- Email: [email protected]
SCEs induced by ionizing radiation are not the result of exchanges between homologous chromosomes
Mutation Research, 121 (1983) 205-210
205
Elsevier MRLett 0422
SCEs induced by ionizing radiation are not the result of exchanges between homologous chromosomes R o b e r t B. Pai nt er and William F. M o r g a n Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, San Francisco, CA 94143 (U.S.A.)
(Accepted 11 April 1983)
There is some dispute about the effects of ionizing radiation on formation of sister-chromatid exchanges (SCEs). In general it has been observed that ionizing radiation is a poor inducer of SCEs; however, the extent of stimulation of SCE by radiation varies from no increases to as high as 2-3-fold (Wolff et al., 1974; Perry and Evans, 1975; Galloway, 1977; Abranovsky et al., 1978; Littlefield et al., 1979; Livingston and Dethlefsen, 1979; Morgan and Crossen, 1980). In reviewing the protocols for these experiments, it is evident that the highest increase in SCEs after ionizing radiation is observed when bromouracil deoxyriboside (BrdUrd) is in the cell cultures for a generation or so before the irradiation (Perry and Evans, 1975; Morgan and Crossen, 1980). This increase in SCE formation has generally been attributed to a sensitization to strand breakage by the BrdUrd; however, other interpretations are possible. It is conceivable that X-ray-induced exchanges are really not SCEs at all but are complete symmetrical exchanges between homologous chromosomes. There are already indications in the literature that chromatid exchanges between homologous chromosomes occur in mammalian cells; for instance, in Bloom's syndrome cells quadriradial exchanges are observed (German, 1969). If there were an exchange between two homologous chromosomes in G~ phase and then each of the chromosomes replicates during S, both homologous chromosomes could have an apparent SCE at the same site when they arrive at mitosis (Fig. 1). Note that BrdUrd substitution before the exchange would be necessary to observe the exchange. The increase in X-ray-induced SCEs is of low enough frequency that such an exchange could go unnoticed in human and other cells with many chromosomes. To determine if there is a high frequency of simultaneous SCEs in homologs, we irradiated human lymphocytes and measured the frequency of simultaneous SCEs in homologs of chromosomes 1, 2 and 3, which are relatively easily recognized. 0165-7992/83/$ 03.00 © 1983 ElsevierScience Publishers B.V.
206
Homolog A
HornologB
Replication
;, /l Fig. 1. Scheme for reciprocal exchanges between homologs before replication. Cell had previously gone through one S phase in medium with BrdUrd so that one of the two DNA strands is substituted, as designated by wavy line. The exchange before the next replication in BrdUrd medium results in an apparent SCE in both homologs, as seen in the next mitosis (observed M).
Materials and methods P e r i p h e r a l b l o o d l y m p h o c y t e s f r o m a n o r m a l , h e a l t h y m a l e were c u l t u r e d in R P M I 1640 m e d i u m s u p p l e m e n t e d with 10070 fetal c a l f serum, penicillin (50 u n i t s / m l ) , s t r e p t o m y c i n (50 ~ g / m l ) , 2 m M L-glutamine a n d 2 % p h y t o h a e m a g glutinin. B r d U r d was a d d e d at a final c o n c e n t r a t i o n o f 2 × 10 - 5 M a n d care was t a k e n at all times to k e e p the cultures o u t o f t h e light. T w o h o u r s p r i o r to harvest, C o l c e m i d was a d d e d at a final c o n c e n t r a t i o n o f 10 - 7 M , a n d c h r o m o s o m e p r e p a r a tions m a d e as p r e v i o u s l y d e s c r i b e d ( M o r g a n a n d C r o s s e n , 1980). C u l t u r e s were i r r a d i a t e d with 150 r a d o f X - r a y s f r o m a G e n e r a l Electric M a x i t r o n 300 (300 k V p ; 20 m A , n o m i n a l H V L , 2.0 m m Cu). I r r a d i a t i o n o c c u r r e d either at 28 h a f t e r i n i t i a t i o n o f c u l t u r e (just b e f o r e a d d i t i o n o f B r d U r d - c o n t a i n i n g m e d i u m )
207 or after about one generation in BrdUrd-containing medium at 16 h before fixation. All fixations were at 64 h after initiation. Differential staining was by the method of Perry and W o l f f (1974), slightly modified. Slides were stained in 33258 Hoechst solution (5 /~g/ml) for 20 min, mounted in Sorensen's buffer (pH = 6.8), exposed to black fluorescent light on a 58°C hot plate for 6 - 8 min, and stained with 507o Giemsa. The incidence of SCEs was determined in 50 second-division cells from each of two replicate cultures, for a total o f 100 cells at each point. Since homologs of chromosome pairs 1, 2 and 3 can be unequivocally identified, SCEs in these 6 chromosomes were noted separately. Metaphase cells with both homologs showing an exchange at approximately the same position were photographed. The precise location of the SCE in both homologs was determined by careful measurement and homologous exchanges were noted separately.
Results When cells were irradiated with 150 rad and then incubated for two cell cycles in BrdUrd (36 h), the SCE frequency was not statistically different from the unirradiated controls (Table 1). When the irradiation followed approximately one BrdUrd labeling cycle (i.e., irradiation 48 h after culture initiation) and preceded the second cycle of BrdUrd labeling, a significant increase in SCEs was observed (p < 0.001, Table 1). Although the frequency of sister-chromatid exchanges was increased approximately 40°7o after X-irradiation when the treatment occurred between 2 rounds of BrdUrd labeling, there was no evidence for increased frequency of homologous TABLE 1 EFFECT OF BrdUrd SUBSTITUTIONON FREQUENCY OF X-RAY-INDUCEDSCE Kind of DNA irradiated
SCE per 100 cells Total
Only in chromosome pairs 1 + 2 + 3
Simultaneously in homologsc
Control; not irradiated
726
189
3
Unsubstituted a
739
199
0
1028d
278e
0
BrdUrd-substitutedb
alrradiation immediately before addition of BrdUrd to medium. blrradiation 16 h before harvest. CNumber of identical SCE in homologs of chromosomes 1, 2 or 3. dSignificantly different from control (p < 0.001). eSignificantly different from control (p < 0.01).
O0
209
¢
l
Fig. 2. Identical SCE in homologs. (A) No. 1 chromosomes; (B) No. 2 chromosomes; (C) No. 3 chromosomes.
chromosome exchange (i.e., simultaneous exchanges on both homologs) in Xirradiated cells (Table 1). Fig. 2 shows examples of simultaneously occurring SCEs in homologs, which might have arisen in exchanges between homologous chromosomes during G1; however, these were observed only in unirradiated populations and not in either of the irradiated populations (Table 1).
Discussion
If the increase in SCEs after irradiation of cells containing BrdUrd in their DNA were all due to exchange between homologs, about 44 more simultaneous SCEs would have been observed among the three large chromosomes in the 100 cells scored. This is based on the fact that 89 SCEs were induced (278-189) and on the assumption that about one-half of the homologs would have the correct polarity for the exchanges to be seen. Instead there were no simultaneous SCEs in homologs in the samples of irradiated cells. Consequently, the small increase in exchange frequency observed after X-irradiation is probably due to true SCEs. The most abundant lesion induced by X-rays is the single strand break, the majority of which are
210 r a p i d l y r e j o i n e d a n d are t h o u g h t to be repaired b e f o r e they c a n elicit a n S C E response. W h e n the p o l y ( A D P - r i b o s e ) p o l y m e r a s e i n h i b i t o r , 3 - a m i n o b e n z a m i d e , was used to delay the r e j o i n i n g o f X - r a y - i n d u c e d single-strand breaks (Zwelling et al., 1982) n o increase in SCE f r e q u e n c y was observed ( M o r g a n et al., 1983), suggesting some o t h e r lesion gives rise to X - r a y - i n d u c e d SCEs.
References Abranovsky, I., G. Vorsanger and K. Hirschorn (1978) Sister chromatid exchange induced by X-ray of human lymphocytes and the effect of L-cysteine, Mutation Res., 50, 93-100. Galloway, S.M. (1977) Ataxia telangiectasia: The effect of chemical mutagens and X-rays on sisterchromatid exchanges in blood lymphocytes, Mutation Res., 45, 343-349. German, J. (1969) Bloom's syndrome, I. Genetical and clinical observations in the first twenty-seven patients, Am. J. Hum. Genet., 21, 196-227. Littlefield, L.G., S.P. Colyer, E.E. Joiner and R.J. Dufrain (1979) Sister chromatid exchanges in human lymphocytes exposed to ionizing radiation during Go, Radiation Res., 78, 514-521. Livingston, G.K., and L.A. Dethlefsen (1979) Effects of hyperthermia and X-irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells, Radiation Res., 77, 512-520. Morgan, W.F., and P.E. Crossen (1980) X irradiation and sister chromatid exchange in cultured human lymphocytes, Environ. Mutagen., 2, 149-155. Morgan, W.F., J.L. Schwartz, J.P. Murnane and S. Wolff (1983) Effect of 3-aminobenzamide on sister chromatid exchange frequency in X-irradiated cells, Radiation Res., in press. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-157. Wolff, S., J. Bodycote and R.B. Painter (1974) Sister-chromatid exchanges induced in Chinese hamster cells by UV irradiation of different stages of the cell cycle: The necessity for cells to pass through S, Mutation Res., 25, 73-81. Zwelling, L.A., D. Kerrigan and Y. Pommier (1982) lnhibitors of poly(adenosine diphosphoribose) synthesis slow the resealing rate of X-ray-induced DNA strand breaks, Biochem. Biophys. Res. Commun., 104, 897-902.
205
Elsevier MRLett 0422
SCEs induced by ionizing radiation are not the result of exchanges between homologous chromosomes R o b e r t B. Pai nt er and William F. M o r g a n Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, San Francisco, CA 94143 (U.S.A.)
(Accepted 11 April 1983)
There is some dispute about the effects of ionizing radiation on formation of sister-chromatid exchanges (SCEs). In general it has been observed that ionizing radiation is a poor inducer of SCEs; however, the extent of stimulation of SCE by radiation varies from no increases to as high as 2-3-fold (Wolff et al., 1974; Perry and Evans, 1975; Galloway, 1977; Abranovsky et al., 1978; Littlefield et al., 1979; Livingston and Dethlefsen, 1979; Morgan and Crossen, 1980). In reviewing the protocols for these experiments, it is evident that the highest increase in SCEs after ionizing radiation is observed when bromouracil deoxyriboside (BrdUrd) is in the cell cultures for a generation or so before the irradiation (Perry and Evans, 1975; Morgan and Crossen, 1980). This increase in SCE formation has generally been attributed to a sensitization to strand breakage by the BrdUrd; however, other interpretations are possible. It is conceivable that X-ray-induced exchanges are really not SCEs at all but are complete symmetrical exchanges between homologous chromosomes. There are already indications in the literature that chromatid exchanges between homologous chromosomes occur in mammalian cells; for instance, in Bloom's syndrome cells quadriradial exchanges are observed (German, 1969). If there were an exchange between two homologous chromosomes in G~ phase and then each of the chromosomes replicates during S, both homologous chromosomes could have an apparent SCE at the same site when they arrive at mitosis (Fig. 1). Note that BrdUrd substitution before the exchange would be necessary to observe the exchange. The increase in X-ray-induced SCEs is of low enough frequency that such an exchange could go unnoticed in human and other cells with many chromosomes. To determine if there is a high frequency of simultaneous SCEs in homologs, we irradiated human lymphocytes and measured the frequency of simultaneous SCEs in homologs of chromosomes 1, 2 and 3, which are relatively easily recognized. 0165-7992/83/$ 03.00 © 1983 ElsevierScience Publishers B.V.
206
Homolog A
HornologB
Replication
;, /l Fig. 1. Scheme for reciprocal exchanges between homologs before replication. Cell had previously gone through one S phase in medium with BrdUrd so that one of the two DNA strands is substituted, as designated by wavy line. The exchange before the next replication in BrdUrd medium results in an apparent SCE in both homologs, as seen in the next mitosis (observed M).
Materials and methods P e r i p h e r a l b l o o d l y m p h o c y t e s f r o m a n o r m a l , h e a l t h y m a l e were c u l t u r e d in R P M I 1640 m e d i u m s u p p l e m e n t e d with 10070 fetal c a l f serum, penicillin (50 u n i t s / m l ) , s t r e p t o m y c i n (50 ~ g / m l ) , 2 m M L-glutamine a n d 2 % p h y t o h a e m a g glutinin. B r d U r d was a d d e d at a final c o n c e n t r a t i o n o f 2 × 10 - 5 M a n d care was t a k e n at all times to k e e p the cultures o u t o f t h e light. T w o h o u r s p r i o r to harvest, C o l c e m i d was a d d e d at a final c o n c e n t r a t i o n o f 10 - 7 M , a n d c h r o m o s o m e p r e p a r a tions m a d e as p r e v i o u s l y d e s c r i b e d ( M o r g a n a n d C r o s s e n , 1980). C u l t u r e s were i r r a d i a t e d with 150 r a d o f X - r a y s f r o m a G e n e r a l Electric M a x i t r o n 300 (300 k V p ; 20 m A , n o m i n a l H V L , 2.0 m m Cu). I r r a d i a t i o n o c c u r r e d either at 28 h a f t e r i n i t i a t i o n o f c u l t u r e (just b e f o r e a d d i t i o n o f B r d U r d - c o n t a i n i n g m e d i u m )
207 or after about one generation in BrdUrd-containing medium at 16 h before fixation. All fixations were at 64 h after initiation. Differential staining was by the method of Perry and W o l f f (1974), slightly modified. Slides were stained in 33258 Hoechst solution (5 /~g/ml) for 20 min, mounted in Sorensen's buffer (pH = 6.8), exposed to black fluorescent light on a 58°C hot plate for 6 - 8 min, and stained with 507o Giemsa. The incidence of SCEs was determined in 50 second-division cells from each of two replicate cultures, for a total o f 100 cells at each point. Since homologs of chromosome pairs 1, 2 and 3 can be unequivocally identified, SCEs in these 6 chromosomes were noted separately. Metaphase cells with both homologs showing an exchange at approximately the same position were photographed. The precise location of the SCE in both homologs was determined by careful measurement and homologous exchanges were noted separately.
Results When cells were irradiated with 150 rad and then incubated for two cell cycles in BrdUrd (36 h), the SCE frequency was not statistically different from the unirradiated controls (Table 1). When the irradiation followed approximately one BrdUrd labeling cycle (i.e., irradiation 48 h after culture initiation) and preceded the second cycle of BrdUrd labeling, a significant increase in SCEs was observed (p < 0.001, Table 1). Although the frequency of sister-chromatid exchanges was increased approximately 40°7o after X-irradiation when the treatment occurred between 2 rounds of BrdUrd labeling, there was no evidence for increased frequency of homologous TABLE 1 EFFECT OF BrdUrd SUBSTITUTIONON FREQUENCY OF X-RAY-INDUCEDSCE Kind of DNA irradiated
SCE per 100 cells Total
Only in chromosome pairs 1 + 2 + 3
Simultaneously in homologsc
Control; not irradiated
726
189
3
Unsubstituted a
739
199
0
1028d
278e
0
BrdUrd-substitutedb
alrradiation immediately before addition of BrdUrd to medium. blrradiation 16 h before harvest. CNumber of identical SCE in homologs of chromosomes 1, 2 or 3. dSignificantly different from control (p < 0.001). eSignificantly different from control (p < 0.01).
O0
209
¢
l
Fig. 2. Identical SCE in homologs. (A) No. 1 chromosomes; (B) No. 2 chromosomes; (C) No. 3 chromosomes.
chromosome exchange (i.e., simultaneous exchanges on both homologs) in Xirradiated cells (Table 1). Fig. 2 shows examples of simultaneously occurring SCEs in homologs, which might have arisen in exchanges between homologous chromosomes during G1; however, these were observed only in unirradiated populations and not in either of the irradiated populations (Table 1).
Discussion
If the increase in SCEs after irradiation of cells containing BrdUrd in their DNA were all due to exchange between homologs, about 44 more simultaneous SCEs would have been observed among the three large chromosomes in the 100 cells scored. This is based on the fact that 89 SCEs were induced (278-189) and on the assumption that about one-half of the homologs would have the correct polarity for the exchanges to be seen. Instead there were no simultaneous SCEs in homologs in the samples of irradiated cells. Consequently, the small increase in exchange frequency observed after X-irradiation is probably due to true SCEs. The most abundant lesion induced by X-rays is the single strand break, the majority of which are
210 r a p i d l y r e j o i n e d a n d are t h o u g h t to be repaired b e f o r e they c a n elicit a n S C E response. W h e n the p o l y ( A D P - r i b o s e ) p o l y m e r a s e i n h i b i t o r , 3 - a m i n o b e n z a m i d e , was used to delay the r e j o i n i n g o f X - r a y - i n d u c e d single-strand breaks (Zwelling et al., 1982) n o increase in SCE f r e q u e n c y was observed ( M o r g a n et al., 1983), suggesting some o t h e r lesion gives rise to X - r a y - i n d u c e d SCEs.
References Abranovsky, I., G. Vorsanger and K. Hirschorn (1978) Sister chromatid exchange induced by X-ray of human lymphocytes and the effect of L-cysteine, Mutation Res., 50, 93-100. Galloway, S.M. (1977) Ataxia telangiectasia: The effect of chemical mutagens and X-rays on sisterchromatid exchanges in blood lymphocytes, Mutation Res., 45, 343-349. German, J. (1969) Bloom's syndrome, I. Genetical and clinical observations in the first twenty-seven patients, Am. J. Hum. Genet., 21, 196-227. Littlefield, L.G., S.P. Colyer, E.E. Joiner and R.J. Dufrain (1979) Sister chromatid exchanges in human lymphocytes exposed to ionizing radiation during Go, Radiation Res., 78, 514-521. Livingston, G.K., and L.A. Dethlefsen (1979) Effects of hyperthermia and X-irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells, Radiation Res., 77, 512-520. Morgan, W.F., and P.E. Crossen (1980) X irradiation and sister chromatid exchange in cultured human lymphocytes, Environ. Mutagen., 2, 149-155. Morgan, W.F., J.L. Schwartz, J.P. Murnane and S. Wolff (1983) Effect of 3-aminobenzamide on sister chromatid exchange frequency in X-irradiated cells, Radiation Res., in press. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-157. Wolff, S., J. Bodycote and R.B. Painter (1974) Sister-chromatid exchanges induced in Chinese hamster cells by UV irradiation of different stages of the cell cycle: The necessity for cells to pass through S, Mutation Res., 25, 73-81. Zwelling, L.A., D. Kerrigan and Y. Pommier (1982) lnhibitors of poly(adenosine diphosphoribose) synthesis slow the resealing rate of X-ray-induced DNA strand breaks, Biochem. Biophys. Res. Commun., 104, 897-902.