Mutagenesis, Vol. 17, No. 2, 177-181,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
REVIEW |
Induction of micronuclei in human cell lines and primary cells by combination treatment with 
-radiation and ethyl methanesulfonate
Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany
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Abstract |
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While testing for genotoxicity is usually performed on single chemicals, exposure of humans often involves combinations of agents. Previous results from this laboratory showed supra-additivity for the induction of micronuclei in p53-mutated mouse lymphoma L5178Y cells after combined treatment with
-radiation from a 137Cs source and ethyl methanesulfonate (EMS). The question now was whether supra-additivity was a general phenomenon for the genotoxicity of this combination of a physical and a chemical DNA-damaging agent or whether the result was species- and cell type-specific. The same combination of agents was investigated in two human lymphoblastoid cell lines, TK6 (wild-type p53) and WTK1 (mutated p53), and primary fibroblasts from a fetal human lung. Doses were in the linear dose–effect range, resulting in a 1.5- to 3-fold increase in micronuclei above control. Radiation doses were between 125 and 350 mGy, while the EMS concentrations were 20–50 µg/ml for the cell lines and 250–350 µg/ml for the primary cells. In none of the human test systems was supra-additivity observed. With the WTK1 cells, which are most similar to the mouse cells regarding p53 status, there was even a tendency for a sub-additive combination effect. Possible explanations for the difference to the mouse cells could be related to species-specific aspects, different consequences of the p53 mutations or the presence of additional mutations. It is concluded that caution is advised in the interpretation and extrapolation of experimental results of mixture toxicity data because the outcome could be highly specific for the given selection of agents, doses and assays. |
Introduction |
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Humans are exposed to DNA-damaging agents as largely undefined mixtures. A combination of different carcinogens with possibly different modes of action could result in an additive or non-additive (supra- or sub-additive) effect. In a previous paper (Stopper et al., 2000
) we investigated the genotoxicity of two standard mutagens, -radiation and the DNA alkylator ethyl methanesulfonate (EMS) in L5178Y mouse lymphoma cells. We chose the formation of micronuclei as the genotoxicity end-point because the assay is simple, fast and reliable (Miller et al., 1998; von der Hude et al., 2000). L5178Y cells were chosen based on their frequent use in routine genotoxicity testing (Combes et al., 1995). The combination treatment exerted a supra-additive genotoxic effect in several independent repeat experiments (Stopper et al., 2000).
L5178Y mouse lymphoma cells harbor a p53 tumor suppressor gene mutation (Storer et al., 1997) and p53 protein is known to be involved in various aspects of the cellular response to DNA damage (Agarwal et al., 1998). To see whether supra-additive induction of genotoxicity by -radiation and EMS is independent of the p53 mutation in L5178Y cells, the human lymphoblastoid cell lines TK6 and WTK1 were chosen. These are two B-lymphoblastoid cell lines derived from the same male donor (Levy et al., 1968), but only WTK1 expresses mutant p53 (Xia et al., 1995). Acquisition of the ability for unlimited numbers of cell divisions might induce several changes in cell lines that may or may not be relevant for risk assessment of human exposure to genotoxic agents and mixtures. To include a test system avoiding this general problem, primary human embryonic fibroblasts were added to the study.
The strategy for the experimental design was (i) determination of dose–effect relationships for the individual agents to be tested in combination, (ii) choice of doses to result in an increase by a factor of 1.5–4 of controls, but still within the linear part of the dose–response curve. The latter requirement minimized the danger that analysis of the combination effect could be complicated by non-linearities of the dose–response characteristics of the single agents.
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Materials and methods |
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Cell culture
TK6 and WTK1 cells were cultured in RPMI 1640 medium supplemented with antibiotics, 0.25 mg/ml L-glutamine, 107 µg/ml sodium pyruvate and 10% heat-inactivated horse serum (all from Sigma Chemie, Deisenhofen, Germany). Primary human embryonic fibroblast cells from lung tissue of a 16-week-old fetus were cultured in MEM/Earle's medium supplemented with antibiotics, 0.25 mg/ml L-glutamine, 107 µg/ml sodium pyruvate and 15% heat-inactivated fetal calf serum. Cell cultures were grown in a humidified atmosphere with 5% CO2 in air at 37°C.
In vitro micronucleus test
Exponentially growing cells were irradiated for a short time (dose rate 1 Gy/min) at room temperature using a 137Cs source (662 keV -radiation; Caesa-Gammatron; Siemens, Erlangen, Germany). EMS was added within 10 min after irradiation. EMS was prepared fresh as a 100x stock solution in DMSO before addition to the cell cultures. The vehicle control contained a final concentration of 1% solvent (DMSO). Cytochalasin B was added to a final concentration of 2 µg/ml 1 h later. Four hours after that, cells were centrifuged and the medium was replaced, again containing fresh cytochalasin B. The cells were incubated for 25 (TK6 and WTK1) or 19 h (primary human embryonic fibroblasts). Total time until harvest was thus 30 h for TK6 and WTK1 and 24 h for primary human embryonic fibroblasts. Preliminary experiments had shown these time points to be optimal to achieve high percentages of binucleate cells. For harvesting, the cells were placed on glass slides by cytospin centrifugation and fixed with methanol (–20°C, 1 h). To stain nuclei and micronuclei, the slides were incubated with acridine orange (0.00625% w/v in Sorensen buffer, pH 6.8, 4 min). Slides were washed twice with buffer and mounted for microscopy with buffer. Percentages of binucleate cells were determined. Numbers of nuclei and micronuclei in binucleate cells were scored at a magnification of 500x. For each analysis 1000 cells on each of two slides were evaluated, yielding 2x1000 binucleates per data point.
Statistical evaluation
To test for putative deviations from additivity, a two-factor analysis of variance with interaction was employed, as described in detail in Stopper et al. (2000).
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Results |
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The formation of micronuclei was investigated in the human lymphoblastoid cell line TK6 after treatment with
-radiation and EMS. From the individual dose–response curves (Figure 1A and B) we chose doses in the linear part for the combination treatments. In these experiments each treatment (control, -radiation, EMS and combination) was performed on three independent cultures, with evaluation of 2x1000 cells from two different slides for each culture (Figure 1C). When we combined irradiation doses of 150 or 125 mGy with EMS concentrations of 20 or 25 µg/ml, micronucleus induction was close to the theoretical additive value in both experiments. There was no deviation from additivity (P
1/= 0.2).
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The same procedure was followed with the human lymphoblastoid cell line WTK1 (Figure 2A and B
). Treatment conditions were chosen from the linear part of the dose–response curves, with 350 or 150 mGy -radiation plus 50 µg/ml EMS (Figure 2C). Combination values were slightly below additivity. However, statistical analysis showed no significance for a possible sub-additivity (P = 0.09/0.07). Thus, in both human lymphoblastoid cell lines there was no indication of a deviation from additivity when treated with low dose combinations of -radiation and EMS.
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Finally, primary human embryonic fibroblasts were used. Dose–response experiments (Figure 3A and B
) revealed 250 mGy and 250/350 µg/ml EMS as suitable for the combination experiments (Figure 3C). Again, combination treatments showed no deviation from additivity (P = 0.4/0.3).
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Analysis of the percentage of binucleate cells (Table I
) showed that treatments used for the combination experiments did not (TK6, experiments 1 and 2; WTK-1, experiment 2) or only slightly (WTK-1, experiment 1) reduce cell proliferation. The highest decrease was observed in experiment 1 with primary human fibroblasts, where the combination treatment yielded 62% of the binucleate cells of the control. The second experiment with human primary fibroblasts showed only a decrease to 77% of the control.
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Discussion |
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Micronucleus induction by a combination of
-radiation and EMS in the two human lymphoblastoid cell lines TK6 and WTK1, as well as in primary human embryonic fibroblasts, was found to be additive. Previous work had shown supra-additivity in the p53-mutated mouse lymphoma L5178Y cell line (Stopper et al., 2000). TK6 cells harbor an intact p53 gene (Xia et al., 1995) and mutations in tumor suppressor genes can be assumed to be absent in primary human embryonic cells. Since WTK1 cells also harbor a mutated p53 gene (Xia et al., 1995), a deviation from additivity might have been expected in this line. This was not seen. If at all, the deviation appeared to be sub-additive. Several explanations are conceivable for the difference between mouse L5178Y and human WTK1 cells. Possible confounding differences in cell death or general susceptibility to the genotoxic treatments have to be addressed first. The presence of necrotic and apoptotic cells was assessed and found to be extremely low (~1%) in the dose range from which treatment conditions were chosen for the combinations. The same was true for the combination treatments themselves. Necrotic and apoptotic responses could thus not have influenced the interpretation of the results as being additive. Although L5178Y cells showed a lower frequency of spontaneous micronuclei, variation between experiments was also low in the cell lines used here so that detection of a potential deviation from additivity was not impeded.
In both cell lines the p53 mutation is a point mutation. In L5178Y cells it is a heterozygous point mutation in codon 170 (Storer et al., 1997), while in WTK1 cells it is a homozygous point mutation in codon 237 (Xia et al., 1995). In both cell lines the mutation leads to overexpression of mutated p53 protein. The mutations therefore seem to have equivalent consequences in both cell lines. However, detailed analyses of changes in all of the many p53 functions are not available in these cell lines and a difference in p53 mutant phenotype cannot therefore be excluded. The relevance of different p53 mutant phenotypes to genotoxicity has been supported by recent investigations. WTK1 cells showed a higher spontaneous frequency of chromosomal aberrations than TK6 cells (Geiger et al., 1999). On the other hand, TK6E6, a HPV16E6-transfected TK6 cell line which is negative for p53 function because of rapid degradation of the gene product via the proteasome pathway (Scheffner et al., 1990), were similar to TK6 (Geiger et al., 1999). Chuang et al. created a double p53 knockout cell line from TK6 cells (NH32), which behaved similarly to TK6 but differently from WTK1 in mutation tests using the thymidine kinase locus (Chuang et al., 1999). These findings suggest that mutant p53 as present in WTK1 cells can have a gain of function phenotype. In such cells, certain pathways of apoptosis may be suppressed or cells may be prone to recombination (Wiese et al., 2001). However, the fact that mouse L5178Y cells are also prone to recombination (Liechty et al., 1998; Preisler et al., 2000) may indicate that the difference in reaction to the combination of -radiation and EMS between mouse L5178Y and human WTK1 cells cannot be explained by a different p53 mutant phenotype.
Human cells are more resistant to transformation than rodent cells (Balmain and Harris, 2000). Due to their shorter lifespan mice might have less stringent control mechanisms to avoid tumor development. Therefore, the role of p53 as a `guardian of the genome' (Sigal and Rotter, 2000) and thus the effect of a p53 mutation may have different consequences in a human cell as compared with a mouse cell. In addition, it is probable that L5178Y cells harbor additional mutations in other cell cycle/DNA repair-related genes which interact with the mutated p53 product to generate the supra-additive effects. Finally, it cannot be excluded at this stage that the mutated p53 gene in L5178Y cells is not related to the supra-additive gentoxicity induced by the combination of -radiation and EMS.
Regarding the question of potentiating effects of combination exposures, our results indicate that caution must be exerted in the interpretation and extrapolation of experimental results that are based on a specific selection of agents, doses and assays. Results on deviation from additivity cannot be extrapolated from one cell type and treatment combination to others without experimental support.
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Acknowledgments |
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We thank Dr J.Schlatter, monitoring scientist of the BAG, for valuable advice on all parts of this work, PD Dr Annette Kopp-Schneider for the statistical analysis and Mr Michael Kessler for skilled technical assistance. Primary human embryonic fibroblast cells were provided by Dr D.Schindler (Department of Human Genetics, University of Würzburg, Germany). Financial support by the Swiss Federal Office of Public Health (BAG grant no. FE 00.000265) is gratefully acknowledged.
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Notes |
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1 To whom correspondence should be addressed. Tel: +49 931 201 3427; Fax: +49 931 201 3446; Email: stopper{at}toxi.uni-wuerzburg.de
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Received on July 27, 2001; accepted on November 28, 2001.
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