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Cell kinetics and sister-chromatid-exchange frequency in human lymphocytes
Mutation Research, 120 (1983) 193-199 Elsevier Biomedical Press
193
Cell kinetics and sister-chromatid-exchange frequency in human lymphocytes L. Lamberti, P. Bigatti Ponzetto and G. Ardito Institute o f Anthropology, Via Accademia Albertina 17, 10123 Turin (Italy) (Accepted 1 February 1983)
Summary Repeated cultures from 5 healthy donors were set up in 3 tissue-culture media: TC109, TC199 and TC1640. For each donor, 3 blood samples were taken at 1-week intervals, making a total of 5 x 3 × 3 = 45 cultures. Our results show a large variability in the individual SCE base-line frequency. This variability can be partly attributed to the different tissue-culture media or to the donors themselves. Our results, however, stress the importance of the proliferating rate of the culture in determining the SCE frequency.
Observation of the frequency of sister-chromatid exchange (SCE) in short-term cultured peripheral lymphocytes is considered a useful technique for the assay of effects of chemical mutagens and/or carcinogens on eukaryotic chromosomes. Its utility, however, is potentially limited by the large variability attributable to many factors under cell-culture conditions as well as to the individual base-line which can also differ in repeated cultures from the same individual at similar concentrations of BrdU. As regards culture conditions, the variability of SCE frequency has been reported according to the type of serum or tissue-culture medium used (Bianchi et al., 1979; Ghosh and Nand, 1979; Kato and Sandberg, 1977; Morgan and Crossen, 1981), to the amount of BrdU available for lymphocytes (Carrano et al., 1980; Lambert et al., 1976; Latt and Juergens, 1977) and to many other factors such as duration of the cell cycle (Snope and Rary, 1979; Abdel-Fadil et al., 1982) or the harvesting time (Beek and Obe, 1979; Giulotto et al., 1980). However, as to the variability in the individual SCE base-line, apart from the variation due to the age of the subject (de Arce, 1981; Ardito et al., 1980; Galloway and Evans, 1975; Lambert, 1980), it has been hypothesized that the different B and 0165-7992/83/0000-0000/$ 03.00 © 1983 Elsevier Science Publishers
194 T l y m p h o c y t e s u b - p o p u l a t i o n s c o u l d d i f f e r in b o t h t h e i r p r o l i f e r a t i n g r a t e a n d S C E f r e q u e n c y ( R i e d e l a n d O b e , 1980; S a n t e s s o n et a l . , 1979). S h o u l d this b e t r u e , t h e d i f f e r e n t S C E f r e q u e n c i e s s o m e t i m e s o b s e r v e d in c u l t u r e s h a r v e s t e d at s u b s e q u e n t i n t e r v a l s m i g h t b e s i m p l y r e l a t e d t o d i f f e r e n c e s in t h e a m o u n t
of B and T lym-
p h o c y t e s o c c u r r i n g f r o m t i m e t o t i m e ( A r d i t o et a l . , 1981). L i n d b l a d a n d L a m b e r t (1981) f o u n d
t h a t t h e cell p r o l i f e r a t i n g r a t e is a l s o p o s s i b l y i n v o l v e d in S C E
frequency. To contribute to the analysis of possible factors responsible for the SCE base-line f r e q u e n c y w e h a r v e s t e d r e p e a t e d l y m p h o c y t e c u l t u r e s f r o m t h e s a m e d o n o r s in various culture media.
Materials and methods 15 s a m p l e s o f p e r i p h e r a l h e p a r i n i z e d b l o o d f r o m 5 h e a l t h y d o n o r s o f b o t h sexes (3 o ' a n d 2 9 ) w e r e set u p f o r l y m p h o c y t e c u l t u r e s a c c o r d i n g t o t r a d i t i o n a l t e c h n i q u e s (6 m l t i s s u e - c u l t u r e m e d i u m , 2 m l f o e t a l c a l f s e r u m , 0.2 m l P H A ,
0.3 m l
whole blood, antibiotics).
TABLE 1 MEAN FREQUENCIES OF SCE IN DIFFERENT TISSUE-CULTURE MEDIA Donor A
D
TC NCTCI09
TC199
TC RPMII640
X ±
X ±
X ±
t7
~
o
1st week 2nd week 3rd week
6.00 ± 1.71 5.20 ± 2.42 5.45 +_ 2.28
8.17 ± 3.09 4.23 ± 1.81 4.77 ± 2.49
4.90 + 1.84 4.63 ± 2.82 4.37 ± 2.24
1st week 2nd week 3rd week
7.12 ± 2.86 5.57 ± 2.42 -
7.37 _+ 3.16 7.43 ± 2.55 7.20 + 2.51
9.37 + 2.76 6.67 ± 2.73 6.60 ± 2.55
1st week 2nd week 3rd week
6.36 :l: 2.28 4.87 ± 2.64 6.63 + 2.33
6.97 _+ 2.91 4.73 ± 2.24 4.73 ± 2.24
6.50 :l: 2.56 5.57 ± 2.28 6.57 ± 2.88
1st week 2nd week 3rd week
6.50 + 2.54 9.07 + 3.68 6.17 _+ 2.75
8.80 + 3.23 8.63 + 2.93 8.17 ± 3.50
8.30 ± 3.66 6.40 + 2.51
1st week 2nd week 3rd week
4.63 ± 1.73 4.63 + 1.90 4.00 ± 1.56
6.50 ± 3.33 6.70 + 3.27 5.83 ± 2.61
4.30 ± 1.71 4.33 ± 1.69 3.93 + 1.72
From each donor (A to E), 3 blood samples were taken at 1-week intervals (lst, 2nd and 3rd weeks). The bars indicate culture failures.
195 C u l t u r e s were set up in 3 d i f f e r e n t tissue-culture m e d i a , n a m e l y D i f c o T C N C T C 109, D i f c o T C 1 9 9 a n d D i f c o T C R P M I 1 6 4 0 . F r o m each d o n o r , 3 b l o o d sam p l es were t a k e n at 1-week intervals, u p to a t o t a l n u m b e r o f 45 cultures. A f t e r 24 h, B r d U was a d d e d to the cultures at a final c o n c e n t r a t i o n o f 5 / ~ g / m l . C u l t u r e s were kept in the d ar k . C o l c e m i d at a final c o n c e n t r a t i o n o f 0.25 # g / m l was a d d e d 2 h b e f o r e the h ar v es t i n g . Slides were stained a c c o r d i n g to t h e p r o c e d u r e o f B r o g g e r et al. (1979). Slides were c o d e d a n d s c o r e d b l in d by t w o researchers. 30 s e c o n d - d i v i s i o n cells f r o m each c u l t u r e were scored f o r S C E , a n d t h e n u m b e r o f cells in the first (M1), s e c o n d (ME) a n d t h i r d (M3) division were c o u n t e d in 100 m e t a p h a s e s .
Results and d i s c u s s i o n T h e S C E f r e q u e n c i e s in the cultures are r e p o r t e d in T a b l e 1. B o t h the rows (i.e. cultures h a r v e s t e d f r o m th e same d o n o r in d i f f e r e n t tissue-culture m e d i a ) an d the c o l u m n s (i.e. cultures h a r v e s t e d in the s a m e tissue-culture m e d i u m b u t f r o m dif-
TABLE 2 NUMER OF 1st, 2nd AND 3rd OR FURTHER (COMBINED CLASS) MITOSES IN REPEATED CULTURES Donor
TC NCTC109
TC199
TC RPMII640
Ml
M2 M3
PRI
Ml
M2 M3 PRI
MI
M2 M3 PRI
A 1st week 2nd week 3rd week
8 25 23
21 39 28
71 36 49
(2.63) (2.11) (2.26)
19 7 10
52 17 19
29 76 71
(2.10) (2.69) (2.61)
l0 8 7
19 23 29
71 69 64
(2.61) (2.61) (2.57)
1st week 2nd week 3rd week
24 17
36 47
40 36
(2.16) (2.19)
19 13 14
34 38 30
47 49 56
(2.28) (2.36) (2.42)
35 14 18
40 32 39
25 54 43
(1.90) (2.40) (2.25)
C 1st week 2nd week 3rd week
14 14 22
38 40 39
48 46 39
(2.34) (2.32) (2.17)
22 13 9
56 42 38
22 45 53
(2.00) (2.32) (2.44)
14 12 19
53 32 38
33 56 43
(2.19) (2.44) (2.24)
D 1st week 2nd week 3rd week
17 29 20
29 39 32
54 32 48
(2.37) (2.03) (2.28)
16 51 28
43 43 48
41 6 24
(2.25) (1.55) (1.96)
56 16
26 32
18 52
(1.62) (2.36)
1st week 2nd week 3rd week
12 9 21
21 25 31
67 66 48
(2.55) (2.57) (2.27)
29 27 19
45 57 42
26 16 39
(1.97) (1.89) (2.20)
9 22 9
27 35 22
64 43 69
(2.55) (2.21) (2.60)
B
E
The numbers in parentheses are the Proliferating Rate Indices of the cultures. (For PRI definition see text.) The bars indicate culture failures.
196
ferent .samples) show a large variability in SCE frequency. This variability was evaluated according to the bayesian viewpoint, building the 'a posteriori' density function for the mean-square error between the SCE mean values relative to the tissue-culture media and to the donors, assuming that the 'a priori' density function is 'non-informative' (i.e. a 'local uniform density function'). In this way it is possible to evaluate the main effects o f these two factors on total variance. This procedure is, except for slight modifications, the same as described in Box and Tiao (1973, Chapter 6, pp. 329-340) for the Cross Classification Random Model. The interaction between these two factors was considered irrelevant. In Fig. 1 the density function concerning the effects of culture medium and donor are reported. This figure shows that the second factor contributed more to the total variability than the first factor. Moreover, Table 1 indicates that some individuals showed values that differed significantly not only in the different culture media but also in different cultures harvested in the same medium. Subject A, for example, showed an SCE frequency o f 8.17 + 3.09 in TC199 in the first week, but a value of 4.23 _+ 1.81 during the second week ( P < 0.01, t test). Similarly, subject B in TC1640 showed an SCE value o f 9.37 +_ 2.76 during the first week and 6.67 _+ 2.73 during the second week ( p < 0 . 0 1 , t test). In other cases, the SCE frequency was almost constant (i.e. individual E in TC1640 or individual B in TC199). There is controversy on this topic in the literature. Some authors report a good constancy of SCE frequency in repeated cultures from the same subject harvested in different months (Morgan and Crossen, 1977; Cohen et al., 1982) whereas other authors do not agree with this conclusion (Latt and Juergens, 1977). The better to clarify the variability o f SCE frequency, since Lindblad and f(,~
0.~
O.E
0.~
0.2
0
1
2
3
4
Fig. 1. Density f u n c t i o n of the effect of culture m e d i u m (
5
6) a n d d o n o r ( . . . . ).
197
Lambert (1981) have hypothesized that the proliferating rate o f the cultures might be involved in SCE frequency, we analysed this variable in our cultures. The data are reported in Table 2. Accordingly, we correlated the SCE frequency with the number of 2nd (M2) and 3rd (M3) divisions. We found a positive correlation (r = 0.50, p < 0 . 0 1 ) between M2 and SCE frequency and a negative correlation (r = - 0 . 6 9 , p < 0.01) between M3 and SCE frequency. These results fully confirm those o f Lindblad and Lambert (1981). However, the better to define the cellular kinetics, a parameter that considers at the same time the number of M1, M2 and M3 must be envisaged. For this purpose we considered different indices, and chose the following Proliferating Rate Index (PRI) which, in our opinion, is the most suitable: PRI
=
1M1 + 2 M 2 + 3 M 3 100
We then calculated the correlation between this index and the SCE frequency (Fig. 2). The results (r = - 0 . 7 0 , p < 0 . 0 1 ) seem to confirm that this correlation exists: the higher the PRI value, the lower the SCE frequency. Since a 2-way analysis of variance of the data in Table 1 shows that the SCE frequency differed significantly in the 3 tissue-culture media (0.05 > p > 0.01) and in the 5 donors (p<0.01), we wondered whether these differences could be due to differences in the lymphocyte-proliferating rates. Indeed, the X2 test shows that there was a significant difference in the cell proliferation both between the tissue-culture media (p < 0.005) and between the donors (17< 0.005).
SC~cell
95" 90856.0757065-
.~..
60 55 50
•
45 40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2,60
"PR
I"
Fig. 2. Correlation between Proliferating Rate Index (PRI) and SCE frequency. Each point represents the average number of SCE in 30 cells analysed•
198 These results indicate that the rate o f cell p r o l i f e r a t i o n in the culture is a n i m p o r t a n t d e t e r m i n a n t o f SCE frequency. This o b s e r v a t i o n is in agreement with the results o f L i n d b l a d a n d L a m b e r t (1981). O u r m a i n c o n c l u s i o n is that, even t h o u g h o t h e r possible d e t e r m i n a n t s such as the d i f f e r e n t l y m p h o c y t e s u b - p o p u l a t i o n s s h o u l d n o t be u n d e r e s t i m a t e d , w h e n we are faced with the possible effects o f m u t a g e n s a n d / o r carcinogens, the l y m p h o c y t e - p r o l i f e r a t i n g rate o f the culture c a n n o longer be disregarded.
Acknowledgements W e are grateful to M. Di Bacco, Professor o f M e t h o d o l o g i c a l Statistics, B o l o g n a University, a n d to Professor G. Allasia, Ist. Calcoli N u m e r i c i , T u r i n University. This work was s u p p o r t e d b y the I t a l i a n M i n i s t r y o f P u b l i c H e a l t h (60°7o grants).
References Abdel-Fadil, M.R., C.G. Palmer and N. Heerema (1982) Effect of temperature variation on sisterchromatid exchanges and cell-cycle duration in cultured human lymphocytes, Mutation Res., 104, 267-273. Arce de, M.A. (1981) The effect of donor sex and age on the number of sister-chromatid exchanges in human lymphocytes growing in vitro, Hum. Genet., 57, 83-85. Ardito, G., L. Lamberti, E. Ansaldi and P. Ponzetto (1980) Sister-chromatid exchanges in cigarettesmoking human females and their newborns, Mutation Res., 78, 209-212. Ardito, G., L. Lamberti, R. Stanyon and G. Benedetti (1981) Cellular kinetics in lymphocytes of 4 species of macaques measured by BrdU sister-chromatid differential staining, Mutation Res., 91, 141-146. Beek, B., and G. Obe (1979) Sister chromatid exchanges in human leukocyte chromosomes: spontaneous and induced frequencies in early- and late-proliferating cells in vitro, Hum. Genet., 49, 51-61. Bianchi, N.O., S.M. Bianchi and M. Larramendy (1979) Kinetics of human lymphocyte division and chromosomal radiosensitivity, Mutation Res., 63, 317-324. Box, G.E.P., and G.C. Tiao (1973) Bayesian Inference in Statistical Analysis, Addison-Wesley Publ. Comp. Bragger, A., G. Ardito and H. Wakswik (1979) No synergism between caffeine and saccharin in the induction of sister chromatid exchanges in human lymphocytes, Hereditas, 91, 135-138. Carrano, A.V., J.L. Minkler, D.G. Stetka and D.H. Moore II (1980) Variation in the baseline sister chromatid exchange frequency in human lymphocytes, Environ. Mutagen., 2, 325-337. Cohen, M.M., A.O. Martin, C. Ober and S.J. Simpson (1982) A family study of spontaneous sister chromatid exchange frequency, Am. J. Hum. Genet., 34, 294-306. Galloway, S.M., and H.J. Evans (1975) Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia, Cytogenet. Cell Genet., 15, 17-29. Gosh, P.K., and R. Nand (1979) Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum, Hum. Genet., 51, 167-170. Guilotto, E., A. Mottura, R. Giorgi, L. de Carli and F. Nuzzo (1980) Frequencies of sister-chromatid exchanges in relation to cell kinetics in lymphocyte cultures, Mutation Res., 70, 343-350.
199 Kato, H., and A.A. Sandberg (1977) The effect of sera on sister chromatid exchanges in vitro, Exp. Cell Res., 109, 445-448. Lambert, B. (1980) Sister chromatid exchange in human lymphocytes as an indicator of DNA damage and repair in vivo, in: A. Castellani (Ed.), Lymphocyte Stimulation, Plenum, New York, pp. 119-129. Lambert, B., K. Hansson, J. Lindsten, M. Sten and B. Warelius (1976) Bromodeoxyuridine-induced sister chromatid exchanges in human lymphocytes, Hereditas, 83, 163-174. Latt, S.A., and L.A. Juergens (1977) Determinants of sister chromatid exchange frequencies in human chromosomes, in: E.B. Hook and I.H. Porter (Eds.), Population Cytogenetics, Academic Press, New York, pp. 217-236. Lindblad, A., and B. Lambert (1981) Relation between sister chromatid exchange, cell proliferation and proportion of B and T cells in human lymphocyte cultures, Hum. Genet., 57, 31-34. Morgan, W.F., and P.E. Crossen (1977) The incidence of sister chromatid exchanges in cultured human lymphocytes, Mutation Res., 42, 305-312. Morgan, W.F., and P.E. Crossen (1981) Factors influencing sister-chromatid exchange rate in cultured human lymphocytes, Mutation Res., 81, 395-402. Riedel, L., and G. Obe (1980) Trenimon-induced SCEs and structural chromosomal aberrations in earlyand late-dividing lymphocytes, Mutation Res., 73, 125-131. Santesson, B., K. Lindahl-Kiessling and A. Mattsson (1979) SCE in B and T lymphocytes. Possible implications for Bloom's syndrome, Clin. Genet., 16, 133-135. Snope, A.J., and J.M. Rary (1979) Cell-cycle duration and sister chromatid exchange frequency in cultured human lymphocytes, Mutation Res., 63, 345-349.
193
Cell kinetics and sister-chromatid-exchange frequency in human lymphocytes L. Lamberti, P. Bigatti Ponzetto and G. Ardito Institute o f Anthropology, Via Accademia Albertina 17, 10123 Turin (Italy) (Accepted 1 February 1983)
Summary Repeated cultures from 5 healthy donors were set up in 3 tissue-culture media: TC109, TC199 and TC1640. For each donor, 3 blood samples were taken at 1-week intervals, making a total of 5 x 3 × 3 = 45 cultures. Our results show a large variability in the individual SCE base-line frequency. This variability can be partly attributed to the different tissue-culture media or to the donors themselves. Our results, however, stress the importance of the proliferating rate of the culture in determining the SCE frequency.
Observation of the frequency of sister-chromatid exchange (SCE) in short-term cultured peripheral lymphocytes is considered a useful technique for the assay of effects of chemical mutagens and/or carcinogens on eukaryotic chromosomes. Its utility, however, is potentially limited by the large variability attributable to many factors under cell-culture conditions as well as to the individual base-line which can also differ in repeated cultures from the same individual at similar concentrations of BrdU. As regards culture conditions, the variability of SCE frequency has been reported according to the type of serum or tissue-culture medium used (Bianchi et al., 1979; Ghosh and Nand, 1979; Kato and Sandberg, 1977; Morgan and Crossen, 1981), to the amount of BrdU available for lymphocytes (Carrano et al., 1980; Lambert et al., 1976; Latt and Juergens, 1977) and to many other factors such as duration of the cell cycle (Snope and Rary, 1979; Abdel-Fadil et al., 1982) or the harvesting time (Beek and Obe, 1979; Giulotto et al., 1980). However, as to the variability in the individual SCE base-line, apart from the variation due to the age of the subject (de Arce, 1981; Ardito et al., 1980; Galloway and Evans, 1975; Lambert, 1980), it has been hypothesized that the different B and 0165-7992/83/0000-0000/$ 03.00 © 1983 Elsevier Science Publishers
194 T l y m p h o c y t e s u b - p o p u l a t i o n s c o u l d d i f f e r in b o t h t h e i r p r o l i f e r a t i n g r a t e a n d S C E f r e q u e n c y ( R i e d e l a n d O b e , 1980; S a n t e s s o n et a l . , 1979). S h o u l d this b e t r u e , t h e d i f f e r e n t S C E f r e q u e n c i e s s o m e t i m e s o b s e r v e d in c u l t u r e s h a r v e s t e d at s u b s e q u e n t i n t e r v a l s m i g h t b e s i m p l y r e l a t e d t o d i f f e r e n c e s in t h e a m o u n t
of B and T lym-
p h o c y t e s o c c u r r i n g f r o m t i m e t o t i m e ( A r d i t o et a l . , 1981). L i n d b l a d a n d L a m b e r t (1981) f o u n d
t h a t t h e cell p r o l i f e r a t i n g r a t e is a l s o p o s s i b l y i n v o l v e d in S C E
frequency. To contribute to the analysis of possible factors responsible for the SCE base-line f r e q u e n c y w e h a r v e s t e d r e p e a t e d l y m p h o c y t e c u l t u r e s f r o m t h e s a m e d o n o r s in various culture media.
Materials and methods 15 s a m p l e s o f p e r i p h e r a l h e p a r i n i z e d b l o o d f r o m 5 h e a l t h y d o n o r s o f b o t h sexes (3 o ' a n d 2 9 ) w e r e set u p f o r l y m p h o c y t e c u l t u r e s a c c o r d i n g t o t r a d i t i o n a l t e c h n i q u e s (6 m l t i s s u e - c u l t u r e m e d i u m , 2 m l f o e t a l c a l f s e r u m , 0.2 m l P H A ,
0.3 m l
whole blood, antibiotics).
TABLE 1 MEAN FREQUENCIES OF SCE IN DIFFERENT TISSUE-CULTURE MEDIA Donor A
D
TC NCTCI09
TC199
TC RPMII640
X ±
X ±
X ±
t7
~
o
1st week 2nd week 3rd week
6.00 ± 1.71 5.20 ± 2.42 5.45 +_ 2.28
8.17 ± 3.09 4.23 ± 1.81 4.77 ± 2.49
4.90 + 1.84 4.63 ± 2.82 4.37 ± 2.24
1st week 2nd week 3rd week
7.12 ± 2.86 5.57 ± 2.42 -
7.37 _+ 3.16 7.43 ± 2.55 7.20 + 2.51
9.37 + 2.76 6.67 ± 2.73 6.60 ± 2.55
1st week 2nd week 3rd week
6.36 :l: 2.28 4.87 ± 2.64 6.63 + 2.33
6.97 _+ 2.91 4.73 ± 2.24 4.73 ± 2.24
6.50 :l: 2.56 5.57 ± 2.28 6.57 ± 2.88
1st week 2nd week 3rd week
6.50 + 2.54 9.07 + 3.68 6.17 _+ 2.75
8.80 + 3.23 8.63 + 2.93 8.17 ± 3.50
8.30 ± 3.66 6.40 + 2.51
1st week 2nd week 3rd week
4.63 ± 1.73 4.63 + 1.90 4.00 ± 1.56
6.50 ± 3.33 6.70 + 3.27 5.83 ± 2.61
4.30 ± 1.71 4.33 ± 1.69 3.93 + 1.72
From each donor (A to E), 3 blood samples were taken at 1-week intervals (lst, 2nd and 3rd weeks). The bars indicate culture failures.
195 C u l t u r e s were set up in 3 d i f f e r e n t tissue-culture m e d i a , n a m e l y D i f c o T C N C T C 109, D i f c o T C 1 9 9 a n d D i f c o T C R P M I 1 6 4 0 . F r o m each d o n o r , 3 b l o o d sam p l es were t a k e n at 1-week intervals, u p to a t o t a l n u m b e r o f 45 cultures. A f t e r 24 h, B r d U was a d d e d to the cultures at a final c o n c e n t r a t i o n o f 5 / ~ g / m l . C u l t u r e s were kept in the d ar k . C o l c e m i d at a final c o n c e n t r a t i o n o f 0.25 # g / m l was a d d e d 2 h b e f o r e the h ar v es t i n g . Slides were stained a c c o r d i n g to t h e p r o c e d u r e o f B r o g g e r et al. (1979). Slides were c o d e d a n d s c o r e d b l in d by t w o researchers. 30 s e c o n d - d i v i s i o n cells f r o m each c u l t u r e were scored f o r S C E , a n d t h e n u m b e r o f cells in the first (M1), s e c o n d (ME) a n d t h i r d (M3) division were c o u n t e d in 100 m e t a p h a s e s .
Results and d i s c u s s i o n T h e S C E f r e q u e n c i e s in the cultures are r e p o r t e d in T a b l e 1. B o t h the rows (i.e. cultures h a r v e s t e d f r o m th e same d o n o r in d i f f e r e n t tissue-culture m e d i a ) an d the c o l u m n s (i.e. cultures h a r v e s t e d in the s a m e tissue-culture m e d i u m b u t f r o m dif-
TABLE 2 NUMER OF 1st, 2nd AND 3rd OR FURTHER (COMBINED CLASS) MITOSES IN REPEATED CULTURES Donor
TC NCTC109
TC199
TC RPMII640
Ml
M2 M3
PRI
Ml
M2 M3 PRI
MI
M2 M3 PRI
A 1st week 2nd week 3rd week
8 25 23
21 39 28
71 36 49
(2.63) (2.11) (2.26)
19 7 10
52 17 19
29 76 71
(2.10) (2.69) (2.61)
l0 8 7
19 23 29
71 69 64
(2.61) (2.61) (2.57)
1st week 2nd week 3rd week
24 17
36 47
40 36
(2.16) (2.19)
19 13 14
34 38 30
47 49 56
(2.28) (2.36) (2.42)
35 14 18
40 32 39
25 54 43
(1.90) (2.40) (2.25)
C 1st week 2nd week 3rd week
14 14 22
38 40 39
48 46 39
(2.34) (2.32) (2.17)
22 13 9
56 42 38
22 45 53
(2.00) (2.32) (2.44)
14 12 19
53 32 38
33 56 43
(2.19) (2.44) (2.24)
D 1st week 2nd week 3rd week
17 29 20
29 39 32
54 32 48
(2.37) (2.03) (2.28)
16 51 28
43 43 48
41 6 24
(2.25) (1.55) (1.96)
56 16
26 32
18 52
(1.62) (2.36)
1st week 2nd week 3rd week
12 9 21
21 25 31
67 66 48
(2.55) (2.57) (2.27)
29 27 19
45 57 42
26 16 39
(1.97) (1.89) (2.20)
9 22 9
27 35 22
64 43 69
(2.55) (2.21) (2.60)
B
E
The numbers in parentheses are the Proliferating Rate Indices of the cultures. (For PRI definition see text.) The bars indicate culture failures.
196
ferent .samples) show a large variability in SCE frequency. This variability was evaluated according to the bayesian viewpoint, building the 'a posteriori' density function for the mean-square error between the SCE mean values relative to the tissue-culture media and to the donors, assuming that the 'a priori' density function is 'non-informative' (i.e. a 'local uniform density function'). In this way it is possible to evaluate the main effects o f these two factors on total variance. This procedure is, except for slight modifications, the same as described in Box and Tiao (1973, Chapter 6, pp. 329-340) for the Cross Classification Random Model. The interaction between these two factors was considered irrelevant. In Fig. 1 the density function concerning the effects of culture medium and donor are reported. This figure shows that the second factor contributed more to the total variability than the first factor. Moreover, Table 1 indicates that some individuals showed values that differed significantly not only in the different culture media but also in different cultures harvested in the same medium. Subject A, for example, showed an SCE frequency o f 8.17 + 3.09 in TC199 in the first week, but a value of 4.23 _+ 1.81 during the second week ( P < 0.01, t test). Similarly, subject B in TC1640 showed an SCE value o f 9.37 +_ 2.76 during the first week and 6.67 _+ 2.73 during the second week ( p < 0 . 0 1 , t test). In other cases, the SCE frequency was almost constant (i.e. individual E in TC1640 or individual B in TC199). There is controversy on this topic in the literature. Some authors report a good constancy of SCE frequency in repeated cultures from the same subject harvested in different months (Morgan and Crossen, 1977; Cohen et al., 1982) whereas other authors do not agree with this conclusion (Latt and Juergens, 1977). The better to clarify the variability o f SCE frequency, since Lindblad and f(,~
0.~
O.E
0.~
0.2
0
1
2
3
4
Fig. 1. Density f u n c t i o n of the effect of culture m e d i u m (
5
6) a n d d o n o r ( . . . . ).
197
Lambert (1981) have hypothesized that the proliferating rate o f the cultures might be involved in SCE frequency, we analysed this variable in our cultures. The data are reported in Table 2. Accordingly, we correlated the SCE frequency with the number of 2nd (M2) and 3rd (M3) divisions. We found a positive correlation (r = 0.50, p < 0 . 0 1 ) between M2 and SCE frequency and a negative correlation (r = - 0 . 6 9 , p < 0.01) between M3 and SCE frequency. These results fully confirm those o f Lindblad and Lambert (1981). However, the better to define the cellular kinetics, a parameter that considers at the same time the number of M1, M2 and M3 must be envisaged. For this purpose we considered different indices, and chose the following Proliferating Rate Index (PRI) which, in our opinion, is the most suitable: PRI
=
1M1 + 2 M 2 + 3 M 3 100
We then calculated the correlation between this index and the SCE frequency (Fig. 2). The results (r = - 0 . 7 0 , p < 0 . 0 1 ) seem to confirm that this correlation exists: the higher the PRI value, the lower the SCE frequency. Since a 2-way analysis of variance of the data in Table 1 shows that the SCE frequency differed significantly in the 3 tissue-culture media (0.05 > p > 0.01) and in the 5 donors (p<0.01), we wondered whether these differences could be due to differences in the lymphocyte-proliferating rates. Indeed, the X2 test shows that there was a significant difference in the cell proliferation both between the tissue-culture media (p < 0.005) and between the donors (17< 0.005).
SC~cell
95" 90856.0757065-
.~..
60 55 50
•
45 40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2,60
"PR
I"
Fig. 2. Correlation between Proliferating Rate Index (PRI) and SCE frequency. Each point represents the average number of SCE in 30 cells analysed•
198 These results indicate that the rate o f cell p r o l i f e r a t i o n in the culture is a n i m p o r t a n t d e t e r m i n a n t o f SCE frequency. This o b s e r v a t i o n is in agreement with the results o f L i n d b l a d a n d L a m b e r t (1981). O u r m a i n c o n c l u s i o n is that, even t h o u g h o t h e r possible d e t e r m i n a n t s such as the d i f f e r e n t l y m p h o c y t e s u b - p o p u l a t i o n s s h o u l d n o t be u n d e r e s t i m a t e d , w h e n we are faced with the possible effects o f m u t a g e n s a n d / o r carcinogens, the l y m p h o c y t e - p r o l i f e r a t i n g rate o f the culture c a n n o longer be disregarded.
Acknowledgements W e are grateful to M. Di Bacco, Professor o f M e t h o d o l o g i c a l Statistics, B o l o g n a University, a n d to Professor G. Allasia, Ist. Calcoli N u m e r i c i , T u r i n University. This work was s u p p o r t e d b y the I t a l i a n M i n i s t r y o f P u b l i c H e a l t h (60°7o grants).
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199 Kato, H., and A.A. Sandberg (1977) The effect of sera on sister chromatid exchanges in vitro, Exp. Cell Res., 109, 445-448. Lambert, B. (1980) Sister chromatid exchange in human lymphocytes as an indicator of DNA damage and repair in vivo, in: A. Castellani (Ed.), Lymphocyte Stimulation, Plenum, New York, pp. 119-129. Lambert, B., K. Hansson, J. Lindsten, M. Sten and B. Warelius (1976) Bromodeoxyuridine-induced sister chromatid exchanges in human lymphocytes, Hereditas, 83, 163-174. Latt, S.A., and L.A. Juergens (1977) Determinants of sister chromatid exchange frequencies in human chromosomes, in: E.B. Hook and I.H. Porter (Eds.), Population Cytogenetics, Academic Press, New York, pp. 217-236. Lindblad, A., and B. Lambert (1981) Relation between sister chromatid exchange, cell proliferation and proportion of B and T cells in human lymphocyte cultures, Hum. Genet., 57, 31-34. Morgan, W.F., and P.E. Crossen (1977) The incidence of sister chromatid exchanges in cultured human lymphocytes, Mutation Res., 42, 305-312. Morgan, W.F., and P.E. Crossen (1981) Factors influencing sister-chromatid exchange rate in cultured human lymphocytes, Mutation Res., 81, 395-402. Riedel, L., and G. Obe (1980) Trenimon-induced SCEs and structural chromosomal aberrations in earlyand late-dividing lymphocytes, Mutation Res., 73, 125-131. Santesson, B., K. Lindahl-Kiessling and A. Mattsson (1979) SCE in B and T lymphocytes. Possible implications for Bloom's syndrome, Clin. Genet., 16, 133-135. Snope, A.J., and J.M. Rary (1979) Cell-cycle duration and sister chromatid exchange frequency in cultured human lymphocytes, Mutation Res., 63, 345-349.