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Radiation-induced chromosome damage in human peripheral blood lymphocytes in vitro I. RBE and dose-rate studies with fast neutrons
Mutation Research
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
367
R A D I A T I O N - I N D U C E D CHROMOSOME DAMAGE IN HUMAN P E R I P H E R A L BLOOD LYMPHOCYTES I N
VITRO
I. RBE AND DOSE-RATE STUDIES W I T H FAST N E U T R O N S D. SCOTT, HEATHER SHARPE*, A. L. BATCHELOR, H. J. EVANS ANDD. G. PAPWORTH Medical Research Council, Radiobiological Research Unit, and *Atomic Energy Research Establishment, Harwell, Didcot, Berks. (Great Britain)
(Received May 28th, 1969)
SUMMARY
Human peripheral blood lymphocytes were irradiated in vitro, before or after stimulation with phytohemagglutinin, with neutrons from uranium fission, at different doses and dose rates, or with acute X-rays (250 kV). Cells were examined at their first mitotic division in culture for chromosome aberrations. An almost IOOO-fold difference in neutron dose rate (0.057-50 rad/inin, doses up to 15o rad) did not change the frequency of aberrations in cells irradiated after stimulation. A small dose-rate effect was observed for cells irradiated in the nonstimulated condition but this was thought to be due to a differential stimulation of less heavily radiation-damaged cells. The data for the induction of dicentric aberrations by X-rays were in agreement with EVANS' earlier findings of an almost linear dose-response curve. Since the dose-response relationship after neutrons was linear, a mean relative biological effectiveness value of approx. 3.5, which did not vary much over the wide range of doses used, was obtained for the induction of dicentrics by fast neutrons (mean energy approx. 0, 7 MeV) relative to X-rays.
INTRODUCTION There are many difficulties attendant upon any attempt to obtain accurate data on the relationship between radiation dose and the amount of chromosome damage induced in human cells in vivo. Observations are restricted to individuals who have been accidentally or therapeutically irradiated and rarely do such individuals receive homogeneous whole-body irradiation of known dose. Thus, it is often impossible to specify accurately the dose which has been received by the cells which are examined for chromosomal abnormalities. A further problem is that cells in different phases of the mitotic and meiotic cycles m a y vary considerably in their radiosensitivity 42 so that if asynchronously Abbreviations: PHA, phytohemagglutinin; RBE, relative biological effectiveness. Mutation Res., 8 (1969) 367-381
368
D. SCOTTet al.
developing populations of cells are irradiated, different levels of chromosome damage will be found in dividing cells at different times after irradiation. However, it has been found that normal human peripheral blood lymphocytes are all at the same stage of the mitotic cycle" and can be stinmlated in vitro with PHA to undergo transformation and mitosis 3°, When such cells are irradiated in vivo or in vitro, then stinmlated with P H A in vitro, they are found to contain chromosome as opposed to chromatid aberrations when observed at mitosis and are thus in the G1, or pre-DNA-synthetic phase of the mitotic cycle at the time of irradiationS, ". We have obtained, from experiments which will be reported in this and in later papers, accurate data on the dose-response relationships for chromosonle aberrations induced by different qualities of radiation delivered at various doses and dose rates to cultures of human lymphocytes. There have been numerous reports of the results of experiments in which doses of X- or 7-rays have been delivered, in vitro, to lymphocytes in the G~ stage of the mitotic cycle. Unfortunately, however, in most of these experimentsl,4,~,2~, 2a determinations of the amount of chromosome damage induced by the radiation were made on cells at 72 or more hours after stimulation, at which time cells are now known to be undergoing their second and subsequent mitotic divisions in cultureS,9,2°, 32. It is essential that cells be examined at their first mitosis after irradiation since cells containing certain types of chromosomal aberration have a lesser capacity to undergo subsequent mitoses than those not containing aberrations 33. Dose-response determinations in which cells have been observed at their first mitosis after X- or y-irradiation of G~ cells in vitro have been reported by several workers ~2,~a,~v,29,aa,4ubut their results are generally not in agreement with one another either from the point of view of the amount of chromosome damage induced by a given radiation dose or in the shapes of the dose-response curves~, ~8. Attempts are being made in this and other laboratories to ascertain the reasons for these conflicting results. However, EVANS1~ la has obtained consistent results with his experimental technique which has therefore been used in the experiments which are reported here in which cultured human lymphocytes were irradiated, before or after stimulation with PHA, with fast neutrons at different dose rates, and a comparison has been made of the effectiveness of neutrons relative to X-rays in the induction of chromosome aberrations observed at the first mitotic division in vitro. Preliminary results of the experiments have been reported previously s~. MATERIALS AND
METHODS
The same male blood donor was used for all experiments and, immediately after being taken, the heparinised whole venous blood was used to set up a large number of microcultures n each consisting of 0.3 ml whole blood in 5.0 ml of medium, with or without P H A (Type M, Difco Laboratories, Detroit, Mich.), in a tissue-culture flask (Falcon Plastics, Los Angeles, Calif.). Cells were, thus, irradiated whilst in the culture medium, in the stimulated ( + P H A ) or non-stimulated (--PHA) condition, several microcultures being used for each radiation dose. Cultures were kept at a constant temperature of 37 a I ° and fixed between 48 and 56 h from the time of adding PHA. o.I ml of IO-~ M colcemid was added to each Mutation Res., 8 (1969) 367-381
HUMAN CHROMOSOME ABERRATIONS
369
culture 4 h before fixation. I t has been shown s0 that, with the culture technique employed in these experiments, cells which undergo mitosis before 56 h after P H A stimulation are at their first division in vitroS,~, 3~. Further details of culture medium, fixation procedure, etc. are given elsewhere n. For scoring purposes all slides were coded and randomised, and only cells with a complete chromosome complement (i.e. having 46 centromeres) were scored 38. Cells were examined for the presence of dicentric aberrations, centric rings, acentric rings, interstitial deletions (minutes) and terminal deletions 1~. Because of their low frequency and the difficulty, in m a n y cases, of distinguishing between them, acentric rings, minutes and terminal deletions have been combined and classified as fragments in Tables I - I V . We have found that the scoring of the frequency of dicentrics after a given radiation dose is very consistent from one experiment to another and from one observer to another, whereas scoring of other aberration types is less reliable 38. In calculating the R B E of different types of radiation and in determining dose-rate effects from the present study we have, therefore, used the frequency of dicentrics as our standard of comparison but the frequency of other aberrations has also been tabulated (Tables I-V). The ATLAS computer (Harwell) was used for statistical analysis of the data. Standard errors for aberration yields (shown in graphs) have been calculated on the assumption of a Poisson distribution of aberrations between cells. Experiment z. Acute X-irradiation I h after the addition of blood to microcultures containing PHA, the cultures were exposed to 196, 297 or 400 rad of X-rays (250 kV, H V T 1.2 m m Cu, all doses given in 2 rain) and fixed before 56 h as described above. Experiment 2. Acute neutron irradiation Stimulated and non-stimulated cultures were irradiated, I h after the addition of blood, with various doses of fast neutrons (mean energy approx. 0. 7 MeV) produced from a uranium converter plate 3 installed in the Atomic Energy Research Establishment (A.E.R.E.) reactor BEPO (British Experimental Pile), at a dose rate of approximately 50 rad/min, y-Contamination was approximately lO% of the fast-neutron dose. P H A was added 30 min after irradiation to cultures which had been irradiated in the non-stimulated condition. Experiment 3. Chronic neutron irradiation Cells were irradiated with fast neutrons produced from a uranium converter plate ~7 in the A.E.R.E. Graphite Low Energy Experimental Pile (GLEEP). The converter plate in GLEEP was of similar construction to that in BEPO, SO the neutron spectrum for the chronic exposures was similar to that for the acute exposures in BEPO. y-Contamination was approximately 15% of the fast-neutron dose. Part I of this experiment was performed with the reactor operating at 32 kW to give a dose rate of 6.75 rad/h. Cultures were irradiated for approximately o, 3, 5.75, 8.5, 11.25, 14, 17, 19.75 and 22.5 h in order to achieve the required doses (Table IV), the irradiation being interrupted every 3 h for about IO rain for the addition or removal of culture flasks from the irradiation chamber. The irradiation of stimulated cells for periods of up to 24 h after the addition Mutation Res., 8 (1969) 367-381
37 °
D. SCOTT et al.
of P H A is j u s t i f e d , since the GI phase, which has a m i n i m u m d u r a t i o n s of a p p r o x i m a t e l y 24 h, has a c o n s t a n t r a d i o s e n s i t i v i t y as far as the i n d u c t i o n of chromosome a b e r r a t i o n s is concerned 12. I r r a d i a t i o n of all the s t i n m l a t e d cultures b e g a n I h after t h e blood h a d been a d d e d to the culture m e d i u m containing P H A . The n o n - s t i m u l a t e d cultures were i n t r o d u c e d into the i r r a d i a t i o n c h a m b e r a t successive intervals after the blood h a d been a d d e d so t h a t t h e y were all p r e s e n t in the c h a m b e r a t the end of the i r r a d i a t i o n period, e.g. n o n - s t i m u l a t e d cultures to be i r r a d i a t e d for 22.5 h were p u t in the c h a m ber a t the same t i m e as all the s t i n m l a t e d cultures, whereas those requiring 14 h i r r a d i a t i o n were p u t in 8.5 h later. I n this w a y P H A could be a d d e d to all t h e nons t i m u l a t e d cultures at the same time, a p p r o x i m a t e l y 3o min after t h e y h a d all finished being i r r a d i a t e d . This p r o c e d u r e for i r r a d i a t i n g n o n - s t i m u l a t e d cells will be referred to as Method A. I n P a r t 2 of t h e e x p e r i m e n t (performed 2 d a y s after P a r t i) the reactor was o p e r a t i n g a t 16 k W to give a dose r a t e of 3.41 r a d / h a n d cultures were i r r a d i a t e d for periods of a p p r o x i m a t e l y 5-75, 11-75, 17.75 a n d 23.5 h (Table V). The i r r a d i a t i o n p r o c e d u r e for t h e s t i m u l a t e d a n d for half of the n o n - s t i m u l a t e d cultures was t h e same as in P a r t I (i.e. Method A for n o n - s t i m u l a t e d cultures) b u t i r r a d i a t i o n of the other half of t h e n o n - s t i m u l a t e d cultures began at t h e same time as all the s t i m u l a t e d cultures, i.e. I h after s e t t i n g u p cultures, b u t P H A was n o t a d d e d to a n y of these cultures u n t i l 24 h later. Thus, for m o s t of these cultures there was a t i m e i n t e r v a l between t h e end of i r r a d i a t i o n a n d t h e t i m e of s t i m u l a t i o n (Method B). RESULTS (5) E x p e r i m e n t I . A cute X - i r r a d i a t i o n
T a b l e I gives t h e frequencies of various t y p e s of a b e r r a t i o n o b s e r v e d in cells exposed, in t h e s t i m u l a t e d condition, to different doses of X - r a y s , a n d Fig. I shows t h e f r e q u e n c y of dicentrics after these doses c o m p a r e d w i t h t h e results of e x p e r i m e n t s b y EVANS 13 on s t i m u l a t e d cells in which a different blood donor was used. I n c o m m o n TABLE I F R E Q U E N C Y OF V A R I O U S A B E R R A T I O N T Y P E S A F T E R A C U T E X - R A Y I R R A D I A T I O N
Stimulated or nonstimulated
Dose in vad
Number of cells scored
Dicentrics a
Centric rings
Fragments
Stimulated Stimulated Stimulated
196 297 400
i oo ioo i oo
49 97 i 16
14 16 3°
19 37 7~
a Tricentric regarded as 2 dicentrics, tetracentric as 3, etc, w i t h our previous findings as no significant difference b e t w e e n t h e i n vitro X - r a y s e n s i t i v i t y of the l y m p h o c y t e s of t h e two donors can be d e m o n s t r a t e d either when a c o m p a r i s o n is m a d e on a dose-for-dose basis (P = 0.32, s e e YATES 44) or when the slopes of the b e s t fitting curves are c o m p a r e d (P ~ o.22, s e e A N D E R S O N 2) using a q u a d r a t i c response m o d e l 24. In calculating R B E values for fast neutrons relative to X - r a y s (Table VI) t h e X - r a y d a t a o b t a i n e d in t h e p r e s e n t e x p e r i m e n t h a v e therefore been c o m b i n e d with EVANS' previous d a t a . Mutation Res., 8 (1969) 367-38I
371
HUMAN CHROMOSOME ABERRATIONS
(2) Experiment 2. Acute neutron irradiation Table II gives the aberration frequencies in cells irradiated in the stimulated or non-stimulated condition with doses of fast neutrons delivered at 50 rad/min, and Table III shows that the dose-response relationship for all aberration types can adequately be described by a linear model. Fig. 2 shows the fitted regression lines to 1.5 "
2.C
O 1.C
~ 1.5
to/ I.-
D-
~ tc .o r~
0.5 °"~ 0.5
121
~o
~6o 26o 360 460
Dose (tad)
Dose (tad)
16o
~o
Fig. I. Best fitting q u a d r a t i c curve (with s t a n d a r d error) to t h e d a t a of EVANS 13 for X - i r r a d i a t i o n of s t i m u l a t e d cells (. . . . ) and results of Expt. I for X - i r r a d i a t e d s t i m u l a t e d cells (O). Fig. 2. A c u t e (5 ° r a d / m i n ) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (0) and n o n - s t i m u l a t e d (0) cells. Linear regression lines fitted.
the data on dicentric frequencies and a comparison of the slopes reveals that, on average, there are significantly (P = 0.032) more dicentrics in stimulated that in non-stimulated cells. This is confirmed by a dose-for-dose comparison which gives a ratio of 1.19 (Table VII). TABLE
II
FREQUENCY
OF VARIOUS ABERRATION
T Y P E S A F T E R A C U T E (5 °
rad/min)
NEUTRON
IRRADIATION
Stimulated or nonstimulated
Dose in rad
Number of cells
Dicentrics
Centric rings
Fragments
Stimulated
o 25 5° 75 IOO 125 15°
96 116 88 14° 83 88 59
o 34 36 89 89 III 98
(o.oo) (0"29) (O.41) (O.64) (1.O7) (1.26) (1.66)
o 8 IO 22 14 22 22
(o.oo) (O.O7) (O. II) (O.16) (O'17) (0.25) (0.37)
o II 28 51 44 75 41
(o.oo) (0.09) (0.32) (0.36) (0-53) (0.85) (0.69)
Non-stimulated
o 25 5° 75 IOO 125 15o
ioo 56 55 60 62 61 8I
o 8 I6 45 59 65 IOI
(o.oo) (o. I4) (0.29) (0.75) (0.95) (1.o7) (1.25)
o 4 4 12 II 16 23
(o.oo) (o-07) (0.07) (o.2o) (o.18) (0.26) (0.28)
o 6 8 14 18 28 47
(o.oo) (o. II) (o.15) (0.23) (0.29) (0.46) (0.58)
A b e r r a t i o n yields per cell s h o w n in brackets.
(3) Experiment 3. Chronic neutron irradiation Part z. Dose rate 6.75 rad/h. The results of this experiment are summarized in Tables III and IV and in Fig. 3- The frequency of dicentrics in non-stimulated cells is a good fit to a linear dose-response model whereas the data for stimulated cells are Mutation Res., 8 (1969) 367-381
D. SCOTT et al.
372
T A B L E 11 l DOSE--RESPONSE RELATIONSHIPS FOR NEUTRON-IRRADIATED CELLS
Dose rate
Stimulated or non-stimulated
Aberration type
5° r a d / m i n
Stimulated
Dicentrics Centric rings Fragments
6.24 1.96 7.72
5 5 5
0.28 0.85 °.17
9-95 : 0.47 2.13 :z 0.22 5.44 : 0.34
Non-
Dieentrics Centric rings Fragments
5 .61 2.16 1.88
5 5 5
0.35 0.83 0.86
8.49 ~: 0.50 2.02 2:0.24 3.49 :: o.32
Stimulated
Dicentrics Centrie rings Fragments
13.39 6.1o 5 .86
5 5 5
{).02 0.30 0-32
lO.32 [: 0,64 3.07 :!2 o,2I 4 .80 7!7 0,27
~orl-
Dicentrics Centric rings Fragments
3.38 2.97 2.11
5 5 5
0.64 0-70 0-83
lO.2O [ 0.47 2.70 [ : ° - 2 4 4.98 0.33
Stimulated
Dicentrics Centrie rings Fragments
2.Ol 1.62 1-65
3 3 3
0.57 0-65 0-65
lO.87 :: 0.53 2,87 ~: 0.27 5,20 !: 0.37
2%To rl-
Dicentrics Centric rings Fragments
2-17 0.96 O.16
3 3 3
0.54 o.81 0.98
8,24 ! : o . 4 o 2.11 :-Lc0.20 4,OI :} 0.28
stimulated 6.75 r a d / h
stimulated 3.41 r a d / h
stimulated
Goodness-of-fit to linear model Slope Z~ ~.f.--p - ( × zo 3)
a poor fit (Table III) for although the yields follow a linear response up to about 6o rad (approx. 0. 7 per cell) there are signs of saturation at higher doses. However, a dose-for-dose comparison of the yields of dicentrics in stimulated as opposed to nonstimulated cells reveals no overall significant difference (P = o.86) although, on average, their frequency is slightly higher in stimulated cells (Table VII). P a r t 2. Dose rate 3.4 r rad/h. The results are given in Tables n I and V and Figs. 4 and 5. The irradiation of non-stimulated cells by Methods A and B gives comparable results (Table V, Fig. 4) and the relationship between aberration frequency and dose is linear for both stimulated and non-stimulated cells (Table III, TABLE IV F R E Q U E N C Y OF VARIOUS ABERRATION TYPES AFTER CHRONIC ( 6 . 7 5 r a d / h )
Stimulated or non-stimulated Stimulated
Dose in rad o
19.8 38.3 56.8 75.8 114.o 152.2 Non-stimulated (Method A)
o.o 19.1 38.2 56.3 76,4 113. 9 I52,2
Dicentrics
Centric rings
Fragments
.So 142 162 149 164 133 147
o 28 7° lO6 147 151 194
o 12 24 32 32 47 60
o (o.oo) 18 (o.13) 27 (o. I7)
5° IOO IOO IOO IOO IOO IOO
A b e r r a t i o n yields per cell s h o w n in brackets. Mutation Res., 8 (1969) 367-381
NEUTRON IRRADIATION
Number of cells scored
(o.oo) (o.2o) (o.43) (o.7I) (0.90) (1.14) (I.32)
o (o.oo) I9 49 54 75 12o 148
(o. I9) (°,49) (0-54) (0.75) (1.2o) (1.48)
(o,oo) (o,o8) (o.15) (o.2I) (0.20) (0.35) (o.4I)
o (o.oo) 4 I3 17 25 27 37
(0'°4) (o. I3) (°.17) (0.25) (0.27) (0.37)
3 6 (0.24)
72 (o.44) 74 (0.56) 97 (o.66)
o (o.oo) I3 19 27 37 61 7°
(°'I3) (o. I9) (0.27) (0.37) (o.61) (°.7°)
HUMAN CHROMOSOME ABERRATIONS
373
2.0 1.5
~
1.0
U
"~ 0.5 t5 50 D o s e (r,ad)
Fig. 3. Chronic (6.75 r a d / h ) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (~) a n d n o n - s t i m u l a t e d (ll) cells.
Figs. 4 and 5). Again, however, there are more aberrations in stimulated than in nonstimulated cells (P ---- 0.000016 for dicentrics, Fig. 5, Table VII).
(4) Gamma-ray contamination The doses shown in Tables II, IV and V and Figs. 2-7 are neutron doses only. A small contribution to the aberration frequencies will be due to the additional yrays to which the cells were exposed (see MATERIALSAND METHODS).However, recent experiments (unpublished) in the laboratory, with cobalt-6o y-rays, demonstrate that, on the assumption that there is no interaction between chromosome breaks induced by the neutrons and y-raysIg, 4~, the y-ray contribution to yield will, at most, be less than 30/0. In the following determinations of dose-rate effect and R B E the y-contamination has therefore been ignored.
(5) The effect of dose rate (a) Stimulated cells. When the aberration frequencies induced in stimulated TABLE V FREQUENCY OF VARIOUS ABERRATION TYPES AFTER CHRONIC (3.41 r a d / h ) NEUTRON IRRADIATION Stimulated or non-stimulated
Dose in rad
N u m b e r of cells scored
Dicentrics
Centric rings
Fragments
Stimulated
o 20.0 40.3 60. 3 80.0
13o 192 161 208 197
i 49 73 144 16o
(o.oi) (0.26) (0-45) (0.69) (o.81)
o 13 14 38 46
(o.oo) (0.07) (0.09) (o.18) (0.23)
o 17 31 73 80
(o.oo) (0.09) (o.19) (0.35) (o.41)
Non-stimulated (Method A)
o 19.7 39.7 6o.0 8o.0
93 209 183 158 144
o 27 52 84 ioo
(o.oo) (o. 13) (0.28) (0.53) (0.69)
o 4 II 17 27
(o,oo) (0.02) (0.06) (o. i i ) (o.19)
o 16 32 39 48
(o.oo) (0.08) (o.17) (0.25) (0.33)
Non-stimulated (Method A)
o 20.0 40.3 60. 3 80.0
5° ioo ioo ioo lOO
o 18 37 39 75
(o.oo) (o.18) (0.37) (0.39) (0.75)
o 7 IO 18 17
(o.oo) (0-07) (o.Io) (o.18) (o.17)
o 9 14 21 3°
(o.oo) (0.09) (o.14) (o.21) (0.30)
A b e r r a t i o n yields p e r cell s h o w n in b r a c k e t s . Mutation Res., 8 (1969) 367-381
n. SCOTT et al.
374 2.(3 2.0
1.5
1.5
~I.0 "F_.
o
~o.5
,~--l"
~u O.5
16o
5O Dose (rod)
o
50 Dose (rod)
16o
Fig. 4- Chronic (3.41 rad/h) n e u t r o n d o s e - r e s p o n s e d a t a for n o n - s t i m u l a t e d cells irradiated by Method A, all irradiations ending at same time (@), or Method B, all irradiations starting at same time (V). Linear regression line fitted to pooled data. Fig. 5. Chronic (3.41 rad]h) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (&) and n o n - s t i m u l a t e d (A) cells. Linear regression lines fitted.
ceils at the three different neutron dose rates are compared, no significant difference is obtained (P = o.50 for dicentrics from a comparison of regression lines). Thus, an almost IOOO-fold range of dose rate (o.o57-5o rad/min, doses up to 15o rad) does not influence the aberration yield (Fig. 6, Table III). ~.0
1.5 0 [3
CL 1.o
o&
.~-
~ 0.5
DA
~o
0
o
Dose
(rod)
lO0
15o
Fig. 6. N e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d cells irradiated at 5 ° r a d / m i n (O) and 6.75 r a d / h (~), a n d 3.41 r a d / h (&).
(b) Non-stimulated cells. The results reported above showed that at two of the three dose rates used (50 rad/min and 3.41 rad/h) significantly less aberrations were found in non-stimulated than in stimulated cells, whereas in the third case (6.75 rad/h) no significant difference was obtained. As a result of this, and the fact that no dose-rate effect exists for stimulated cells, in comparing the aberration frequencies in cells irradiated in the non-stimulated condition one finds that the level of damage in cells irradiated at 6.75 rad/h is significantly higher than in cells irradiated at the higher dose rate of 50 rad/min (P = o.o13) or the lower dose rate of 3.41 rad/h (P ~ o.oo15) and that there is no difference (P ~ 0.69) between the yields induced at the latter two dose rates (Fig. 7). We believe this dose-rate effect to be more apparent than real and possibly due to a differential stimulation by PHA of less heavily radiation-damaged cells (see DISCUSSION). Mutation Res., 8 (1969) 367-38t
375
HUMAN CHROMOSOME ABERRATIONS
2.0
_
1.5
f
.g8 .~ 0.5 0
i
50
100
Dose (r'ad)
150
Fig. 7. N e u t r o n d o s e - r e s p o n s e d a t a for n o n - s t i m u l a t e d cells irradiated at 5 ° t a d / r a i n (@), 6.75 r a d / h (11), and 3.41 r a d / h (&). Linear regression lines fitted.
(6) RBE Since no dose-rate effect was observed for cells irradiated in the stimulated condition our calculations of the relative efficiency of neutrons and X - r a y s in inducing chromosome damage are restricted to cells which were irradiated in this condition (Table VI, Fig. 8). W h e n the X - r a y data obtained in Expt. I are combined with T A B L E VI RBE
OF FAST NEUTRON'S
COMPARED
WITH
X-RAYS
Comparison
Yield of dicentrics (per cell)
Dose of X-rays (Dx) (rad)
Dose of neutrons (Dn) (rad)
RBE D x -~- S.E. Dn ---
X - r a y s ( E x p t . I + d a t a o f EVANS13) c o m p a r e d with combined n e u t r o n d a t a at 50 r a d / m i n , 6.75 r a d / h and 3.41 r a d / h for stimulated cells
o.oooi o. 5 i.o 1. 5
o.°396 172.7 ± 3.5 312-7 ~ 5.9 433.7 ~ 12.8
o-oo97 48.3 ~_ 1.2 96. 7 ~ 2. 4 145.° ± 3-7
4- IO 3.57 ~ o.12 3.24 ~ o.Io 2.99 -~ o.12
X - r a y s (as above) c o m p a r e d w i t h n e u t r o n d a t a at 5 ° r a d / m i n for s t i m u l a t e d cells
o.oooi 0. 5 I.O 1. 5
0.0396 172.7 ~ 3-5 312.7 • 5.9 433-7 ± 12.8
O.OLOI 50.3 zk 2.4 lOO.5 i 4-7 15o.8 :L- 7.1
3.94 3-44 ~ o.18 3.11 ~ o.16 2.88 ± o.16
EVANS' results 13 for stimulated cells, the combined data for dicentric aberrations can be described (goodness-of-fit P = o.14) b y the quadratic expressionS4; y
where
and from which
--_ ~ D + ~ D
2
Y = the yield of aberrations per cell = one-track component of yield /3 : - two-track component of yield D = radiation dose ~ ~ (2.52 ~ o.15)" l O - 3 /3 ~ (2.16 ~- 0 . 5 8 ) . l O - 6
These values of ~ and fl have been used in Table VI to calculate the doses of X - r a y s which are required to induce a given level of chromosome damage. For calculations of the neutron doses which are required to produce the same levels of darnMutation Res., 8 (1969) 367-381
376
D.
~.5
j,
SCOTT et
al.
o~,Z /
2
O'
100
200
300
Dose(rod)
4.00
500
Fig. 8. X - R a y d o s e - r e s p o n s e c u r v e (with s t a n d a r d error) f r o m c o m b i n e d d a t a of EVANS 13 for s t i m u l a t e d cells a n d of our E x p t . I, fitted to q u a d r a t i c model. L i n e a r regression line (with s t a n d a r d error/ for n e u t r o n d a t a in s t i m u l a t e d cells also s h o w n .
age, the results from the experiments on stimulated cells at the three different dose rates have been combined. The slope of the regression line for these combined data is (lO.34 ~ o.26), lO -3 but, because of the inclusion of the 6.75 rad/h data, the fit to linearity is poor (P = o.o8o). Therefore, Table VI shows the R B E values obtained by comparing the X-ray yields with those obtained from the acute neutron experiment only (stimulated cells) in which the fit to linearity is better (Table I I I , Fig. z). There is, in fact, little difference between the R B E values which are obtained in the two cases. It should be noted that over the wide range of radiation doses which were used, the R B E value does not vary much, having a mean value of about 3.5- Even upon extrapolation to very low levels of damage, e.g. lO-4 dieentrics/cell, the R B E only increases to about 4 (see DISCUSSION). DISCUSSION
(z) Aberration frequencies in cells irradiated in the stimulated or non-stimulated condition It is our experience in culturing human peripheral blood lymphocytes that, from one experiment to another, the degree of mitotic activity obtained in the cultures m a y v a r y considerably, even though the same donor and the same type of culture medium is used. This is presumably a reflection of the variable extent to which the lymphocytes transform and undergo mitosis under the influence of PHA. Just what factors, in our experiments, determine the proportion of lymphocytes which respond to the PHA, is not clear although we have evidence which suggests that the pH of the culture medium, the ~liet and differential blood count of the donor, and the particular batch of P H A used are of some importance 3~. A further observation which we have made is that irradiation of the lymphocytes in vitro causes a reduction in mitotic activity in the cultures relative to that found in non-irradiated cultures (Fig. 9) and that when lymphocytes are irradiated in the non-stimulated condition, the amount of mitotic activity in PHA-stimulated cultures of such cells is sometimes, but not always, less than that in cultures of cells which are irradiated in the stimulated condition (in G1) with the same dose 33. I t would appear therefore that the irradiation of lymphocytes may, under certain conditions, Mutation Res., 8 (I969) 367-381
377
HUMAN CHROMOSOME ABERRATIONS
1.5
1.C
6.O 5.0
~
£L
4.0 £.
•-~ &O
o5
~5
2.0 1.0 36
42 48 54 Fixation time (h)
60
66
72
160 260 360 460 461
O'
Dose (oed)
F i g . 9. M i t o t i c i n d e x , a t d i f f e r e n t t i m e s a f t e r s t i m u l a t i o n , o f w h o l e b l o o d m i c r o c u l t u r e s g i v e n 3 0 0 t a d X - r a y s (©) o r u n i r r a d i a t e d ( e ) . C u l t u r e s w e r e i r r a d i a t e d i h a f t e r s t i m u l a t i o n a n d m i t o t i c cells w e r e a c c u m u l a t e d w i t h c o l c e m i d f o r 6 h p r i o r t o e a c h f i x a t i o n t i m e s h o w n . 2 0 0 0 cells s c o r e d per point. F i g . IO. X - R a y d o s e - r e s p o n s e Quadratic curves fitted.
d a t a f r o m EVANS 18 f o r s t i m u l a t e d
(©) a n d n o n - s t i m u l a t e d
(O) cells.
reduce their capacity to respond to PHA but, again, we do not know what these conditions are. Such observations may help to explain the results of our comparisons of the amount of chromosome damage in cells irradiated in the stimulated or non-stimulated state. In all our neutron experiments there were, on average, more aberrations in stimulated than in non-stimulated cells although the difference was only significant at two of the three dose rates used. This difference was also found in an experiment of EVANS13 with X-irradiation (Fig. IO) although it was not statistically significant (P = o.12). The ratio (k) of the yields in stimulated and non-stimulated cells differs significantly from one experiment to another (P = o.o19, Table VII). It is tempting to suggest that for cells irradiated in the non-stimulated condition the more heavily radiation-damaged cells (i.e. those which would contain relatively high aberration yields) may fail to respond to the PHA and therefore never be observed at mitosis, and that the degree of response failure varies from one experiment TABLE
VII
RATIO (k) OF FREQUENCIES STIMULATED CONDITION
OF DICENTRICS
IN CELLS
IRRADIATED
Experiment
Ratio k (maximum likelihood)
Test for k = •
X - r a y s (EVANS 13) N e u t r o n s (5 ° r a d / m i n ) N e u t r o n s (6,75 r a d / h ) N e u t r o n s (3.41 r a d / h )
1.149 1.19o i.oti 1.343
U u u u
Overall Yields at doses common compared.
--L O.102 ~- o . o 9 1 ~ o.o61 ~ 0.092
= -= =
1.56 ; 2.28; o.177; 4.3I;
u = 4,o3; to both experiments
P P 19 19
IN THE
STIMULATED
AND NON-
Homogeneity of k between doses Z2 d.f. t9 = = = =
o,12o 0.023 0.860 0.000o16
P = o.oooo56
of a p a i r ( s t i m u l a t e d
5 .26 6.49 6.08 4.72
3 5 5 3
O. 15 0.26 0.30 o.19
22.55
16
o. 13
and non-stimulated)
were
M u t a t i o n Res., 8 (1969) 3 6 7 - 3 8 1
378
D. SCOTT et al.
to another. Thus, one m i g h t expect t h e r e to be a positive correlation between values of k in different e x p e r i m e n t s a n d the m a g n i t u d e of t h e difference in m i t o t i c a c t i v i t y in cultures of cells i r r a d i a t e d in t h e s t i m u l a t e d as o p p o s e d to the n o n - s t i m u l a t e d condition in these experiments. This h y p o t h e s i s is at p r e s e n t u n d e r test in our l a b o r a t o r y . If this selective s t i n m l a t i o n of less h e a v i l y r a d i a t i o n - d a m a g e d cells does occur a n d t h e e x t e n t to which it occurs varies from one e x p e r i m e n t to a n o t h e r for reasons which are at p r e s e n t u n k n o w n , this will be of considerable i m p o r t a n c e in assessing the level of c h r o m o s o m e d a m a g e in i n d i v i d u a l s i r r a d i a t e d in vivo either accidentally or t h e r a p e u t i c a l l y since, of course, t h e l y n l p h o c y t e s in vivo will be i r r a d i a t e d in t h e nons t i m u l a t e d condition. The level of c h r o m o s o m e d a m a g e o b s e r v e d in c u l t u r e d l y m p h o eytes from such individuals, which m i g h t be used to e s t i m a t e the r a d i a t i o n dose received ~5, would t h e n d e p e n d u p o n their degree of response to P H A from one occasion to another. I t is therefore of considerable i m p o r t a n c e to d e t e r m i n e w h e t h e r or not the difference in a b e r r a t i o n yields in s t i n m l a t e d a n d n o n - s t i m u l a t e d ceils is due to selective s t i m u l a t i o n , for although the n~axinmm difference which we h a v e y e t o b s e r v e d is only 25°/o (3.41 r a d / h , E x p t . , Fig. 5), p o t e n t i a l l y the difference m a y be m u c h greater. There m a y be s u b - p o p u l a t i o n s of small l y m p h o c y t e s in t h e p e r i p h e r a l blood with different radiosensitivities a n d with different capacities to respond to P H A . Certainly on criteria o t h e r t h a n r a d i o s e n s i t i v i t y the small l y m p h o c y t e s are a heterogeneous collection of cells 4~ a n d RICHTER AND NASPITZ at have r e c e n t l y shown t h a t different p o p u l a t i o n s of small l y m p h o c y t e s show different degrees of s e n s i t i v i t y to P H A .
(2)
Comparison with the results of other workers EVANS la a n d SHARPE et al. 38 h a v e d r a w n a t t e n t i o n to the m a r k e d q u a n t i t a t i v e differences b e t w e e n results o b t a i n e d in different l a b o r a t o r i e s on the frequencies of c h r o m o s o m e a b e r r a t i o n s in h u m a n leucocytes after X- or ),-irradiation in vitro. I t should be p o i n t e d out t h a t large discrepancies are still found even when only those cells which are undergoing their first m i t o t i c division in culture are scored (cf. ref. 13 w i t h refs. 29 a n d 4o). The possibility t h a t different l a b o r a t o r i e s are effectively exam i n i n g different p o p u l a t i o n s of small l y m p h o c y t e s , as referred to above, is at present u n d e r investigation. GoocH et al. TM i r r a d i a t e d n o n - s t i n m l a t e d h u m a n l y m p h o e y t e s w i t h I4.I-MeV n e u t r o n s at a dose r a t e of 6 r a d / m i n a n d Fig. I I shows t h a t t h e frequencies of dicen2.0
1,5 0 0 .1.0
cgx
"E.
E 0.5 i:5
AO
•
o A
•
•
•
O&
v
50 Dose (rods)
•
,w
100
,.-J
150 200
Fig. i i. Neutron dose-response data from the present experiment for stimulated cells irradiated at 5o rad/min (©), 6.75 rad/h ([]) and 3.41 rad/h (L\), and for non-stimulated cells irradiated at 5° rad/min (O), 6.75 rad/h (Ul) and 3.41 rad/h (A). Also, dose-response data of GoocH et al. 18 for non-stimulated cells irradiated at 6 rad/min with I4.I-MeV neutrons (w). Mutation Res., 8 (1969) 367-381
HUMAN CHROMOSOME ABERRATIONS
379
trics which they obtained at different doses is, on average, some io times lower than the frequencies obtained in the present experiment with neutrons of approximately o.7-MeV mean energy. Part of the difference may be attributable to the differences in the quality of neutrons used and to the fact that their fixations were made at 72 h, but the major part of the discrepancy remains unexplained at the present time as do the differences between the various X-ray results. Not only were the frequencies of aberrations induced by the I4.I-MeV neutrons much lower than in our experiments but the dose-response curve was non-linear having a dose exponent of 1.42 4- o.2o on a power-law model (see DISCUSSION,section 3).
(3) RBE An unusual feature of the X-ray dose-response curves of EVANS13 and that obtained in Expt. I in the present investigation, is that they are almost linear (Fig. I), the combined data having a dose exponent of 1.15 -L o.o4. Exchange aberrations (e.g. dicentrics and rings) have been shown to increase approximately as the square of the close after X+irradiation of plant cells3S,3~,~1, and NORMAN AND SASAK129have found this to be the case for human lymphocytes. On the other hand, with densely-ionizing radiations, e.g. neutrons, such aberrations are generally found to increase linearly with dosel6,~9,~3. Since the shapes of the doseresponse curves for sparsely and densely-ionizing radiations are usually found to differ, a constant RBE value is not obtained. However, since in the experiments reported here we have obtained a linear relationship between neutron dose and aberration frequency and an almost linear response for X-irradiated cells, the R B E value does not change markedly over a wide range of doses (Table VI) so that a single value of 3.5 is a meaningful estimate. From preliminary experiments with 2.5-MeV neutrons, GoocH el al. TM have quoted an R B E value, relative to 25o-kV X-rays, of between 4 and 5 for chromosome terminal deletions in human lymphocytes irradiated in vitro. Our value of 3.5 for dicentrics is rather lower than the majority of values which have been obtained for the induction of chromosomal aberrations by fast neutrons and X- or 7-rays in plant cellsl°,n, ~2,2+ and is also somewhat lower than the R B E obtained for the inhibition of colony formation in kidney cells of human origin with 3-MeV monochromatic neutrons and 25o-kV X-rays ~.
(4) Dose-rate effect Our demonstration of the lack of a dose-rate effect after neutron irradiation of stimulated cells is in agreement with observations made on plant cells 1~. These observations, and the demonstration of a linear dose-response for exchange aberrations after neutron irradiation (see references above) have been interpreted as showing that with high LET radiation both the breaks which are necessary to produce a chromosome exchange (e.g. dicentric) are produced by a single ionizing track, whereas, after low LET radiation each of the two breaks is produced by an independent track since the yield of exchanges increases as the square of the dose and a dose-rate effect is observed ~3. On this basis one would interpret our near-linear dose-response curve for X-rayinduced aberrations in human lymphocytes as indicating that, in general, only a single ionizing track is required to produce both the breaks required for the ex-
Mutation Res., 8 (1969) 367-38I
It. SCOTT et al.
380 change~:L However, a pronounced
recent
w o r k in t h i s l a b o r a t o r y
dose-rate effect for the induction
w h i c h is c o n t r a r y
to expectation
if e x c h a n g e s
(unpublished)
has demonstrated
of d i c e n t r i c s a f t e r l o w L E T i r r a d i a t i o n are the result of one-track
events.
ACKNOWLEDGEMENTS The authors invaluable
would
technical
BARNES f o r t a k i n g the A.E.R.E.
like to express
assistance,
blood samples,
reactors
their thanks
t o M r . T . R . L. BIGGER f o r
D r . G. T . BUNGAY, o u r b l o o d d o n o r , D r . D . W . H . M r . M. CORP f o r X - i r r a d i a t i o n s ,
GLEEP a n d BEPO f o r t h e f a s t - n e u t r o n
and the staff of
irradiations.
REFERENCES I j'AN-CHI, W., AND C. SHING-TING, T h e effect of r a d i a t i o n dose rate on t h e f r e q u e n c y of 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 e u k o c y t e s i r r a d i a t e d in vitro, Kexue Tongbao, 17 (1966) 136 . 2 ANDERSON, T. W., Introduction to Multivariate Statistical Analysis, W'iley, N e w York, 1958. 3 BATCHELOR, A. L., AND P. \V. EDMONDSON, F a s t - n e u t r o n i r r a d i a t i o n of large animals, Biological Effects of Neutron and Proton Irradiations, Vol. 2, I n t e r n a t i o n a l A t o m i c E n e r g y Agency, Vienna, 1964, p. 34 BELL, A. G., AND D. G. BAKER, I r r a d i a t i o n - i n d u c e d c h r o m o s o m e a b e r r a t i o n s in n o r m a l h u m a n leucocytes in culture, Can. J. Genet. Cytol., 4 (1962) 34 o. 5 BENDER, M. A., AND P. C. GOOCH, T y p e s a n d r a t e s of X - r a y - i n d u c e d c h r o m o s o m e a b e r r a t i o n s in h u m a n blood i r r a d i a t e d in vitro, Proc. Natl. Acad. Sci. (U.S.), 48 (r962) 522. 6 BENDER, M. A., AND D. M. PRESCOTT, D N A s y n t h e s i s a n d m i t o s i s in cultures of h u m a n peripheral leucocytes, Exptl. Cell Res., 27 (1962) 221. 7 BRORRSE, J. J., AND G. W. BARRNDSEN, Effects of m o n o e n e r g e t i c n e u t r o n r a d i a t i o n on h u m a n cells in tissue culture, Biological Effects of Neutron and Proton Irradiations, Vol. i, I n t e r n a t i o n a l A t o m i c E n e r g y Agency, Vienna, 1964, p. 3o9. 8 BUCKTON, K. E., AND M. C. PIKE, C h r o m o s o m e i n v e s t i g a t i o n s on l y m p h o c y t e s f r o m irrad i a t e d p a t i e n t s : effects of t i m e in culture, Nature, 2o2 (1964) 714 . 9 BUCKTON, K. E., .aND M. C. PIKE, T i m e in culture, A n i m p o r t a n t variable in s t u d y i n g in vivo r a d i a t i o n - i n d u c e d c h r o m o s o m e d a m a g e in m a n , Intern. J. Radiation Biol., 8 (i964) 439. IO CONGER, A. D., M. C. RANDOLPH, C. W. SHEPPARD AND n . g . LUIPPOLD, Q u a n t i t a t i v e relation of R B E in Tradescantia a n d a v e r a g e L E T of g a m m a - r a y s , X - r a y s , a n d 1.3-I.5-, a n d 14. I-MeV f a s t n e u t r o n s , Radiation Res., 9 (1958) 525 • I i EVANS, H. J., A simple m i c r o t e c h n i q u e for o b t a i n i n g h u m a n c h r o m o s o m e p r e p a r a t i o n s with s o m e c o m m e n t s on D N A replication in sex c h r o m o s o m e s of t h e goat, cow a n d pig, Exptl. Cell Res., 38 (1965) 511. 12 EVA.~S, H. J., R e p a i r a n d r e c o v e r y f r o m c h r o m o s o m e d a m a g e after f r a c t i o n a t e d X - r a y dosage, Genetical Aspects of Radiosensitivity, I n t e r n a t i o n a l A t o m i c E n e r g y A g e n c y , Vienna, 1966, p. 3 i. 13 EVANS, H. J., D o s e - r e s p o n s e relations from in vitro studies, in H. J. EVANS, W. M. COURT BROWN AND A. S. McLEAN (Eds.), Human Radiation Cytogenetics, N o r t h - H o l l a n d , A m s t e r d a m , 1967, p. 2o. 14 EVANS, H. J., G. J. NEARY AND F. S. WILLIAS~SON, T h e relative biological efficiency of single doses of fast n e u t r o n s a n d g a m n l a - r a y s on Viciafaba roots a n d t h e effect of oxygen, P a r t lI. C h r o m o s o m e d a m a g e : t h e p r o d u c t i o n of nlicronuclei, Intern. J. Radiation Biol., i (1959) 2 ~6. 15 EVANS, H. J., W. M. COURT BROWN AND A. S. McLEAN (Eds.), Human Radiation Cytogenetics, N o r t h - H o l l a n d , A m s t e r d a m , 1967 . 16 GILES, N. H., C o m p a r a t i v e s t u d i e s of t h e c y t o g e n e t i c effects of n e u t r o n s a n d X - r a y s , Genetics, 28 (1943) 398. 17 GOLOB, E., P. FISCHER AND E. KUNZE-MOHL, Einfluss der K u l t u r d a u e r a u f s t r a h l e n i n d u z i e r t e C h r o m o s o m e n a b e r r a t i o n e n m e n s c h l i c h e r L y m p h o z y t e n des p e r i p h e r e n Blutes, Strahlentherapie, 137 (1969) 63. 18 GOOCH, P. C., M. A. BENDER AND M. C. RANDOLPH, 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 s o m a t i c cells b y n e u t r o n s , Biological Effects of Neutron and Proton Irradiations, Vol. i, hItern a t i o n a l A t o m i c E n e r g y A g e n c y , Vienna, 1964, p. 325 . 19 MEDDLE, J. A., L a c k of i n t e r a c t i o n of X - r a y - a n d n e u t r o n - i n d u c e d c h r o m o s o m e b r e a k s in barley, Mutation Res., 2 (1965) 149.
34utation Res., 8 (1969) 367-381
HUMAN CHROMOSOME ABERRATIONS
381
20 HEDDLE, J. A., H. J. EVANS AND D. SCOTT, S a m p l i n g t i m e a n d t h e c o m p l e x i t y of t h e h u m a n l e u c o c y t e c u l t u r e s y s t e m , in H. J. EVANS, W. M. COURT BROWN AND A. S. MCLEAN (Eds.), N o r t h - H o l l a n d , A m s t e r d a m , 1967, p. 6. 2I KELLY, S., AND ~. D. BROWN, C h r o m o s o m e a b e r r a t i o n s as a biological d o s i m e t e r , Am. J. Public Health, 55 (1965) 141922 KIRBY-SMITH, J. S., AND C. P. SWANSON, T h e effects of f a s t n e u t r o n s f r o m a n u c l e a r d e t o n a t i o n on c h r o m o s o m e b r e a k a g e in Tradescantia, Science, 119 (I954) 42. 23 LEA, D. E., Actions of Radiations on Living Cells, U n i v e r s i t y Press, Cambridge, 1947. 24 LEA, D. E., AND D. G. CATCHESIDE, I n d u c t i o n b y r a d i a t i o n of c h r o m o s o m e a b e r r a t i o n s in Tradescantia, J. Genet., 44 (I942) 216. 25 LING, N. R., Lymphocyte Stimulation, N o r t h - H o l l a n d , A m s t e r d a m , 1968. 26 MARSHAK, A., Effects of fast n e u t r o n s on c h r o m o s o m e s in mitosis, Proc. Soc. Exptl. Biol. :VIed., 41 (1939) 176. 27 NEARY, G. J., R. J. MUNSON AND R. I-L MOLE, Chronic Radiation Hazards, P e r g a m o n , Oxford, 195728 NORMAN, A., R. E. OTTMAN, M. SASAKI AND R. C. VEOMETT, T h e f r e q u e n c y of dicentrics in h u m a n l e u c o c y t e s i r r a d i a t e d in vivo a n d in vitro, Radiology, 83 (1964) lO8. 29 NORMAN, A., AND M. S. SASAKI, C h r o m o s o m e - e x c h a n g e a b e r r a t i o n s in h u m a n l y m p h o c y t e s , Intern. J. Radiation Biol., II (1966) 321. 3 ° NO,YELL, P. C., P h y t o h e m a g g l u t i n i n : a n i n i t i a t o r of m i t o s i s in c u l t u r e s of n o r m a l h u m a n leucocytes, Cancer Res., 20 (I96O) 462. 31 I~_ICHTER, ~¥~., AND C. K. NASPITZ, T h e effects of v a r y i n g c o n c e n t r a t i o n s of p h y t o h e m a g g l u t i n i n a n d c o n d i t i o n s of i n c u b a t i o n on t h e e n h a n c e d s u r v i v a l of h u m a n p e r i p h e r a l l y m p h o c y t e s in vitro, Blood, 32 (1968) 134. 32 SASAKI, M. S., SND A. NORMAN, Proliferation of h u m a n l y m p h o c y t e s in culture, Nature, 21o (1966) 913. 33 SASAKI, M. S., AND A. NORMAN, Selection a g a i n s t c h r o m o s o m e a b e r r a t i o n s in h u m a n l y m p h o cytes, Nature, 214 (1967) 502. 34 SAVANKAYEV, A. V., AND N. P. BOCHKOV, T h e effect of g a m m a - r a y s on h u m a n c h r o m o s o m e s , I. T h e d e p e n d e n c e of c h r o m o s o m e a b e r r a t i o n f r e q u e n c y u p o n t h e dose w i t h i r r a d i a t i o n in vitro, Genetika, 4 (1968) 13o. 35 SAX, K., T y p e s a n d frequencies of c h r o m o s o m a l a b e r r a t i o n s i n d u c e d b y X - r a y s , Cold Spring Harbor Syrup. Quant. Biol., 9 (1941) 9336 SCHREK, R., AND S. STEFANI, R a d i o r e s i s t a n e e of p h y t o h e m a g g l u t i n i n t r e a t e d n o r m a l a n d l e u k e m i c l y m p h o c y t e s , J. Natl. Cancer Inst., 32 (1964) 507 . 37 SCOTT, D., A. L. BATCHELOR, H. SHARPE AND H. J. EVANS, R B E for f a s t n e u t r o n s a n d dose rate s t u d i e s u s i n g fast n e u t r o n irradiation, in H. J. EVANS, W. M. COURT BROWN AND A. S. McLEAN" (Eds.), H u m a n Radiation Cytogenetics, N o r t h - H o l l a n d , A m s t e r d a m , I967, p. 3738 SHARPE, H. B. A., D. SCOTT AND G. W. DOLPHIN, 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 b y X - i r r a d i a t i o n in vitro: T h e effect of c u l t u r e t e c h n i q u e s a n d blood d o n o r s on a b e r r a t i o n yield, Mutation Res., 7 (1969) 453-46I. 39 THODAY, J. M., Effects of n e u t r o n s a n d X - r a y s on c h r o m o s o m e s of Tradescantia, J. Genet., 43 (1942 ) 189. 4 ° VISSELDT, J., R a d i a t i o n - i n d u c e d c h r o m o s o m e a b e r r a t i o n s in h u m a n cells, Doctoral Thesis, p u b l i s h e d as R i s 6 R e p o r t No. 117, D a n i s h A t o m i c E n e r g y Commission, 1966. 41 WOLFF, S., D e l a y of c h r o m o s o m e rejoining in Vieia faba i n d u c e d b y irradiation, Nature, 173 (1954) 5 ol. 42 WOLFF, S., C h r o m o s o m e a b e r r a t i o n s a n d t h e cell cycle, Radiation Res., 33 (1968) 609. 43 WOLFF, S., K. C. ATWOOD, M. L. RANDOLPH ANn H. E. LUIPPOLD, F a c t o r s limiting t h e n u m b e r of r a d i a t i o n - i n d u c e d c h r o m o s o m e e x c h a n g e s , I. D i s t a n c e : evidence from n o n - i n t e r a c t i o n oi X - r a y - a n d n e u t r o n - i n d u c e d breaks, dr. Biophys. Biochem. Cytol., 4 (1958) 365 • 44 YATES, F., T h e use of t r a n s f o r m a t i o n s a n d m a x i m u m likelihood in t h e a n a l y s i s of q u a n t a l e x p e r i m e n t s i n v o l v i n g two t r e a t m e n t s , Biometrika, 42 (1955) 382. 45 YOFPEY, J. M., The Lymphocyte in Immunology and Haemopoiesis, Arnold, L o n d o n , 1967, p. 7.
Mutation Res., 8 (1969) 367-381
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
367
R A D I A T I O N - I N D U C E D CHROMOSOME DAMAGE IN HUMAN P E R I P H E R A L BLOOD LYMPHOCYTES I N
VITRO
I. RBE AND DOSE-RATE STUDIES W I T H FAST N E U T R O N S D. SCOTT, HEATHER SHARPE*, A. L. BATCHELOR, H. J. EVANS ANDD. G. PAPWORTH Medical Research Council, Radiobiological Research Unit, and *Atomic Energy Research Establishment, Harwell, Didcot, Berks. (Great Britain)
(Received May 28th, 1969)
SUMMARY
Human peripheral blood lymphocytes were irradiated in vitro, before or after stimulation with phytohemagglutinin, with neutrons from uranium fission, at different doses and dose rates, or with acute X-rays (250 kV). Cells were examined at their first mitotic division in culture for chromosome aberrations. An almost IOOO-fold difference in neutron dose rate (0.057-50 rad/inin, doses up to 15o rad) did not change the frequency of aberrations in cells irradiated after stimulation. A small dose-rate effect was observed for cells irradiated in the nonstimulated condition but this was thought to be due to a differential stimulation of less heavily radiation-damaged cells. The data for the induction of dicentric aberrations by X-rays were in agreement with EVANS' earlier findings of an almost linear dose-response curve. Since the dose-response relationship after neutrons was linear, a mean relative biological effectiveness value of approx. 3.5, which did not vary much over the wide range of doses used, was obtained for the induction of dicentrics by fast neutrons (mean energy approx. 0, 7 MeV) relative to X-rays.
INTRODUCTION There are many difficulties attendant upon any attempt to obtain accurate data on the relationship between radiation dose and the amount of chromosome damage induced in human cells in vivo. Observations are restricted to individuals who have been accidentally or therapeutically irradiated and rarely do such individuals receive homogeneous whole-body irradiation of known dose. Thus, it is often impossible to specify accurately the dose which has been received by the cells which are examined for chromosomal abnormalities. A further problem is that cells in different phases of the mitotic and meiotic cycles m a y vary considerably in their radiosensitivity 42 so that if asynchronously Abbreviations: PHA, phytohemagglutinin; RBE, relative biological effectiveness. Mutation Res., 8 (1969) 367-381
368
D. SCOTTet al.
developing populations of cells are irradiated, different levels of chromosome damage will be found in dividing cells at different times after irradiation. However, it has been found that normal human peripheral blood lymphocytes are all at the same stage of the mitotic cycle" and can be stinmlated in vitro with PHA to undergo transformation and mitosis 3°, When such cells are irradiated in vivo or in vitro, then stinmlated with P H A in vitro, they are found to contain chromosome as opposed to chromatid aberrations when observed at mitosis and are thus in the G1, or pre-DNA-synthetic phase of the mitotic cycle at the time of irradiationS, ". We have obtained, from experiments which will be reported in this and in later papers, accurate data on the dose-response relationships for chromosonle aberrations induced by different qualities of radiation delivered at various doses and dose rates to cultures of human lymphocytes. There have been numerous reports of the results of experiments in which doses of X- or 7-rays have been delivered, in vitro, to lymphocytes in the G~ stage of the mitotic cycle. Unfortunately, however, in most of these experimentsl,4,~,2~, 2a determinations of the amount of chromosome damage induced by the radiation were made on cells at 72 or more hours after stimulation, at which time cells are now known to be undergoing their second and subsequent mitotic divisions in cultureS,9,2°, 32. It is essential that cells be examined at their first mitosis after irradiation since cells containing certain types of chromosomal aberration have a lesser capacity to undergo subsequent mitoses than those not containing aberrations 33. Dose-response determinations in which cells have been observed at their first mitosis after X- or y-irradiation of G~ cells in vitro have been reported by several workers ~2,~a,~v,29,aa,4ubut their results are generally not in agreement with one another either from the point of view of the amount of chromosome damage induced by a given radiation dose or in the shapes of the dose-response curves~, ~8. Attempts are being made in this and other laboratories to ascertain the reasons for these conflicting results. However, EVANS1~ la has obtained consistent results with his experimental technique which has therefore been used in the experiments which are reported here in which cultured human lymphocytes were irradiated, before or after stimulation with PHA, with fast neutrons at different dose rates, and a comparison has been made of the effectiveness of neutrons relative to X-rays in the induction of chromosome aberrations observed at the first mitotic division in vitro. Preliminary results of the experiments have been reported previously s~. MATERIALS AND
METHODS
The same male blood donor was used for all experiments and, immediately after being taken, the heparinised whole venous blood was used to set up a large number of microcultures n each consisting of 0.3 ml whole blood in 5.0 ml of medium, with or without P H A (Type M, Difco Laboratories, Detroit, Mich.), in a tissue-culture flask (Falcon Plastics, Los Angeles, Calif.). Cells were, thus, irradiated whilst in the culture medium, in the stimulated ( + P H A ) or non-stimulated (--PHA) condition, several microcultures being used for each radiation dose. Cultures were kept at a constant temperature of 37 a I ° and fixed between 48 and 56 h from the time of adding PHA. o.I ml of IO-~ M colcemid was added to each Mutation Res., 8 (1969) 367-381
HUMAN CHROMOSOME ABERRATIONS
369
culture 4 h before fixation. I t has been shown s0 that, with the culture technique employed in these experiments, cells which undergo mitosis before 56 h after P H A stimulation are at their first division in vitroS,~, 3~. Further details of culture medium, fixation procedure, etc. are given elsewhere n. For scoring purposes all slides were coded and randomised, and only cells with a complete chromosome complement (i.e. having 46 centromeres) were scored 38. Cells were examined for the presence of dicentric aberrations, centric rings, acentric rings, interstitial deletions (minutes) and terminal deletions 1~. Because of their low frequency and the difficulty, in m a n y cases, of distinguishing between them, acentric rings, minutes and terminal deletions have been combined and classified as fragments in Tables I - I V . We have found that the scoring of the frequency of dicentrics after a given radiation dose is very consistent from one experiment to another and from one observer to another, whereas scoring of other aberration types is less reliable 38. In calculating the R B E of different types of radiation and in determining dose-rate effects from the present study we have, therefore, used the frequency of dicentrics as our standard of comparison but the frequency of other aberrations has also been tabulated (Tables I-V). The ATLAS computer (Harwell) was used for statistical analysis of the data. Standard errors for aberration yields (shown in graphs) have been calculated on the assumption of a Poisson distribution of aberrations between cells. Experiment z. Acute X-irradiation I h after the addition of blood to microcultures containing PHA, the cultures were exposed to 196, 297 or 400 rad of X-rays (250 kV, H V T 1.2 m m Cu, all doses given in 2 rain) and fixed before 56 h as described above. Experiment 2. Acute neutron irradiation Stimulated and non-stimulated cultures were irradiated, I h after the addition of blood, with various doses of fast neutrons (mean energy approx. 0. 7 MeV) produced from a uranium converter plate 3 installed in the Atomic Energy Research Establishment (A.E.R.E.) reactor BEPO (British Experimental Pile), at a dose rate of approximately 50 rad/min, y-Contamination was approximately lO% of the fast-neutron dose. P H A was added 30 min after irradiation to cultures which had been irradiated in the non-stimulated condition. Experiment 3. Chronic neutron irradiation Cells were irradiated with fast neutrons produced from a uranium converter plate ~7 in the A.E.R.E. Graphite Low Energy Experimental Pile (GLEEP). The converter plate in GLEEP was of similar construction to that in BEPO, SO the neutron spectrum for the chronic exposures was similar to that for the acute exposures in BEPO. y-Contamination was approximately 15% of the fast-neutron dose. Part I of this experiment was performed with the reactor operating at 32 kW to give a dose rate of 6.75 rad/h. Cultures were irradiated for approximately o, 3, 5.75, 8.5, 11.25, 14, 17, 19.75 and 22.5 h in order to achieve the required doses (Table IV), the irradiation being interrupted every 3 h for about IO rain for the addition or removal of culture flasks from the irradiation chamber. The irradiation of stimulated cells for periods of up to 24 h after the addition Mutation Res., 8 (1969) 367-381
37 °
D. SCOTT et al.
of P H A is j u s t i f e d , since the GI phase, which has a m i n i m u m d u r a t i o n s of a p p r o x i m a t e l y 24 h, has a c o n s t a n t r a d i o s e n s i t i v i t y as far as the i n d u c t i o n of chromosome a b e r r a t i o n s is concerned 12. I r r a d i a t i o n of all the s t i n m l a t e d cultures b e g a n I h after t h e blood h a d been a d d e d to the culture m e d i u m containing P H A . The n o n - s t i m u l a t e d cultures were i n t r o d u c e d into the i r r a d i a t i o n c h a m b e r a t successive intervals after the blood h a d been a d d e d so t h a t t h e y were all p r e s e n t in the c h a m b e r a t the end of the i r r a d i a t i o n period, e.g. n o n - s t i m u l a t e d cultures to be i r r a d i a t e d for 22.5 h were p u t in the c h a m ber a t the same t i m e as all the s t i n m l a t e d cultures, whereas those requiring 14 h i r r a d i a t i o n were p u t in 8.5 h later. I n this w a y P H A could be a d d e d to all t h e nons t i m u l a t e d cultures at the same time, a p p r o x i m a t e l y 3o min after t h e y h a d all finished being i r r a d i a t e d . This p r o c e d u r e for i r r a d i a t i n g n o n - s t i m u l a t e d cells will be referred to as Method A. I n P a r t 2 of t h e e x p e r i m e n t (performed 2 d a y s after P a r t i) the reactor was o p e r a t i n g a t 16 k W to give a dose r a t e of 3.41 r a d / h a n d cultures were i r r a d i a t e d for periods of a p p r o x i m a t e l y 5-75, 11-75, 17.75 a n d 23.5 h (Table V). The i r r a d i a t i o n p r o c e d u r e for t h e s t i m u l a t e d a n d for half of the n o n - s t i m u l a t e d cultures was t h e same as in P a r t I (i.e. Method A for n o n - s t i m u l a t e d cultures) b u t i r r a d i a t i o n of the other half of t h e n o n - s t i m u l a t e d cultures began at t h e same time as all the s t i m u l a t e d cultures, i.e. I h after s e t t i n g u p cultures, b u t P H A was n o t a d d e d to a n y of these cultures u n t i l 24 h later. Thus, for m o s t of these cultures there was a t i m e i n t e r v a l between t h e end of i r r a d i a t i o n a n d t h e t i m e of s t i m u l a t i o n (Method B). RESULTS (5) E x p e r i m e n t I . A cute X - i r r a d i a t i o n
T a b l e I gives t h e frequencies of various t y p e s of a b e r r a t i o n o b s e r v e d in cells exposed, in t h e s t i m u l a t e d condition, to different doses of X - r a y s , a n d Fig. I shows t h e f r e q u e n c y of dicentrics after these doses c o m p a r e d w i t h t h e results of e x p e r i m e n t s b y EVANS 13 on s t i m u l a t e d cells in which a different blood donor was used. I n c o m m o n TABLE I F R E Q U E N C Y OF V A R I O U S A B E R R A T I O N T Y P E S A F T E R A C U T E X - R A Y I R R A D I A T I O N
Stimulated or nonstimulated
Dose in vad
Number of cells scored
Dicentrics a
Centric rings
Fragments
Stimulated Stimulated Stimulated
196 297 400
i oo ioo i oo
49 97 i 16
14 16 3°
19 37 7~
a Tricentric regarded as 2 dicentrics, tetracentric as 3, etc, w i t h our previous findings as no significant difference b e t w e e n t h e i n vitro X - r a y s e n s i t i v i t y of the l y m p h o c y t e s of t h e two donors can be d e m o n s t r a t e d either when a c o m p a r i s o n is m a d e on a dose-for-dose basis (P = 0.32, s e e YATES 44) or when the slopes of the b e s t fitting curves are c o m p a r e d (P ~ o.22, s e e A N D E R S O N 2) using a q u a d r a t i c response m o d e l 24. In calculating R B E values for fast neutrons relative to X - r a y s (Table VI) t h e X - r a y d a t a o b t a i n e d in t h e p r e s e n t e x p e r i m e n t h a v e therefore been c o m b i n e d with EVANS' previous d a t a . Mutation Res., 8 (1969) 367-38I
371
HUMAN CHROMOSOME ABERRATIONS
(2) Experiment 2. Acute neutron irradiation Table II gives the aberration frequencies in cells irradiated in the stimulated or non-stimulated condition with doses of fast neutrons delivered at 50 rad/min, and Table III shows that the dose-response relationship for all aberration types can adequately be described by a linear model. Fig. 2 shows the fitted regression lines to 1.5 "
2.C
O 1.C
~ 1.5
to/ I.-
D-
~ tc .o r~
0.5 °"~ 0.5
121
~o
~6o 26o 360 460
Dose (tad)
Dose (tad)
16o
~o
Fig. I. Best fitting q u a d r a t i c curve (with s t a n d a r d error) to t h e d a t a of EVANS 13 for X - i r r a d i a t i o n of s t i m u l a t e d cells (. . . . ) and results of Expt. I for X - i r r a d i a t e d s t i m u l a t e d cells (O). Fig. 2. A c u t e (5 ° r a d / m i n ) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (0) and n o n - s t i m u l a t e d (0) cells. Linear regression lines fitted.
the data on dicentric frequencies and a comparison of the slopes reveals that, on average, there are significantly (P = 0.032) more dicentrics in stimulated that in non-stimulated cells. This is confirmed by a dose-for-dose comparison which gives a ratio of 1.19 (Table VII). TABLE
II
FREQUENCY
OF VARIOUS ABERRATION
T Y P E S A F T E R A C U T E (5 °
rad/min)
NEUTRON
IRRADIATION
Stimulated or nonstimulated
Dose in rad
Number of cells
Dicentrics
Centric rings
Fragments
Stimulated
o 25 5° 75 IOO 125 15°
96 116 88 14° 83 88 59
o 34 36 89 89 III 98
(o.oo) (0"29) (O.41) (O.64) (1.O7) (1.26) (1.66)
o 8 IO 22 14 22 22
(o.oo) (O.O7) (O. II) (O.16) (O'17) (0.25) (0.37)
o II 28 51 44 75 41
(o.oo) (0.09) (0.32) (0.36) (0-53) (0.85) (0.69)
Non-stimulated
o 25 5° 75 IOO 125 15o
ioo 56 55 60 62 61 8I
o 8 I6 45 59 65 IOI
(o.oo) (o. I4) (0.29) (0.75) (0.95) (1.o7) (1.25)
o 4 4 12 II 16 23
(o.oo) (o-07) (0.07) (o.2o) (o.18) (0.26) (0.28)
o 6 8 14 18 28 47
(o.oo) (o. II) (o.15) (0.23) (0.29) (0.46) (0.58)
A b e r r a t i o n yields per cell s h o w n in brackets.
(3) Experiment 3. Chronic neutron irradiation Part z. Dose rate 6.75 rad/h. The results of this experiment are summarized in Tables III and IV and in Fig. 3- The frequency of dicentrics in non-stimulated cells is a good fit to a linear dose-response model whereas the data for stimulated cells are Mutation Res., 8 (1969) 367-381
D. SCOTT et al.
372
T A B L E 11 l DOSE--RESPONSE RELATIONSHIPS FOR NEUTRON-IRRADIATED CELLS
Dose rate
Stimulated or non-stimulated
Aberration type
5° r a d / m i n
Stimulated
Dicentrics Centric rings Fragments
6.24 1.96 7.72
5 5 5
0.28 0.85 °.17
9-95 : 0.47 2.13 :z 0.22 5.44 : 0.34
Non-
Dieentrics Centric rings Fragments
5 .61 2.16 1.88
5 5 5
0.35 0.83 0.86
8.49 ~: 0.50 2.02 2:0.24 3.49 :: o.32
Stimulated
Dicentrics Centrie rings Fragments
13.39 6.1o 5 .86
5 5 5
{).02 0.30 0-32
lO.32 [: 0,64 3.07 :!2 o,2I 4 .80 7!7 0,27
~orl-
Dicentrics Centric rings Fragments
3.38 2.97 2.11
5 5 5
0.64 0-70 0-83
lO.2O [ 0.47 2.70 [ : ° - 2 4 4.98 0.33
Stimulated
Dicentrics Centrie rings Fragments
2.Ol 1.62 1-65
3 3 3
0.57 0-65 0-65
lO.87 :: 0.53 2,87 ~: 0.27 5,20 !: 0.37
2%To rl-
Dicentrics Centric rings Fragments
2-17 0.96 O.16
3 3 3
0.54 o.81 0.98
8,24 ! : o . 4 o 2.11 :-Lc0.20 4,OI :} 0.28
stimulated 6.75 r a d / h
stimulated 3.41 r a d / h
stimulated
Goodness-of-fit to linear model Slope Z~ ~.f.--p - ( × zo 3)
a poor fit (Table III) for although the yields follow a linear response up to about 6o rad (approx. 0. 7 per cell) there are signs of saturation at higher doses. However, a dose-for-dose comparison of the yields of dicentrics in stimulated as opposed to nonstimulated cells reveals no overall significant difference (P = o.86) although, on average, their frequency is slightly higher in stimulated cells (Table VII). P a r t 2. Dose rate 3.4 r rad/h. The results are given in Tables n I and V and Figs. 4 and 5. The irradiation of non-stimulated cells by Methods A and B gives comparable results (Table V, Fig. 4) and the relationship between aberration frequency and dose is linear for both stimulated and non-stimulated cells (Table III, TABLE IV F R E Q U E N C Y OF VARIOUS ABERRATION TYPES AFTER CHRONIC ( 6 . 7 5 r a d / h )
Stimulated or non-stimulated Stimulated
Dose in rad o
19.8 38.3 56.8 75.8 114.o 152.2 Non-stimulated (Method A)
o.o 19.1 38.2 56.3 76,4 113. 9 I52,2
Dicentrics
Centric rings
Fragments
.So 142 162 149 164 133 147
o 28 7° lO6 147 151 194
o 12 24 32 32 47 60
o (o.oo) 18 (o.13) 27 (o. I7)
5° IOO IOO IOO IOO IOO IOO
A b e r r a t i o n yields per cell s h o w n in brackets. Mutation Res., 8 (1969) 367-381
NEUTRON IRRADIATION
Number of cells scored
(o.oo) (o.2o) (o.43) (o.7I) (0.90) (1.14) (I.32)
o (o.oo) I9 49 54 75 12o 148
(o. I9) (°,49) (0-54) (0.75) (1.2o) (1.48)
(o,oo) (o,o8) (o.15) (o.2I) (0.20) (0.35) (o.4I)
o (o.oo) 4 I3 17 25 27 37
(0'°4) (o. I3) (°.17) (0.25) (0.27) (0.37)
3 6 (0.24)
72 (o.44) 74 (0.56) 97 (o.66)
o (o.oo) I3 19 27 37 61 7°
(°'I3) (o. I9) (0.27) (0.37) (o.61) (°.7°)
HUMAN CHROMOSOME ABERRATIONS
373
2.0 1.5
~
1.0
U
"~ 0.5 t5 50 D o s e (r,ad)
Fig. 3. Chronic (6.75 r a d / h ) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (~) a n d n o n - s t i m u l a t e d (ll) cells.
Figs. 4 and 5). Again, however, there are more aberrations in stimulated than in nonstimulated cells (P ---- 0.000016 for dicentrics, Fig. 5, Table VII).
(4) Gamma-ray contamination The doses shown in Tables II, IV and V and Figs. 2-7 are neutron doses only. A small contribution to the aberration frequencies will be due to the additional yrays to which the cells were exposed (see MATERIALSAND METHODS).However, recent experiments (unpublished) in the laboratory, with cobalt-6o y-rays, demonstrate that, on the assumption that there is no interaction between chromosome breaks induced by the neutrons and y-raysIg, 4~, the y-ray contribution to yield will, at most, be less than 30/0. In the following determinations of dose-rate effect and R B E the y-contamination has therefore been ignored.
(5) The effect of dose rate (a) Stimulated cells. When the aberration frequencies induced in stimulated TABLE V FREQUENCY OF VARIOUS ABERRATION TYPES AFTER CHRONIC (3.41 r a d / h ) NEUTRON IRRADIATION Stimulated or non-stimulated
Dose in rad
N u m b e r of cells scored
Dicentrics
Centric rings
Fragments
Stimulated
o 20.0 40.3 60. 3 80.0
13o 192 161 208 197
i 49 73 144 16o
(o.oi) (0.26) (0-45) (0.69) (o.81)
o 13 14 38 46
(o.oo) (0.07) (0.09) (o.18) (0.23)
o 17 31 73 80
(o.oo) (0.09) (o.19) (0.35) (o.41)
Non-stimulated (Method A)
o 19.7 39.7 6o.0 8o.0
93 209 183 158 144
o 27 52 84 ioo
(o.oo) (o. 13) (0.28) (0.53) (0.69)
o 4 II 17 27
(o,oo) (0.02) (0.06) (o. i i ) (o.19)
o 16 32 39 48
(o.oo) (0.08) (o.17) (0.25) (0.33)
Non-stimulated (Method A)
o 20.0 40.3 60. 3 80.0
5° ioo ioo ioo lOO
o 18 37 39 75
(o.oo) (o.18) (0.37) (0.39) (0.75)
o 7 IO 18 17
(o.oo) (0-07) (o.Io) (o.18) (o.17)
o 9 14 21 3°
(o.oo) (0.09) (o.14) (o.21) (0.30)
A b e r r a t i o n yields p e r cell s h o w n in b r a c k e t s . Mutation Res., 8 (1969) 367-381
n. SCOTT et al.
374 2.(3 2.0
1.5
1.5
~I.0 "F_.
o
~o.5
,~--l"
~u O.5
16o
5O Dose (rod)
o
50 Dose (rod)
16o
Fig. 4- Chronic (3.41 rad/h) n e u t r o n d o s e - r e s p o n s e d a t a for n o n - s t i m u l a t e d cells irradiated by Method A, all irradiations ending at same time (@), or Method B, all irradiations starting at same time (V). Linear regression line fitted to pooled data. Fig. 5. Chronic (3.41 rad]h) n e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d (&) and n o n - s t i m u l a t e d (A) cells. Linear regression lines fitted.
ceils at the three different neutron dose rates are compared, no significant difference is obtained (P = o.50 for dicentrics from a comparison of regression lines). Thus, an almost IOOO-fold range of dose rate (o.o57-5o rad/min, doses up to 15o rad) does not influence the aberration yield (Fig. 6, Table III). ~.0
1.5 0 [3
CL 1.o
o&
.~-
~ 0.5
DA
~o
0
o
Dose
(rod)
lO0
15o
Fig. 6. N e u t r o n d o s e - r e s p o n s e d a t a for s t i m u l a t e d cells irradiated at 5 ° r a d / m i n (O) and 6.75 r a d / h (~), a n d 3.41 r a d / h (&).
(b) Non-stimulated cells. The results reported above showed that at two of the three dose rates used (50 rad/min and 3.41 rad/h) significantly less aberrations were found in non-stimulated than in stimulated cells, whereas in the third case (6.75 rad/h) no significant difference was obtained. As a result of this, and the fact that no dose-rate effect exists for stimulated cells, in comparing the aberration frequencies in cells irradiated in the non-stimulated condition one finds that the level of damage in cells irradiated at 6.75 rad/h is significantly higher than in cells irradiated at the higher dose rate of 50 rad/min (P = o.o13) or the lower dose rate of 3.41 rad/h (P ~ o.oo15) and that there is no difference (P ~ 0.69) between the yields induced at the latter two dose rates (Fig. 7). We believe this dose-rate effect to be more apparent than real and possibly due to a differential stimulation by PHA of less heavily radiation-damaged cells (see DISCUSSION). Mutation Res., 8 (1969) 367-38t
375
HUMAN CHROMOSOME ABERRATIONS
2.0
_
1.5
f
.g8 .~ 0.5 0
i
50
100
Dose (r'ad)
150
Fig. 7. N e u t r o n d o s e - r e s p o n s e d a t a for n o n - s t i m u l a t e d cells irradiated at 5 ° t a d / r a i n (@), 6.75 r a d / h (11), and 3.41 r a d / h (&). Linear regression lines fitted.
(6) RBE Since no dose-rate effect was observed for cells irradiated in the stimulated condition our calculations of the relative efficiency of neutrons and X - r a y s in inducing chromosome damage are restricted to cells which were irradiated in this condition (Table VI, Fig. 8). W h e n the X - r a y data obtained in Expt. I are combined with T A B L E VI RBE
OF FAST NEUTRON'S
COMPARED
WITH
X-RAYS
Comparison
Yield of dicentrics (per cell)
Dose of X-rays (Dx) (rad)
Dose of neutrons (Dn) (rad)
RBE D x -~- S.E. Dn ---
X - r a y s ( E x p t . I + d a t a o f EVANS13) c o m p a r e d with combined n e u t r o n d a t a at 50 r a d / m i n , 6.75 r a d / h and 3.41 r a d / h for stimulated cells
o.oooi o. 5 i.o 1. 5
o.°396 172.7 ± 3.5 312-7 ~ 5.9 433.7 ~ 12.8
o-oo97 48.3 ~_ 1.2 96. 7 ~ 2. 4 145.° ± 3-7
4- IO 3.57 ~ o.12 3.24 ~ o.Io 2.99 -~ o.12
X - r a y s (as above) c o m p a r e d w i t h n e u t r o n d a t a at 5 ° r a d / m i n for s t i m u l a t e d cells
o.oooi 0. 5 I.O 1. 5
0.0396 172.7 ~ 3-5 312.7 • 5.9 433-7 ± 12.8
O.OLOI 50.3 zk 2.4 lOO.5 i 4-7 15o.8 :L- 7.1
3.94 3-44 ~ o.18 3.11 ~ o.16 2.88 ± o.16
EVANS' results 13 for stimulated cells, the combined data for dicentric aberrations can be described (goodness-of-fit P = o.14) b y the quadratic expressionS4; y
where
and from which
--_ ~ D + ~ D
2
Y = the yield of aberrations per cell = one-track component of yield /3 : - two-track component of yield D = radiation dose ~ ~ (2.52 ~ o.15)" l O - 3 /3 ~ (2.16 ~- 0 . 5 8 ) . l O - 6
These values of ~ and fl have been used in Table VI to calculate the doses of X - r a y s which are required to induce a given level of chromosome damage. For calculations of the neutron doses which are required to produce the same levels of darnMutation Res., 8 (1969) 367-381
376
D.
~.5
j,
SCOTT et
al.
o~,Z /
2
O'
100
200
300
Dose(rod)
4.00
500
Fig. 8. X - R a y d o s e - r e s p o n s e c u r v e (with s t a n d a r d error) f r o m c o m b i n e d d a t a of EVANS 13 for s t i m u l a t e d cells a n d of our E x p t . I, fitted to q u a d r a t i c model. L i n e a r regression line (with s t a n d a r d error/ for n e u t r o n d a t a in s t i m u l a t e d cells also s h o w n .
age, the results from the experiments on stimulated cells at the three different dose rates have been combined. The slope of the regression line for these combined data is (lO.34 ~ o.26), lO -3 but, because of the inclusion of the 6.75 rad/h data, the fit to linearity is poor (P = o.o8o). Therefore, Table VI shows the R B E values obtained by comparing the X-ray yields with those obtained from the acute neutron experiment only (stimulated cells) in which the fit to linearity is better (Table I I I , Fig. z). There is, in fact, little difference between the R B E values which are obtained in the two cases. It should be noted that over the wide range of radiation doses which were used, the R B E value does not vary much, having a mean value of about 3.5- Even upon extrapolation to very low levels of damage, e.g. lO-4 dieentrics/cell, the R B E only increases to about 4 (see DISCUSSION). DISCUSSION
(z) Aberration frequencies in cells irradiated in the stimulated or non-stimulated condition It is our experience in culturing human peripheral blood lymphocytes that, from one experiment to another, the degree of mitotic activity obtained in the cultures m a y v a r y considerably, even though the same donor and the same type of culture medium is used. This is presumably a reflection of the variable extent to which the lymphocytes transform and undergo mitosis under the influence of PHA. Just what factors, in our experiments, determine the proportion of lymphocytes which respond to the PHA, is not clear although we have evidence which suggests that the pH of the culture medium, the ~liet and differential blood count of the donor, and the particular batch of P H A used are of some importance 3~. A further observation which we have made is that irradiation of the lymphocytes in vitro causes a reduction in mitotic activity in the cultures relative to that found in non-irradiated cultures (Fig. 9) and that when lymphocytes are irradiated in the non-stimulated condition, the amount of mitotic activity in PHA-stimulated cultures of such cells is sometimes, but not always, less than that in cultures of cells which are irradiated in the stimulated condition (in G1) with the same dose 33. I t would appear therefore that the irradiation of lymphocytes may, under certain conditions, Mutation Res., 8 (I969) 367-381
377
HUMAN CHROMOSOME ABERRATIONS
1.5
1.C
6.O 5.0
~
£L
4.0 £.
•-~ &O
o5
~5
2.0 1.0 36
42 48 54 Fixation time (h)
60
66
72
160 260 360 460 461
O'
Dose (oed)
F i g . 9. M i t o t i c i n d e x , a t d i f f e r e n t t i m e s a f t e r s t i m u l a t i o n , o f w h o l e b l o o d m i c r o c u l t u r e s g i v e n 3 0 0 t a d X - r a y s (©) o r u n i r r a d i a t e d ( e ) . C u l t u r e s w e r e i r r a d i a t e d i h a f t e r s t i m u l a t i o n a n d m i t o t i c cells w e r e a c c u m u l a t e d w i t h c o l c e m i d f o r 6 h p r i o r t o e a c h f i x a t i o n t i m e s h o w n . 2 0 0 0 cells s c o r e d per point. F i g . IO. X - R a y d o s e - r e s p o n s e Quadratic curves fitted.
d a t a f r o m EVANS 18 f o r s t i m u l a t e d
(©) a n d n o n - s t i m u l a t e d
(O) cells.
reduce their capacity to respond to PHA but, again, we do not know what these conditions are. Such observations may help to explain the results of our comparisons of the amount of chromosome damage in cells irradiated in the stimulated or non-stimulated state. In all our neutron experiments there were, on average, more aberrations in stimulated than in non-stimulated cells although the difference was only significant at two of the three dose rates used. This difference was also found in an experiment of EVANS13 with X-irradiation (Fig. IO) although it was not statistically significant (P = o.12). The ratio (k) of the yields in stimulated and non-stimulated cells differs significantly from one experiment to another (P = o.o19, Table VII). It is tempting to suggest that for cells irradiated in the non-stimulated condition the more heavily radiation-damaged cells (i.e. those which would contain relatively high aberration yields) may fail to respond to the PHA and therefore never be observed at mitosis, and that the degree of response failure varies from one experiment TABLE
VII
RATIO (k) OF FREQUENCIES STIMULATED CONDITION
OF DICENTRICS
IN CELLS
IRRADIATED
Experiment
Ratio k (maximum likelihood)
Test for k = •
X - r a y s (EVANS 13) N e u t r o n s (5 ° r a d / m i n ) N e u t r o n s (6,75 r a d / h ) N e u t r o n s (3.41 r a d / h )
1.149 1.19o i.oti 1.343
U u u u
Overall Yields at doses common compared.
--L O.102 ~- o . o 9 1 ~ o.o61 ~ 0.092
= -= =
1.56 ; 2.28; o.177; 4.3I;
u = 4,o3; to both experiments
P P 19 19
IN THE
STIMULATED
AND NON-
Homogeneity of k between doses Z2 d.f. t9 = = = =
o,12o 0.023 0.860 0.000o16
P = o.oooo56
of a p a i r ( s t i m u l a t e d
5 .26 6.49 6.08 4.72
3 5 5 3
O. 15 0.26 0.30 o.19
22.55
16
o. 13
and non-stimulated)
were
M u t a t i o n Res., 8 (1969) 3 6 7 - 3 8 1
378
D. SCOTT et al.
to another. Thus, one m i g h t expect t h e r e to be a positive correlation between values of k in different e x p e r i m e n t s a n d the m a g n i t u d e of t h e difference in m i t o t i c a c t i v i t y in cultures of cells i r r a d i a t e d in t h e s t i m u l a t e d as o p p o s e d to the n o n - s t i m u l a t e d condition in these experiments. This h y p o t h e s i s is at p r e s e n t u n d e r test in our l a b o r a t o r y . If this selective s t i n m l a t i o n of less h e a v i l y r a d i a t i o n - d a m a g e d cells does occur a n d t h e e x t e n t to which it occurs varies from one e x p e r i m e n t to a n o t h e r for reasons which are at p r e s e n t u n k n o w n , this will be of considerable i m p o r t a n c e in assessing the level of c h r o m o s o m e d a m a g e in i n d i v i d u a l s i r r a d i a t e d in vivo either accidentally or t h e r a p e u t i c a l l y since, of course, t h e l y n l p h o c y t e s in vivo will be i r r a d i a t e d in t h e nons t i m u l a t e d condition. The level of c h r o m o s o m e d a m a g e o b s e r v e d in c u l t u r e d l y m p h o eytes from such individuals, which m i g h t be used to e s t i m a t e the r a d i a t i o n dose received ~5, would t h e n d e p e n d u p o n their degree of response to P H A from one occasion to another. I t is therefore of considerable i m p o r t a n c e to d e t e r m i n e w h e t h e r or not the difference in a b e r r a t i o n yields in s t i n m l a t e d a n d n o n - s t i m u l a t e d ceils is due to selective s t i m u l a t i o n , for although the n~axinmm difference which we h a v e y e t o b s e r v e d is only 25°/o (3.41 r a d / h , E x p t . , Fig. 5), p o t e n t i a l l y the difference m a y be m u c h greater. There m a y be s u b - p o p u l a t i o n s of small l y m p h o c y t e s in t h e p e r i p h e r a l blood with different radiosensitivities a n d with different capacities to respond to P H A . Certainly on criteria o t h e r t h a n r a d i o s e n s i t i v i t y the small l y m p h o c y t e s are a heterogeneous collection of cells 4~ a n d RICHTER AND NASPITZ at have r e c e n t l y shown t h a t different p o p u l a t i o n s of small l y m p h o c y t e s show different degrees of s e n s i t i v i t y to P H A .
(2)
Comparison with the results of other workers EVANS la a n d SHARPE et al. 38 h a v e d r a w n a t t e n t i o n to the m a r k e d q u a n t i t a t i v e differences b e t w e e n results o b t a i n e d in different l a b o r a t o r i e s on the frequencies of c h r o m o s o m e a b e r r a t i o n s in h u m a n leucocytes after X- or ),-irradiation in vitro. I t should be p o i n t e d out t h a t large discrepancies are still found even when only those cells which are undergoing their first m i t o t i c division in culture are scored (cf. ref. 13 w i t h refs. 29 a n d 4o). The possibility t h a t different l a b o r a t o r i e s are effectively exam i n i n g different p o p u l a t i o n s of small l y m p h o c y t e s , as referred to above, is at present u n d e r investigation. GoocH et al. TM i r r a d i a t e d n o n - s t i n m l a t e d h u m a n l y m p h o e y t e s w i t h I4.I-MeV n e u t r o n s at a dose r a t e of 6 r a d / m i n a n d Fig. I I shows t h a t t h e frequencies of dicen2.0
1,5 0 0 .1.0
cgx
"E.
E 0.5 i:5
AO
•
o A
•
•
•
O&
v
50 Dose (rods)
•
,w
100
,.-J
150 200
Fig. i i. Neutron dose-response data from the present experiment for stimulated cells irradiated at 5o rad/min (©), 6.75 rad/h ([]) and 3.41 rad/h (L\), and for non-stimulated cells irradiated at 5° rad/min (O), 6.75 rad/h (Ul) and 3.41 rad/h (A). Also, dose-response data of GoocH et al. 18 for non-stimulated cells irradiated at 6 rad/min with I4.I-MeV neutrons (w). Mutation Res., 8 (1969) 367-381
HUMAN CHROMOSOME ABERRATIONS
379
trics which they obtained at different doses is, on average, some io times lower than the frequencies obtained in the present experiment with neutrons of approximately o.7-MeV mean energy. Part of the difference may be attributable to the differences in the quality of neutrons used and to the fact that their fixations were made at 72 h, but the major part of the discrepancy remains unexplained at the present time as do the differences between the various X-ray results. Not only were the frequencies of aberrations induced by the I4.I-MeV neutrons much lower than in our experiments but the dose-response curve was non-linear having a dose exponent of 1.42 4- o.2o on a power-law model (see DISCUSSION,section 3).
(3) RBE An unusual feature of the X-ray dose-response curves of EVANS13 and that obtained in Expt. I in the present investigation, is that they are almost linear (Fig. I), the combined data having a dose exponent of 1.15 -L o.o4. Exchange aberrations (e.g. dicentrics and rings) have been shown to increase approximately as the square of the close after X+irradiation of plant cells3S,3~,~1, and NORMAN AND SASAK129have found this to be the case for human lymphocytes. On the other hand, with densely-ionizing radiations, e.g. neutrons, such aberrations are generally found to increase linearly with dosel6,~9,~3. Since the shapes of the doseresponse curves for sparsely and densely-ionizing radiations are usually found to differ, a constant RBE value is not obtained. However, since in the experiments reported here we have obtained a linear relationship between neutron dose and aberration frequency and an almost linear response for X-irradiated cells, the R B E value does not change markedly over a wide range of doses (Table VI) so that a single value of 3.5 is a meaningful estimate. From preliminary experiments with 2.5-MeV neutrons, GoocH el al. TM have quoted an R B E value, relative to 25o-kV X-rays, of between 4 and 5 for chromosome terminal deletions in human lymphocytes irradiated in vitro. Our value of 3.5 for dicentrics is rather lower than the majority of values which have been obtained for the induction of chromosomal aberrations by fast neutrons and X- or 7-rays in plant cellsl°,n, ~2,2+ and is also somewhat lower than the R B E obtained for the inhibition of colony formation in kidney cells of human origin with 3-MeV monochromatic neutrons and 25o-kV X-rays ~.
(4) Dose-rate effect Our demonstration of the lack of a dose-rate effect after neutron irradiation of stimulated cells is in agreement with observations made on plant cells 1~. These observations, and the demonstration of a linear dose-response for exchange aberrations after neutron irradiation (see references above) have been interpreted as showing that with high LET radiation both the breaks which are necessary to produce a chromosome exchange (e.g. dicentric) are produced by a single ionizing track, whereas, after low LET radiation each of the two breaks is produced by an independent track since the yield of exchanges increases as the square of the dose and a dose-rate effect is observed ~3. On this basis one would interpret our near-linear dose-response curve for X-rayinduced aberrations in human lymphocytes as indicating that, in general, only a single ionizing track is required to produce both the breaks required for the ex-
Mutation Res., 8 (1969) 367-38I
It. SCOTT et al.
380 change~:L However, a pronounced
recent
w o r k in t h i s l a b o r a t o r y
dose-rate effect for the induction
w h i c h is c o n t r a r y
to expectation
if e x c h a n g e s
(unpublished)
has demonstrated
of d i c e n t r i c s a f t e r l o w L E T i r r a d i a t i o n are the result of one-track
events.
ACKNOWLEDGEMENTS The authors invaluable
would
technical
BARNES f o r t a k i n g the A.E.R.E.
like to express
assistance,
blood samples,
reactors
their thanks
t o M r . T . R . L. BIGGER f o r
D r . G. T . BUNGAY, o u r b l o o d d o n o r , D r . D . W . H . M r . M. CORP f o r X - i r r a d i a t i o n s ,
GLEEP a n d BEPO f o r t h e f a s t - n e u t r o n
and the staff of
irradiations.
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