . We found the best fit was to the linear regression fordicentrics plus centric rings and is described by the equation y=(0. 193 +0. 195)10 -2+ (0. 488 --0.061)10 -3D. The combined data curve with 95 per cent confidence limitsis shown in figure 3 .

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on the regression line are shown) ..

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We believe that our results and the pooled calibration curve may be of some valueto other laboratories using cytogenetic dosimetry in radiological protection .

Figure 2 . Dose-response curve of acentrics (95 per cent confidence limits on the regressionline are shown) .

Correspondence

•

Vutpis et al . 1976∎ Ku6erov6 et al. 1972•

Present data

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Figure 3. Dose-response curve of dicentrics plus centric rings from combined data (95per cent confidence limits on the regression line are shown) .

ReferencesEVANS, H. J ., 1965, Expl Cell Res., 38, 511 .KucEROVA, M., ANDERSON, A. J . B ., BUCKTON, K. E., and EVANS, H. J ., 1972, Int . J. Radiat .

Biol ., 21, 389 .

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LLOYD, D . C., PURROTT, R. J ., DOLPHIN, G . W., BOLTON, D., EDWARDS, A. A., and CORP,M. J ., 1975, Int. J. Radiat . Biol ., 28, 75 .

LuCHNIK, N. V., and SEVANKAEV, A . V ., 1976, Mutation Res ., 36, 363 .VAN DER VOORT, E., and HALLEUX, J. P ., 1973, An Algorithm for Non-Linear Data Fit by the

Least Squares Method, Rep. EUR 4959 e .VULPIS, N ., PANETTA, G ., and ToGNACCI, L ., 1976, Int. J . Radiat . Biol ., 29, 595 .

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