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Mitomycin C-induced damage and repair in human and pig lymphocytes
Mutation Research, 160 (1986) 27-32 Elsevier
27
MTR 04150
Mitomycin C-induced damage and repair in human and pig lymphocytes * Martha S. Bianchi, Marcelo L. Larramendy and Nrstor O. Bianchi Instituto Multidisciplinario de Biologla Celular (IMB1CE), C. C. 403, 1900 La Plata (Argentina) (Received 16 April 1985) (Revision received 22 July 1985) (Accepted 6 September 1985)
Summary Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the GO period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into inono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be repaired between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.
It has been demonstrated that chromosomes from different animal species may exhibit a variable radiosensitivity to ionizing radiation (Sankaranarayanan, 1976), which can be ascribed to variations in nuclear volume, DNA content and DNA repair efficiency (Griffin et al., 1970; Abrahamson et al., 1973; Sasaki, 1975). On the other hand, although there is ample information on the action of chemical compounds in the chromosomes of different species, a valid interspecies comparison of clastogenesis by these agents is not possible at the present time. Chromosomes from pig lymphocytes are more resistant to X-ray damage than those from human lymphocytes (Bianchi et al., 1979, 1981). Moreover, pig lymphocytes labeled with BrdU have been reported to have lower basal frequencies of SCEs than human lymphocytes (Bianchi et al., 1977). Therefore, we have employed human and
*
This work was supported by grants from the CONICET and CIC.
pig lymphocytes to analyse the chromosomal response to mitomycin C (MMC), a known crosslinking alkylating agent. The results obtained indicate that there are no significant differences in the sensitivity to MMC or in the efficiency of repair of MMC-induced lesions, responsible for inducing SCEs, between human and pig chromosomes. Material and methods Blood cultures were set up in a medium containing Ham F10 80%, fetal calf serum 17%, phytohemagglutinin (PHA) 3% and BrdU 10 /~g/ml. For pig lymphocytes the culture medium was supplemented with 0.2 mg/ml of glutamine and 0.13 mg/ml of arginine. During the last 3 h before termination, cultures were treated with colchicine 0.1 ~g/ml. Harvesting was performed at 48 and 72 h for pig and human lymphocytes respectively. It has been previously reported that pig lymphocytes in culture undergo in 48 h a number of cell cycles equivalent to those occurring in human lymphocytes in 72 h (Lezana et al., 1978). For analysis of
0027-5107/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
28
SCEs, slides were processed by the FPG technique (Perry and Wolff, 1974). MMC treatments lasted 20 min and were carried out at the moment of starting the cultures. After the treatments, cells were washed twice with culture medium and incubated in MMC-free medium up to the time of harvesting. MMC doses employed are detailed in Tables 1-3. The total number of individuals analysed were 6 normal male human beings and 6 male pigs. Frequencies of SCEs were estimated in 20 second mitoses per individual and per MMC dose point. 2 human donors and 4 pigs were employed for liquid-holding experiments. Control cells and cells treated with MMC (1.92 × 10 -6 M) received PHA at 24-h intervals from 0 to 120 h after starting the culture (Evans and Vijayalaxmi, 1980; Obe et al., 1982). Cultures were harvested at 48 and 72 h after PHA addition, for pig and human lymphocytes, respectively. The analysis of SCEs per cell cycle was performed in two pigs and two humans using a method described elsewhere (Bianchi and Larramendy, 1983). Control and MMC-treated (1.92 × 10 6 M) lymphocytes were grown for 30 h for pigs and 48 h for humans in a culture medium free of BrdU but containing 0.5 /~Ci/ml of 3HTdR (sp. act. 6.7 Ci/mmole). At the end of the 3HTdR treatment the cells were washed 3 times in Hanks' solution, resuspended in culture medium and grown for an additional 48 h in the presence of 10 /~g/ml of
BrdU and harvested. Chromosome preparations were processed using the FPG method. 30 metaphases in second division after the BrdU treatment (third divison after starting the culture) were photographed. Afterwards, slides were washed in xylene, restained with carbol-fuchsin, mounted with AR10 Kodak stripping film, exposed for 15 20 days and developed. Metaphases were then relocated and photographed. The analysis of silver grain distribution on harlequin chromosomes allowed the identification of SCEs occurring in the first, second and third cell cycles (Bianchi and Larramendy, 1983). Results and discussion
Frequency of MMC-induced SCEs. The frequency of SCEs in control cells and in cells treated with increasing doses of MMC is presented in Table 1 for humans and in Table 2 for pigs. The chi-square test was employed to analyse ~he interindividual variation for each MMC dose. In both species, variation between individuals was not significant except for the highest MMC concentration (Tables 1, 2). At this dose of the drug, however, interindividual variations in mitotic indices were also found. This suggests that heavily damaged cells may have failed to reach mitoses or to complete two rounds of replication. Accordingly, variation in SCE rates may have resulted from an indirect action of the drug on the cell
TABLE 1 F R E Q U E N C Y OF SCEs IN H U M A N LYMPHOCYTES PULS E-TR EA TED WITH I N C R E A S I N G DOSES OF MMC MMC
Donor " A
chi-square B
0
7.10+_0.83
7.80+_0.69
1.2 x l 0 7 M
9.65+_0.60
14.80+_0.96
C 8.25+_0.99
D
E
F
7.90+_0.70
8.95+0.73
7.15+0.56
ns
10.10_+0.73
10.00_+0.87
13.00+0.90
10.75+_0.58
ns
2.4 x l 0
vM
11.85+_0.88
17.10+_0.86
11.30_+0.86
13.10+_0.64
13.45+_0.62
11.50_+0.54
ns
4.8 x l 0
~M
13.30+-0.83
19.55+-1.37
14.40+_0.94
13.60+_0.76
18.40_+0.98
14.85+0.90
ns
9.6 × 1 0
VM
18.45+_0.85
22.15_+1.56
16.15_+1.06
17.60_+0.63
23.65_+1.15
18.85_+0.91
ns
1.92×10
6M
20.10+-0.84
28.95+1.66
22.75_+1.06
23.25+-1.17
37.10+1.36
30.05+1.64
ns
3.84x10
6M
35.50+_1.38
35.80+_1.36
33.25_+1.42
29.20_+0.73
61.00+- 1.46
53.94_+ 1.94
p < 0.01
~' Average and standard errors.
29 TABLE 2 F R E Q U E N C Y OF SCEs IN PIG LYMPHOCYTES PULSE-TREATED WITH I N C R E A S I N G DOSES OF MMC MMC
Donor a
chi-square
A 0
B
C
D
E
F
7.00_+0.51
6.25_+0.62
5.90_+0.53
8.15_+0.62
7.80_+0.75
6.05_+0.61
ns
1.2 x l O -7 M
11.05_+0.73
11.75_+0.79
9.30_+0.59
11.60_+0.85
9.75_+0.62
10.35_+0.62
ns
2.4 )<10 7 M
12.10_+0.79
13.30-+0.88
10.65-+0.57
11.70_+1.04
12.40_+1.03
12.85_+0.69
ns
4.8 )<10 -7 M
15.65-+1.11
15.90-+1.18
16.10-+0.76
14.35-+0.79
13.15_+0.96
14.75+_0.81
ns
9.6 )<10 -7 M
16.70_+0.96
21.95_+1.45
20.85_+1.08
20.35_+1.25
15.65_+0.94
21.30_+0.83
ns
1.92 × 10 -6 M
24.80_+1.20
32.55_+1.32
26.05_+1.68
22.00_+1.30
34.50 _+1.77
ns
3.84)<10 6 M
42.65_+2.28
31.20_+0.96
50.30 _+1.74
p < 0.01
78.70_+3.76
52.70_+1.44
-
a Average and standard errors.
cycle progression and not as the consequence of the primary action on chromosomal DNA. Since no interindividual variations were detected, results from humans and results from pigs were averaged. The correlation test showed that SCE frequencies increase as a linear function of MMC doses in both human ( r = 0 . 9 2 , y = 10.52 + 24.73x) and pig lymphocytes (r = 0.89, y = 8.69 + 32.66x). The Snedecor test showed that interspecies differences were not significant (Table
3).
10 6 M of MMC (the highest dose does not induce detectable changes in mitotic indices) and stored in GO from 0 to 96-120 h with intervals of 24 h. Figs. 1 and 2 illustrate the results in human and pig donors. GO synchronization of human and pig lymphocytes was checked by incubating nonstimulated lymphocytes during 4 h in the presence of 20 /~Ci/ml of 3HTdR (sp. act. 20 Ci/mmole) and determining the radioactivity incorporated by autoradiography. In both species more than 98%
Liquid holding. In the experiments reported here lymphocytes were pulse-treated with 1.92 × 30
TABLE 3
IA
30
AVERAGE FREQUENCIES OF SCEs IN H U M A N A N D PIG LYMPHOCYTES PULSE-TREATED WITH INCREASI N G DOSES OF MMC MMC
Human
Pig se
.~
20
20
I0
10
Snedecor se
test
F=1.98 0
7.86+0.28
6.86_+0.39
ns
1.2 )<10 -7 M
11.38_+0.84
10.63_+0.41
ns
2.4 ×10 -7 M
13.05_+0.88
12.17_+0.38
ns
4.8 )<10 7 M
15.68_+1.07
14.98___0.46
ns
9.6 ×10 -7 M
19.47_+1.16
19.47_+1.07
ns
1.92×10 - 6 M
27.03_+2.55
27.98_+2.38
ns
3.84x10
41.45_+5.24
51.11_+7.85
ns
.-L .... 4 ...... ÷. . . . ~"--. ~
24
48
72
96
2~,
1,8
71
96
LH(hs)
6M
Fig. 1. Liquid-holding experiments. H u m a n donors A and B. Broken and solid lines represent control and M M C - i n d u c e d
SCEs respectively.
30 repaired. Working with MMC and its monofunctional derivative decarbamoyl-MMC, Linnainmaa and Wolff (1982) found that monoadducts, rather than interstrand biadducts, are responsible for SCE formation. In light of all this information, the increase in SCEs detected in MMC-liquid holding experiments with human donor B and with pig donors A and B can be interpreted as a half-repair of cross-linking which changes bi- into mono-adducts with a consequent rise in exchanges. The lack of a consistent decrease in SCEs below levels at 0-h holding time in lymphocytes challenged with MMC, apparently indicates that repair of monoadducts does not take place during the GO period. Similar conclusions were also reached by Evans and Vijayalaxmi (1980) for human lymphocytes. SCEs per cell wcle. Experiments performed with CHO cells and human lymphocytes indicate that the increase in SCEs induced by MMC does not last more than one cell cycle (Linnainmaa and Wolff, 1982; Littlefield et al., 1983; Natarajan et al., 1983). Our results are in general agreement with these data. Table 4 illustrates the frequency of SCEs after each cell cycle in human and pig lymphocytes
of cells did not incorporate ~HTdR and were assumed to be in GO. In both species, at 0-h holding time MMCtreated lymphocytes showed 3-6-fold increases of SCEs over those found in control cells. Human donor B and pig donors C and D showed no consistent variations in SCE frequencies in relation to holding time. On the other hand, human donor A and pig donors A and B exhibited significant and persistent increases in SCEs as the storage time increased. In human A and pig A the increases were evident at 72-h holding time. Pig B already showed a consistent rise in SCEs at 24-h holding time (Figs. 1, 2). Other authors have also observed increases in SCEs in human lymphocytes stored in GO from 6 to 9 days (Evans and Vijayalaxmi, 1980). It is known that MMC alkylates the 06 position of guanine-inducing DNA monoadducts and interstrand biadducts in a 10/1 ratio (Tomasz et al., 1974). Fujiwara and Tatsumi (1975) have reported that repair of DNA cross-links induced by MMC in mammalian cells is a two-step process. In the first stage there is a half repair which changes the interstrand biadducts into monoadducts, then, in a second and slower step monoadducts are
401
/ • T
// /,
/
//
/ /
/
/
/
/J /
/ 7
w
,/
2o
w 2o
J
// 10"
i i 0
24
48
72
24
96
LH(hs)
48
72
31
'°tD
I0 "
10 -
-_~,,
24
j4- .....
48
72
96
"
-[
120
2t,
t,8
72
96
120
LH (hs)
Fig. 2. Liquid-holding experiments. Pig donors A, B, C and D. Broken and solid lines represent control and MMC-induced SCEs respectively.
respectively. Both species show an increase in SCEs above control levels in first mitoses of lymphocytes pulse-treated with MMC. Conversely, in sec-
ond and third mitoses the frequency of exchanges reached control or near-control levels. If DNA adducts induced by MMC are not
TABLE4 FREQUENCY OF S C E s P E R CELL C Y C L E I N H U M A N AND PIG LYMPHOCYTES Species
Treatments
1st cell cycle SCEs per cell
Human A
Pig A
2nd cell cycle Induced SCEs a
3rd cell cycle
SCEs per cell
Induced SCEs
SCEs per cell
Induced SCEs
Control MMC 1.92×10 -6 M
15.77 _+1.00 24.76 _4-1.49
8.99
3.17 + 0.38 6.20 _+0.34
3.03
4.90 + 0.43 8.03_+0.50
3.13
Control MMC 1.92x 10 -6 M
13.64 -+ 0.97 29.23-+1.67
15.59
3.09 + 0.29 7.07_+0.51
3.98
5.22_+0.37 7.70+_0.67
2.48
Control MMC 1.92×10 -6 M
6.90 -+ 0.66 15.19 + 1.36
8.29
4.90 -+0.46 6.45 -+0.51
1.55
7.30 _+0.64 7.13_+0.52
-0.17
Control MMC 1.92×10 -6 M
8.73_+0.68 14.31 -+ 1.05
5.58
5.65-+0.52 4.94 _+0.48
- 0.71
6.59_+0.49 6.37_+0.69
-0.22
a Induced SCEs = MMC-induced S C E s - c o n t r o l SCEs.
32
repaired, the frequency of SCEs is expected to halve after each cell cycle. On the other hand, if some sort of repair takes place during or after the first round of chromosome replication, SCEs in second mitoses will decrease to a frequency significantly lower than half the rate detected in first mitoses. In the experiments reported here, the frequency of SCEs is the result of the combined action of 3HTdR and MMC in the first cell cycle and BrdU and MMC (if adducts are long-lived), or BrdU alone (if adducts are short-lived) in subsequent cycles. Therefore, to obtain the frequency of exchanges induced by MMC alone we have to subtract the basal levels of SCEs observed in control cells from the frequency of exchanges detected in MMC-challenged cells (Table 4). Comparison of the induced frequencies of SCEs shows in both species a marked fall of exchanges between the first and second mitoses. Since repair of monoadducts produced by MMC does not occur in the GO period, the decrease in exchanges should result from some process of adduct repair taking place during the S or G2 periods of the first cell cycle or during the G1 period of the second cycle. At the present time there is no information on the mechanism of this repair. The persistence of a slight increase in real SCEs in the second and third cycle (Table 4) is probably due to the persistence of some small amount of long lasting MMC-induced lesions. Linnainmaa and Wolff (1982) have found this to be the case when high doses of MMC, such as the one employed in this experiment, are used to treat the cells.
Acknowledgements We thank Lic. Liliana Cort6s and Lic. Miguel Reigosa for technical assistance. References Abrahamson, S., M.A Bender and A.L. Conger (1973) Uniformity of radiation-induced mutation rates among different species, Nature (London), 245,460-462. Bianchi, N.O.. and M.L. Larramendy (1983) The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges, Chromosoma. 88, 11 15.
Bianchi, N.O., M.S. Bianchi, E.A. Lezana and J.E. ZabalaSuarez (1977) lnterspecies variation in the frequency of SCE, in: A. de la Chapelle and M. Sorsa (Eds.), Chromosomes Today, Vol. 6, Elsevier/North-Holland, Amsterdam, pp. 307 314. Bianchi, N.O., M.S. Bianchi and M.L. Larramendy (1979) Kinetics of h u m a n lymphocyte division and chromosomal radiosensitivity, Mutation Res.. 63, 317 324. Bianchi, M., N.O. Bianchi, M.L. Larramendy and J. GarciaHeras (1981) Chromosomal radiosensitivity of pig leucocytes in relation to sampling time. Mutation Res., 80, 313 320. Evans, H.J., and Vijayalaxmi (1980) Storage enhances chromosome damage after exposure of human leukocytes to mitomycin C, Nature (London), 284, 370-372. Fujiwara, Y., and M. Tatsumi (1975) Repair of mitomycin (7 damage to D N A in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun., 66, 592 598. Griffin, C.S., D. Scott and D.G. Papworth (1970) The influence of D N A content and nuclear volume on the frequency of radiation-induced chromosome aberrations in Bufo species, Chromosoma, 30, 228-230. Lezana, E.A., M.S. Bianchi and N.O. Bianchi (1978) Kinetics of division of PHA stimulated pig lymphocytes, Experientia, 34, 30-31. Linnainmaa, K., and S. Wolff (1982) Sister chromatid exchange induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen. Environ. Mutagen., 4, 239-247. Littlefield, L.G., S.P. Colyer and R.J. Du Frain (1983) SCE evaluations in h u m a n lymphocytes after GO exposure to Mitomycin (', Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions, Mutation Res., 107, 119 130. Natarajan, A.T., A.D. Tates, M. Meijers, I. Neuteboom and N. de Vogel (1983) Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations by mitomycin C and methyl methanesulfonate in Chinese hamster ovary cells, Mutation Res., 121, 211-223. Obe, G., S. Kalweit, C. Nowak and F. Ali-Osman (1982) Liquid holding experiments with h u m a n peripheral lymphocytes, 1. Effects of liquid holding on sister chromatid exchanges induced by trenimon, diepoxybutane, bleomycin and X-rays, Biol. Zbl., 101, 97 113. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Sankaranarayanan, K. (1976) Evaluation and re-evaluation of genetic radiation hazards in man, II. The arm number hypothesis and the induction of reciprocal translocation in man, Mutation Res., 35, 371-386. Sasaki, M.S. (1975) A comparison of chromosomal radiosensitivities of somatic cells of mouse and man, Mutation Res., 29, 433-448. Tomasz, M., C.M. Mercado, J. Olson and N. Chatterjie (1974) The mode of interaction of Mitomycin C with deoxyribonucleic acid and other polynucleotides in vitro, Biochemistry, 13, 4878-4887.
27
MTR 04150
Mitomycin C-induced damage and repair in human and pig lymphocytes * Martha S. Bianchi, Marcelo L. Larramendy and Nrstor O. Bianchi Instituto Multidisciplinario de Biologla Celular (IMB1CE), C. C. 403, 1900 La Plata (Argentina) (Received 16 April 1985) (Revision received 22 July 1985) (Accepted 6 September 1985)
Summary Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the GO period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into inono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be repaired between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.
It has been demonstrated that chromosomes from different animal species may exhibit a variable radiosensitivity to ionizing radiation (Sankaranarayanan, 1976), which can be ascribed to variations in nuclear volume, DNA content and DNA repair efficiency (Griffin et al., 1970; Abrahamson et al., 1973; Sasaki, 1975). On the other hand, although there is ample information on the action of chemical compounds in the chromosomes of different species, a valid interspecies comparison of clastogenesis by these agents is not possible at the present time. Chromosomes from pig lymphocytes are more resistant to X-ray damage than those from human lymphocytes (Bianchi et al., 1979, 1981). Moreover, pig lymphocytes labeled with BrdU have been reported to have lower basal frequencies of SCEs than human lymphocytes (Bianchi et al., 1977). Therefore, we have employed human and
*
This work was supported by grants from the CONICET and CIC.
pig lymphocytes to analyse the chromosomal response to mitomycin C (MMC), a known crosslinking alkylating agent. The results obtained indicate that there are no significant differences in the sensitivity to MMC or in the efficiency of repair of MMC-induced lesions, responsible for inducing SCEs, between human and pig chromosomes. Material and methods Blood cultures were set up in a medium containing Ham F10 80%, fetal calf serum 17%, phytohemagglutinin (PHA) 3% and BrdU 10 /~g/ml. For pig lymphocytes the culture medium was supplemented with 0.2 mg/ml of glutamine and 0.13 mg/ml of arginine. During the last 3 h before termination, cultures were treated with colchicine 0.1 ~g/ml. Harvesting was performed at 48 and 72 h for pig and human lymphocytes respectively. It has been previously reported that pig lymphocytes in culture undergo in 48 h a number of cell cycles equivalent to those occurring in human lymphocytes in 72 h (Lezana et al., 1978). For analysis of
0027-5107/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
28
SCEs, slides were processed by the FPG technique (Perry and Wolff, 1974). MMC treatments lasted 20 min and were carried out at the moment of starting the cultures. After the treatments, cells were washed twice with culture medium and incubated in MMC-free medium up to the time of harvesting. MMC doses employed are detailed in Tables 1-3. The total number of individuals analysed were 6 normal male human beings and 6 male pigs. Frequencies of SCEs were estimated in 20 second mitoses per individual and per MMC dose point. 2 human donors and 4 pigs were employed for liquid-holding experiments. Control cells and cells treated with MMC (1.92 × 10 -6 M) received PHA at 24-h intervals from 0 to 120 h after starting the culture (Evans and Vijayalaxmi, 1980; Obe et al., 1982). Cultures were harvested at 48 and 72 h after PHA addition, for pig and human lymphocytes, respectively. The analysis of SCEs per cell cycle was performed in two pigs and two humans using a method described elsewhere (Bianchi and Larramendy, 1983). Control and MMC-treated (1.92 × 10 6 M) lymphocytes were grown for 30 h for pigs and 48 h for humans in a culture medium free of BrdU but containing 0.5 /~Ci/ml of 3HTdR (sp. act. 6.7 Ci/mmole). At the end of the 3HTdR treatment the cells were washed 3 times in Hanks' solution, resuspended in culture medium and grown for an additional 48 h in the presence of 10 /~g/ml of
BrdU and harvested. Chromosome preparations were processed using the FPG method. 30 metaphases in second division after the BrdU treatment (third divison after starting the culture) were photographed. Afterwards, slides were washed in xylene, restained with carbol-fuchsin, mounted with AR10 Kodak stripping film, exposed for 15 20 days and developed. Metaphases were then relocated and photographed. The analysis of silver grain distribution on harlequin chromosomes allowed the identification of SCEs occurring in the first, second and third cell cycles (Bianchi and Larramendy, 1983). Results and discussion
Frequency of MMC-induced SCEs. The frequency of SCEs in control cells and in cells treated with increasing doses of MMC is presented in Table 1 for humans and in Table 2 for pigs. The chi-square test was employed to analyse ~he interindividual variation for each MMC dose. In both species, variation between individuals was not significant except for the highest MMC concentration (Tables 1, 2). At this dose of the drug, however, interindividual variations in mitotic indices were also found. This suggests that heavily damaged cells may have failed to reach mitoses or to complete two rounds of replication. Accordingly, variation in SCE rates may have resulted from an indirect action of the drug on the cell
TABLE 1 F R E Q U E N C Y OF SCEs IN H U M A N LYMPHOCYTES PULS E-TR EA TED WITH I N C R E A S I N G DOSES OF MMC MMC
Donor " A
chi-square B
0
7.10+_0.83
7.80+_0.69
1.2 x l 0 7 M
9.65+_0.60
14.80+_0.96
C 8.25+_0.99
D
E
F
7.90+_0.70
8.95+0.73
7.15+0.56
ns
10.10_+0.73
10.00_+0.87
13.00+0.90
10.75+_0.58
ns
2.4 x l 0
vM
11.85+_0.88
17.10+_0.86
11.30_+0.86
13.10+_0.64
13.45+_0.62
11.50_+0.54
ns
4.8 x l 0
~M
13.30+-0.83
19.55+-1.37
14.40+_0.94
13.60+_0.76
18.40_+0.98
14.85+0.90
ns
9.6 × 1 0
VM
18.45+_0.85
22.15_+1.56
16.15_+1.06
17.60_+0.63
23.65_+1.15
18.85_+0.91
ns
1.92×10
6M
20.10+-0.84
28.95+1.66
22.75_+1.06
23.25+-1.17
37.10+1.36
30.05+1.64
ns
3.84x10
6M
35.50+_1.38
35.80+_1.36
33.25_+1.42
29.20_+0.73
61.00+- 1.46
53.94_+ 1.94
p < 0.01
~' Average and standard errors.
29 TABLE 2 F R E Q U E N C Y OF SCEs IN PIG LYMPHOCYTES PULSE-TREATED WITH I N C R E A S I N G DOSES OF MMC MMC
Donor a
chi-square
A 0
B
C
D
E
F
7.00_+0.51
6.25_+0.62
5.90_+0.53
8.15_+0.62
7.80_+0.75
6.05_+0.61
ns
1.2 x l O -7 M
11.05_+0.73
11.75_+0.79
9.30_+0.59
11.60_+0.85
9.75_+0.62
10.35_+0.62
ns
2.4 )<10 7 M
12.10_+0.79
13.30-+0.88
10.65-+0.57
11.70_+1.04
12.40_+1.03
12.85_+0.69
ns
4.8 )<10 -7 M
15.65-+1.11
15.90-+1.18
16.10-+0.76
14.35-+0.79
13.15_+0.96
14.75+_0.81
ns
9.6 )<10 -7 M
16.70_+0.96
21.95_+1.45
20.85_+1.08
20.35_+1.25
15.65_+0.94
21.30_+0.83
ns
1.92 × 10 -6 M
24.80_+1.20
32.55_+1.32
26.05_+1.68
22.00_+1.30
34.50 _+1.77
ns
3.84)<10 6 M
42.65_+2.28
31.20_+0.96
50.30 _+1.74
p < 0.01
78.70_+3.76
52.70_+1.44
-
a Average and standard errors.
cycle progression and not as the consequence of the primary action on chromosomal DNA. Since no interindividual variations were detected, results from humans and results from pigs were averaged. The correlation test showed that SCE frequencies increase as a linear function of MMC doses in both human ( r = 0 . 9 2 , y = 10.52 + 24.73x) and pig lymphocytes (r = 0.89, y = 8.69 + 32.66x). The Snedecor test showed that interspecies differences were not significant (Table
3).
10 6 M of MMC (the highest dose does not induce detectable changes in mitotic indices) and stored in GO from 0 to 96-120 h with intervals of 24 h. Figs. 1 and 2 illustrate the results in human and pig donors. GO synchronization of human and pig lymphocytes was checked by incubating nonstimulated lymphocytes during 4 h in the presence of 20 /~Ci/ml of 3HTdR (sp. act. 20 Ci/mmole) and determining the radioactivity incorporated by autoradiography. In both species more than 98%
Liquid holding. In the experiments reported here lymphocytes were pulse-treated with 1.92 × 30
TABLE 3
IA
30
AVERAGE FREQUENCIES OF SCEs IN H U M A N A N D PIG LYMPHOCYTES PULSE-TREATED WITH INCREASI N G DOSES OF MMC MMC
Human
Pig se
.~
20
20
I0
10
Snedecor se
test
F=1.98 0
7.86+0.28
6.86_+0.39
ns
1.2 )<10 -7 M
11.38_+0.84
10.63_+0.41
ns
2.4 ×10 -7 M
13.05_+0.88
12.17_+0.38
ns
4.8 )<10 7 M
15.68_+1.07
14.98___0.46
ns
9.6 ×10 -7 M
19.47_+1.16
19.47_+1.07
ns
1.92×10 - 6 M
27.03_+2.55
27.98_+2.38
ns
3.84x10
41.45_+5.24
51.11_+7.85
ns
.-L .... 4 ...... ÷. . . . ~"--. ~
24
48
72
96
2~,
1,8
71
96
LH(hs)
6M
Fig. 1. Liquid-holding experiments. H u m a n donors A and B. Broken and solid lines represent control and M M C - i n d u c e d
SCEs respectively.
30 repaired. Working with MMC and its monofunctional derivative decarbamoyl-MMC, Linnainmaa and Wolff (1982) found that monoadducts, rather than interstrand biadducts, are responsible for SCE formation. In light of all this information, the increase in SCEs detected in MMC-liquid holding experiments with human donor B and with pig donors A and B can be interpreted as a half-repair of cross-linking which changes bi- into mono-adducts with a consequent rise in exchanges. The lack of a consistent decrease in SCEs below levels at 0-h holding time in lymphocytes challenged with MMC, apparently indicates that repair of monoadducts does not take place during the GO period. Similar conclusions were also reached by Evans and Vijayalaxmi (1980) for human lymphocytes. SCEs per cell wcle. Experiments performed with CHO cells and human lymphocytes indicate that the increase in SCEs induced by MMC does not last more than one cell cycle (Linnainmaa and Wolff, 1982; Littlefield et al., 1983; Natarajan et al., 1983). Our results are in general agreement with these data. Table 4 illustrates the frequency of SCEs after each cell cycle in human and pig lymphocytes
of cells did not incorporate ~HTdR and were assumed to be in GO. In both species, at 0-h holding time MMCtreated lymphocytes showed 3-6-fold increases of SCEs over those found in control cells. Human donor B and pig donors C and D showed no consistent variations in SCE frequencies in relation to holding time. On the other hand, human donor A and pig donors A and B exhibited significant and persistent increases in SCEs as the storage time increased. In human A and pig A the increases were evident at 72-h holding time. Pig B already showed a consistent rise in SCEs at 24-h holding time (Figs. 1, 2). Other authors have also observed increases in SCEs in human lymphocytes stored in GO from 6 to 9 days (Evans and Vijayalaxmi, 1980). It is known that MMC alkylates the 06 position of guanine-inducing DNA monoadducts and interstrand biadducts in a 10/1 ratio (Tomasz et al., 1974). Fujiwara and Tatsumi (1975) have reported that repair of DNA cross-links induced by MMC in mammalian cells is a two-step process. In the first stage there is a half repair which changes the interstrand biadducts into monoadducts, then, in a second and slower step monoadducts are
401
/ • T
// /,
/
//
/ /
/
/
/
/J /
/ 7
w
,/
2o
w 2o
J
// 10"
i i 0
24
48
72
24
96
LH(hs)
48
72
31
'°tD
I0 "
10 -
-_~,,
24
j4- .....
48
72
96
"
-[
120
2t,
t,8
72
96
120
LH (hs)
Fig. 2. Liquid-holding experiments. Pig donors A, B, C and D. Broken and solid lines represent control and MMC-induced SCEs respectively.
respectively. Both species show an increase in SCEs above control levels in first mitoses of lymphocytes pulse-treated with MMC. Conversely, in sec-
ond and third mitoses the frequency of exchanges reached control or near-control levels. If DNA adducts induced by MMC are not
TABLE4 FREQUENCY OF S C E s P E R CELL C Y C L E I N H U M A N AND PIG LYMPHOCYTES Species
Treatments
1st cell cycle SCEs per cell
Human A
Pig A
2nd cell cycle Induced SCEs a
3rd cell cycle
SCEs per cell
Induced SCEs
SCEs per cell
Induced SCEs
Control MMC 1.92×10 -6 M
15.77 _+1.00 24.76 _4-1.49
8.99
3.17 + 0.38 6.20 _+0.34
3.03
4.90 + 0.43 8.03_+0.50
3.13
Control MMC 1.92x 10 -6 M
13.64 -+ 0.97 29.23-+1.67
15.59
3.09 + 0.29 7.07_+0.51
3.98
5.22_+0.37 7.70+_0.67
2.48
Control MMC 1.92×10 -6 M
6.90 -+ 0.66 15.19 + 1.36
8.29
4.90 -+0.46 6.45 -+0.51
1.55
7.30 _+0.64 7.13_+0.52
-0.17
Control MMC 1.92×10 -6 M
8.73_+0.68 14.31 -+ 1.05
5.58
5.65-+0.52 4.94 _+0.48
- 0.71
6.59_+0.49 6.37_+0.69
-0.22
a Induced SCEs = MMC-induced S C E s - c o n t r o l SCEs.
32
repaired, the frequency of SCEs is expected to halve after each cell cycle. On the other hand, if some sort of repair takes place during or after the first round of chromosome replication, SCEs in second mitoses will decrease to a frequency significantly lower than half the rate detected in first mitoses. In the experiments reported here, the frequency of SCEs is the result of the combined action of 3HTdR and MMC in the first cell cycle and BrdU and MMC (if adducts are long-lived), or BrdU alone (if adducts are short-lived) in subsequent cycles. Therefore, to obtain the frequency of exchanges induced by MMC alone we have to subtract the basal levels of SCEs observed in control cells from the frequency of exchanges detected in MMC-challenged cells (Table 4). Comparison of the induced frequencies of SCEs shows in both species a marked fall of exchanges between the first and second mitoses. Since repair of monoadducts produced by MMC does not occur in the GO period, the decrease in exchanges should result from some process of adduct repair taking place during the S or G2 periods of the first cell cycle or during the G1 period of the second cycle. At the present time there is no information on the mechanism of this repair. The persistence of a slight increase in real SCEs in the second and third cycle (Table 4) is probably due to the persistence of some small amount of long lasting MMC-induced lesions. Linnainmaa and Wolff (1982) have found this to be the case when high doses of MMC, such as the one employed in this experiment, are used to treat the cells.
Acknowledgements We thank Lic. Liliana Cort6s and Lic. Miguel Reigosa for technical assistance. References Abrahamson, S., M.A Bender and A.L. Conger (1973) Uniformity of radiation-induced mutation rates among different species, Nature (London), 245,460-462. Bianchi, N.O.. and M.L. Larramendy (1983) The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges, Chromosoma. 88, 11 15.
Bianchi, N.O., M.S. Bianchi, E.A. Lezana and J.E. ZabalaSuarez (1977) lnterspecies variation in the frequency of SCE, in: A. de la Chapelle and M. Sorsa (Eds.), Chromosomes Today, Vol. 6, Elsevier/North-Holland, Amsterdam, pp. 307 314. Bianchi, N.O., M.S. Bianchi and M.L. Larramendy (1979) Kinetics of h u m a n lymphocyte division and chromosomal radiosensitivity, Mutation Res.. 63, 317 324. Bianchi, M., N.O. Bianchi, M.L. Larramendy and J. GarciaHeras (1981) Chromosomal radiosensitivity of pig leucocytes in relation to sampling time. Mutation Res., 80, 313 320. Evans, H.J., and Vijayalaxmi (1980) Storage enhances chromosome damage after exposure of human leukocytes to mitomycin C, Nature (London), 284, 370-372. Fujiwara, Y., and M. Tatsumi (1975) Repair of mitomycin (7 damage to D N A in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun., 66, 592 598. Griffin, C.S., D. Scott and D.G. Papworth (1970) The influence of D N A content and nuclear volume on the frequency of radiation-induced chromosome aberrations in Bufo species, Chromosoma, 30, 228-230. Lezana, E.A., M.S. Bianchi and N.O. Bianchi (1978) Kinetics of division of PHA stimulated pig lymphocytes, Experientia, 34, 30-31. Linnainmaa, K., and S. Wolff (1982) Sister chromatid exchange induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen. Environ. Mutagen., 4, 239-247. Littlefield, L.G., S.P. Colyer and R.J. Du Frain (1983) SCE evaluations in h u m a n lymphocytes after GO exposure to Mitomycin (', Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions, Mutation Res., 107, 119 130. Natarajan, A.T., A.D. Tates, M. Meijers, I. Neuteboom and N. de Vogel (1983) Induction of sister-chromatid exchanges (SCEs) and chromosomal aberrations by mitomycin C and methyl methanesulfonate in Chinese hamster ovary cells, Mutation Res., 121, 211-223. Obe, G., S. Kalweit, C. Nowak and F. Ali-Osman (1982) Liquid holding experiments with h u m a n peripheral lymphocytes, 1. Effects of liquid holding on sister chromatid exchanges induced by trenimon, diepoxybutane, bleomycin and X-rays, Biol. Zbl., 101, 97 113. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Sankaranarayanan, K. (1976) Evaluation and re-evaluation of genetic radiation hazards in man, II. The arm number hypothesis and the induction of reciprocal translocation in man, Mutation Res., 35, 371-386. Sasaki, M.S. (1975) A comparison of chromosomal radiosensitivities of somatic cells of mouse and man, Mutation Res., 29, 433-448. Tomasz, M., C.M. Mercado, J. Olson and N. Chatterjie (1974) The mode of interaction of Mitomycin C with deoxyribonucleic acid and other polynucleotides in vitro, Biochemistry, 13, 4878-4887.