Mutagenesis vol. 18 no. 6 pp. 539-543,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
A new cytogenetic approach for the evaluation of mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase
1Health Physics and Environmental Hygiene, National Centre for Scientific Research ‘Demokritos’, Agia Paraskevi, Athens and 2National Institute for Occupational Medicine and Safety, Athens, Greece
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
The aim of the present study was to develop and standardize a cytogenetic approach for evaluation of the mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase. Even though cytogenetic end-points such as sister chromatid exchange (SCE) have been extensively used to indirectly assess the DNA-damaging potential of various chemicals, they are based on metaphase chromosome analysis. Cells delayed in G2 phase after chemical exposure are not included in conventional SCE analysis. The yield of SCEs obtained, therefore, can be biased, since predominantly undamaged cells proceed to metaphase without delay. To overcome this shortcoming of conventional SCE analysis, the use of a new cytogenetic approach for genotoxic studies is presented that enables the analysis of SCEs directly in G2 phase using drug-induced premature chromosome condensation in cultured peripheral blood lymphocytes. By means of this method, firstly, the possibility that SCE analysis in metaphase chromosomes underestimates the mutagenic potential of various chemicals was tested. Secondly, whether the genotoxic potential of suspected carcinogens could be evaluated using SCE analysis in G2 phase, even at exposures that arrest cells in G2 phase, was examined. Thirdly, whether an important part of the background variation in SCE frequency among individuals is due to the delay of affected cells in G2 phase, rather than to a true biological variation in the cytogenetic end-point used, was tested. The results showed that a higher SCE frequency was scored in G2 phase than in metaphase. Subsequently, the mutagenic potential of chemicals that temporarily arrest cells in G2 phase could now be evaluated more accurately. In addition, it may be of interest to further examine the involvement of cell cycle kinetics in the baseline SCE variation among individuals since a lesser SCE variability was observed when the analysis was carried out in G2 phase rather than at metaphase.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Sister chromatid exchange (SCE) analysis in human peripheral blood lymphocytes has often been applied as a cytogenetic assay for biomonitoring and genotoxicity testing of potentially mutagenic and carcinogenic chemicals (Natarajan, 2002
). At present, however, there are two main practical problems with respect to the use of SCE analysis. First, the SCE frequencies after exposure to genotoxic chemicals are obtained scoring only cells that have reached metaphase following exposure to non-cytotoxic chemical doses. Affected cells that have been arrested in G2 phase of the cell cycle are not included in the analysis. Since predominantly undamaged cells proceed to metaphase without delay, conventional SCE analysis may underestimate the clastogenic and mutagenic potential of various chemicals considered as possible human carcinogens. Thus, there is a need for a method to score SCEs directly in G2 phase prematurely condensed chromosomes (G2 PCCs) and more accurately evaluate the mutagenic potential of chemicals that induce cell cycle arrest in G2 phase. Second, the SCE baseline fluctuates among individuals and between studies (Schwartz et al., 1990). It depends on the concentration of incorporated bromodeoxyuridine (BrdU) in the DNA (Natarajan et al., 1986) and such a variation may sometimes be higher than the effect associated with exposure to genotoxic carcinogens. Since only cells that proceed to metaphase can presently be analyzed using SCEs, it could be of interest to examine whether a major part of this background variation is due to differences in cell cycle kinetics rather than due to a true biological variation in the end-point used. A method to simultaneously visualize interphase and metaphase chromosomes, thus enabling independent SCE scoring in G2 phase and metaphase cells, could facilitate the elucidation of this issue.
The visualization of interphase chromosomes in peripheral blood lymphocytes and their use for biomonitoring purposes following exposure to genotoxic agents first became possible using a method for cell fusion and premature chromosome condensation (PCC) induction (Pantelias and Maillie, 1983, 1984). Thereafter, researchers have examined interphase chromosomal damage in lymphocytes using the PCC methodology, which has proved to be a powerful cytogenetic tool for the identification of factors involved in the conversion of DNA damage into chromosomal damage (Terzoudi and Pantelias, 1997), thus affecting sensitivity to genotoxic agents (Terzoudi et al., 2000).
In this report, a simple and easy protocol is presented for the analysis of SCEs directly in G2 phase peripheral blood lymphocytes. The methodology is based on PCC induction using calyculin A, a potent inhibitor of protein phosphatases types 1 and 2A (Asakawa and Gotoh, 1997; Coco-Martin and Begg, 1997; Durante et al., 1998; Gotoh et al., 1999), and the visualization of SCEs by applying the fluorescence-plus-Giemsa (FPG) technique (Perry and Wolff, 1974; Jan et al., 1982) in G2 PCCs. This cytogenetic approach may be more sensitive than conventional SCE analysis of metaphase cells since it overcomes some of its shortcomings and may be unique for screening possible human carcinogens that induce cell cycle arrest in G2 phase, with respect to their genotoxic activity.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Culture conditions and premature chromosome condensation induction in G2 phase
Peripheral blood was taken with heparinized syringes from healthy individuals. An aliquot of 0.5 ml of whole blood was added to each culture tube containing 5 ml of McCoy’s 5A medium supplemented with 10% fetal calf serum, 1% glutamine, 1% antibiotics (penicillin and streptomycin), 1% phytohemaglutinin and incubated at 37°C for 72 h in a humidified incubator in an atmosphere of 5% CO2/95% air. For PCC induction in G2 phase lymphocytes, calyculin A (Sigma-Aldrich) was used. In order to determine the optimum conditions for PCC induction and scoring, calyculin A was added to the whole blood cultures at doses of 10, 50 and 100 nM during the final 0.5, 1 or 3 h of incubation. Replicate cultures were also made containing 0.05 µg/ml colcemid during the last 3 h of culture but not treated with calyculin A. The frequency of cells with PCCs expressed as a percentage of all nuclei observed was scored at low magnification (x200) along lines crossing the center of the spread drop of chromosome preparations. About 300 cells per experimental point were analyzed. The frequencies of cells with fully condensed or partially condensed chromosomes or unaffected by calyculin A, as well as cells at metaphase, were scored for each dose and treatment time.
Sister chromatid exchanges in G2 and M phase lymphocytes
5-BrdU (Sigma) was added at a final concentration of 20 µM 24 h after culture initiation. Cultures were incubated at 37°C for 72 h prior to cell harvest. During this culture period, incorporation of BrdU into replicating cells allows for the unequivocal identification of second division metaphase cells. The cultured cells were treated with hypotonic (0.075 M) KCl, fixed with methanol/acetic acid (3:1) and 20 µl of cell suspension was dropped on wet slides. Air dried slides were stored in the dark. For visualization of SCEs, the slides were stained by the FPG technique according to the Perry and Wolff (1974) and Jan et al. (1982) protocols. A few drops of Hoechst 33258 (5 µg/ml) in Sorensen buffer (pH 6.8) were placed on each slide and covered with coverslips. They were then placed on a slide warmer set at 55°C and exposed to a black light fluorescent lamp (Radium SupraBlack HBT 125-281) at a distance of 2 cm for 10 min. Coverslips were removed by soaking the slides in Sorensen’s buffer and the slides were stained with 3% Giemsa solution (Gurr R66 in Sorensen’s buffer) for 15 min. The slides were finally mounted with coverslips and coded for analysis to avoid bias. For SCE scoring, the criteria suggested by Carrano and Natarajan (1988) were applied. Only second division metaphases and G2 PCCs, identifiable by their uniform differential staining pattern, containing 46 chromosomes were analyzed.
To test whether the mutagenic potential of possible genotoxic agents may be underestimated when the conventional SCE analysis is applied and also for the assessment of exposures that arrest cells at G2 phase, whole blood cultures were treated for the last 24 h of the total 72 h culture period with the following chemicals: the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) was used at concentrations of 92.7, 463 and 927 µM; the herbicide gramoxone, also known as paraquat dichloride (1,1-dimethyl-4,4-bipyridium) at a concentration of 500 µM; hydroquinone (1,4 benzenediol) at concentrations of 50 and 200 µM. All chemicals were obtained from Sigma-Aldrich, Germany. Mitomycin C (Kyowa Hakko Kogyo Co. Ltd, Japan) was prepared in RPMI medium and used as a positive control at a final concentration of 0.3 µM. Calyculin A was dissolved in absolute ethanol, atrazine was prepared in dimethyl sulfoxide (DMSO), paraquat was dissolved in distilled water and hydroquinone in phosphate buffer solution (PBS).
For each experiment and chemical concentration within an experimental set, a minimum of three lymphocyte cultures were run. Routinely, 30–50 cells from each culture were scored for SCEs. Standard deviations of the mean values from three independent experiments were calculated for each experimental point. Data were evaluated statistically by Student’s t-test.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Using cytogenetic end-points such as chromosomal damage and SCE analysis, a large number of studies have been carried out on the in vivo and in vitro genotoxicity of possible human carcinogens. The results, however, are not always conclusive and sometimes conflicting cytogenetic findings have been reported. Particularly, when the frequency of SCEs is slightly increased with respect to the controls after exposure in the range of non-cytotoxic doses, the chemical activity is characterized as minimal (Kligerman et al., 2000
). Even though the use of higher chemical doses could clarify whether the induced SCE frequency is dose-dependent, they cannot be applied since the affected cells will be arrested in G2 phase and not proceed to metaphase, at least temporarily, preventing their analysis using the conventional SCE methodology.
The proposed cytogenetic approach for genotoxic studies in this report overcomes this shortcoming of the conventional method and is based on the visualization and analysis of SCEs in lymphocyte G2 PCCs. Examples of SCEs as visualized in G2 PCCs are shown in Figures 1 and 2. In both control (Figure 1A) and exposed chromosome spreads (Figures 1B and 2A) the sister chromatids in each chromosome are aligned in close contact, parallel to each other, and the centromeres are not clearly visible. As a result, dicentrics and acentric fragments cannot be easily identified and this is a major shortcoming when drug-induced G2 PCCs and solid Giemsa stain are used for conventional chromosome aberration analysis (Gotoh and Asakawa, 1996; Kanda et al., 1999). For SCE analysis and its use for genotoxic studies, however, the fact that the centromere position becomes unclear in drug-induced G2 PCCs seems not to be a problem. On the contrary, this feature differentiates the appearance of G2 PCCs from that of chromosomes at metaphase (Figure 2B), thus enabling the simultaneous analysis of SCEs in G2 PCCs and in metaphase chromosomes on the same chromosome preparations after exposure to genotoxic agents.
|
|
The experiments conducted here aim at the standardization of a simple protocol for SCE analysis in G2 PCCs, verification of a possible underestimation of the mutagenic potential of various chemicals when the conventional SCE analysis is applied and also the assessment of exposures that arrest cells in G2 phase. Four sets of experiments were carried out.
In the first set the appropriate conditions for PCC induction and visualization of SCEs in G2 PCCs of peripheral blood lymphocytes were obtained. As shown in Table I, treatments with 50 nM calyculin A for 2 h as well as with 100 nM for 1 h resulted in the highest percentage of cells with PCCs. However, the chromosomes in the majority of the cells became fuzzy and shortened when the 72 h blood cultures were subsequently treated with calyculin A for >1 h. For this reason, considering chromosome morphology as well, a 1 h treatment with 50 nM calyculin A was chosen as optimum for PCC induction and SCE analysis in G2 phase lymphocytes.
|
To test whether the mutagenic potential of possible carcinogens could be carried out even at exposures that arrest lymphocytes in G2 phase, and particularly in order to test whether the induction of SCEs is dose-dependent at high doses, a second set of experiments was conducted, and the results are shown in Table II. When the chemicals atrazine and hydroquinone were used in blood cultures at very high concentrations (toxic), SCEs could not be scored using conventional SCE analysis as no cells at mitosis were present at this experimental point. However, as is shown in Table II, by using premature chromosome condensation, a genotoxic assessment and the yields of SCEs per cell in G2 phase were easily obtained, even at doses exceeding the toxic limits. For the case of atrazine, an increase in concentration from 463 to 927 µM did not increase the frequency of SCEs, as scored in lymphocyte G2 PCCs. This finding does not support a genotoxic mode of action of this chemical.
|
In the third set of experiments the proposed cytogenetic approach was applied to test whether SCE analysis in metaphase chromosomes is the most sensitive method to estimate the genotoxic potential of various suspected carcinogens. The results and their statistical analysis are presented in Table III. A much higher SCE yield per cell was scored in G2 PCCs than in cells at metaphase. These results suggest that using conventional SCE analysis at metaphase cells, the mutagenic potential of chemicals that temporarily arrest cells in the G2 phase of the cell cycle could have been underestimated.
|
In the fourth set of experiments, the involvement of cell cycle kinetics in the variation in baseline SCEs among individuals was examined. The results and their statistical analysis are shown in Table IV. On average the SCE frequency obtained in G2 phase is significantly higher (0.01 < P < 0.001) than that obtained in metaphase. In addition, a lesser SCE variability was observed when the analysis was carried out in G2 PCCs than in metaphase cells. The range for spontaneous SCEs per cell among healthy individuals was 4.5–10.5, with a coefficient of variation (CV) of 28.3%, when analyzed in cells at metaphase, whereas the range was 7.0–8.5, with a CV of 7.6%, when SCEs were scored in G2 PCCs. These results suggest that an important part of the background variation in the frequency of SCEs observed among individuals may be due to differences in cell cycle kinetics rather than to a true biological variation in the end-point used.
|
In conclusion, in this work a simple protocol for SCE analysis in G2 PCCs is presented that overcomes some of the disadvantages of conventional SCE analysis in metaphase cells. This cytogenetic approach enables the analysis of SCEs in cells arrested in the G2 phase of the cell cycle and can be easily applied since it requires only standard cytogenetic laboratory equipment. Therefore, it can be performed in most biomonitoring laboratories in order to assess the genotoxic effect of chemical exposures even at doses that arrest cells in G2 phase. In particular, it may be a unique method for investigating whether conventional SCE analysis in peripheral blood lymphocytes underestimates the clastogenic and mutagenic potential of various chemicals considered as possible human carcinogens, as well as for elucidating the biological basis of the variability in the frequency of SCEs observed among individuals.
|
Notes |
|---|
3To whom correspondence should be addressed. Tel: +30 210 6503865; Fax: +30 210 6534710; Email: georgia@ipta.demokritos.gr
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
-
Asakawa,Y. and Gotoh,E. (1997) A method for detecting sister chromatid exchanges using prematurely condensed chromosomes and immunogold-silver staining. Mutagenesis, 12, 175–177.
Carrano,A.V. and Natarajan,A.T. (1988) Consideration for population monitoring using cytogenetic techniques. International Commission for Protection against Environmental Mutagens and Carcinogens publication no. 14. Mutat. Res., 204, 397–406.
Coco-Martin,J.M. and Begg,A.C. (1997) Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumour cell lines with different radiosensitivities. Int. J. Radiat. Biol., 71, 265–273.[CrossRef][Web of Science][Medline]
Durante,M., Furusawa,Y. and Gotoh,E. (1998) A simple method for simultaneous interphase-metaphase chromosome analysis in biodosimetry. Int. J. Radiat. Biol., 74, 457–462.[CrossRef][Web of Science][Medline]
Gotoh,E. and Asakawa,Y. (1996) Detection and evaluation of chromosomal aberrations induced by high doses of 
-irradiation using immunogold-silver painting of prematurely condensed chromosomes. Int. J. Radiat. Biol., 70, 517–520.[CrossRef][Web of Science][Medline]
Gotoh,E., Kawata,T. and Durante,M. (1999) Chromatid break rejoining and exchange aberration formation following -ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation. Int. J. Radiat. Biol., 75, 1129–1135.[CrossRef][Web of Science][Medline]
Jan,K.Y., Wang-Wuu,S. and Wen,W.N. (1982) A simplified fluorescence plus Giemsa method for consistent differential staining of sister chromoatids. Stain Technol., 57, 45–46.[Web of Science][Medline]
Kanda,R., Hayata,I. and Lloyd,D.C. (1999) Easy biodosimetry for high-dose radiation exposures using drug-induced, prematurely condensed chromosomes. Int. J. Radiat. Biol.,75, 441–446.
Kligerman,A.D., Doerr,C.L., Tennant,A.H. and Zucker,R.M. (2000) Cytogenetic studies of three triazine herbicides. I. In vitro studies. Mutat. Res., 465, 53–59.[Web of Science][Medline]
Natarajan,A.T. (2002) Chromosome aberrations: past, present and future. Mutat. Res., 504, 3–16.[Web of Science][Medline]
Natarajan,A.T., Rotteveel,A.H., van Pieterson,J. and Schliermann,M.G. (1986) Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister chromatid exchanges, detected by immunological methods. Mutat. Res., 163, 51–55.[Web of Science][Medline]
Pantelias,G.E. and Maillie,H.D. (1983) A simple method for premature chromosome condensation induction in primary human and rodent cells using polyethylene glycol. Somat. Cell Genet., 9, 533–547.
Pantelias,G.E. and Maillie,H.D. (1984) The use of peripheral blood mononuclear cell prematurely condensed chromosomes for biological dosimetry. Radiat. Res., 99, 140–150.[Web of Science][Medline]
Perry,P. and Wolff,S. (1974) New Giemsa method for the differential staining of sister chromatids. Nature, 251, 156–158.[CrossRef][Medline]
Schwartz,S., Astemborski,J.A., Budacz,A.P., Boughman,J.A., Wasserman,S.S. and Cohen,M.M. (1990) Repeated measurement of spontaneous and clastogen-induced sister-chromatid exchange. Mutat. Res., 234, 51–59.[Web of Science][Medline]
Terzoudi,G.I. and Pantelias,G.E. (1997) Conversion of DNA damage into chromosome damage in response to cell cycle regulation of chromatin condensation after irradiation. Mutagenesis, 12, 271–276.
Terzoudi,G.I., Jung,T., Hain,J., Vrouvas,J., Margaritis,K., Donta-Bakoyianni,C., Makropoulos,V., Angelakis, Ph. and Pantelias,G.E. (2000) Increased G2 chromosomal radiosensitivity in cancer patients: The role of cdk1/cyclinB activity level in the mechanisms involved. Int. J. Radiat. Biol., 76, 607–616.[CrossRef][Web of Science][Medline]
Received on May 15, 2003;
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  V. I. Hatzi, G. I. Terzoudi, V. Makropoulos, C. Maravelias, and G. E. Pantelias Pre-irradiation exposure of peripheral blood lymphocytes to glutaraldehyde induces radiosensitization by increasing the initial yield of radiation-induced chromosomal aberrations Mutagenesis, March 1, 2008; 23(2): 101 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Swain, J. Ding, D. L. Brautigan, E. Villa-Moruzzi, and G. D. Smith Proper Chromatin Condensation and Maintenance of Histone H3 Phosphorylation During Mouse Oocyte Meiosis Requires Protein Phosphatase Activity Biol Reprod, April 1, 2007; 76(4): 628 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. I. Terzoudi, K. N. Manola, G. E. Pantelias, and G. Iliakis Checkpoint Abrogation in G2 Compromises Repair of Chromosomal Breaks in Ataxia Telangiectasia Cells Cancer Res., December 15, 2005; 65(24): 11292 - 11296. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||