Mutagenesis, Vol. 15, No. 3, 235-238,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Supra-additive genotoxicity of a combination of 
-irradiation and ethyl methanesulfonate in mouse lymphoma L5178Y cells
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 always comprises a number of genotoxic agents. The investigation of potentially synergistic effects of combinations therefore is an important issue in toxicology. Combinations of 511 keV
-radiation with the chemical alkylating agent ethyl methane-sulfonate were investigated in the in vitro micronucleus test in mouse lymphoma L5178Y cells. With combinations in the low dose linear effect range for the individual agents (0.25–2 Gy and 0.8–3.2 mM, respectively), supra-additivity by 34–86% was seen. The synergism was more pronounced at the higher dose levels. Supra-additivity was confirmed in experiments using cytochalasin B and analyzing binucleate cells only, to control for putative effects on the cell cycle. Statistical significance was shown by a 2-factor analysis of variance with interaction. The results indicate that damage to DNA by -radiation and alkylation could affect different rate limiting steps in the formation of micronuclei. Further investigations will have to show whether the observations are of general validity, in particular, whether other end-points of genotoxicity produce the same results and whether the degree of supra-additivity is always dose dependent. The latter would have a strong impact on risk assessment for mixtures at low doses. |
Introduction |
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Humans are exposed to DNA-damaging agents as undefined mixtures. A combination of different carcinogens with possibly different modes of action could result in an additive or non-additive (supra- or infra-additive) effect. Burkart and Jung (1998) recently published theoretical considerations about possible deviation from additivity of combinations of damaging agents. First of all they caution that synergism should not be postulated in situations termed `isoaddition', as observed for agents that act by the same mechanism and exhibit sublinear (convex) dose–response curves. Under such conditions, a combination effect could appear to be supra-additive because the increment in effect exerted by the second agent produces a steeper slope of the dose–response curve of the first. Nevertheless, they see a potential for `true' synergistic interactions in multistep processes (such as carcinogenesis) if two or more consecutive rate limiting steps are affected by different agents. This is due to the understanding that the dose–response relationship for the two steps taken together appears as a product of the two single steps and may become exponential (Lutz, 1990
). Another mechanistic possibility for synergistic effects has been suggested by Elmazar et al. (1997). They extended previous in vitro data to in vivo experiments in mice and obtained synergistic teratogenic damage if certain ligands for retinoid receptors were co-administered. Their explanation was that two different receptor types were activated by the ligands to form a heterodimer, which then led to the induction of teratogenic effects. A similar situation could be hypothesized for other reactions (e.g. in carcinogenesis) that only occur if two distinct proteins are enabled to form an active complex by two specific compounds.
Combination exposure to ionizing radiation and a chemical mutagen is not only of relevance for environmental mutagenesis and carcinogenesis but is of interest also for combination cancer therapy, though with an opposing sign. Therefore, in this paper we investigated the genotoxicity of two standard mutagens, -irradiation and the DNA alkylator ethyl methane-sulfonate (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. The usefulness of this end-point has been described (Kirsch-Volders, 1997; Miller et al., 1998; W.Von der Hude et al., in preparation). L5178Y cells were chosen based on their frequent use in routine genotoxicity testing. 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 within the low dose linear part of the dose–response curve such that analysis of the combination effect would not be complicated by non-linearities of the dose–response effect of the single agents.
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Materials and methods |
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Cell culture
Cells were cultured in suspension in RPMI 1640 supplemented with antibiotics, 0.25 mg/ml L-glutamine, 107 µg/ml sodium pyruvate and 10% heat-inactivated horse serum (all from Sigma Chemie GmbH, Deisenhofen, Germany). Cell cultures were grown in a humidified atmosphere with 5% CO2 in air at 37°C.
In vitro micronucleus test
Exponentially growing L5178Y mouse lymphoma cells were irradiated for a short time (25–120 s) at room temperature with Cs radiation using a 511 keV Caesa-Gammatron (Siemens, Erlangen, Germany). Dose rates were 0.6 Gy/min for 0.25 Gy and 1 Gy/min for 0.5, 1 and 2 Gy. 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). After 4 h, cells were centrifuged and the medium was replaced. When indicated, cytochalasin B was added to a final concentration of 5 µg/ml at this time. The cells were then incubated for 15 h. For harvesting, the cells were brought onto glass slides by cytospin centrifugation and fixed with methanol (–20°C, 1 h). To stain nuclei and micronuclei, the slides were incubated with Hoechst 33258 (5 µg/ml in phosphate-buffered saline, 3 min) in experiments without cytochalasin B and with acridine orange (0.00625% w/v in Sorensen buffer, pH 6.8, 4 min) in experiments with cytochalasin B. Slides were washed twice with buffer and mounted for microscopy, using mounting medium (Linaris, Germany) for Hoechst stain and buffer for acridine orange stain. Numbers of nuclei and micronuclei were scored at a magnification of 500x. Without the use of cytochalasin B, the frequency of spontaneous binucleate and multinucleate cells was determined to be 0.4%. In experiments with cytochalasin B, the percentage of binucleate cells was evaluated as a cell proliferation marker. Dose–response experiments and the combination experiment without cytochalasin B were performed three times, and one representative experiment is shown. The repeat experiments for the combination using the cytochalasin B protocol are all shown.
Statistical evaluation
For the testing of putative supra-additive or infra-additive effects of combination treatment, the data were evaluated with a 2-factor analysis of variance with interaction. The underlying model is described by the equation 
, where y is the observed micronucleus frequency, ctr is the control (solvent control) micronucleus frequency, a and b are the frequencies for the single treatments and c is the interaction term for simultaneous administration of the two treatments, describing the additional positive or negative effect obtained by simultaneous administration. Hence, the expected micronucleus frequency for simultaneous administration of the agents is ctr + a + b + c. d is the error term accounting for the variation within groups. The error is assumed to have a normal distribution with mean 0 and identical standard deviation for all treatment groups. The P value is reported for the test of the hypothesis that c = 0. It describes the probability that the observed difference between the effect of the mixture and the sum of the individual effects is different from 0 by chance alone. A P value of <0.05 is considered to represent significance.
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Results |
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Using L5178Y mouse lymphoma cells we investigated the formation of micronuclei after treatment with
-irradiation and EMS. From the individual dose–response curves (Figure 1) we chose doses in the linear part of the dose–response effects for combination treatments. When we combined irradiation doses between 0.25 and 1 Gy with EMS concentrations between 0.8 and 3.2 mM, we found an induction of micronuclei that was higher than would be expected if the effects of both treatments were additive (Figure 2). The extent of supra-additivity appeared to increase with dose and came close to a factor of 2.
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Two questions could be raised that required further experiments. First, possible effects on cell cycle progression should be controlled for and, second, the detected supra-additivity needed statistical evaluation.
Therefore, we designed an experiment that included the use of cytochalasin B in the protocol. Cytochalasin B prevents cells from separating after mitosis, resulting in binucleate cells. The micronucleus evaluation could now be limited to the binucleate (i.e. actively proliferating) cell population in each treated cell culture, thus normalizing the micronucleus frequency to the progression of cell proliferation. Furthermore, the percentage of binucleate cells in the population was determined as a marker for cell proliferation changes. In this experiment each treatment (control, irradiation, EMS and combination) was performed on three independent cultures to enable proper statistical evaluation.
Percentages of binucleate cells showed that 0.5 Gy irradiation did not affect cell proliferation of L5178Y mouse lymphoma cells compared with the solvent control while treatment with 3.2 mM EMS caused a reduction in cell proliferation (Table I, experiment a). The combination treatment also caused a reduction in proliferation which was similar to that induced by EMS alone. Thus, no supra-additive change in cell cycle progression was observed. Micronucleus frequencies for the combination treatment were again supra-additive compared with the single treatments. Statistical analysis revealed significance (P = 0.002) for the supra-additivity of the group of combination treatments compared with the groups of single treatments. Two independent repeat experiments (Table I, experiments b and c) also showed supra-additive micronucleus induction, to further support the results of experiment a. The smaller effect of EMS in experiment b could be due to more advanced degradation in the commercial batch used.
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Discussion |
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Combination treatment with
-irradiation and the DNA alkylating agent EMS exerted a supra-additive genotoxic effect in L5178Y mouse lymphoma cells with micronucleus formation chosen as the biological end-point. The over-additivity was demonstrated in several independent repeat experiments as well as in one experiment that included independent replicates and allowed statistical analysis of the data. This result was obtained from doses within the linear part of the dose–response curves of the individual agents so that the supra-additivity is unlikely to be the result of `isoaddition' in the case of a non-linear dose–response, as cautioned in the Introduction.
According to Burkart and Jung (1988), effects on two rate limiting steps in a multistep process are most likely to yield synergistic results. In the genotoxicity of DNA-damaging agents like EMS or -irradiation the probability of DNA damage becoming fixed as a heritable genetic change is dependent on the relative rates of DNA repair and cell division (Lutz, 1990). These two processes can be regarded as rate limiting and it might be conceivable that irradiation and alkylation affect different rate limiting steps. In fact, it has been demonstrated that there is specificity for multiple G2/M cell cycle progression checkpoints in response to different types of DNA damage and that radiation induces a type of G2/M arrest that is different from that seen with most other stress stimuli, such as genotoxic chemicals (Abbott and Holt, 1999; Wang et al., 1999).
Cell cycle control checkpoints and DNA repair have been shown to be important for micronucleus formation. We found in mutant hamster cell lines that a line with features of ataxia telangiectasia showing a defect in cell cycle regulation (but normal DNA strand break repair) as well as another cell line with a DNA double-strand break repair defect were more sensitive to micronucleus induction than the parental cell lines (Stopper et al., 1997). The cell line used here, L5178Y mouse lymphoma, harbors a p53 tumor suppressor gene mutation (Storer et al., 1997) and p53 is known to be involved in cell cycle regulation in response to induced DNA damage. For instance, it was recently shown that down-regulation of cell cycle-regulated genes after low dose irradiation was p53-dependent (De Toledo et al., 1998). It has recently been discussed in the literature whether tumor suppressor gene mutations such as this p53 mutation are of general relevance for genotoxicity studies. Therefore, it will be interesting to determine the role of this gene in the reaction of cells to such a combination treatment by studying various cell lines with and without p53 mutations as well as primary cells. Overall, to identify the mechanism responsible for the observed supra-additivity, further experiments will be necessary.
The agents used here, -irradiation and EMS, have not only been used as model compounds for genotoxicity studies, but are of practical relevance. A combination of radiation and chemotherapy is used in cancer treatment. Spindle poisons such as taxol have often been used as radiosensitizers, on the assumption that these compounds inhibit cell cycle progression such that cells are arrested or delayed in their most sensitive cell cycle phase (G2/S phase), which increases the radiosensitivity of this cell population (Tishler et al., 1992). However, cell lines so far investigated for a radiosensitzing effect of taxol in cytotoxicity or cloning efficiency assays have given inconclusive results (Preisler et al., 1998, and references therein) and when induction of micronuclei was analyzed there was no apparent deviation from additivity (Preisler et al., 1999). Recently other agents, such as mitomycin (Haffty et al., 1997), have also been used in combination with radiation in cancer treatment. Based on our data it may be hypothesized that alkylators may not only yield additive effects in treatment, but may in some cases act as radiosensitizers.
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Acknowledgments |
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We thank Dr J.Schlatter, monitoring scientist of the BAG, for valuable advice in all parts of this work, Dr Annette Kopp-Schneider for the statistical analysis and Mrs M.Ruppert, Mrs M.Gerhard and Mr M.Kessler for their skilled technical assistance. Financial support by the Swiss Federal Office of Public Health (BAG grant no. FE 316.97.0606) is gratefully acknowledged.
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Notes |
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1 To whom correspondence should be addressed. Tel: +49 0931 201 3427; Fax: +49 0931 201 3446; Email: stopper{at}toxi.uni-wuerzburg.de
2 Present address: NIEHS/NIH, Research Triangle Park, NC, USA
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Received on October 11, 1999; accepted on December 21, 1999.
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