Mutagenesis, Vol. 15, No. 2, 99-104,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Combined effects of 
-radiation and ethylene oxide in human diploid fibroblasts
Department of Molecular Genome Research, Stockholm University, SE-106 91 Stockholm, Sweden and 1 Department of Molecular Genetics, Cancer Research Institute, 833 91 Bratislava, Slovak Republic
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Abstract |
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Human diploid VH-10 fibroblasts were pre-exposed to
-rays and then treated with ethylene oxide (EtO). In the reverse experiment, the cells were pretreated with EtO and then exposed to -rays. Two different dose rates of -rays were used: a low dose rate (LDR, 0.66 Gy/min) and a high dose rate (HDR, 10 Gy/min). Cell killing, mutagenicity and DNA double-strand breakage were studied in both types of experiment. The induction of mutations in the HPRT locus was studied by selection in medium containing 6-thioguanine. DNA double-strand breakage, measured as fraction of activity released (FAR), was investigated using pulsed field gel electrophoresis. Concerning mutagenesis, it was found that pre-exposure of the cells to -radiation (1 Gy) followed by treatment with EtO (2.5 mMh) led to an additive co-interaction, irrespective of dose rate. On the other hand, the reverse experimental procedure (pretreatment with EtO followed by -ray exposure) resulted in an antagonistic effect, which was most pronounced when the HDR was applied. In the latter case, the resultant mutant frequency was two times lower than the sum of the mutant frequencies after the individual treatments. However, the effect of the combined treatment on FAR was different: FAR increased with both combinations of agents used compared with the separate and hypothetically expected effects. Moreover, the HDR exposure led to an additional increase in FAR compared with the LDR one. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Humans are living in a complex environment where different factors can interfere with each other. Among them, ionizing radiation and genotoxic chemicals with carcinogenic potential play an important role. Chemical carcinogens, which are often tumor promoters, can be present in the food, air and water, in cigarette smoke, car exhausts, etc. Knowledge about the resulting effects and mechanisms of co-interaction of different mutagenic/carcinogenic agents is limited.
Combined exposure to ionizing radiation and chemicals occurs frequently in daily life. As an example, an increased lung cancer incidence in smokers exposed to radon can be mentioned (Health Effects of Exposure to Radon, BEIR VI, 1999
). The situation in large areas of Belarus and Ukraine, contaminated with radionuclides following the Chernobyl catastrophe (Samner et al., 1994), also strongly supports the need for a better understanding of the influence of low doses of ionizing radiation in combination with chemical carcinogens on humans.
It is generally known that ionizing radiation can lead to induction of neoplastic cell transformation (Terzaghi and Little, 1976; reviewed in United Nations Scientific Committee on the Effects of Atomic Radiation, 1986) and gene mutation (Thacker, 1986; O'Neill et al., 1990) in mammalian cells. Higher doses of ionizing radiation induce large deletions at the HPRT locus in Chinese hamster cells (Vrieling et al., 1985) and in human lymphocytes (Skulimowski et al., 1986; reviewed by Bastlová et al., 1993).
An environmental pollutant and known mutagen and carcinogen in experimental animals and in humans, ethylene oxide (EtO) is a widely used industrial chemical and sterilant of medical equipment (Ehrenberg and Hussain, 1981; Kolman et al., 1986; Dellarco et al., 1990). The ability of EtO to transform mouse embryo fibroblasts (C3H/10T1/2 cells) in vitro was demonstrated earlier and its effect was compared with that of -radiation (Kolman et al., 1989, 1990). It was also shown that EtO induces mutations in the HPRT locus in human diploid fibroblasts in vitro and that a great majority (~50%) of these mutations are large deletions (Bastlová et al., 1993).
EtO is also an inducer of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in human diploid fibroblasts (Nygren et al., 1994). The SSB/DSB ratio was found to be ~23 (Kolman et al., 1997). It was shown that normal human fibroblasts are able to repair ~50% of DSBs within 18 h after treatment with EtO (Nygren et al., 1994).
In this paper the combined action of two mutagenic agents, -radiation and EtO, was studied in human normal fibroblasts, using different orders of their application (first exposure to -rays and then treatment with EtO or the opposite order). It was investigated whether such different experimental conditions may influence the mutation frequency at the HPRT locus, as well as what the consequence is of such a combined treatment in terms of DNA damage measured by pulsed field gel electrophoresis (PFGE).
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Materials and methods |
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Materials
EtO was obtained from Fluka (Buchs, Switzerland). Ham's F-10 medium was obtained from the Swedish National Veterinary Institute (Uppsala, Sweden). Fetal calf serum (FCS), phosphate-buffered saline (PBS) and antibiotics were obtained from Flow Laboratories (Rickmansworth, UK). [14C]Thymidine was obtained from Amersham (Aylesbury, UK). Pulsed field certified agarose was from Bio-Rad (Richmond, CA). Agarose for PFGE sample preparation and 6-thioguanine (6-TG) were purchased from Sigma (St Louis, MO).
Cell culture
Normal human diploid fibroblasts (strain VH-10, passages 8–11) were cultured in 100 mm plastic tissue culture plates in Ham's F-10 medium supplemented with 15% heat-inactivated FCS, 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin and modified by omission of hypoxanthine and thymidine, in a humidified 3% CO2 atmosphere at 37°C. The cells were seeded at 3–5x105 per dish and subcultured until 75% confluency was reached.
Combined treatment with -rays and EtO
The cells, growing in Ham's F-10 medium containing 15% FCS, were washed with PBS, trypsinized and resuspended in 4.5 ml of the same medium without serum. In each experiment the cells (2x106 per sample) were separately exposed to -rays and to EtO and were also treated with a combination of these agents as described below.
The cells were irradiated using two different 137Cs sources: (i) a model IC 900 irradiation chamber, providing a dose rate of 0.66 Gy/min (low dose rate, LDR); (ii) a model Gammacell 1000, providing a dose rate of 10 Gy/min (high dose rate, HDR). Doses between 1 and 4 Gy were used to study cell survival and 6-TG-resistant mutant frequency. The lowest dose of 1 Gy was chosen for the combined treatments.
A stock solution of EtO (100 mM) in PBS was prepared by weighing in a tightly closed, glass screw-cap tube. The cells were treated with different concentrations of EtO (2.5–7.5 mM) for 1 h in a 37°C shaking water bath (Kolman et al., 1992). A concentration of 2.5 mM was used throughout the experiments described in this paper.
The cells (4.5 ml of cell suspension) were pre-exposed to 1 Gy of -radiation and then treated for 1 h with 2.5 mM EtO (0.5 ml of a 25 mM solution of EtO was added to the cell suspension). After treatment, the medium was removed by centrifugation and the cells rinsed once with 5 ml PBS, resuspended in 5 ml of Ham's F-10 medium with 15% FCS and distributed on plates for estimation of survival and expression of 6-TG-resistant mutants. In other samples the cells were pretreated with 2.5 mM of EtO for 1 h. After treatment, the medium was removed and the cells were washed as described above. Afterwards, the cells were resuspended in 5 ml of medium and irradiated with 1 Gy. The following procedure was the same as that for the reverse treatment conditions.
For estimation of survival, 300 cells were seeded per 60 mm dish in triplicate. The medium was renewed after 1 week and after one more week the cells were fixed with methanol, washed and stained with 5% Giemsa reagent. The colonies were counted and survival was evaluated.
Estimation of 6-TG-resistant mutant frequency
The frequency of 6-TG-resistant mutants was estimated as described previously (Kolman et al., 1992). The expression time for induced mutants was estimated to be 9–11 days. Within this time the cells were reseeded twice at a density of 5x105 cells/dish and grown to 75% confluence. At the end of the expression period 70 000 cells were seeded on 50 plates in F-10 medium containing 5 µg/ml 6-TG (5 mg/ml dissolved in 0.5% Na2CO3 and boiled for 3–5 min). The medium was renewed after 10 days and after an additional 10 days colonies of 6-TG-resistant cells were fixed with methanol, stained with 5% Giemsa reagent and counted. Cloning efficiency was determined in Ham's F-10 medium without 6-TG, in 60 mm dishes, in triplicate (survival estimation as above).
Pulsed field gel electrophoresis (PFGE)
After treatment, the cells were washed twice with PBS. Afterwards, the cell pellets were resuspended in a buffer containing 20 mM NaCl, 50 mM EDTA and 10 mM Tris–HCl (pH 7.2) to a density of 6x106 cells/ml. The cell suspensions obtained were mixed 1:1 with 1% low melting point agarose. The agarose mixtures (100 µl, 3x105 cells) were pipetted into plug moulds and cooled to 4°C until solidified. Subsequently, the plugs were lysed at 37°C for 72 h in 0.5 M EDTA (pH 8.0), 1% sarcosyl and 1 mg/ml proteinase K. Before insertion into wells in a 0.9% agarose gel, the plugs were washed three times with TE buffer (pH 8.0) (Maniatis et al., 1982). After plug addition, the wells were sealed with 0.5% low melting point agarose. Electrophoresis was carried out at 14°C in 0.5x TBE buffer (Maniatis et al., 1982) with a CHEF-DR II apparatus (Bio-Rad) using the following steps: (i) 48 h at 35 V; (ii) 48 h at 50 V, with a 96 h pulse ramp overlapping these two steps with pulses decreasing from 90 to 45 min; (iii) 48 h at 60 V, with a pulse ramp from 45 min to 2 s. After electrophoresis, the gels were stained with 0.5 µg/ml ethidium bromide, destained in deionized water and visualized on a UV trans-illuminator.
Due to the homogeneous field characteristics, straight lanes containing DNA were obtained. Lanes were separated from the wells by a cut introduced as close as possible to the well. Subsequently, lanes were separated from each other. For each sample, two slices were obtained: the first contained the DNA left in the plug, while the other contained the DNA released into the gel. The agarose slices were then melted and hydrolyzed in 2.5 ml of 4 M HCl at 90°C for 25 min. The volume of all samples was adjusted with water to attain a 1 M concentration of HCl. Radioactivity was determined by liquid scintillation counting in 0.5 ml of each sample.
To evaluate the PFGE data, the fraction of activity released (FAR) from the plugs was calculated. FAR as a measure of the amount of DNA DSBs has already been used by many authors (Iliakis et al., 1991; Metzger and Iliakis, 1991; Rydberg et al., 1994). FAR was calculated as lane c.p.m. divided by total (lane + plug) c.p.m. In each experiment, one part of the cells was left untreated and used as a control to determine the background (FAR for the untreated sample). The average background was found to be 26%. The background was always subtracted prior to plotting or statistical analysis of the obtained results. All data presented here were confirmed in at least three independent experiments and the results shown are means ± SD.
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Results |
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The effect of
-radiation at the two different dose rates on survival of VH-10 cells exposed in suspension is shown in Figure 1. As is obvious, the HDR exposure led to a greater cytotoxic effect, especially at doses of 2–4 Gy, than the LDR one. At the dose of 2 Gy, survival after HDR exposure was two times lower compared with that after LDR exposure (30 versus 60%). At the lower dose of 1 Gy there was a small, but insignificant, difference between the HDR and LDR exposures (Figure 1). A dose-dependent increase in 6-TG-resistant mutants was demonstrated after both LDR and HDR -ray exposures (Figure 2). HDR irradiation induced 6-TG-resistant mutants more effectively than the LDR one: the mutant frequency was about two and four times higher after doses of 1 and 2 Gy, respectively, when the HDR was applied.
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, 10 Gy/min). Data are from three independent experiments with standard deviations.
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The dose of EtO (mMh) is given as initial concentration (mM)xtime of treatment (h) (Ehrenberg et al., 1983
). Dose–response curves for cell survival and for induced mutant frequency at the HPRT locus after EtO treatment in suspension were published previously (Kolman et al., 1992). The data obtained in this study (Table I) show that after 2.5 mMh EtO treatment, cell survival was 73.1 ± 10.7% and the average mutant frequency was 27.1 (95% CI, 20.47–35.20) per 106 survivors.
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The effects of the combined action of
-radiation and EtO on cell survival and on the induction of 6-TG-resistant mutants are summarized in Table I
. There was no significant difference between the average survival data for the two types of combined treatment, however, cell survival after the combined treatments was always somewhat lower than that after single treatments. The effect of the combined treatments was more pronounced in respect of induction of HPRT mutants. When the cells had first been pre-exposed to HDR -radiation and then treated with EtO, the resultant mutant frequency was approximately the sum of the mutant frequencies obtained with separate treatments. In the case of pre-exposure to LDR -radiation, it was 1.3 times lower than that expected from summation. On the other hand, when the cells were first pretreated with EtO and then exposed to -rays, the resultant mutant frequencies at both dose rates were approximately two times lower than the summed values of the separate treatments.
Figures 3 and 4 show DNA DSBs presented as a percentage of FAR plotted against the type of treatment. Individual treatment with EtO or exposure to -rays induced very low, if any, FAR: 2.5 mMh EtO treatment led to FAR of ~2% and irradiation with 1 Gy followed by 1 h incubation caused practically non-detectable FAR, irrespective of the dose rate used. However, a difference between the single and the combined actions of -rays and EtO was observed: the combined action of -rays and EtO led to an increase in FAR compared with the single actions of these agents. Moreover, a difference between the combined actions was also found depending upon the dose rate used: about 2.4 and 1.8 times higher FAR after 2.5 mMh EtO + 1 Gy and 1 Gy + 2.5 mMh, respectively, was detected for HDR exposure (Figure 4) when compared with LDR exposure (Figure 3). In addition, the use of HDR exposure led to a 1.8 times higher FAR after 1 Gy + 2.5 mMh EtO compared with 2.5 mMh EtO + 1 Gy. Approximately the same FAR values were observed after LDR exposure with both combinations used (Figure 3).
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Discussion |
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The results demonstrate that the combined action of the two mutagenic agents, ionizing radiation and the radiomimetic alkylating agent EtO, may have different influences on various end-points, i.e. cell survival, induction of 6-TG-resistant mutants and DNA DSBs. With respect to survival, cell killing caused by the two agents is always slightly higher than that after individual treatments, however, it seems to be independent of the order of their application. At the same time, a difference between the two dose rates (by a factor of 15) does not significantly influence the total killing effect.
The estimation of mutant frequencies was performed using a 9–11 day expression time, which showed optimal yields of mutants in our previous studies (Kolman et al., 1992). Spontaneous mutant frequency under these conditions was 4.6 (95% CI, 1.85–9.48) per 106 survivors. There were some variations in mutant frequencies between individual experiments with the same treatment. Such difficulties with mutation measurements can possibly be explained by different clonogenic abilities of 6-TG-resistant mutants. As we observed earlier, mutants with large deletions often have a low growth capacity and can easily be lost.
The order of application plays an important role in the induction of 6-TG-resistant mutants. The results indicate that pre-exposure to -rays leads to increased mutant frequencies irrespective of the dose rate. The co-interactive effect of the two agents seems in this case to be additive, which is manifested by approximately summed numbers of HPRT mutants. However, when EtO was applied first, the character of the co-interaction was quite different, being rather antagonistic: the number of HPRT mutants for both the LDR and HDR exposures was two times lower than one would expect from a simple summation of the mutant frequencies of the separate treatments. One possible explanation of such a difference in the co-interactive effects of the two types of combined treatment might be suggested: DNA damage induced by pre-exposure to -radiation persists within the 1 h EtO treatment. It cannot be excluded that EtO prevents DNA repair enzymes operating and, therefore, both agents probably contribute to induction of mutations. However, when -radiation is applied after EtO treatment (the EtO was removed by washing; see Materials and methods), the cells might be able to repair, at least in part, the promutagenic lesions induced by the -rays.
It should also be noted that the mutant frequency does not correlate with DNA fragmentation: although combined EtO + -rays cause higher DNA fragmentation than that after the individual treatments, the induction of 6-TG mutants is even lower than after EtO treatment or -ray exposure separately. This fact could possibly be explained by the different nature of DSBs induced by ionizing radiation and EtO (Nygren et al., 1994); DSBs induced by the two agents could be processed in distinct ways with respect to correct and/or incorrect DSB rejoining. Furthermore, the repair kinetics could also be different.
No DNA DSBs were detected in the case of -rays only, irrespective of the dose rate used. This is not unexpected since the cells were collected after 1 h incubation in Ham's F-10 medium, allowing DNA DSBs to be repaired. Experiments confirming this were indeed carried out (data not shown).
In this study we have chosen doses of both agents which are not very toxic to the cells (in our experiments survival for the separate treatments was in the range 45–89%) but, at the same time, produce a substantial mutagenic effect. We have further tried to extend this study using higher doses of these agents, i.e. 5 mMh EtO treatment and exposure to 2 Gy of -radiation (data not shown), however, such experimental conditions greatly reduced cell survival, which was in the range 10–20%, so that 6-TG-resistant mutants could easily be lost in these experiments.
The combined action of two mutagenic agents may be theoretically classified as additive, synergistic or antagonistic (Ager and Haynes, 1988). For example, the interaction between UV light and ionizing radiation, earlier studied in yeast cells and in other microorganisms (Elkind and Sutton, 1959; Bhaumik and Bhattacharjee, 1968; also reviewed by Ager and Haynes, 1988), has mostly been described as synergistic. A mathematical description of survival curves obtained after the combined action of UV light and X-rays in Escherichia coli B/r was presented and a synergistic interaction was demonstrated (Ager and Haynes, 1987). A synergistic character of the interaction of these two mutagenic agents with respect to induced mutations was also shown (Davies et al., 1967). Additionally, a synergistic effect on cell killing was found in E.coli B/r pretreated with the radiomimetic alkylating agent nitrogen mustard followed by exposure to X-rays (Ager and Haynes, 1988).
On the other hand, the combination of UV light and a radiomimetic agent may lead to a decreased mutation frequency. In our earlier study (Kolman and Näslund, 1983) we described strongly decreased forward mutation frequencies in the lacI gene of E.coli strains with different abilities to repair DNA damage after combined treatment with UV light and EtO. Similar results were obtained by Hotz (1977) using a combination of UV light and ethylmethanesulfonate. The authors (Hotz, 1977; Kolman and Näslund, 1983) explained these findings by the capacity of low doses of UV light to `switch on' different DNA repair systems participating in the elimination of damage induced by the chemical mutagen.
The data presented in this paper indicate an increase in the effectiveness of the DNA damaging effect of the combined treatment, as measured by PFGE, irrespective of the order in which -rays and EtO were applied. Moreover, a considerable increase in FAR on HDR exposure after both combined treatments (Figure 4) can be interpreted as a synergistic effect. However, mutation induction might be considerably influenced by the order of their application, probably depending on induction of DNA repair functions. Additional experiments are necessary for a better understanding of the combined action of -radiation and radiomimetic agents. Such experiments are now in progress in our laboratory.
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Acknowledgments |
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This work was financially supported by the Swedish Radiation Protection Institute, the Swedish Fund for Research Without Animal Experiments and the Slovak Grant Agency for Science (grant no. 2/4029/97).
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Notes |
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2 To whom correspondence should be addressed. Tel: +46 8 164 038; Fax: +46 8 732 5561; Email: ada.kolman{at}molgen.su.se
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References |
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Received on February 9, 1999; accepted on November 23, 1999.
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