Mutation Research, 245 (1990) 5-9
5
Elsevier MUTLET 0387
Hypotonic treatment leads to chromosomal aberrations but not to sisterchromatid exchanges in human lymphocytes S. K a l w e i t 1, C. N o w a k 1 a n d G. O b e 1' 2 1Freie Universitiit Berlin, lnstitut fiir Genetik, D-IO00 Berlin 33 (Germany) and 2University GHS Essen, FB 9, Department of Genetics, D-4300 Essen I (F.R.G.)
(Accepted9 April 1990)
Keywords: Hypotonictreatment;Chromosomeaberration;Humanlymphocytes
Summary Human peripheral lymphocytes were isolated from whole blood and exposed to culture medium of reduced osmolality. This hypotonic treatment led to a significant increase in the frequencies of chromosomal aberrations when the osmolality was reduced to 60 mOsm/kg H20 and below. Maximum damage occurred when the hypotonic treatment was done 27 or 30 h after starting the cultures. We also looked for the induction of sister-chromatid exchanges (SCE) by hypotonic culture conditions, but the SCE frequencies were not influenced.
During the past years it has become obvious that osmotic stress can damage mammalian cells. Various biological effects, such as radiosensitization (Raaphorst and Kruuv, 1976, 1977a,b; Raaphorst et al., 1977), cytotoxicity (Galloway et al., 1987), gene mutations (Seeberg et al., 1988), cell transformation (Garry et al., 1981), or chromosomal aberrations (Ashby and Ishidate, 1986; Brusick, 1986; Galloway et al., 1985, 1987; Ishidate et al., 1984; Nowak, 1987; Nowak and Kalweit, 1987; Tuschy and Obe, 1988), have been observed following hyper- or hypo-tonic treatment.
Studies on the induction of chromosomal aberrations by hypotonic culture conditions have so far been done with V79 hamster cells (Nowak, 1987; Nowak and Kalweit, 1987). When the osmolality of the medium, which is usually 300 mOsm/kg H20, was reduced to 60 mOsm/kg H20, high frequencies of chromosomal aberrations and totally damaged cells were found. In this study we investigated the effect of hypotonic treatment conditions on the chromosomes of human lymphocytes in vitro. Two genetic endpoints, namely chromosomal aberrations and sister-chromatid exchanges, were analyzed, following exposure to media of different osmolalities.
Correspondence: Prof. Dr. G. Obe, Universityof Essen GHS, FB 9, Departmentof Genetics,P.O. Box 103764, D-4300Essen 1 (F.R.G.).
Materials and methods Human peripheral lymphocytes were isolated
0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)
from whole blood by sucrose gradient centrifugation. 1 × 106 cells were seeded into 2.5-ml cultures, containing 2.0 ml M c C o y ' s medium (Seromed), 20°7o fetal calf serum (Seromed), 0.06 ml phytohemagglutinin (PHA, Seromed) and 0.05 ml bromodeoxyuridine (BrdUrd, final concentration 2 × 10 -~ M, Serva). The cells were cultivated for 50 or 72 h including a 5-h treatment with Colcemid (0.08/zg/ml). Preparations were made following a routine protocol and slides were stained differentially using 5% Giemsa stain. For chromosomal aberrations (CA) at least 100 uniformly stained (M1) metaphases from 50-h cultures and for sisterchromatid exchanges (SCE) 25 differentially stained (M2) metaphases from 72-h cultures were analyzed. Mitotic indices (MIs) were determined by scoring 1000 cells per entry and are given as percent mitoses. In 72-h cultures the frequencies of polyploid metaphases were determined in the population o f Mz metaphases. The experiments were performed 2-5 times.
Hypotonic treatment M c C o y ' s 5A medium has an osmolality o f 300
m O s m / k g H20. The osmolality was reduced by adding distilled water and the resulting osmolalities were measured with an osmometer. A dilution o f 1 part medium with 9 parts distilled water results in an osmolality of 30 m O s m / k g H20. The pH of the M c C o y ' s medium changed upon dilution. Undiluted medium had a pH of 7.2, dilution to 200 m O s m / k g H 2 0 had no influence on pH, but the p H dropped to 6.0 in medium o f 45 m O s m / k g H20. Lymphocytes were cultured for at least 21 h, pelleted and the supernatant was kept at 37°C as conditioned medium. The cells were incubated for 60 min in 2.5 ml serum-free prewarmed hypotonic medium. The hypotonic medium was discarded and the cells were recovered in conditioned medium. In order to analyze whether a reduction of the pH has any influence on the frequencies o f chromosomal aberrations the cells were exposed at 27 h culture time to 2.5 ml serum-free prewarmed medium o f p H 7.0, 6.5 and 6.0 for 60 min. The pH was adjusted by addition of 1 N HCI and measured with a pH meter. The cells were recovered after treatment for 22 h in complete medium of pH 7.2.
TABLE 1 CHROMOSOMALABERRATIONSOBSERVEDIN METAPHASESOF HUMAN LYMPHOCYTESFOLLOWINGEXPOSURE TO DILUTED McCOY'S MEDIUM The cells were cultured in G1 (27 h) for 60 min in hypotonic medium and recovered in isotonic medium for 22 h. 2 independent experiments are given, 100 metaphases per experiment were scored. Osmolality
o7oCells showing
of hypotonic medium (mOsm/kg H20)
Chromatid breaks
300 (control) 180 150 120 90 75 60 45 30
Mitotic index (°70)
Chromatid exchanges
Chromosome exchanges
0.5
0.0
0.0
1.0
1.0
5,2
1.0 0.0 1.5 0.0 0.5 0.5 0.0 1.0 1.0 1.5 13.0 4.5 9.0 9.0 No metaphases
0.0 0.0 0.0 0.0 0.0 0.5 1.0
0.0 0.0 0.0 0.0 0.5 0.0 0.0
1.0 1.5 0.5 1.0 3.0 14.0 15,0
1.0 1.5 1.0 1.0 3.5 18.5 20.0
6.8 3.9 5.9 4.2 2,7 1.9 0.8 0.0
0.5
All aberrations
Total number of breaks
Isochromatid breaks
The total number of breaks was calculated as follows: chromatid and isochromatid breaks = 1; chromatid and chromosome exchanges = 2.
Results and discussion The frequencies of chromosomal aberrations and mitotic indices are compiled in Tables 1-3. When lymphocytes are exposed to hypotonic culture conditions 27 h after stimulation (Table 1), which probably represents a G~ population of cells, the frequencies of chromosomal aberrations are in the control range until an osmolality of 75 mOsm/kg H20. Hypotonic treatment with 60 and 45 mOsm/kg H20 leads to a clear increase in chromosomal aberrations. When we treat the cells with 30 mOsm/kg H20 a total suppression of the mitotic index is found. For further experiments we chose 60 mOsm/kg H20, an osmolality which induces chromosomal aberrations at reasonable toxicity. Table 2 shows the results of a hypotonic treatment with 60 mOsm/kg H20 at different stages of the cell cycle. Up to a culture time of 21 h, representing cells in their Go and early GI phases, no metaphases are found, the lymphocytes seem to be extremely sensitive to hypotonic culture conditions. Hypotonic treatment 27 or 30 h after stimulation leads to
clearly increased frequencies of chromosomal aberrations. But when the hypotonic treatment is done 36, 39, 42 or 45 h after stimulation chromosomal aberrations are not increased. These results show that hypotonic culture conditions can lead to chromosomal aberrations in human lymphocytes, but the total number of breaks in 100 metaphases never exceeds 20. And it is only possible to induce chromosomal aberrations during a short period of the cell cycle, probably at the end of the G1 phase. Undiluted culture medium has a pH of 7.2, but a hypotonic solution of 45 mOsm/kg H20, prepared by dilution with distilled water, has a pH of 6.0. In order to exclude pH effects on the induction of chromosomal aberrations we cultivated human lymphocytes in isotonic medium with pH values of 7.0, 6.5 and 6.0. Table 3 shows that a pH drop does not lead to chromosomal aberrations when the osmolality is kept normal (300 mOsm/kg H20). A comparison of data from this study with V79 hamster cells reveals some remarkable differences. In V79 cells hypotonic treatment leads to extraordinarily high aberration frequencies
TABLE 2 C H R O M O S O M A L A B E R R A T I O N S OBSERVED IN M E T A P H A S E S OF H U M A N L Y M P H O C Y T E S F O L L O W I N G E X P O S U R E T O D I L U T E D M c C O Y ' S M E D I U M OF 60 m O s m / k g H 2 0 The cells were treated at different culture times for 60 min and fixation was done 50 h after starting the cultures. 2 independent experiments are given, 100 rnetaphases per experiment were scored. Treatment
°7o Cells showing
Total
Mitotic
time (h)
Chro-
Iso-
Chro-
Chro-
All
number
index
matid breaks
chromatid breaks
matid exchanges
mosome exchanges
aberrations
of breaks
(07o)
21 24 27 30 33 36 39 42 45
Nomethaphases 6.0 2.0 4.5 3.0 7.5 6.0 3.5 3.0 2.5 0.5 1.0 1.0 2.0 1.0 4.0 1.0
0.0 1.5 1.0 0.5 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
8.0 8.5 8.5 5.0 2.5 2.0 1.5 4.0
8.0 10.5 15.5 7.5 3.0 2.0 3.0 5.0
0.0 1.2 2.1 2.3 5.5 3.2 6.3 5.4 6.9
Control
2.0
0.0
0.0
3.0
3.0
7.9
1.0
TABLE 3 C H R O M O S O M A L ABERRATIONS OBSERVED IN METAPHASES OF H U M A N LYMPHOCYTES FOLLOWING EXPOSURE TO DECREASED pH The cells were cultured in G1 (27 h) for 60 min in medium of decreased pH and recovered in medium of normal pH for 22 h. 100 metaphases were scored, pH
Chromatid breaks
7.2 (control) 7.0 6.5 6.0
Total number of breaks
% Cells showing
1 . 0
0.0 2.0 0.0
Isochromatid breaks
Chromatid exchanges
Chromosome exchanges
All aberrations
0,0
0.0
0.0
1.0
1.0
0.0 0.0 1.0
0,0 0.0 0.0
0,0 0.0 0.0
0.0 2.0 1.0
0.0 2,0 1.0
the S phase (Nowak, 1989), in human lymphocytes we could not find such an S-independent mode of action. The mechanisms by which hypotonic treatment acts on the chromatin and subsequently leads to chromosomal aberrations are as yet unknown. In previous papers (Nowak, 1987, 1989) we discussed
(Nowak, 1987) while human lymphocytes seem to be less sensitive to the induction of chromosomal aberrations, but when we compare toxicity, V79 hamster cells and human lymphocytes react quite similarly. In V79 hamster cells we could clearly show that the induction of chromosomal aberrations after hypotonic treatment is independent of TABLE 4
S I S T E R - C H R O M A T I D E X C H A N G E S AND P O L Y P L O I D M2 CELLS OBSERVED IN METAPHASES OF H U M A N LYMPHOCYTES F O L L O W I N G E X P O S U R E TO D I L U T E D McCOY'S MEDIUM The cells were cultured for 27 h, followed by a 60-rain treatment in hypotonic medium, Fixation was done 44 h later. 25 metaphases were scored per experiment and concentration. Experiment A
Experiment B
Experiment C
Experiment D
Experiment E
of treatment
SCE
Poly-
SCE
Poly-
SCE
Poly
medium ( m O s m / k g H20)
per meta-
ploids (070)
per meta-
ploids (070)
per meta-
ploids (o70)
SCE per meta-
SCE per meta-
Osmolality
phase
phase
Polyploids (°7o)
phase
phase
Polyploids (07o)
phase
300 (control)
4.2
0.0
6.8
0.0
8.7
0,0
6.5
0.0
6.8
0.0
90 75 60
6.9 5.7 6.1
0.0 0,0 3.0
7,1 7,0 7.4
0.0 0,0 5.0
_a 6.8
2.0
55 50 45
toxic
40 35 30
. toxic
2.0 1.0 3.0 5.0 7.0 9.0
-
7.2 6.4 6.9 7.8 7.1 8.1 toxic toxic
4,0 0.0 2.0 4.0 2,0 5.0
25
6.3 6.4 6.1 7.3 6.4 7.8 toxic toxic
a Not tested.
8.8 .
-
. -
-
7.6
-
8.0 -
. 10.3
. 3.0 -
1.0
-
7,6 toxic
3.0
possible mechanisms and came to the conclusion that probably lysosomal damage is involved. Lysosomal damage can occur because of the lowered osmolality, leading to a release of enzymes such as DNase, which subsequently act on the DNA. Whether such a mechanism is also responsible for the aberrations found in human lymphocytes we do not know yet. We did 5 independent experiments on the induction of sister-chromatid exchanges by hypotonic culture conditions (Table 4). The results clearly show that hypotonic treatment does not lead to increased frequencies of sister-chromatid exchanges. Galloway et al. (1987) treated CHO cells with hypertonic media and found a slight increase in SCEs, and they speculated that this increase could be due to a reduction in cell density of growing cells by cell killing and cell-cycle delay. They also speculated about an increased BrdUrd uptake because of the reduced number of living cells. Following treatment in medium with low osmolalities we observed increased frequencies of differentially stained polyploid metaphases (Table 4). Polyploid metaphases were also found in CHO cells cultured in hypertonic media (Galloway et al., 1987). Electron microscopic observations of Brinkley et al. (1980) showed that hypotonic treatment leads to spindle destruction, depolymerization of spindle microtubules, and disrupted kinetochores. These findings may indicate that the proper assembly of spindle microtubules is disturbed by hypotonic conditions. References Ashby, J., and M. Ishidate (1986) Clastogenicity in vitro of the Na, K, and Mg salts of saccharin; and of magnesium chloride; consideration of significance, Mutation Res., 163, 63-73. Brinkley, B.R., S.M. Cox and D.A. Pepper (1980) Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro, Cytogenet. Cell. Genet., 26, 165-175.
Brusick, D. (1986) Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations, Environ. Mol. Mutagen., 8, 879-886. Galloway, S.M., C.L. Bean, M.A. Armstrong, D. Deasy, A. Kraynak and M.O. Bradley (1985) False positive in vitro chromosome aberration tests with non-mutagens at high ion concentrations and osmolalities, Environ. Mol. Mutagen., 7, 1-51. Galloway, S.M., A. Deasy, C.L. Bean, A.R. Kraynak, M.J. Armstrong and M.O. Bradley (1987) Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks and the relation to toxicity, Mutation Res., 189, 15-25. Garry, R.F., M.P. Moyer, J.M. Bishop, R.C. Moyer and M.R.F. Waite (1981) Transformation paremeters induced in chick cells by incubation in media of altered NaC1 concentration, Virology, 111,427-439. Ishidate, M., T. Sofuni, K. Yoshikawa, M. Hayashi, T. Nohmi, M. Sawada and A. Matsuoka (1984) Primary mutagenicity screening of food additives currently used in Japan, Food Chem. Toxicol., 22, 623-636. Nowak, C. (1987) Studies on the ability of hypotonic solutions to induce chromosomal aberrations in V79 cells, Teratogen. Mutagen. Carcinogen., 7, 515-525. Nowak, C. (1989) Induction of chromosomal aberrations by hypotonic culture conditions is independent of the S-phase in V79 hamster cells, Env. Mol. Mutagen., 13, 44-49. Nowak, C., and S. Kalweit (1987) Anisotonic culture conditions induce chromosomal aberrations in V79 hamster cells, Mutation Res., 182, 275-293. Raaphorst, G.P., and J. Kruuv (1976) Effect of tonicity on radiosensitivity of mammalian cells, Int. J. Radiat. Biol., 29, 493-500. Raaphorst, G.P., and J. Kruuv (1977a) Effect of salt solutions on radiosensitivity of mammalian ceils, Int. J. Radiat. Biol., 32, 71-88. Raaphorst, G.P., and J. Kruuv (1977b) Effect of salt solutions on radiosensitivity of mammalian cells. 11. Treatment with hypotonic solutions, Int. J. Radiat. Biol., 32, 89-101. Raaphorst, G.P., H.E. Frey and J. Kruuv (1977) Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions, Int. J. Radiat. Biol., 32, 109-126. Seeberg, A.H., P. Mosesso and R. Forster (1988) High doselevel-effects in mutagenicity assays utilising mammalian cells in culture, Mutagenesis, 3,213-218. Tuschy, S., and G. Obe (1988) Potentiation of Alu l-induced chromosome aberrations by high salt concentrations in Chinese hamster ovary cells, Mutation Res., 207, 83-87. Communicated by F.H. Sobels
5
Elsevier MUTLET 0387
Hypotonic treatment leads to chromosomal aberrations but not to sisterchromatid exchanges in human lymphocytes S. K a l w e i t 1, C. N o w a k 1 a n d G. O b e 1' 2 1Freie Universitiit Berlin, lnstitut fiir Genetik, D-IO00 Berlin 33 (Germany) and 2University GHS Essen, FB 9, Department of Genetics, D-4300 Essen I (F.R.G.)
(Accepted9 April 1990)
Keywords: Hypotonictreatment;Chromosomeaberration;Humanlymphocytes
Summary Human peripheral lymphocytes were isolated from whole blood and exposed to culture medium of reduced osmolality. This hypotonic treatment led to a significant increase in the frequencies of chromosomal aberrations when the osmolality was reduced to 60 mOsm/kg H20 and below. Maximum damage occurred when the hypotonic treatment was done 27 or 30 h after starting the cultures. We also looked for the induction of sister-chromatid exchanges (SCE) by hypotonic culture conditions, but the SCE frequencies were not influenced.
During the past years it has become obvious that osmotic stress can damage mammalian cells. Various biological effects, such as radiosensitization (Raaphorst and Kruuv, 1976, 1977a,b; Raaphorst et al., 1977), cytotoxicity (Galloway et al., 1987), gene mutations (Seeberg et al., 1988), cell transformation (Garry et al., 1981), or chromosomal aberrations (Ashby and Ishidate, 1986; Brusick, 1986; Galloway et al., 1985, 1987; Ishidate et al., 1984; Nowak, 1987; Nowak and Kalweit, 1987; Tuschy and Obe, 1988), have been observed following hyper- or hypo-tonic treatment.
Studies on the induction of chromosomal aberrations by hypotonic culture conditions have so far been done with V79 hamster cells (Nowak, 1987; Nowak and Kalweit, 1987). When the osmolality of the medium, which is usually 300 mOsm/kg H20, was reduced to 60 mOsm/kg H20, high frequencies of chromosomal aberrations and totally damaged cells were found. In this study we investigated the effect of hypotonic treatment conditions on the chromosomes of human lymphocytes in vitro. Two genetic endpoints, namely chromosomal aberrations and sister-chromatid exchanges, were analyzed, following exposure to media of different osmolalities.
Correspondence: Prof. Dr. G. Obe, Universityof Essen GHS, FB 9, Departmentof Genetics,P.O. Box 103764, D-4300Essen 1 (F.R.G.).
Materials and methods Human peripheral lymphocytes were isolated
0165-7992/90/$ 03.50 © 1990ElsevierSciencePublishers B.V. (BiomedicalDivision)
from whole blood by sucrose gradient centrifugation. 1 × 106 cells were seeded into 2.5-ml cultures, containing 2.0 ml M c C o y ' s medium (Seromed), 20°7o fetal calf serum (Seromed), 0.06 ml phytohemagglutinin (PHA, Seromed) and 0.05 ml bromodeoxyuridine (BrdUrd, final concentration 2 × 10 -~ M, Serva). The cells were cultivated for 50 or 72 h including a 5-h treatment with Colcemid (0.08/zg/ml). Preparations were made following a routine protocol and slides were stained differentially using 5% Giemsa stain. For chromosomal aberrations (CA) at least 100 uniformly stained (M1) metaphases from 50-h cultures and for sisterchromatid exchanges (SCE) 25 differentially stained (M2) metaphases from 72-h cultures were analyzed. Mitotic indices (MIs) were determined by scoring 1000 cells per entry and are given as percent mitoses. In 72-h cultures the frequencies of polyploid metaphases were determined in the population o f Mz metaphases. The experiments were performed 2-5 times.
Hypotonic treatment M c C o y ' s 5A medium has an osmolality o f 300
m O s m / k g H20. The osmolality was reduced by adding distilled water and the resulting osmolalities were measured with an osmometer. A dilution o f 1 part medium with 9 parts distilled water results in an osmolality of 30 m O s m / k g H20. The pH of the M c C o y ' s medium changed upon dilution. Undiluted medium had a pH of 7.2, dilution to 200 m O s m / k g H 2 0 had no influence on pH, but the p H dropped to 6.0 in medium o f 45 m O s m / k g H20. Lymphocytes were cultured for at least 21 h, pelleted and the supernatant was kept at 37°C as conditioned medium. The cells were incubated for 60 min in 2.5 ml serum-free prewarmed hypotonic medium. The hypotonic medium was discarded and the cells were recovered in conditioned medium. In order to analyze whether a reduction of the pH has any influence on the frequencies o f chromosomal aberrations the cells were exposed at 27 h culture time to 2.5 ml serum-free prewarmed medium o f p H 7.0, 6.5 and 6.0 for 60 min. The pH was adjusted by addition of 1 N HCI and measured with a pH meter. The cells were recovered after treatment for 22 h in complete medium of pH 7.2.
TABLE 1 CHROMOSOMALABERRATIONSOBSERVEDIN METAPHASESOF HUMAN LYMPHOCYTESFOLLOWINGEXPOSURE TO DILUTED McCOY'S MEDIUM The cells were cultured in G1 (27 h) for 60 min in hypotonic medium and recovered in isotonic medium for 22 h. 2 independent experiments are given, 100 metaphases per experiment were scored. Osmolality
o7oCells showing
of hypotonic medium (mOsm/kg H20)
Chromatid breaks
300 (control) 180 150 120 90 75 60 45 30
Mitotic index (°70)
Chromatid exchanges
Chromosome exchanges
0.5
0.0
0.0
1.0
1.0
5,2
1.0 0.0 1.5 0.0 0.5 0.5 0.0 1.0 1.0 1.5 13.0 4.5 9.0 9.0 No metaphases
0.0 0.0 0.0 0.0 0.0 0.5 1.0
0.0 0.0 0.0 0.0 0.5 0.0 0.0
1.0 1.5 0.5 1.0 3.0 14.0 15,0
1.0 1.5 1.0 1.0 3.5 18.5 20.0
6.8 3.9 5.9 4.2 2,7 1.9 0.8 0.0
0.5
All aberrations
Total number of breaks
Isochromatid breaks
The total number of breaks was calculated as follows: chromatid and isochromatid breaks = 1; chromatid and chromosome exchanges = 2.
Results and discussion The frequencies of chromosomal aberrations and mitotic indices are compiled in Tables 1-3. When lymphocytes are exposed to hypotonic culture conditions 27 h after stimulation (Table 1), which probably represents a G~ population of cells, the frequencies of chromosomal aberrations are in the control range until an osmolality of 75 mOsm/kg H20. Hypotonic treatment with 60 and 45 mOsm/kg H20 leads to a clear increase in chromosomal aberrations. When we treat the cells with 30 mOsm/kg H20 a total suppression of the mitotic index is found. For further experiments we chose 60 mOsm/kg H20, an osmolality which induces chromosomal aberrations at reasonable toxicity. Table 2 shows the results of a hypotonic treatment with 60 mOsm/kg H20 at different stages of the cell cycle. Up to a culture time of 21 h, representing cells in their Go and early GI phases, no metaphases are found, the lymphocytes seem to be extremely sensitive to hypotonic culture conditions. Hypotonic treatment 27 or 30 h after stimulation leads to
clearly increased frequencies of chromosomal aberrations. But when the hypotonic treatment is done 36, 39, 42 or 45 h after stimulation chromosomal aberrations are not increased. These results show that hypotonic culture conditions can lead to chromosomal aberrations in human lymphocytes, but the total number of breaks in 100 metaphases never exceeds 20. And it is only possible to induce chromosomal aberrations during a short period of the cell cycle, probably at the end of the G1 phase. Undiluted culture medium has a pH of 7.2, but a hypotonic solution of 45 mOsm/kg H20, prepared by dilution with distilled water, has a pH of 6.0. In order to exclude pH effects on the induction of chromosomal aberrations we cultivated human lymphocytes in isotonic medium with pH values of 7.0, 6.5 and 6.0. Table 3 shows that a pH drop does not lead to chromosomal aberrations when the osmolality is kept normal (300 mOsm/kg H20). A comparison of data from this study with V79 hamster cells reveals some remarkable differences. In V79 cells hypotonic treatment leads to extraordinarily high aberration frequencies
TABLE 2 C H R O M O S O M A L A B E R R A T I O N S OBSERVED IN M E T A P H A S E S OF H U M A N L Y M P H O C Y T E S F O L L O W I N G E X P O S U R E T O D I L U T E D M c C O Y ' S M E D I U M OF 60 m O s m / k g H 2 0 The cells were treated at different culture times for 60 min and fixation was done 50 h after starting the cultures. 2 independent experiments are given, 100 rnetaphases per experiment were scored. Treatment
°7o Cells showing
Total
Mitotic
time (h)
Chro-
Iso-
Chro-
Chro-
All
number
index
matid breaks
chromatid breaks
matid exchanges
mosome exchanges
aberrations
of breaks
(07o)
21 24 27 30 33 36 39 42 45
Nomethaphases 6.0 2.0 4.5 3.0 7.5 6.0 3.5 3.0 2.5 0.5 1.0 1.0 2.0 1.0 4.0 1.0
0.0 1.5 1.0 0.5 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
8.0 8.5 8.5 5.0 2.5 2.0 1.5 4.0
8.0 10.5 15.5 7.5 3.0 2.0 3.0 5.0
0.0 1.2 2.1 2.3 5.5 3.2 6.3 5.4 6.9
Control
2.0
0.0
0.0
3.0
3.0
7.9
1.0
TABLE 3 C H R O M O S O M A L ABERRATIONS OBSERVED IN METAPHASES OF H U M A N LYMPHOCYTES FOLLOWING EXPOSURE TO DECREASED pH The cells were cultured in G1 (27 h) for 60 min in medium of decreased pH and recovered in medium of normal pH for 22 h. 100 metaphases were scored, pH
Chromatid breaks
7.2 (control) 7.0 6.5 6.0
Total number of breaks
% Cells showing
1 . 0
0.0 2.0 0.0
Isochromatid breaks
Chromatid exchanges
Chromosome exchanges
All aberrations
0,0
0.0
0.0
1.0
1.0
0.0 0.0 1.0
0,0 0.0 0.0
0,0 0.0 0.0
0.0 2.0 1.0
0.0 2,0 1.0
the S phase (Nowak, 1989), in human lymphocytes we could not find such an S-independent mode of action. The mechanisms by which hypotonic treatment acts on the chromatin and subsequently leads to chromosomal aberrations are as yet unknown. In previous papers (Nowak, 1987, 1989) we discussed
(Nowak, 1987) while human lymphocytes seem to be less sensitive to the induction of chromosomal aberrations, but when we compare toxicity, V79 hamster cells and human lymphocytes react quite similarly. In V79 hamster cells we could clearly show that the induction of chromosomal aberrations after hypotonic treatment is independent of TABLE 4
S I S T E R - C H R O M A T I D E X C H A N G E S AND P O L Y P L O I D M2 CELLS OBSERVED IN METAPHASES OF H U M A N LYMPHOCYTES F O L L O W I N G E X P O S U R E TO D I L U T E D McCOY'S MEDIUM The cells were cultured for 27 h, followed by a 60-rain treatment in hypotonic medium, Fixation was done 44 h later. 25 metaphases were scored per experiment and concentration. Experiment A
Experiment B
Experiment C
Experiment D
Experiment E
of treatment
SCE
Poly-
SCE
Poly-
SCE
Poly
medium ( m O s m / k g H20)
per meta-
ploids (070)
per meta-
ploids (070)
per meta-
ploids (o70)
SCE per meta-
SCE per meta-
Osmolality
phase
phase
Polyploids (°7o)
phase
phase
Polyploids (07o)
phase
300 (control)
4.2
0.0
6.8
0.0
8.7
0,0
6.5
0.0
6.8
0.0
90 75 60
6.9 5.7 6.1
0.0 0,0 3.0
7,1 7,0 7.4
0.0 0,0 5.0
_a 6.8
2.0
55 50 45
toxic
40 35 30
. toxic
2.0 1.0 3.0 5.0 7.0 9.0
-
7.2 6.4 6.9 7.8 7.1 8.1 toxic toxic
4,0 0.0 2.0 4.0 2,0 5.0
25
6.3 6.4 6.1 7.3 6.4 7.8 toxic toxic
a Not tested.
8.8 .
-
. -
-
7.6
-
8.0 -
. 10.3
. 3.0 -
1.0
-
7,6 toxic
3.0
possible mechanisms and came to the conclusion that probably lysosomal damage is involved. Lysosomal damage can occur because of the lowered osmolality, leading to a release of enzymes such as DNase, which subsequently act on the DNA. Whether such a mechanism is also responsible for the aberrations found in human lymphocytes we do not know yet. We did 5 independent experiments on the induction of sister-chromatid exchanges by hypotonic culture conditions (Table 4). The results clearly show that hypotonic treatment does not lead to increased frequencies of sister-chromatid exchanges. Galloway et al. (1987) treated CHO cells with hypertonic media and found a slight increase in SCEs, and they speculated that this increase could be due to a reduction in cell density of growing cells by cell killing and cell-cycle delay. They also speculated about an increased BrdUrd uptake because of the reduced number of living cells. Following treatment in medium with low osmolalities we observed increased frequencies of differentially stained polyploid metaphases (Table 4). Polyploid metaphases were also found in CHO cells cultured in hypertonic media (Galloway et al., 1987). Electron microscopic observations of Brinkley et al. (1980) showed that hypotonic treatment leads to spindle destruction, depolymerization of spindle microtubules, and disrupted kinetochores. These findings may indicate that the proper assembly of spindle microtubules is disturbed by hypotonic conditions. References Ashby, J., and M. Ishidate (1986) Clastogenicity in vitro of the Na, K, and Mg salts of saccharin; and of magnesium chloride; consideration of significance, Mutation Res., 163, 63-73. Brinkley, B.R., S.M. Cox and D.A. Pepper (1980) Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro, Cytogenet. Cell. Genet., 26, 165-175.
Brusick, D. (1986) Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations, Environ. Mol. Mutagen., 8, 879-886. Galloway, S.M., C.L. Bean, M.A. Armstrong, D. Deasy, A. Kraynak and M.O. Bradley (1985) False positive in vitro chromosome aberration tests with non-mutagens at high ion concentrations and osmolalities, Environ. Mol. Mutagen., 7, 1-51. Galloway, S.M., A. Deasy, C.L. Bean, A.R. Kraynak, M.J. Armstrong and M.O. Bradley (1987) Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks and the relation to toxicity, Mutation Res., 189, 15-25. Garry, R.F., M.P. Moyer, J.M. Bishop, R.C. Moyer and M.R.F. Waite (1981) Transformation paremeters induced in chick cells by incubation in media of altered NaC1 concentration, Virology, 111,427-439. Ishidate, M., T. Sofuni, K. Yoshikawa, M. Hayashi, T. Nohmi, M. Sawada and A. Matsuoka (1984) Primary mutagenicity screening of food additives currently used in Japan, Food Chem. Toxicol., 22, 623-636. Nowak, C. (1987) Studies on the ability of hypotonic solutions to induce chromosomal aberrations in V79 cells, Teratogen. Mutagen. Carcinogen., 7, 515-525. Nowak, C. (1989) Induction of chromosomal aberrations by hypotonic culture conditions is independent of the S-phase in V79 hamster cells, Env. Mol. Mutagen., 13, 44-49. Nowak, C., and S. Kalweit (1987) Anisotonic culture conditions induce chromosomal aberrations in V79 hamster cells, Mutation Res., 182, 275-293. Raaphorst, G.P., and J. Kruuv (1976) Effect of tonicity on radiosensitivity of mammalian cells, Int. J. Radiat. Biol., 29, 493-500. Raaphorst, G.P., and J. Kruuv (1977a) Effect of salt solutions on radiosensitivity of mammalian ceils, Int. J. Radiat. Biol., 32, 71-88. Raaphorst, G.P., and J. Kruuv (1977b) Effect of salt solutions on radiosensitivity of mammalian cells. 11. Treatment with hypotonic solutions, Int. J. Radiat. Biol., 32, 89-101. Raaphorst, G.P., H.E. Frey and J. Kruuv (1977) Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions, Int. J. Radiat. Biol., 32, 109-126. Seeberg, A.H., P. Mosesso and R. Forster (1988) High doselevel-effects in mutagenicity assays utilising mammalian cells in culture, Mutagenesis, 3,213-218. Tuschy, S., and G. Obe (1988) Potentiation of Alu l-induced chromosome aberrations by high salt concentrations in Chinese hamster ovary cells, Mutation Res., 207, 83-87. Communicated by F.H. Sobels
