Mutagenesis, Vol. 15, No. 1, 85-90,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction and repair of formaldehyde-induced DNA–protein crosslinks in repair-deficient human cell lines
Universitätsklinikum Ulm, Abteilung Medizinische Genetik, D-89070 Ulm, Germany
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Abstract |
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We have previously shown that the alkaline Comet assay (single cell gel electrophoresis) in a modified version is a sensitive test for the detection of formaldehyde-induced DNA–protein crosslinks (DPC). Our results also indicated that formaldehyde-induced DPC are related to the formation of chromosomal effects such as micronuclei and sister chromatid exchanges. To better understand the genetic consequences of formaldehyde-induced DPC we have now investigated the induction and removal of DPC in relationship to the formation of micronuclei in normal and repair-deficient human cell lines. We did not find significant differences between normal cells, a xeroderma pigmentosum (XP) cell line and a Fanconi anaemia (FA) cell line with respect to the induction and removal of DPC. However, the induction of micronuclei was enhanced in both repair-deficient cell lines, particularly in XP cells, under the same treatment conditions. Comparative investigations with the DNA–DNA crosslinker mitomycin C (MMC) revealed a delayed removal of crosslinks and enhanced induction of micronuclei in both repair-deficient cell lines. FA cells were found to be particularly hypersensitive to micronucleus induction by MMC. In contrast to the results with formaldehyde, induction of micronuclei by MMC occurred at much lower concentrations than the effects in the Comet assay. Our results suggest that more than one repair pathway can be involved in the repair of crosslinks and that disturbed excision repair has more severe consequences with regard to the formation of chromosomal aberrations after formaldehyde treatment than has disturbed crosslink repair.
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Introduction |
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Numerous studies have shown that formaldehyde is genotoxic and mutagenic to mammalian cells and that it induces a broad spectrum of genetic effects (Ma and Harris, 1988
; IARC, 1995; Conaway et al., 1996). Animal studies demonstrated that high concentrations of formaldehyde can cause irreversible damage to the nasal epithelium of rats and that in some cases rats exposed to these concentrations developed neoplasia (IARC, 1995). The primary genotoxic effect of formaldehyde seems to be the formation of DNA–protein crosslinks (DPC) in target tissues. In vivo experiments with rats and monkeys indicated that the rate of DPC formation is proportional to the tissue concentration of formaldehyde (Casanova et al., 1991, 1994). It has therefore been suggested that the rate of formation of DPC can be regarded as a surrogate for the delivered concentration of formaldehyde and that the determination of DPC levels might improve human cancer risk estimates (Casanova et al., 1991; Conolly and Andersen, 1993). However, the biological significance of DPC for mutagenesis and carcinogenesis is at present poorly understood. Although genotoxicity of formaldehyde has been demonstrated in various assays, it is not clear whether formaldehyde-induced DPC are directly involved in the formation of mutations and what kind of mutations might be responsible for formaldehyde-induced carcinogenesis. Our comparative studies on the induction of DPC and other genetic end-points in V79 cells showed that formaldehyde significantly induced DPC, sister chromatid exchanges (SCE) and micronuclei (MN) in the same range of concentrations but did not induce gene mutations in the HPRT test (Merk and Speit, 1998). We used a modification of the alkaline Comet assay to measure the induction and removal of formaldehyde-induced DPC. DPC were significantly induced by formaldehyde at concentrations that caused only low cytotoxicity and were removed within 24 h, with considerable persistence during the first hours after treatment. A comparison of the dose–response curves for DPC and MN suggested a causal connection. Possibly, replication and/or incomplete repair of DPC-containing DNA might lead to gaps and, consequently, to chromosomal aberrations.
The repair of DPC is not completely understood. There are studies indicating that DPC are repaired by excision repair mechanisms (Fornace and Seres, 1982; Gantt, 1987; Oleinick et al., 1987), in contrast to DNA–DNA crosslinks, for which a more specific crosslink repair has been suggested (Sasaki, 1975; Fujiwara, 1982; Lambert et al., 1997). One approach to better understand the relationship between induced DNA lesions, their repair and mutagenic consequences is the use of cell lines deficient in specific pathways of DNA repair (Fornace and Seres, 1982; Collins, 1993; Digweed, 1993; Helbig and Speit, 1997; Merk and Speit, 1997). We therefore investigated the induction and removal of formaldehyde-induced DPC in a normal human cell line in comparison with a xeroderma pigmentosum (XP) cell line and a Fanconi anaemia (FA) cell line. The XP cell line is deficient in nucleotide excision repair (Van Duin et al., 1989; Speit and Hartmann, 1995) while the FA cell line has a genetic defect leading to hypersensitivity towards DNA–DNA crosslinkers like MMC (Duckworth-Rysiecki et al., 1986; Saito et al., 1993). In addition to formaldehyde we studied the effects of MMC in these cell lines to see whether specific differences exist in the repair of different kinds of crosslinks.
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Materials and methods |
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Cells and chemicals
The normal human cell line MRC5CV1, the XP cell line XP12ROSV (complementation group A) and the FA cell line GM06914 (complementation group A) were used for the experiments. These cell lines are derived from male donors and are SV40 transformed. Cells were cultivated in minimal essential medium (MEM) with Earle's salts, supplemented with 10% foetal calf serum, 2 mM glutamine and antibiotics. Cells were maintained in a humidified incubator at 37°C with 5% CO2 at pH 7.2 and harvested with 0.15% trypsin and 0.08% EDTA (Speit and Hartmann, 1995
). Formaldehyde was purchased from Merck (Darmstadt, Germany) and MMC was from Sigma (Munich, Germany). Both substances were dissolved immediately before use in Hank's balanced salt solution (HBSS). Agarose (MEEO) was supplied by Roth (Karlsruhe, Germany) and low melting point agarose (LMA) (Sea plaque) was from Biozym (Hameln, Germany). Cell culture media were obtained from Biochrom (Berlin, Germany) and the other chemicals were purchased from Sigma (Munich, Germany).
Comet assay
Cultures were treated with formaldehyde or MMC in serum-free medium for 2 h. Treated cells and controls were trypsinized and kept on ice to inhibit repair. For the detection of crosslinks with the Comet assay (Merk and Speit, 1999), ~4x105 cells in 500 µl were then exposed to 3 Gy of 60Co 
-rays (Gammacell 2000; Nuclear Data, Frankfurt, Germany) at 4 Gy/min. In the presence of crosslinks, -ray-induced DNA migration is reduced. The reduction in -ray-induced DNA migration is used as a measure of crosslinks.
Cells were processed in the alkaline version of the Comet assay as described in detail earlier (Speit et al., 1998). About 104 cells in 10 µl were mixed with 120 µl of LMA (0.5%) and added to a slide precoated with agarose. Lysis was performed overnight at pH 10. After that cells were placed in an electrophoresis chamber (in an ice bath at 4°C), exposed to alkali (pH 13) for 25 min and then electrophoresis was performed for 25 min at 25 V (0.86 V/cm) and 300 mA. Slides were neutralized, dried with 100% ethanol, stained with ethidium bromide (20 µg/ml) and analysed using a fluorescence microscope and image analysis (Comet Assay II V1.02; Perceptive Instruments, Haverhill, UK). Fifty cells per slide were evaluated and the mean of the tail moment (DNA migrationxtail intensity) was taken as a measure of DNA damage. For a direct comparison of the three cell lines, the relative tail moment is given, i.e. the difference expressed as a percentage in comparison with a culture which was only irradiated (= 100%).
Micronucleus test
To analyse the frequency of cells with MN, cultures were treated with formaldehyde or MMC in serum-free medium for 2 h, washed twice and further cultivated for 48 h. Trypsinized cells were exposed briefly to a hypotonic solution (0.4% KCl), then fixed three times (15 min) with methanol/acetic acid and carefully dropped onto a slide. Air-dried slides were stained with acridine orange (50 µg/ml) and 1500 cells/slide were analysed for the presence of MN.
All tests were repeated in independent experiments. Differences between the control and other values were tested for significance using Student's t-test.
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Results |
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Formaldehyde treatment of the three cell lines did not induce DNA migration in the Comet assay under standard test conditions (data not shown).
-Irradiation (3 Gy) alone led to a strongly increased tail moment: from 0.4 ± 0.2 to 6.0 ± 1.8 in MRC5CV1 cells, from 0.2 ± 0.1 to 6.5 ± 1.2 in GM06914 and from 0.3 ± 0.0 to 6.5 ± 0.2 in XP12ROSV (mean of two experiments ± SEM). When formaldehyde-treated (2 h) cells were irradiated with -radiation at the end of the formaldehyde treatment and directly analysed in the Comet assay, a clear effect on DNA migration can be seen (Figure 1). Formaldehyde caused a concentration-related decrease in radiation-induced DNA migration (expressed as a percentage, i.e. 100% value represents irradiation alone) with a significant (P < 0.05) reduction at 125 µM. At 500 µM formaldehyde, DNA migration was completely inhibited in most of the cells. The results do not indicate any significant difference in the induction of DPC between the three cell lines.
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Figure 2
shows the time course for the elimination of formaldehyde-induced DPC measured with the modified Comet assay. Cells were treated with formaldehyde (125 µM) for 2 h and irradiated with -radiation (3 Gy) immediately at the end of the formaldehyde treatment or 2, 4, 8 or 24 h later. Each culture was analysed in the Comet assay immediately after irradiation. The figure gives the relative reduction in DNA migration (tail moment) compared with irradiated controls without formaldehyde treatment (= 100%; see above). The increase in radiation-induced DNA migration with time reflects the repair of DPC. It can be seen that 24 h after formaldehyde treatment there is no longer any inhibition of DNA migration, indicating complete removal of DPC in all three cell lines.
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However, clear differences were found between the three cell lines with respect to the induction of MN (Figure 3
). The frequency of MN was increased after treatment with formaldehyde in a concentration-related manner for the same range of concentrations in all cell lines. The induction of MN was stronger in both repair-deficient cell lines than in normal cells. The background level of MN was subtracted for a direct comparison of the three cell lines. The XP cell line had a higher background frequency of MN (10%) than the other two cell lines (3%) but also showed the highest production of MN after formaldehyde treatment. Besides higher induced frequencies of micronucleated cells, the frequency of cells with higher numbers of MN per cell was increased in the repair-deficient cell lines, particularly in XP cells (Figure 4).
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Comparative investigations with the DNA–DNA crosslinker MMC revealed a slight induction of DNA migration in the Comet assay under standard conditions at cytotoxic concentrations, with a maximum effect at 200 µM (Figure 5A
). Despite this small inducing effect, MMC clearly reduced -ray-induced DNA migration in accordance with our results for V79 cells (Merk and Speit, 1999). No significant difference was found between the three human cell lines (Figure 5B). In contrast, the efficiency of removal of MMC-induced crosslinks seems to be different (Figure 6). No significant cancellation of the inhibition of -radiation induced DNA migration was seen in XP and FA cells in the course of the first 8 h after the end of MMC treatment (10 µM). After 24 h, no inhibition of induced DNA migration was found in MRC5CV1 and FA cells, while there was still significant inhibition in XP cells.
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The induction of MN by MMC showed characteristic differences between the cell lines (Figure 7
). The two repair-deficient cell lines also showed hypersensitivity towards MMC. The difference between normal and XP cells was similar to after treatment with formaldehyde. However, in FA cells MN frequencies increased steeply at low MMC concentrations and reached a plateau at 0.1 µM. Higher concentrations completely inhibited cell growth and could not be evaluated. The enhanced induction of MN in the repair-deficient cell lines was again due to a higher frequency of micronucleated cells and to a higher frequency of micronuclei per cell (Figure 8). In contrast to the results obtained with formaldehyde, MMC induced MN in all three cell lines at much lower concentrations than those at which it reduced DNA migration in the Comet assay.
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Discussion |
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Crosslinks (DNA–DNA and DNA–protein) are induced by various chemical and physical agents, many of them known or suspected carcinogens. However, it is not clear to what extent the different types of crosslinks are directly responsible for the formation of mutations and cancer (Ma and Harris, 1988
; Zamble and Lippard, 1995; Merk and Speit, 1999). We have recently shown that the Comet assay in a modified version is a sensitive test for the detection of formaldehyde-induced DPC but less sensitive for the evaluation of DNA–DNA crosslinkers (Merk and Speit, 1998, 1999). The results also indicated that the relationship between crosslinking and mutagenicity seems to be different for the different types of crosslinks (Merk and Speit, 1999). For formaldehyde, a causal connection between the induction of DPC and the formation of MN was suggested and it was proposed that replication and/or incomplete repair of DPC-containing DNA might lead to gaps and, consequently to chromosomal aberrations (Merk and Speit, 1998). Our comparative investigations with normal and repair-deficient human cell lines have further elucidated the relationship between the induction and repair of DPC and the formation of MN. First, we could show that there is no significant difference in the induction and removal of DPC between the cell lines as measured with the Comet assay. There also seems to be no difference between the results obtained with these human cell lines (Figures 1 and 2
) and those previously published for V79 cells (Merk and Speit, 1998). The present data suggest that neither defective nucleotide excision repair (XP cells) nor defective crosslink repair (FA cells) seems to cause significantly delayed removal of formaldehyde-induced DPC. In accordance with our results, DPC induced by trans-platinum were also repaired in excision-deficient XP cells (Fornace and Seres, 1982; Gantt, 1987). However, using alkaline elution, it was found that the removal of trans-platinum-induced DPC was slower in XP than in normal cells (Fornace and Seres, 1982). Up to now not much is known about the repair of DPC in mammalian cells and it is unclear whether different types of DPC are repaired in the same manner. In general, DPC are repaired more slowly than DNA single-strand breaks (Sugiyama et al., 1986; Oleinick et al., 1987) and excision repair mechanisms have been proposed (Fornace and Seres, 1982; Oleinick et al., 1987). However, our findings with the Comet assay and earlier studies using the alkaline elution technique (Grafstrom et al., 1984; Gantt, 1987) suggest that cells deficient in excision repair can efficiently remove DPC and imply a further repair pathway.
While our studies using the Comet assay did not indicate any significant difference between normal and repair-deficient cells with regard to the repair capacities for formaldehyde-induced DPC, clear differences were found with the MN test. The Comet assay results (i.e. the similar abolition of reduced DNA migration) suggest that the first step(s) in DPC repair (i.e. removal of the DNA-bound protein or the crosslinked DNA base from the DNA) takes place at a normal rate in the repair-deficient cell lines. However, both of the repair-deficient cell lines were hypersensitive towards the formation of formaldehyde-induced MN. Assuming that MN are the result of incorrect DNA repair, it can be concluded that both repair pathways are involved in the error-free repair of formaldehyde-induced DNA lesions. If DPC are responsible for the formation of MN, as suggested earlier (Merk and Speit, 1998), both repair defects seem to reduce the fidelity of DPC repair. However, it cannot be excluded at present that another formaldehyde-induced minor DNA lesion is involved in formaldehyde-induced MN.
FA cells are known to be hypersensitive to MMC and other DNA–DNA crosslinkers, leading to reduced cell survival and increased chromosome damage (Sasaki and Tonomura, 1973; Saito et al., 1993). The molecular basis for this sensitivity has not been fully elucidated yet but a direct or indirect defect in the crosslink repair pathway is likely (Buchwald and Moustacchi, 1998). For the FA cell line used in our experiments, reduced cell survival after MMC treatment has been shown previously (Duckworth-Rysiecki et al., 1986; Saito et al., 1993) and this sensitivity towards MMC was confirmed in our MN experiments. In contrast to the results with formaldehyde, removal of MMC-induced crosslinks as measured with the Comet assay showed some differences between the cell lines. We observed almost no crosslink removal in both repair-deficient cell lines in the course of the first 8 h after treatment. Twenty-four hours after the end of the treatment, complete removal of crosslinks was achieved in normal cells and FA cells, while in XP cells the majority of crosslinks still persisted. These results indicate that both repair pathways are involved in the resolution of DNA–DNA crosslinks and point to a clear difference in the repair of this type of crosslink on the one hand and DPC on the other hand. The delayed removal of crosslinks and the enhanced induction of MN by MMC in the XP cell line is in accordance with recent results showing that repair proteins which are involved in nucleotide excision repair specifically recognize and bind to MMC crosslinks (Warren et al., 1998). Although delay in DNA crosslink removal is more pronounced in XP cells, the defect in FA cells seems to be more relevant for the formation of MN after MMC treatment. In contrast, the defect in XP cells has a stronger impact on formaldehyde-induced MN despite an apparent normal rate of DPC removal. It is still unclear to what extent formaldehyde-induced DPC are directly responsible for MN formation. However, our comparative investigations suggest that disturbed excision repair has more severe consequences with regard to the formation of chromosomal aberrations after formaldehyde treatment than has disturbed crosslink repair.
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Acknowledgments |
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This work was supported by the program Environment and Health (PUG) at the Forschungszentrum Karlsruhe with funds from the Department for the Environment Baden-Württemberg.
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Notes |
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1 To whom correspondence should be addressed.Tel: +49 731 5023429; Fax: +49 731 5023438; Email: guenter.speit{at}medizin.uni-ulm.de
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Received on July 26, 1999; accepted on September 27, 1999.
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