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Comparison of in vivo and in vitro SCE induction
Mutation Research, 106 (1982) 85-90 Elsevier Biomedical Press
85
Comparison of in vivo and in vitro SCE induction E d w a r d L. S c h n e i d e r a n d J a n e t Lewis Section on Cellular Aging and Genetics, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224 (U.S.A.)
(Received 13 April 1981) (Revision received 3 April 1982) (Accepted 5 April 1982)
Summary In vivo and in vitro sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.
The introduction of the BrdU differential chromatid staining techniques has greatly facilitated the detection of sister-chromatid exchanges (SCE) (Kato, 1974; Latt, 1974; Perry and Wolff, 1974). These techniques can be utilized in vivo (Allen and Latt, 1976; Schneider et al., 1976; Vogel and Bauknecht, 1976) as well as in vitro for the analysis of SCE induction. There are obvious advantages of each approach for the screening of compounds. In vivo SCE analyses simulate human in vivo exposure, can detect compounds which require metabolic activation and utilize normal cell populations. In vitro SCE analyses are more simple, rapid and may be able to detect SCE induction at lower compound concentrations. In this report we have examined in vivo and in vitro SCE induction by 4 representative mutagens. In order to insure that we were not dealing with differential cellular response in vivo and in vitro, we have examined the same cell type, the P388 tumor cell in both environments.
Materials and methods
Cells and compounds P388 cells were obtained from the Mason Research Institute, Worchester, MA. 0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
86 An aliquot of these cells were tested for mycoplasma contamination by the Institute for Medical Research, Cambden, NJ and was negative. DBA mice were obtained from the Jackson Laboratories, Bar Harbor, ME. Mitomycin C (MMC) and daunomycin (DM) were obtained from Sigma Chemicals, adriamycin (ADM) from Adria Labs. and 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) from Bristol Labs. In vitro exposure of cells to mutagens P388 cells were cultured in Eagle's MEM supplemented with non-essential amino acids, glutamine and 10% fetal calf serum (Flow). 1 day after seeding into 75-mm plastic flasks (Falcon), the P388 cells were exposed for an additional 30h to the mutagen to be examined and 10 # g / m l bromodeoxyuridine (BrdU, Sigma). Colcemid (Gibco), 2 #g, was added to each flask 2 h prior to cell culture harvest. Cells were then removed from their monolayers, fixed and stained as previously described (Schneider et al., 1978). For examination of the effect of breakdown products, parallel cultures were either exposed to BCNU or MMC for 24 h or 1 h. The cells exposed for 1 h were washed with media and then cultured in media containing BrdU only. In vivo exposure of cells to mutagens To examine P388 cells in vivo, 2 x 10 6 cells were injected into the peritoneal cavities of six 8-month-old DBA mice. On the following day, the mice were placed into modified Bollman restrainers and an intravenous infusion of BrdU at 50 m g / k g w t . / h in phosphate-buffered saline (PBS) begun as previously described (Schneider et al., 1976). The compound to be tested was administered by the same intravenous route 1 h after the start of the infusion in 0.2 ml PBS. 2 h prior to the termination of the infusion 2 #g of Colcemid was injected intravenously. The animals were sacrificed 26 h after the start of the infusion and the P388 cells were collected from the peritoneal cavity. The cells were then fixed and stained as previously described for other in vivo cell populations (Schneider et al., 1978). Determinations of S C E and cell-cycle kinetics 25 well spread metaphase cells were analyzed for the frequencies of SCE in each cell culture and from each animal. The same techniques which permit the enumeration of SCE also allow the analysis of cell replication kinetics (Schneider et al., 1977; Tice et al., 1976). Cells which have replicated for 0-1, 2 or 3 and subsequent cell cycles in the presence of BrdU can be distinguished by the differential staining properties of their metaphase chromosomes. For the examination of cell-replication kinetics, 100 well spread metaphase cells were classified into 0-1, 2 or 3 + replication cycles. A replication index (RI) was formulated where RI : % zero and first replication cycle cells X 1 + % second replication cycle cells X 2 + % third and subsequent replication cycle cells X 3/100. This index is reproducible as long as there are minimal numbers of fourth replication cycle cells present. The current experiments are designed to minimize the number of cells which have replicated more than 3 times. This index is used here for comparative rather than quantitative purposes.
87 Results
and discussion
The induction of SCE by several mutagens in P388 cells in vivo and in vitro is summarized in Tables 1 and 2. In vivo, M M C is the most potent inducer of SCE followed by B C N U and ADM.. D M is clearly the least potent inducer of SCE in vivo. In vitro, A D M and D M are the most potent inducers of SCE followed closely by MMC. B C N U is the least effective inducer of SCE in vitro. Inhibition of cell-replication kinetics, indicated by a decrease in the frequency of 3 + cells and a corresponding increase in 0-1 replication cycle cells or as a decline in the replication index (RI) closely correlates with the induction of SCE by these compounds in vivo and in vitro. M M C produced the greatest inhibition of replication kinetics in vivo while A D M and D M produced the only inhibition of replication kinetics in vitro. P388 cell replication kinetics were similar in vivo and in vitro. The 3-fold increase in background SCE levels in vitro confirms our previous studies in which we have observed higher background SCE levels in diploid cell populations in vivo than in vitro ( K r a m et al., 1979). This probably reflects the reported effects of light (Ikushima and Wolff, 1974) and tissue culture nutrients (Kato and Sandberg, 1977) on SCE induction in vitro. There are m a n y possible explanations for the varying potencies of these compounds in vivo and in vitro. While concentrations of mutagen which reach the cell can be closely regulated in vitro, there are m a n y possible mechanisms which might result in differing access to cells in vivo. First, transport of the compound from the
TABLE 1 SCE INDUCTION BY 4 MUTAGENS IN VIVO IN P388 TUMOR CELLS Compound (dose, mg/kg)
SCE/chrom.
Replication cycle (%)
RIb
0-1
2
3+
0.161 --+0.013a 0.167--+0.016 0.173--0.021
5 3 15
67 80 55
28 17 30
2.23 2.14 2.15
BCNU, 1.5 BCNU, 3.0
1.118-----0.053 1.884---0.059
11 21
89 79
0 0
1.89 1.79
MMC, 1.5 MMC, 3.0
i.639-+0.079 2.355-+0.135
10 45
88 55
2 0
1.92 1.55
ADM, ADM, DM, DM,
1.300-+0.077 1.833-+0.118 0.526-+0.039 0.867-+0.048
11 19 6 30
89 81 90 64
0 0 4 6
1.89 1.81 1.98 1.76
0 0 0
3.0 4.0 2.0 4.0
a Mean_~standard error of the mean. b RI = % 0 - 1 replication cycle cells X 1 + % 2nd replication cycle ceils x 2 + % 3 +
ceils× 3/100.
repfication cycle
88 TABLE 2 SCE I N D U C T I O N BY 4 M U T A G E N S IN VITRO IN P388 T U M O R CELLS Compound (dose, n g / m l )
SCE/chrom.
Replication cycle (%) 0-1
0 0
RIb
2
3+
0.445 ± 0.044 a 0.439 ± 0.025
0 0
76 96
24 4
2.24 2.04
B C N U , 100 B C N U , 200 B C N U , 500
0.540-----0.034 0.536-+-0.040 0.627---+0.033
4 0 0
56 58 74
40 42 26
2.36 2.42 2.26
MMC, MMC, MMC,
5 10 15
0.641 ±0.053 0.745±0.035 1.038±0.057
0 2 0
94 98 100
6 0 0
2.06 1.98 2.00
ADM, ADM, ADM,
5 10 15
0.744±0.045 0.835±0.046 1.226±0.069
0 0 10
100 100 90
0 0 0
2.00 2.00 1.90
DM, DM,
5 20
0.742±0.046 1.465 --+0.057
0 16
78 84
22 0
2.22 1.84
Mean__+ standard error of the mean. b R I = % 0-1 replication cycle c e l l s X l + % 2nd replication cycle c e l l s X 2 + % 3 + cells/100. a
replication cyck
1.0
o o
E 2 6 0.5 (/)
I 100
I 1 I 200 300 400 BCNU (ng/ml)
I 500
Fig. 1. Induction of SCE by B C N U in vitro; the effect of 1 h vs. 24 h B C N U incubation. Note that SCE induction was significantly higher in P388 cells that had a l-h exposure (closed circles) than in cells left in the presence of B C N U (open circles). Each point represents the mean SCE value per 25 metaphase cells. The bars are the standard errors of the means.
89 b l o o d to the p e r i t o n e a l cavity m a y differ b e t w e e n c o m p o u n d s . Secondly, certain c o m p o u n d s m a y b e b o u n d to s e r u m c o m p o n e n t s a n d n o t b e released easily. F i n a l l y , the m u t a g e n s m a y have different rates of excretion. It is also p o s s i b l e that cellular a c t i v a t i o n of these c o m p o u n d s m a y b e different in vivo a n d in vitro. I n the case of B C N U , there is a n o t h e r i n t r i g u i n g m e c h a n i s m for the low S C E i n d u c t i o n in vitro. It has been suggested that b r e a k d o w n p r o d u c t s o f this c o m p o u n d i n h i b i t D N A r e p a i r (Erickson et al., 1977,' 1978, K a n n et al., 1974). T o e x a m i n e if B C N U b r e a k d o w n p r o d u c t s or the d r u g itself i n h i b i t e d S C E i n d u c t i o n in vitro, P388 cells were e x p o s e d to various c o n c e n t r a t i o n s of B C N U for 1 h a n d then the m o n o l a y e r was w a s h e d free of this c o m p o u n d . This p r o c e d u r e resulted in a signific a n t increase in S C E i n d u c t i o n b y B C N U (Fig. 1). Similar t r e a t m e n t of cell cultures w i t h M M C ( e x p o s u r e a n d then r e m o v a l of the m u t a g e n ) d i d n o t significantly alter S C E i n d u c t i o n in P388 cells. R e g a r d l e s s of the m e c h a n i s m s for the differing i n d u c t i o n of S C E in vivo a n d in vitro, these results d o i n d i c a t e that c a u t i o n should be a p p l i e d b e f o r e e q u a t i n g in vivo a n d in vitro results of m u t a g e n screening.
Acknowledgement W e a c k n o w l e d g e the technical s u p p o r t a n d suggestions of R o b e r t M o n t i c o n e .
References Allen, J.W., and S.A. Latt (1976) Analysis of sister chromatid exchange formation in vivo in mouse spermatogonia as a new test system for environmental mutagens, Nature (London), 260, 449-451. Erickson, L.C., M.O. Bradley and K.W. Kohn (1977) Stand breaks in DNA from normal and transformed human cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, Cancer Res., 37, 3744-3750. Erickson, L.C., M.O. Bradley and K.W. Kohn (1978) Differential inhibition of the rejoining of X-ray-induced DNA strand breaks in normal and transformed human fibroblasts treated with 1,3-bis(2-chloroethyl)-l-nitrosourea in vitro, Cancer Res., 38, 672-677. Ikushima, T., and S. Wolff (1974) Sister chromatid exchanges induced by light flashes to 5-bromodeoxyuridine and 5-iododeoxyuridine substituted Chinese hamster chromosomes, Exp. Cell Res., 87, 15-19. Kann, H.E., K.W. Kohn and J.M. Lyles (1974) Inhibition of DNA repair by the 1,3-bis(2-chloroethyl)-lnitrosourea breakdown product, 2-chlorocthyl isocyanate, Cancer Res., 34, 398-402. Kato, H. (1974) Spontaneous sister chromatid exchanges detected by a BudR-labelling method, Nature (London), 251, 70-72. Kato, H., and A.A. Sandberg (1977) The effect of sera on sister chromatid exchanges in vitro, Exp. Cell Res., 109, 445-448. Kram, D., E.L. Schneider, G.C. Scnula and Y. Nakanishi (1979) Spontaneous and mitomycin-C induced sister chromatid exchanges: comparison of in vivo and in vitro systems, Mutation Res., 60, 339-347. Latt, S.A. (1974) Sister chromatid exchanges, indices of human chromosome damage and repair: Detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158.
90 Schneider, E.L., J.R. Chaillet and R.R. Tice (1976) In vivo BUdr labeling of mammalian chromosomes, Exp. Cell Res., 100, 396-399. Schneider, E.L., H. Sternberg and R.R. Tice (1977) In vivo analysis of cellular replication, Pro¢. Natl. Acad. Sci. (U.S.A.), 74, 2041-2044. Schneider, E.L., R.R. Tice and D. Kram (1978) Bromodeoxyuridine differential chromatid staining technique: A new approach to examining sister chromatid exchange and cell replication kinetics, Meth. Cell Biol., 20, 379-409. Tice, R.R., E.L. Schneider and J.M. Rary (1976) The utifization of bromodeoxyuridine incorporation into DNA for the analysis of cellular kinetics, Exp. Cell Res., 102, 232-236. Vogel, W., and T. Banknecht (1976) Differential chromatid staining by in vivo treatment as a mutagenicity test system, Nature (London), 260, 448-449.
85
Comparison of in vivo and in vitro SCE induction E d w a r d L. S c h n e i d e r a n d J a n e t Lewis Section on Cellular Aging and Genetics, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224 (U.S.A.)
(Received 13 April 1981) (Revision received 3 April 1982) (Accepted 5 April 1982)
Summary In vivo and in vitro sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.
The introduction of the BrdU differential chromatid staining techniques has greatly facilitated the detection of sister-chromatid exchanges (SCE) (Kato, 1974; Latt, 1974; Perry and Wolff, 1974). These techniques can be utilized in vivo (Allen and Latt, 1976; Schneider et al., 1976; Vogel and Bauknecht, 1976) as well as in vitro for the analysis of SCE induction. There are obvious advantages of each approach for the screening of compounds. In vivo SCE analyses simulate human in vivo exposure, can detect compounds which require metabolic activation and utilize normal cell populations. In vitro SCE analyses are more simple, rapid and may be able to detect SCE induction at lower compound concentrations. In this report we have examined in vivo and in vitro SCE induction by 4 representative mutagens. In order to insure that we were not dealing with differential cellular response in vivo and in vitro, we have examined the same cell type, the P388 tumor cell in both environments.
Materials and methods
Cells and compounds P388 cells were obtained from the Mason Research Institute, Worchester, MA. 0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
86 An aliquot of these cells were tested for mycoplasma contamination by the Institute for Medical Research, Cambden, NJ and was negative. DBA mice were obtained from the Jackson Laboratories, Bar Harbor, ME. Mitomycin C (MMC) and daunomycin (DM) were obtained from Sigma Chemicals, adriamycin (ADM) from Adria Labs. and 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) from Bristol Labs. In vitro exposure of cells to mutagens P388 cells were cultured in Eagle's MEM supplemented with non-essential amino acids, glutamine and 10% fetal calf serum (Flow). 1 day after seeding into 75-mm plastic flasks (Falcon), the P388 cells were exposed for an additional 30h to the mutagen to be examined and 10 # g / m l bromodeoxyuridine (BrdU, Sigma). Colcemid (Gibco), 2 #g, was added to each flask 2 h prior to cell culture harvest. Cells were then removed from their monolayers, fixed and stained as previously described (Schneider et al., 1978). For examination of the effect of breakdown products, parallel cultures were either exposed to BCNU or MMC for 24 h or 1 h. The cells exposed for 1 h were washed with media and then cultured in media containing BrdU only. In vivo exposure of cells to mutagens To examine P388 cells in vivo, 2 x 10 6 cells were injected into the peritoneal cavities of six 8-month-old DBA mice. On the following day, the mice were placed into modified Bollman restrainers and an intravenous infusion of BrdU at 50 m g / k g w t . / h in phosphate-buffered saline (PBS) begun as previously described (Schneider et al., 1976). The compound to be tested was administered by the same intravenous route 1 h after the start of the infusion in 0.2 ml PBS. 2 h prior to the termination of the infusion 2 #g of Colcemid was injected intravenously. The animals were sacrificed 26 h after the start of the infusion and the P388 cells were collected from the peritoneal cavity. The cells were then fixed and stained as previously described for other in vivo cell populations (Schneider et al., 1978). Determinations of S C E and cell-cycle kinetics 25 well spread metaphase cells were analyzed for the frequencies of SCE in each cell culture and from each animal. The same techniques which permit the enumeration of SCE also allow the analysis of cell replication kinetics (Schneider et al., 1977; Tice et al., 1976). Cells which have replicated for 0-1, 2 or 3 and subsequent cell cycles in the presence of BrdU can be distinguished by the differential staining properties of their metaphase chromosomes. For the examination of cell-replication kinetics, 100 well spread metaphase cells were classified into 0-1, 2 or 3 + replication cycles. A replication index (RI) was formulated where RI : % zero and first replication cycle cells X 1 + % second replication cycle cells X 2 + % third and subsequent replication cycle cells X 3/100. This index is reproducible as long as there are minimal numbers of fourth replication cycle cells present. The current experiments are designed to minimize the number of cells which have replicated more than 3 times. This index is used here for comparative rather than quantitative purposes.
87 Results
and discussion
The induction of SCE by several mutagens in P388 cells in vivo and in vitro is summarized in Tables 1 and 2. In vivo, M M C is the most potent inducer of SCE followed by B C N U and ADM.. D M is clearly the least potent inducer of SCE in vivo. In vitro, A D M and D M are the most potent inducers of SCE followed closely by MMC. B C N U is the least effective inducer of SCE in vitro. Inhibition of cell-replication kinetics, indicated by a decrease in the frequency of 3 + cells and a corresponding increase in 0-1 replication cycle cells or as a decline in the replication index (RI) closely correlates with the induction of SCE by these compounds in vivo and in vitro. M M C produced the greatest inhibition of replication kinetics in vivo while A D M and D M produced the only inhibition of replication kinetics in vitro. P388 cell replication kinetics were similar in vivo and in vitro. The 3-fold increase in background SCE levels in vitro confirms our previous studies in which we have observed higher background SCE levels in diploid cell populations in vivo than in vitro ( K r a m et al., 1979). This probably reflects the reported effects of light (Ikushima and Wolff, 1974) and tissue culture nutrients (Kato and Sandberg, 1977) on SCE induction in vitro. There are m a n y possible explanations for the varying potencies of these compounds in vivo and in vitro. While concentrations of mutagen which reach the cell can be closely regulated in vitro, there are m a n y possible mechanisms which might result in differing access to cells in vivo. First, transport of the compound from the
TABLE 1 SCE INDUCTION BY 4 MUTAGENS IN VIVO IN P388 TUMOR CELLS Compound (dose, mg/kg)
SCE/chrom.
Replication cycle (%)
RIb
0-1
2
3+
0.161 --+0.013a 0.167--+0.016 0.173--0.021
5 3 15
67 80 55
28 17 30
2.23 2.14 2.15
BCNU, 1.5 BCNU, 3.0
1.118-----0.053 1.884---0.059
11 21
89 79
0 0
1.89 1.79
MMC, 1.5 MMC, 3.0
i.639-+0.079 2.355-+0.135
10 45
88 55
2 0
1.92 1.55
ADM, ADM, DM, DM,
1.300-+0.077 1.833-+0.118 0.526-+0.039 0.867-+0.048
11 19 6 30
89 81 90 64
0 0 4 6
1.89 1.81 1.98 1.76
0 0 0
3.0 4.0 2.0 4.0
a Mean_~standard error of the mean. b RI = % 0 - 1 replication cycle cells X 1 + % 2nd replication cycle ceils x 2 + % 3 +
ceils× 3/100.
repfication cycle
88 TABLE 2 SCE I N D U C T I O N BY 4 M U T A G E N S IN VITRO IN P388 T U M O R CELLS Compound (dose, n g / m l )
SCE/chrom.
Replication cycle (%) 0-1
0 0
RIb
2
3+
0.445 ± 0.044 a 0.439 ± 0.025
0 0
76 96
24 4
2.24 2.04
B C N U , 100 B C N U , 200 B C N U , 500
0.540-----0.034 0.536-+-0.040 0.627---+0.033
4 0 0
56 58 74
40 42 26
2.36 2.42 2.26
MMC, MMC, MMC,
5 10 15
0.641 ±0.053 0.745±0.035 1.038±0.057
0 2 0
94 98 100
6 0 0
2.06 1.98 2.00
ADM, ADM, ADM,
5 10 15
0.744±0.045 0.835±0.046 1.226±0.069
0 0 10
100 100 90
0 0 0
2.00 2.00 1.90
DM, DM,
5 20
0.742±0.046 1.465 --+0.057
0 16
78 84
22 0
2.22 1.84
Mean__+ standard error of the mean. b R I = % 0-1 replication cycle c e l l s X l + % 2nd replication cycle c e l l s X 2 + % 3 + cells/100. a
replication cyck
1.0
o o
E 2 6 0.5 (/)
I 100
I 1 I 200 300 400 BCNU (ng/ml)
I 500
Fig. 1. Induction of SCE by B C N U in vitro; the effect of 1 h vs. 24 h B C N U incubation. Note that SCE induction was significantly higher in P388 cells that had a l-h exposure (closed circles) than in cells left in the presence of B C N U (open circles). Each point represents the mean SCE value per 25 metaphase cells. The bars are the standard errors of the means.
89 b l o o d to the p e r i t o n e a l cavity m a y differ b e t w e e n c o m p o u n d s . Secondly, certain c o m p o u n d s m a y b e b o u n d to s e r u m c o m p o n e n t s a n d n o t b e released easily. F i n a l l y , the m u t a g e n s m a y have different rates of excretion. It is also p o s s i b l e that cellular a c t i v a t i o n of these c o m p o u n d s m a y b e different in vivo a n d in vitro. I n the case of B C N U , there is a n o t h e r i n t r i g u i n g m e c h a n i s m for the low S C E i n d u c t i o n in vitro. It has been suggested that b r e a k d o w n p r o d u c t s o f this c o m p o u n d i n h i b i t D N A r e p a i r (Erickson et al., 1977,' 1978, K a n n et al., 1974). T o e x a m i n e if B C N U b r e a k d o w n p r o d u c t s or the d r u g itself i n h i b i t e d S C E i n d u c t i o n in vitro, P388 cells were e x p o s e d to various c o n c e n t r a t i o n s of B C N U for 1 h a n d then the m o n o l a y e r was w a s h e d free of this c o m p o u n d . This p r o c e d u r e resulted in a signific a n t increase in S C E i n d u c t i o n b y B C N U (Fig. 1). Similar t r e a t m e n t of cell cultures w i t h M M C ( e x p o s u r e a n d then r e m o v a l of the m u t a g e n ) d i d n o t significantly alter S C E i n d u c t i o n in P388 cells. R e g a r d l e s s of the m e c h a n i s m s for the differing i n d u c t i o n of S C E in vivo a n d in vitro, these results d o i n d i c a t e that c a u t i o n should be a p p l i e d b e f o r e e q u a t i n g in vivo a n d in vitro results of m u t a g e n screening.
Acknowledgement W e a c k n o w l e d g e the technical s u p p o r t a n d suggestions of R o b e r t M o n t i c o n e .
References Allen, J.W., and S.A. Latt (1976) Analysis of sister chromatid exchange formation in vivo in mouse spermatogonia as a new test system for environmental mutagens, Nature (London), 260, 449-451. Erickson, L.C., M.O. Bradley and K.W. Kohn (1977) Stand breaks in DNA from normal and transformed human cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, Cancer Res., 37, 3744-3750. Erickson, L.C., M.O. Bradley and K.W. Kohn (1978) Differential inhibition of the rejoining of X-ray-induced DNA strand breaks in normal and transformed human fibroblasts treated with 1,3-bis(2-chloroethyl)-l-nitrosourea in vitro, Cancer Res., 38, 672-677. Ikushima, T., and S. Wolff (1974) Sister chromatid exchanges induced by light flashes to 5-bromodeoxyuridine and 5-iododeoxyuridine substituted Chinese hamster chromosomes, Exp. Cell Res., 87, 15-19. Kann, H.E., K.W. Kohn and J.M. Lyles (1974) Inhibition of DNA repair by the 1,3-bis(2-chloroethyl)-lnitrosourea breakdown product, 2-chlorocthyl isocyanate, Cancer Res., 34, 398-402. Kato, H. (1974) Spontaneous sister chromatid exchanges detected by a BudR-labelling method, Nature (London), 251, 70-72. Kato, H., and A.A. Sandberg (1977) The effect of sera on sister chromatid exchanges in vitro, Exp. Cell Res., 109, 445-448. Kram, D., E.L. Schneider, G.C. Scnula and Y. Nakanishi (1979) Spontaneous and mitomycin-C induced sister chromatid exchanges: comparison of in vivo and in vitro systems, Mutation Res., 60, 339-347. Latt, S.A. (1974) Sister chromatid exchanges, indices of human chromosome damage and repair: Detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158.
90 Schneider, E.L., J.R. Chaillet and R.R. Tice (1976) In vivo BUdr labeling of mammalian chromosomes, Exp. Cell Res., 100, 396-399. Schneider, E.L., H. Sternberg and R.R. Tice (1977) In vivo analysis of cellular replication, Pro¢. Natl. Acad. Sci. (U.S.A.), 74, 2041-2044. Schneider, E.L., R.R. Tice and D. Kram (1978) Bromodeoxyuridine differential chromatid staining technique: A new approach to examining sister chromatid exchange and cell replication kinetics, Meth. Cell Biol., 20, 379-409. Tice, R.R., E.L. Schneider and J.M. Rary (1976) The utifization of bromodeoxyuridine incorporation into DNA for the analysis of cellular kinetics, Exp. Cell Res., 102, 232-236. Vogel, W., and T. Banknecht (1976) Differential chromatid staining by in vivo treatment as a mutagenicity test system, Nature (London), 260, 448-449.