125
Mutation Research, 62 (1979) 125--129 © Elsevier/North-Holland Biomedical Press
THE E F F E C T S OF P-RADIATION ON SISTER-CHROMATID EXCHANGES IN C U L T U R E D HUMAN LYMPHOCYTES
PETER E. CROSSEN and WILLIAM F. MORGAN
Cancer Society of New Zealand, Cytogenetics Unit, Christchurch Hospital, Christchurch (New Zealand) (Received 14 December 1978) (Accepted 26 March 1979}
Summary The incidence of Sister-Chromatid Exchanges (SCEs) due to ~-radiation was investigated in cultured human l y m p h o c y t e s using the BrdU/Giemsa technique. Cultures treated continuously with 0.001 and 0.01 pCi of [3H]uridine showed no increase in either chromosome abnormalities or SCEs. Continuous treatment with 0.1 pCi resulted in a significant increase in chromosome aberrations b u t no increase in SCEs, while treatment with 0.2 gCi gave both an increase in chromosome aberrations and SCEs. Cultures given a 4-h pulse with 1.0 gCi showed a significant increase in both SCEs and chromosome aberrations. The results indicate that low levels of ~-radiation do n o t cause an increase in SCEs in human lymphocytes, and, that a number, if n o t all the exchanges observed at low levels of ~-radiation with autoradiography, may be spontaneous events.
Since Taylor's [ 14] initial demonstration of sister-chromatid exchanges there has been considerable controversy as to whether the exchanges were spontaneous events or due to the endogenous ~-radiation from the incorporated tritium necessary for their detection. Marin and Prescott [10] and Geard and Peacock [5] both found that the yield of exchanges was independent of the dose of tritium, whereas Wolff [16] postulated that there is a m a x i m u m number of sites available for exchange, and that the lowest levels of [3H]thymidine used b y Matin and Prescott [10] would saturate these sites. Later studies suggested that the majority, if not all the exchanges, were caused b y the incorporated tritium [2,6,8] and it has become generally accepted that ~-radiation from incorporated tritium can cause an increase in SCE. Autoradiography has the disadvantages that it lacks precision, and, during the long exposures necessary at low levels of isotope incorporation, the emulsion loses its sensitivity and there is latent image fade. These disadvantages have been overcome with the introduction of the BrdU/fluorescence/Giemsa tech-
126 TABLE 1 I N C I D E N C E O F SCEs A N D M E T A P H A S E S W I T H E I T H E R C H R O M O S O M E O R C H R O M A T I D A B E R R A T I O N S I N C U L T U R E S T R E A T E D W I T H 0 . 0 0 1 A N D 0 . 0 1 p C i / m l [ 3 H I U R I D I N E F O R 48 h Donor
Control
1 2 3 4 5 6
TABLE
0.001 pCi/ml
0.01 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
13.95 10.30 10.00 12.00 11.05 10.85
4 2 2 0 0 1
12.95 9.18 9.65 12.65 11.15 10.55
6 1 1 1 3 1
13.33 9.88 10.70 12.05 11.50 10.25
2 4 3 5 0 1
2
INCIDENCE OF SCEs AND METAPHASES WITH EITHER CHROMOSOME OR CHROMATID ABERRATIONS IN C U L T U R E S TREATED CONTINUOUSLY W I T H 0.1 O R 0.2 pCi/ml [3H] U R I D I N E F O R 48 h D o n o r 7 was o m i t t e d f r o m t h e s t a t i s t i c a l a n a l y s i s . Donor
7 8 9 10 11 12
Control
0.1 ~uCi/ml
0.2 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
13.80 13.75 9.85 9.90 8.20 9.20
4 8 6 4 8 4
14.50 15.50 10.70 11.40 14.80 14.45
32 42 38 22 22 32
_ a 16.20 17.12 17.11 b 13.35 c 14.30
64 46 44 50 30 36
a N o 2nd div~ions. b 9 cels counted. c 8 ceHs counted.
TABLE 3 INCIDENCE OF SCEs AND CHROMOSOME [3H] URIDINE Donor
13 14 15 16 17 18
Control
ABERRATIONS
I N C U L T U R E S G I V E N A 4-h P U L S E O F
0.1 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
8.75 8.00 11.25 8.55 8.05 9.05
0 6 6 4 0 2
11.8 14.05 18.55 14.75 15.55 14.60
42 22 38 48 66 38
127 niques [9,11] which provide far more precise determination of SCEs. We n o w report on the use of the BrdU/Giemsa technique to study the incidence of SCEs due to ~-radiation. Materials and methods Human peripheral blood samples were used throughout the study. The blood samples were defibrinated, sedimented in 3% gelatine and the leucocyte-rich serum cultured in Ham's F10 medium containing phytohaemagglutinin {Burroughs Wellcome) for 72 h. Bromodeoxyuridine (BrdU) was added to all cultures at a final concentration of 10 pg/ml after 24 h and light was excluded from the cultures to avoid photolysis of the BrdU substituted DNA [7]. Aqua Colchin (Parke Davis) was added 4 h prior to harvest and cultures harvested as previously described [4]. Previous studies of the effects of/~-radiation on SCE used [3H]thymidine as the source of/~-radiation. However, both BrdU and [3H]thymidine are incorporated into DNA via the same pathway, and therefore likely to compete, leading to poor differential staining and low labelling. For this reason we used [3H]uridine (Radiochemical Centre, Amersham, spec. act. 26.3 Ci/mmol) as our source of ~-radiation. Although n o t incorporated into chromosomal DNA, [3H]uridine is readily incorporated into R N A and is an efficient inducer of chromosomal aberrations [ 1 ]. Two experiments were carried out. In the first [3H]uridine was added to a series of cultures at varying concentrations at the same time as the BrdU and remained in the cultures for the remaining 48 h. In the second, a series of cultures were pulsed with [3H]uridine at 24 h at a concentration of 1.0 pCi/ml. At the end of the 4-h labelling period the cultures were washed twice in normal saline and reincubated in fresh medium. Slides from the control and [3H]uridine labelled preparations were treated for sister-chromatid differential staining by the FPG technique of Perry and Wolff [11], coded and "scored blind" for the incidence of SCE. 20 well spread metaphases showing distinct differential staining were scored for each treatment. The incidence of chromosome- and chromatid-type aberrations was determined in 50 well spread metaphases. These included 1st, 2nd and 3rd division metaphases. Chromosome aberrations included rings, dicentrics and acentric fragments while chromatid aberrations included gaps, breaks and exchanges. [3H] Uridine can cause both c h r o m o s o m e and chromatid aberrations and metaphases were classified as abnormal if they showed either t y p e of aberration. Results Table 1 shows that there is no increase in the incidence of either sister-chromatid exchanges or chromosome aberrations in cultures exposed continuously to [3H]uridine at doses of 0.001 and 0.01 pCi. The incidence of SCEs and c h r o m o s o m e abnormalities in cultures treated continuously with doses of 0.1 and 0.2 pCi/ml are summarised in Table 2. There was a dramatic increase in the n u m b e r of metaphases with either chro-
128
mosome or chromatid aberrations at both doses and a marked reduction in the number of 2nd divisions in cultures treated with 0.2 ttCi. A two-way analysis of variance of the incidence of SCEs indicated that there was a significant difference between groups in the numbers of SCEs (p < 0.01) and an analysis using Scheffe's test showed a significant difference between the control and cultures treated with 0.2 #Ci/ml (p < 0.01). Data from the cultures pulse-labelled with [3H]uridine for 4 h at a dose of 1.0 pCi/ml are shown in Table 3. All 6 donors showed a significant increase in both SCEs and chromosome aberrations (t = 9.1 5 d f p < 0.001). At concentrations of 2 pCi/ml there was considerable cell death and insufficient metaphases for cytogenetic analysis. Discussion Our results clearly show that there is no increase in SCEs in human lymphocytes exposed to fi-radiation from [3H]uridine at levels between 0.001 and 0.01 pCi. An increase in SCEs due to H-radiation from incorporated [3H]thymidine at levels of 0.003--0.01 #Ci has been shown in Pt-K1 and CHO cells [6,8]. However, in both these studies autoradiography was used to detect the exchanges, and all cells studied had incorporated tritium, whereas we were able to compare the exchange rate in cells t h a t had incorporated tritium with those that had not. Furthermore, autoradiography lacks precision as evidenced by reports of apparent isolabelling, which in fact is an artefact due to image spread [17]. We used [3H]uridine as our source of/3-radiation and this may explain the differing results. [3H]Uridine is not incorporated into chromosomal DNA, but into RNA and might be expected to be less efficient in inducing SCE. However, Bender et al. [ 1] have shown that [3H]uridine is an extremely efficient inducer of chromosome aberrations in cultured human lymphocytes, and we also found a significant n u m b e r of both chromosome and chromatid aberrations indicating that the fi-radiation from the incorporated [3H]uridine can cause chromosomal damage. It is n o t clear from the data of Gibson and Prescott [6] or Kato [8] whether they found an increase in chromosome aberrations in their cultures exposed to [ 3H]thymidine. Wolff has postulated that a chromosome has a m a x i m u m number of sites available for exchanges to occur, and that these are saturated at low levels of incorporated tritium [16]. There is evidence to support this hypothesis [6,8] and our results from subjects 8, 11 and 12 also suggest that saturation occurs. However, higher doses of radiation led to cell death and we were n o t able to obtain the dramatic increase in SCEs that can be obtained with agents such as mitocymin C. It is possible that the observed saturation is due to cell death occurring at higher doses, and not to a limitation on the number of available exchanges sites. Moreover, the results of chemical induction of SCEs indicate that there are virtually unlimited sites on a chromosome available for exchanges
[9]. The majority of experimental studies demonstrate that radiation is a poor inducer of SCEs. Human lymphocytes exposed to 150 rad of T-radiation in G1 show a small but significant increase in SCEs and a slight increase when
129 exposed to 200 rad of X-rays in $2 [13]. In addition a dose--response between X-rays and SCEs has been reported in Chinese hamster cells exposed in G1 or S [12]. It is noteworthy that in these two reports the increase in SCEs was minimal when compared with the incidence of chromosome and chromatid aberrations. Our data supports these results in that we did not find an increase in SCEs until there was considerable chromosome damage. In some respects it is not surprising that radiation is a poor inducer of SCEs because the majority of single- and double-strand breaks caused by radiation are repaired within 30 min
[3]. Our failure to find an increase in SCEs at low levels of ~-radiation implies that a number, if not all the exchanges observed with autoradiography at low levels of ~-radiation, are spontaneous events. The stability of SCEs at low levels of incorporation led Tice et al. [15] to postulate also the existence of spontaneous SCEs in vivo. If SCEs can occur spontaneously as it appears they can, their biological significance is as yet unexplained. Acknowledgements This work was supported by the Medical Research Council of New Zealand and the Canterbury and Westland Division of the Cancer Society of New Zealand. We wish to thank Dr. P.H. Fitzgerald for his review of the manuscript and Mrs. Joanna Stewart for the statistical analysis. References 1 B e n d e r , M.A., P.C. G o o c h a n d D.M. P r e s c o t t , 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 c o c y t e c h r o m o s o m e s by 3H-labelled nueleosides, Cytogeneties, 1 (1962) 65--74. 2 B r e w e n , J . G . , a n d W.J. P e a c o c k , T h e e f f e c t o f t r i t i a t e d t h y m i d i n e on sister c h r o m a t i d e x c h a n g e s in a ring c h r o m o s o m e , M u t a t i o n Res., 7 ( 1 9 6 9 ) 4 3 3 - - 4 4 0 . 3 C o r r y , P.M., a n d A. Cole, D o u b l e s t r a n d r e j o i n i n g in m a m m a l i a n D N A , N a t u r e ( L o n d o n ) , N e w Biol.~ 245 (1973) 100--101. 4 Crossen, P.E., G i e m s a b a n d i n g p a t t e r n s of h u m a n c h r o m o s o m e s , Clin. G e n e t . , 3 ( 1 9 7 2 ) 1 6 9 - - 1 7 9 . 5 G e a r d , C.R., a n d W.J. P e a c o c k , Sister c h r o m a t i d e x c h a n g e s in Vica faba, M u t a t i o n Res., 7 ( 1 9 6 9 ) 215--223. 6 G i b s o n , A . D . , a n d D.M. P r e s c o t t , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s in c h r o m o s o m e s o f r a t kan• g a r o o b y t r i t i u m i n c o r p o r a t e d i n t o D N A , E x p . Cell Res., 74 ( 1 9 7 2 ) 3 9 7 - - 4 0 2 . 7 I k u s h i m a , T., a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y light flashes t o 5 - b r o m o d e o x y u r i dine a n d 5 - i o d o d e o x y u r i d i n e s u b s t i t u t e d Chinese h a m s t e r c h r o m o s o m e s , E x p . Cell Res., 87 ( 1 9 7 4 ) 15--19. 8 K a t o , H., S p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s d e t e c t e d b y a B U d R labelling m e t h o d , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 7 0 - - 7 2 . 9 L a t t , S.A., Sister c h r o m a t i d e x c h a n g e s , indices of h u m a n c h r o m o s o m e d a m a g e a n d repair: D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y m i t o m y c i n C, Proc. Natl. A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 3 1 6 2 - 3166. 10 Marin, G., a n d D.M. P r e s c o t t , T h e f r e q u e n c y o f sister e h r o m a t i d e x c h a n g e s f o l l o w i n g e x p o s u r e to v a r y i n g d o s e s o f 3 H - t h y m i d i n e or X-rays, J. Cell Biol,, 21 ( 1 9 6 4 ) 1 5 9 - - 1 6 7 . 11 P e r r y , P., a n d S. Wolff, N e w G i e m s a m e t h o d for t h e d i f f e r e n t i a l staining of sister c h r o m a t i d s , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 1 5 6 - - 1 5 8 . 12 P e r r y , P., a n d H.J. Evans, C y t o l o g i c a l d e t e c t i o n of m u t a g e n - - c a r c i n o g e n e x p o s u r e b y sister e h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 13 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e s -- A sensitive assay of a g e n t s d a m a g i n g hum a n c h r o m o s o m e s , M u t a t i o n Res., 30 ( 1 9 7 5 ) 2 7 3 - - 2 7 8 . 14 T a y l o r , J . H . , Sister c h r o m a t i d e x c h a n g e s in t r i t i u m labelled c h r o m o s o m e s , G e n e t i c s , 43 ( 1 9 5 8 ) 5 1 5 - 529. 15 Tice, R., J. Chaillet a n d E.L. S c h n e i d e r , D e m o n s t r a t i o n of s p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s in vivo, E x p . Cell Res., 1 0 2 ( 1 9 7 6 ) 4 2 6 - - 4 2 9 . 16 Wolff, S., Are sister c h r o m a t i d e x c h a n g e s sister s t r a n d c r o s s o v e r s or r a d i a t i o n i n d u c e d e x c h a n g e s ? , M u t a t i o n Res., 1 ( 1 9 6 4 ) 3 3 7 - - 3 4 3 . 17 Wolff, S., a n d P. P e r r y , D i f f e r e n t i a l staining of sister c h r o m a t i d s a n d the s t u d y o f sister c h r o m a t i d e x c h a n g e s w i t h o u t a u t o r a d i o g r a p h y , C h r o m o s o m a , 48 ( 1 9 7 4 ) 3 4 1 - - 3 5 3 .
Mutation Research, 62 (1979) 125--129 © Elsevier/North-Holland Biomedical Press
THE E F F E C T S OF P-RADIATION ON SISTER-CHROMATID EXCHANGES IN C U L T U R E D HUMAN LYMPHOCYTES
PETER E. CROSSEN and WILLIAM F. MORGAN
Cancer Society of New Zealand, Cytogenetics Unit, Christchurch Hospital, Christchurch (New Zealand) (Received 14 December 1978) (Accepted 26 March 1979}
Summary The incidence of Sister-Chromatid Exchanges (SCEs) due to ~-radiation was investigated in cultured human l y m p h o c y t e s using the BrdU/Giemsa technique. Cultures treated continuously with 0.001 and 0.01 pCi of [3H]uridine showed no increase in either chromosome abnormalities or SCEs. Continuous treatment with 0.1 pCi resulted in a significant increase in chromosome aberrations b u t no increase in SCEs, while treatment with 0.2 gCi gave both an increase in chromosome aberrations and SCEs. Cultures given a 4-h pulse with 1.0 gCi showed a significant increase in both SCEs and chromosome aberrations. The results indicate that low levels of ~-radiation do n o t cause an increase in SCEs in human lymphocytes, and, that a number, if n o t all the exchanges observed at low levels of ~-radiation with autoradiography, may be spontaneous events.
Since Taylor's [ 14] initial demonstration of sister-chromatid exchanges there has been considerable controversy as to whether the exchanges were spontaneous events or due to the endogenous ~-radiation from the incorporated tritium necessary for their detection. Marin and Prescott [10] and Geard and Peacock [5] both found that the yield of exchanges was independent of the dose of tritium, whereas Wolff [16] postulated that there is a m a x i m u m number of sites available for exchange, and that the lowest levels of [3H]thymidine used b y Matin and Prescott [10] would saturate these sites. Later studies suggested that the majority, if not all the exchanges, were caused b y the incorporated tritium [2,6,8] and it has become generally accepted that ~-radiation from incorporated tritium can cause an increase in SCE. Autoradiography has the disadvantages that it lacks precision, and, during the long exposures necessary at low levels of isotope incorporation, the emulsion loses its sensitivity and there is latent image fade. These disadvantages have been overcome with the introduction of the BrdU/fluorescence/Giemsa tech-
126 TABLE 1 I N C I D E N C E O F SCEs A N D M E T A P H A S E S W I T H E I T H E R C H R O M O S O M E O R C H R O M A T I D A B E R R A T I O N S I N C U L T U R E S T R E A T E D W I T H 0 . 0 0 1 A N D 0 . 0 1 p C i / m l [ 3 H I U R I D I N E F O R 48 h Donor
Control
1 2 3 4 5 6
TABLE
0.001 pCi/ml
0.01 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
13.95 10.30 10.00 12.00 11.05 10.85
4 2 2 0 0 1
12.95 9.18 9.65 12.65 11.15 10.55
6 1 1 1 3 1
13.33 9.88 10.70 12.05 11.50 10.25
2 4 3 5 0 1
2
INCIDENCE OF SCEs AND METAPHASES WITH EITHER CHROMOSOME OR CHROMATID ABERRATIONS IN C U L T U R E S TREATED CONTINUOUSLY W I T H 0.1 O R 0.2 pCi/ml [3H] U R I D I N E F O R 48 h D o n o r 7 was o m i t t e d f r o m t h e s t a t i s t i c a l a n a l y s i s . Donor
7 8 9 10 11 12
Control
0.1 ~uCi/ml
0.2 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
13.80 13.75 9.85 9.90 8.20 9.20
4 8 6 4 8 4
14.50 15.50 10.70 11.40 14.80 14.45
32 42 38 22 22 32
_ a 16.20 17.12 17.11 b 13.35 c 14.30
64 46 44 50 30 36
a N o 2nd div~ions. b 9 cels counted. c 8 ceHs counted.
TABLE 3 INCIDENCE OF SCEs AND CHROMOSOME [3H] URIDINE Donor
13 14 15 16 17 18
Control
ABERRATIONS
I N C U L T U R E S G I V E N A 4-h P U L S E O F
0.1 p C i / m l
SCE
% Aberrant metaphases
SCE
% Aberrant metaphases
8.75 8.00 11.25 8.55 8.05 9.05
0 6 6 4 0 2
11.8 14.05 18.55 14.75 15.55 14.60
42 22 38 48 66 38
127 niques [9,11] which provide far more precise determination of SCEs. We n o w report on the use of the BrdU/Giemsa technique to study the incidence of SCEs due to ~-radiation. Materials and methods Human peripheral blood samples were used throughout the study. The blood samples were defibrinated, sedimented in 3% gelatine and the leucocyte-rich serum cultured in Ham's F10 medium containing phytohaemagglutinin {Burroughs Wellcome) for 72 h. Bromodeoxyuridine (BrdU) was added to all cultures at a final concentration of 10 pg/ml after 24 h and light was excluded from the cultures to avoid photolysis of the BrdU substituted DNA [7]. Aqua Colchin (Parke Davis) was added 4 h prior to harvest and cultures harvested as previously described [4]. Previous studies of the effects of/~-radiation on SCE used [3H]thymidine as the source of/~-radiation. However, both BrdU and [3H]thymidine are incorporated into DNA via the same pathway, and therefore likely to compete, leading to poor differential staining and low labelling. For this reason we used [3H]uridine (Radiochemical Centre, Amersham, spec. act. 26.3 Ci/mmol) as our source of ~-radiation. Although n o t incorporated into chromosomal DNA, [3H]uridine is readily incorporated into R N A and is an efficient inducer of chromosomal aberrations [ 1 ]. Two experiments were carried out. In the first [3H]uridine was added to a series of cultures at varying concentrations at the same time as the BrdU and remained in the cultures for the remaining 48 h. In the second, a series of cultures were pulsed with [3H]uridine at 24 h at a concentration of 1.0 pCi/ml. At the end of the 4-h labelling period the cultures were washed twice in normal saline and reincubated in fresh medium. Slides from the control and [3H]uridine labelled preparations were treated for sister-chromatid differential staining by the FPG technique of Perry and Wolff [11], coded and "scored blind" for the incidence of SCE. 20 well spread metaphases showing distinct differential staining were scored for each treatment. The incidence of chromosome- and chromatid-type aberrations was determined in 50 well spread metaphases. These included 1st, 2nd and 3rd division metaphases. Chromosome aberrations included rings, dicentrics and acentric fragments while chromatid aberrations included gaps, breaks and exchanges. [3H] Uridine can cause both c h r o m o s o m e and chromatid aberrations and metaphases were classified as abnormal if they showed either t y p e of aberration. Results Table 1 shows that there is no increase in the incidence of either sister-chromatid exchanges or chromosome aberrations in cultures exposed continuously to [3H]uridine at doses of 0.001 and 0.01 pCi. The incidence of SCEs and c h r o m o s o m e abnormalities in cultures treated continuously with doses of 0.1 and 0.2 pCi/ml are summarised in Table 2. There was a dramatic increase in the n u m b e r of metaphases with either chro-
128
mosome or chromatid aberrations at both doses and a marked reduction in the number of 2nd divisions in cultures treated with 0.2 ttCi. A two-way analysis of variance of the incidence of SCEs indicated that there was a significant difference between groups in the numbers of SCEs (p < 0.01) and an analysis using Scheffe's test showed a significant difference between the control and cultures treated with 0.2 #Ci/ml (p < 0.01). Data from the cultures pulse-labelled with [3H]uridine for 4 h at a dose of 1.0 pCi/ml are shown in Table 3. All 6 donors showed a significant increase in both SCEs and chromosome aberrations (t = 9.1 5 d f p < 0.001). At concentrations of 2 pCi/ml there was considerable cell death and insufficient metaphases for cytogenetic analysis. Discussion Our results clearly show that there is no increase in SCEs in human lymphocytes exposed to fi-radiation from [3H]uridine at levels between 0.001 and 0.01 pCi. An increase in SCEs due to H-radiation from incorporated [3H]thymidine at levels of 0.003--0.01 #Ci has been shown in Pt-K1 and CHO cells [6,8]. However, in both these studies autoradiography was used to detect the exchanges, and all cells studied had incorporated tritium, whereas we were able to compare the exchange rate in cells t h a t had incorporated tritium with those that had not. Furthermore, autoradiography lacks precision as evidenced by reports of apparent isolabelling, which in fact is an artefact due to image spread [17]. We used [3H]uridine as our source of/3-radiation and this may explain the differing results. [3H]Uridine is not incorporated into chromosomal DNA, but into RNA and might be expected to be less efficient in inducing SCE. However, Bender et al. [ 1] have shown that [3H]uridine is an extremely efficient inducer of chromosome aberrations in cultured human lymphocytes, and we also found a significant n u m b e r of both chromosome and chromatid aberrations indicating that the fi-radiation from the incorporated [3H]uridine can cause chromosomal damage. It is n o t clear from the data of Gibson and Prescott [6] or Kato [8] whether they found an increase in chromosome aberrations in their cultures exposed to [ 3H]thymidine. Wolff has postulated that a chromosome has a m a x i m u m number of sites available for exchanges to occur, and that these are saturated at low levels of incorporated tritium [16]. There is evidence to support this hypothesis [6,8] and our results from subjects 8, 11 and 12 also suggest that saturation occurs. However, higher doses of radiation led to cell death and we were n o t able to obtain the dramatic increase in SCEs that can be obtained with agents such as mitocymin C. It is possible that the observed saturation is due to cell death occurring at higher doses, and not to a limitation on the number of available exchanges sites. Moreover, the results of chemical induction of SCEs indicate that there are virtually unlimited sites on a chromosome available for exchanges
[9]. The majority of experimental studies demonstrate that radiation is a poor inducer of SCEs. Human lymphocytes exposed to 150 rad of T-radiation in G1 show a small but significant increase in SCEs and a slight increase when
129 exposed to 200 rad of X-rays in $2 [13]. In addition a dose--response between X-rays and SCEs has been reported in Chinese hamster cells exposed in G1 or S [12]. It is noteworthy that in these two reports the increase in SCEs was minimal when compared with the incidence of chromosome and chromatid aberrations. Our data supports these results in that we did not find an increase in SCEs until there was considerable chromosome damage. In some respects it is not surprising that radiation is a poor inducer of SCEs because the majority of single- and double-strand breaks caused by radiation are repaired within 30 min
[3]. Our failure to find an increase in SCEs at low levels of ~-radiation implies that a number, if not all the exchanges observed with autoradiography at low levels of ~-radiation, are spontaneous events. The stability of SCEs at low levels of incorporation led Tice et al. [15] to postulate also the existence of spontaneous SCEs in vivo. If SCEs can occur spontaneously as it appears they can, their biological significance is as yet unexplained. Acknowledgements This work was supported by the Medical Research Council of New Zealand and the Canterbury and Westland Division of the Cancer Society of New Zealand. We wish to thank Dr. P.H. Fitzgerald for his review of the manuscript and Mrs. Joanna Stewart for the statistical analysis. References 1 B e n d e r , M.A., P.C. G o o c h a n d D.M. P r e s c o t t , 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 c o c y t e c h r o m o s o m e s by 3H-labelled nueleosides, Cytogeneties, 1 (1962) 65--74. 2 B r e w e n , J . G . , a n d W.J. P e a c o c k , T h e e f f e c t o f t r i t i a t e d t h y m i d i n e on sister c h r o m a t i d e x c h a n g e s in a ring c h r o m o s o m e , M u t a t i o n Res., 7 ( 1 9 6 9 ) 4 3 3 - - 4 4 0 . 3 C o r r y , P.M., a n d A. Cole, D o u b l e s t r a n d r e j o i n i n g in m a m m a l i a n D N A , N a t u r e ( L o n d o n ) , N e w Biol.~ 245 (1973) 100--101. 4 Crossen, P.E., G i e m s a b a n d i n g p a t t e r n s of h u m a n c h r o m o s o m e s , Clin. G e n e t . , 3 ( 1 9 7 2 ) 1 6 9 - - 1 7 9 . 5 G e a r d , C.R., a n d W.J. P e a c o c k , Sister c h r o m a t i d e x c h a n g e s in Vica faba, M u t a t i o n Res., 7 ( 1 9 6 9 ) 215--223. 6 G i b s o n , A . D . , a n d D.M. P r e s c o t t , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s in c h r o m o s o m e s o f r a t kan• g a r o o b y t r i t i u m i n c o r p o r a t e d i n t o D N A , E x p . Cell Res., 74 ( 1 9 7 2 ) 3 9 7 - - 4 0 2 . 7 I k u s h i m a , T., a n d S. Wolff, Sister c h r o m a t i d e x c h a n g e s i n d u c e d b y light flashes t o 5 - b r o m o d e o x y u r i dine a n d 5 - i o d o d e o x y u r i d i n e s u b s t i t u t e d Chinese h a m s t e r c h r o m o s o m e s , E x p . Cell Res., 87 ( 1 9 7 4 ) 15--19. 8 K a t o , H., S p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s d e t e c t e d b y a B U d R labelling m e t h o d , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 7 0 - - 7 2 . 9 L a t t , S.A., Sister c h r o m a t i d e x c h a n g e s , indices of h u m a n c h r o m o s o m e d a m a g e a n d repair: D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y m i t o m y c i n C, Proc. Natl. A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 3 1 6 2 - 3166. 10 Marin, G., a n d D.M. P r e s c o t t , T h e f r e q u e n c y o f sister e h r o m a t i d e x c h a n g e s f o l l o w i n g e x p o s u r e to v a r y i n g d o s e s o f 3 H - t h y m i d i n e or X-rays, J. Cell Biol,, 21 ( 1 9 6 4 ) 1 5 9 - - 1 6 7 . 11 P e r r y , P., a n d S. Wolff, N e w G i e m s a m e t h o d for t h e d i f f e r e n t i a l staining of sister c h r o m a t i d s , N a t u r e ( L o n d o n ) , 251 ( 1 9 7 4 ) 1 5 6 - - 1 5 8 . 12 P e r r y , P., a n d H.J. Evans, C y t o l o g i c a l d e t e c t i o n of m u t a g e n - - c a r c i n o g e n e x p o s u r e b y sister e h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 13 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e s -- A sensitive assay of a g e n t s d a m a g i n g hum a n c h r o m o s o m e s , M u t a t i o n Res., 30 ( 1 9 7 5 ) 2 7 3 - - 2 7 8 . 14 T a y l o r , J . H . , Sister c h r o m a t i d e x c h a n g e s in t r i t i u m labelled c h r o m o s o m e s , G e n e t i c s , 43 ( 1 9 5 8 ) 5 1 5 - 529. 15 Tice, R., J. Chaillet a n d E.L. S c h n e i d e r , D e m o n s t r a t i o n of s p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s in vivo, E x p . Cell Res., 1 0 2 ( 1 9 7 6 ) 4 2 6 - - 4 2 9 . 16 Wolff, S., Are sister c h r o m a t i d e x c h a n g e s sister s t r a n d c r o s s o v e r s or r a d i a t i o n i n d u c e d e x c h a n g e s ? , M u t a t i o n Res., 1 ( 1 9 6 4 ) 3 3 7 - - 3 4 3 . 17 Wolff, S., a n d P. P e r r y , D i f f e r e n t i a l staining of sister c h r o m a t i d s a n d the s t u d y o f sister c h r o m a t i d e x c h a n g e s w i t h o u t a u t o r a d i o g r a p h y , C h r o m o s o m a , 48 ( 1 9 7 4 ) 3 4 1 - - 3 5 3 .
