Mutagenesis vol. 18 no. 6 pp. 545-548,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Genotoxic and protective effects of hyperbaric oxygen in A549 lung cells
Universitätsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm, Germany
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Abstract |
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The human lung cell line A549 is frequently used as an in vitro model for studying lung toxicity and genotoxicity of environmental mutagens and carcinogens. Hyperbaric oxygen (HBO) treatment has been shown to be an excellent model for investigating the genetic consequences of oxidative stress in vitro and in vivo. We have now studied the genotoxic effects of and adaptive protection by HBO in A549 cells. Using the alkaline Comet assay, our results show that HBO exposure directly induces DNA damage but reduces the DNA-damaging effect of a second HBO treatment of the cells 24 h later. HBO pretreatment also reduces potassium chromate-induced genotoxicity in A549 cells. Heme oxygenase-1 (HO-1) protein level is increased 24 h after HBO exposure and inhibition of HO-1 activity by tin-mesoporphyrin increased HBO genotoxicity. These results are in accordance with previous findings suggesting an involvement of HO-1 in the adaptive protection. Our study indicates that A549 cells are an appropriate cell culture system for the evaluation of genetic effects induced by HBO in lung cells.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Hyperbaric oxygen (HBO) treatment as used therapeutically has been shown to induce genotoxic effects in peripheral blood cells of healthy human subjects (for a review see Speit et al., 2002
). Interestingly, the DNA-damaging effect of HBO was only seen immediately after a single HBO exposure. It was not detected after further treatments under the same conditions, indicating an increase in cellular defence against oxidative stress. Further investigations suggested an involvement of heme oxygenase-1 (HO-1) in this adaptive protection (Speit et al., 2000; Rothfuss et al., 2001). Since induction of oxidative DNA damage, DNA repair and formation of mutations, as well as induction of antioxidant defence mechanisms, are biological events on the cellular level, they can be easily and adequately studied in cultured mammalian cells. Therefore, we have established and successfully used an HBO exposure chamber for cell cultures (Rothfuss et al., 1999, 2000; Rothfuss and Speit, 2002). However, the cell culture systems used up to now, i.e. human lymphocytes and V79 cells, showed some limitations with regard to the characterization of potential mutagenic risks caused by HBO exposure. Peripheral human lymphocytes are not the primary target cells of in vivo HBO exposure and do not proliferate in vivo. The V79 cell line is a permanent Chinese hamster cell line derived from lung cells but without specific features of lung cells. V79 cells seem to be over-sensitive to HBO-induced oxidative damage and it has been shown that one important defence system, the antioxidant enzyme HO-1, is not inducible in this cell line (Rothfuss and Speit, 2002).
A549 cells, a human pulmonary epithelial cell line derived from a lung carcinoma (Lieber et al., 1976), have been frequently used to study toxic and genotoxic effects of environmental pollutants because lung cells represent the principal biological target for inhaled (geno)toxins (Lee et al., 1996; Dubrovskaya and Wetterhahn, 1998; Ollikainen et al., 2000; Hodges et al., 2001; Hodges and Chipman, 2002). Two of these studies used the Comet assay to determine oxidative DNA damage in A549 cells (Ollikainen et al., 2000; Hodges et al., 2001). A549 cells have specific cellular features, such as high levels of glutathione (Carmichael et al., 1988) and high (non-induced) HO-1 gene expression (Dubrovskaya and Wetterhahn, 1998), which contribute significantly to the cell response to oxidative stress. To characterize the usefulness of this cell line for the evaluation of mutagenic effects caused by HBO, we have now studied the genotoxic and protective effects of HBO exposure in A549 cells as well as the involvement of HO-1 in antioxidant defence.
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Materials and methods |
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Cell culture
The A549 cell line (American Type Culture Collection, Rockville, MD) is an epithelial-like human lung cell line derived through explant culture of carcinomatous lung tissue from a 58-year-old male Caucasian (Lieber et al., 1976
) and was a gift from Dr Andrea Hartwig (Karlsruhe, Germany). The V79 cell line is a permanent Chinese hamster cell line derived from lung tissue. Adherent cells were cultured in minimal essential medium (MEM), supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and 50 µg/ml gentamicin. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and harvested with 0.15% trypsin and 0.08% EDTA.
Exposure to HBO
HBO exposure was performed in a small, temperature-controlled (37°C) hyperbaric chamber as described previously (Rothfuss et al., 2001). The gas composition was 98% O2 and 2% CO2 in order to maintain physiological pH. Before compression, the chamber was flushed with gas and compressed at 0.2 bar/min until the required pressure was reached. If not otherwise indicated, experiments were conducted for 2 h at 3 bar pressure. After exposure, the chamber was decompressed (0.2 bar/min) to atmospheric pressure. Control experiments with hyperbaric air (21% O2) revealed no induction of DNA damage under these conditions. Cells were exposed to HBO in 25 cm2 cell culture flasks in 3 ml serum-free MEM. After exposure to HBO, the cells were either washed and the medium was replaced by fresh complete medium or harvested with trypsin and immediately processed in the Comet assay.
Mutagen treatment
To induce DNA damage, A549 cells were treated with K2CrO4 (50–100 µM) for 1 h at 37°C. After mutagen treatment cells were harvested with trypsin and immediately processed in the Comet assay.
Tin-mesoporphyrin (SnMP) treatment
A549 cells were treated with freshly prepared 10 and 100 µM SnMP (Prophyrin Products, Logan, UT) for 24 h before HBO exposure. As SnMP is sensitive to light, the metalloporphyrin solution was prepared in a darkened room and the cell culture flasks were wrapped with aluminium paper.
Comet assay
The Comet assay was performed as described earlier (Speit and Hartmann, 1999). The standard Comet assay protocol for A549 and V79 cells was 30 min alkaline denaturation followed by 30 min electrophoresis. Images of 50 randomly selected cells stained with ethidium bromide were analysed by image analysis (Perceptive Instruments, Haverhill, UK). The mean tail moment (percentage of DNA in the tail x tail length) of the individual cells was determined according to the image analysis software (Comet assay II V1.02). Differences between mean values from three independently repeated experiments were tested for statistical significance using Student’s t-test.
Western blot analysis
Protein extracts were obtained from A549 cell cultures before and 24 h after exposure to HBO by sonication in RIPA buffer containing 50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.1% SDS and the protease inhibitor phenylmethylsulphonyl fluoride. The cell extract was incubated at 4°C for 25 min, then centrifuged (15 min, 14 000 r.p.m.), the supernatant was shock-frozen in liquid nitrogen and the protein content of the samples was determined. Prior to analysis, 20 µg of protein was boiled for 5 min in an equal volume of 2x SDS sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 2% ß-mercaptoethanol, 20% glycerol and 0.02% bromphenol blue). Samples were separated on a 12% SDS–polyacrylamide gel for 2.5 h at 30 mA. The proteins were then electroblotted onto a PVDF membrane (Immobilon-P; Millipore, Bedford, MA) and incubated for 1 h in phosphate-buffered saline, 0.1% Tween 20 buffer (PBST) containing 5% non-fat dry milk. Rabbit anti-human HO-1 antibody (StressGen, Victoria, Canada) diluted 1:1000 in blocking solution was added. After incubation for 2 h, the membrane was washed extensively with PBST and incubated with goat anti-rabbit IgG antibody (StressGen) diluted 1:2500 for 1 h. The membrane was washed again and membrane-bound antibodies were visualized by enhanced chemiluminescence (Amersham-Pharmacia Biotech, Freiburg, Germany) according to the manufacturer’s protocol.
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Results |
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Exposure to HBO for 2 h caused a significant increase in DNA migration in A549 cells (Figure 1). However, when HBO-treated cells were exposed to HBO under the same conditions for a second time 24 h later, the DNA-damaging effect of HBO was significantly lower (Figure 1). HBO treatment led to a clear induction of HO-1 (Figure 2). Western blot analysis indicated enhanced HO-1 protein levels 24 h after a single HBO treatment, i.e. at the time point when reduced induction of genotoxic effects by a second HBO treatment (Figure 1) was observed. To determine a possible effect of cell density on HBO-induced DNA effects, we exposed cultures with different cell counts (between 1 and 3 million cells per flask) to a single HBO treatment. DNA damage (tail moment) was measured in the Comet assay using two different times for the electrophoresis (30 and 40 min) in three independently repeated tests. The results clearly indicated that there was no significant difference in DNA damage under these test conditions and excluded an influence of cell density on the second HBO treatment (data not shown).
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Comparative dose–response experiments with V79 cells revealed a significant genotoxic effect of HBO in both cell lines after 2 h exposure and no further increase after 3 h exposure (Figure 3). However, the DNA-damaging effect of HBO in the Comet assay was much more pronounced in V79 cells compared with A549 cells.
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To test whether HO-1 activity in A549 cells is involved in the low genotoxicity of HBO in this cell line, we inhibited HO-1 with SnMP (Figure 4). Treatment with SnMP (10 and 100 µM) alone did not induce DNA effects in the Comet assay. Pretreatment with SnMP for 24 h before exposure to HBO led to increased DNA migration in comparison with HBO treatment alone. This concentration-related effect was observed in three independently repeated experiments. However, the inter-experimental variability of the Comet assay was high and statistical significance of the difference was just missed (P = 0.059 for 100 µM SnMP).
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To study whether HBO pretreatment protects A549 cells against the genotoxic action of other (oxidative) mutagens, we treated cultures 24 h after a single HBO exposure (2 h) with K2CrO4 (Figure 5). The HBO-induced genotoxic effect completely disappeared within 24 h (HBO 24 h). K2CrO4 induced a concentration-related statistically significant (P < 0.01) increase in DNA migration. HBO pretreatment reduced the genotoxic effect of K2CrO4. At 50 µM K2CrO4 a small (non-significant) reduction in the tail moment was observed and the DNA-damaging effect of 100 µM K2CrO4 was significantly (P < 0.05) reduced.
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Discussion |
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Our results clearly indicate that HBO treatment leads to a genotoxic effect in A549 cells and also induces adaptive protection against further HBO exposures. These findings fundamentally confirm our previous studies, but there are some characteristic differences and new aspects.
The genotoxic effect of HBO occurs under the same experimental conditions but is smaller than in other cultured cells tested (Rothfuss et al., 1999). The direct comparison between A549 and V79 cells (Figure 3) reveals that V79 cells are extremely sensitive to HBO-induced oxidative stress. A549 cells have a high activity of HO-1 (Dubrovskaya and Wetterhahn, 1998), which may influence the response to HBO. We did not perform a quantitative comparative measurement of HO-1 protein in the two cell lines, but a higher amount of HO-1 protein in A549 cells was indicated by the observation that under the same western blot conditions the same amount of protein led to a much higher intensity of the HO-1 band for A549 cells. Furthermore, pretreatment of A549 cells with SnMP, a selective inhibitor of HO-1, enhanced HBO-induced genotoxicity (Figure 4) while it had no enhancing effect in normal V79 cells (Rothfuss and Speit, 2002). Our results are in agreement with an earlier investigation in which A549 cells stably transfected with rat HO-1 cDNA exhibited increased HO-1 activity and increased resistance to hyperoxic oxidant insult. Tin-protoporphyrin (which acts in the same way as tin-mesoporphyrin) reversed the increased survival observed in A549 cells overexpressing HO-1 (Lee et al., 1996). However, besides the high amount of HO-1, A549 cells also have a high glutathione content (Carmichael et al., 1988), which also might influence the cell response to oxidative attack.
Despite the high HO-1 gene expression in A549 cells, HBO caused an additional induction of HO-1 protein levels and enhanced protection against the genotoxic effect of a second HBO treatment. In contrast, chromium(VI) induced oxidative stress and elevated expression of the HO-1 gene in normal human lung cells (LL 24) but not in A549 cells (Dubrovskaya and Wetterhahn, 1998). We conclude that HBO is a very strong inducer of HO-1. It is known that HO-1 is induced by various forms of oxidative stress (Choi and Alam, 1996; Elbirt and Bonkovsky, 1999), but not much is known about the relative efficiency of the various HO-1 inducers.
Previous investigations with human lymphocytes have shown that induction of HO-1 by HBO not only protects against genotoxic effects of further HBO exposures but also against the genotoxic effects of other oxidants (Speit et al., 2000). Here we tested the effect of HBO on chromate-induced DNA damage and observed a small but significant protective effect. Recently, Wise and co-workers showed that hexavalent chromium, such as in soluble sodium chromate, is cytotoxic and genotoxic to human lung fibroblasts (Wise et al., 2002). The DNA-damaging action of chromate is complex and DNA–protein crosslinks, DNA strand breaks and oxidative damage via a Fenton-like reaction may also be involved (Luo et al., 1996; Merk et al., 2000). Due to the involvement of crosslinks, the DNA migration-inducing effect in the Comet assay is small, as previously observed in V79 cells (Merk et al., 2000). Although the mechanism(s) by which HO-1 exerts its role in antioxidant protection is not yet clear, induction of ferritin due to an increase in free iron levels could play a role. Increased ferritin levels would restrict redox-active iron from participating in the Fenton reaction and thus protect cells from the induction of oxidative DNA damage (Vile and Tyrrell, 1993; Vile et al., 1994; Speit et al., 2000). It is not known yet whether chromate itself can induce HO-1 in A549 cells. As mentioned above, hexavalent chromium did not induce HO-1 in A549 cells (Dubrovskaya and Wetterhahn, 1998), although it significantly induced oxidative DNA damage in this cell line (Hodges et al., 2001).
In summary, we have examined the effect of HBO treatment on A549 lung cells. A549 cells seem to be less sensitive to the DNA-damaging action of HBO in comparison with human lymphocytes and V79 cells. HBO also leads to the induction of adaptive antioxidant protection in A459 cells. Therefore, this permanently proliferating cell line seems to be a suitable in vitro model for the investigation of the genetic consequences of HBO-induced oxidative stress and a useful surrogate for evaluating the mutagenic risk of HBO treatment in lung cells.
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Acknowledgements |
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We would like to thank Dr Andreas Rothfuss for stimulating discussions and Petra Schütz for excellent technical assistance.
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Notes |
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1To whom correspondence should be addressed. Tel: +49 731 500 23429; Fax: +49 731 500 23438; Email: guenter.speit{at}medizin.uni-ulm.de
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Received on June 16, 2003; accepted on August 14, 2003.
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