Mutagenesis, Vol. 17, No. 3, 183-187,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells
Universitätsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm, Germany
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Abstract |
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Formaldehyde (FA) is known to be a genotoxic substance. FA induces DNA–protein crosslinks (DPC) as the primary DNA lesion. However, the significance of DPC for FA-induced mutations and the mechanism(s) of mutation formation are at present poorly understood. Our previous results indicated that FA-induced DPC seem to be related to cytotoxicity and clastogenicity but do not lead to gene mutations in mammalian cells. We now demonstrate that FA efficiently induces mutations in the mouse lymphoma assay (MLA). Treatment of L5178Y cells with FA for 2 h caused a clear and concentration-related mutagenic effect in the MLA. As this mutagenic effect was mainly due to a strong increase in small colony mutants, we suggest that FA mainly causes mutations by induction of chromosomal aberrations. Molecular characterization of spontaneous and FA-induced mutants by loss of heterozygosity analysis showed an extensive loss of functional tk sequences, supporting a clastogenic mechanism of mutation formation. Whole chromosome fluorescence in situ hybridization was used to further elucidate the mechanism(s) of chromosome mutations. Our results indicate that small-scale chromosomal rearrangements (e.g. deletions or recombinations) are mainly involved in FA-induced mutagenesis in the MLA.
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Introduction |
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Formaldehyde (FA) is a highly reactive chemical which is widely used in the manufacturing processes of various industries and in consumer products. Animal studies have demonstrated that high concentrations of FA can induce tumors in the nasal epithelium of rats and FA is suspected to be a human carcinogen (IARC, 1995
). Numerous studies have shown that FA is genotoxic and mutagenic to mammalian cells (Ma and Harris, 1988; IARC, 1995; Conaway et al., 1996). The primary genotoxic effect seems to be the formation of DNA–protein crosslinks (DPC) in target tissues (Casanova et al., 1994). However, the biological significance of DPC for mutagenesis and carcinogenesis is at present poorly understood. It is still a matter of debate whether DPC are directly involved in the formation of mutations, whether specific types of mutations are induced and whether these are responsible for FA-induced carcinogenesis. We have shown that the Comet assay is well suited to sensitive detection of FA-induced DPC in mammalian cells (Merk and Speit, 1998). Comparative investigations using V79 cells and various end-points for genotoxicty and cytotoxicity showed that FA significantly induced DPC, sister chromatid exchanges and micronuclei in the same range of concentrations, in parallel with the induction of cytotoxicity (relative cloning efficiency). In contrast, treatment with FA did not significantly induce gene mutations in the HPRT test with V79 cells (Merk and Speit, 1998, 1999). Our results suggested that FA-induced DPC seem to be related to cytotoxicity and clastogenicity but do not lead to the formation of gene mutations in directly exposed mammalian cells.
To further characterize the mutagenic potential of FA, we have now analyzed the formation of DPC and possible induction of gene mutations in mouse lymphoma L5178Y cells. In contrast to the HPRT gene mutation test, the mouse lymphoma assay (MLA) additionally detects gross alterations like large deletions and rearrangements (Honma et al., 1999). The extent of genetic alterations leading to a TK-deficient cellular phenotype can be determined by analyzing loss of heterozygosity (LOH) at heteromorphic microsatellite repeats in the chromosomal region harboring the tk gene (Liechty et al., 1998). We therefore investigated the extent of LOH at five polymorphic loci on chromosome 11 of FA-induced mutants to further characterize the mechanism of FA-induced mutagenesis. However, LOH analysis provides limited information because it does not detect translocations and changes in chromosome number and it cannot distinguish between deletion and mitotic recombination. To overcome these shortcomings, we combined the results from LOH analysis with whole chromosome 11 fluorescence in situ hybridization. Using this technique, chromosome 11 sequences in the metaphases of mutant cells can be readily identified and the homologous chromosomes 11 can be differentiated by the different sizes of their centromeric regions (Hozier et al., 1982; Liechty et al., 1998). Chromosome 11a has a smaller centromeric region than chromosome 11b and carries a non-functional tk allele because of a point mutation. Our results suggest that mitotic deletions or recombinations play a major role in FA-induced mutagenesis and support the view that gross genetic alterations but not point mutations could be important for FA-induced carcinogenesis.
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Materials and methods |
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Cell culture
L5178Y tk+/– mouse lymphoma cells (clone 3.7.2C) were cultivated in suspension in RPMI-1640 glutamax medium supplemented with 10% heat-inactivated horse serum and penicillin/streptomycin/kanamycin and maintained at 37°C with 5% CO2. Master stocks were obtained after expansive cultivation and purging of tk-/- mutants (Honma et al., 1999
). Stocks were maintained in liquid nitrogen at a density of 1x106 cells/ml. For the experiments, stock cultures were thawed and used on day 6. Horse serum and all other cell culture media and complements were supplied by Gibco BRL. FA was purchased from Merck (Darmstadt, Germany) and was dissolved immediately before use in Hank's balanced salt solution.
Comet assay
For the detection of crosslinks, the modification of the alkaline Comet assay described by Merk and Speit (1998) was performed. Cultures (treated with FA for 2 h or untreated controls) were trypsinized and kept on ice to inhibit repair. About 4x105 cells were exposed to 3 Gy 60Co 
-rays (Gammacell 2000; Nuclear Data, Germany) at 4 Gy/min. The irradiated cells were further processed according to a standard protocol of the Comet assay (Speit and Hartmann, 1999). The times of alkali denaturation and electrophoresis (0.86 V/cm) were 20 min each. Images of 50 randomly selected cells stained with ethidium bromide were analyzed by image analysis (Comet Assay II v.1.02; Perceptive Instruments, Haverhill, UK) and the mean tail moment of the individual cells was used as a measure of DNA damage.
Mutation assay
For determining mutagenic effects, the microwell method of the MLA was performed according to Honma et al. (1999). Cultures of 1x107 L5178Y cells in 20 ml of RPMI with 5% horse serum were exposed to FA for 2 h and subsequently washed twice with fresh medium. After an expression period of 48 h, during which cell densities were kept at 105–106 cells/ml, cells were diluted to 2000 cells/ml in RPMI 1640 with 20% horse serum containing 3 µg/ml trifluorothymidine (TFT) as selective agent. Aliquots of 200 µl were then plated in quadruplicate 96-well plates and incubated at 37°C with 5% CO2. After 12 days, the number of wells containing either small or large colonies were counted under an inverted microscope. Criteria for scoring and calculations were as follows: large colonies have a diameter of >1/3 of the well and small colonies are <1/3 of the diameter of the well. For determination of cloning efficiency directly after FA treatment and at the end of the expression period, an aliquot of each culture was counted to correct for loss of cells during treatment and diluted to 8 cells/ml with RPMI 1640 containing 20% horse serum; 200 µl of this dilution were plated in duplicate 96-well plates. After incubation at 37°C with 5% CO2 for 10 days, colonies were counted under an inverted microscope. The relative cloning efficiency (CE) in each test culture was determined by comparing cloning efficiencies in test and control cultures: CE (%) = (CEtest/CEcontrol)x100. The mutant frequency was corrected for the relative CE at the end of the expression period and calculated according to Honma et al. (1999) as MF = (CEmutant/CEviable)x106 (i.e. mutants/106 viable cells). The selective agent TFT and the positive control 4-nitroquinoline-1-oxide (4-NQO) were purchased from Sigma
LOH analysis
Forty-seven independent spontaneous large and small colony mutants as well as 37 independently induced large and small colony mutants (after exposure to 125 µM FA for 2 h) were picked from the selective plates of additionally performed experiments. Cells were transferred into 6-well plates and incubated in 5 ml of selective medium (RPMI 1640 with 20% serum and 3 µg/ml TFT). To obtain independent mutants, four parallel cultures per experiment were set up and treated separately (A, B, C and D) and only one large and one small mutant colony per culture were picked. Confluent cultures were further grown for 
14 days in 25 cm2 cell culture flasks with 10 ml of selective medium until 10x106 cells were obtained. DNA was isolated using DNAzol (Gibco BRL) according to the protocol of the manufacturer. DNA was resuspended in H2O and stored at 4°C until analysis. Primer sequences for amplification of the polymorphic locus D11Agl1 on chromosome 11 were taken from Liechty et al. (1996) and the touchdown PCR protocol was taken from Preisler et al. (2000). For the markers D11Mit21, D11Mit29, D11Mit63 and D11Mit69 the primer sequences were obtained from the February 1999 release of the mouse genome database (MGD) on the Mouse Genome Informatics web site of the Jackson Laboratory (Bar Harbor, ME) (http://www.informatics.jax.org). For these primers the PCR procedure was essentially the same, except that the annealing temperature was changed as follows: for the first two cycles 61°C, a ramp from 60 to 52°C for the following 7x2 cycles and 50°C for the last 12 cycles. PCR reactions (20 µl) were prepared by mixing 18 µl of PCR Master Mix [10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.5 U Taq polymerase (Pharmacia), 200 µM dNTP] with 5 pmol each primer and 2 µl of template DNA. Reactions were performed in a Perkin Elmer Model 9600 thermal cycler. PCR products of D11Agl1 were analyzed by electrophoresis on a 1.5% agarose gel in 1x TBE buffer; for the other PCR products a 2% agarose gel was used. All gels were stained with ethidium bromide.
Fluorescence in situ hybridization (FISH)
About 5x106 cells from mutant colonies were incubated for 24 h and then colcemid was added for 2 h. The medium was replaced by a hypotonic solution (0.4% KCl at 37°C). After 30 min the cells were fixed four times in methanol/acetic acid (2:1) for 15 min each. Finally, cells were dropped onto glass slides and the slides air dried.
For in situ hybridization the slides were aged for 10–20 days. Slides were incubated for 1 h with RNase (50 µg/ml at 37°C) and washed twice with 2x SSC to reduce the background. Then the slides were treated with pepsin (20 µg/ml at 37°C) for 7 min 30 s, followed by washing for 2x5 min in 1x phosphate-buffered saline (PBS). Before post-fixation for 15 min in 1x PBS, 50 mM MgCl2, 1% formaldehyde, slides were incubated for 5 min in 1x PBS, 50 mM MgCl2. Next, slides were washed for 2x5 min in 1x PBS followed by dehydration in an ethanol series (70, 70, 90, 95, 99 and 99%, –20°C, 2 min each). After that slides were air dried. Denaturation was done by dipping the slides for 75 s in formamide solution (70% in 2x SSC, pH 7.0) at 72°C. After dehydration in an ethanol series the slides were air dried. Before use the biotinylated mouse chromosome 11-specific paint (Cambio, UK) was denaturated for 12 min at 72°C and incubated for 2 h at 37°C. Slides were placed on a slide warmer at 40°C and 10 µl of chromosome painting probe were added for overnight incubation in a humid chamber at 37°C. After incubation at 45°C for 30 min the slides were washed for 3x10 min in 50% formamide, 2x SSC at 45°C, for 3x5 min in 2x SSC at 45°C and then for 5min in 2x SSC at 72°C. Detection and signal amplification were performed using materials from Vector Laboratories (Burlingame, USA) according to Dixkens et al. (1998). Counterstaining of whole DNA was achieved with DAPI. We limited the analysis to mutants that showed LOH for at least D11Mit67 because mutants with smaller losses would be difficult to differentiate from mutants with normal chromosomes using FISH (Liechty et al., 1998). Twenty metaphases from each mutant colony were analyzed for the number of chromosome 11 homologs, for their length and for chromosome 11 centromere sizes. As an internal standard the lengths of the chromosome 11 homologs were normalized to the length of the 12:13 Robertsonian translocation chromosome, which is characteristic of this cell line. This chromosome was chosen because it is readily identifiable in spreads and it is unlikely to be affected in mutants.
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Results |
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Figure 1
shows the effect of FA treatment (2 h) on -ray-induced DNA migration in mouse lymphoma cells. When FA-treated cells were irradiated with -radiation (3 Gy) at the end of the FA treatment and directly analyzed in the Comet assay, a clear effect on DNA migration was seen. While irradiation alone leads to a strongly increased tail moment (4.47 ± 0.42 compared with 0.49 ± 0.07 in controls), FA (31.25–500 µM) caused a concentration-related decrease in radiation-induced DNA migration. At 500 µM FA DNA migration was completely inhibited in all cells (tail moment 0.26 ± 0.02).
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Figure 2
summarizes the results of the MLA after treatment of L5178Y cells with FA at different concentrations. Total mutant frequencies (MF) (Figure 2A) as well as MFs for large colonies (LC) and small colonies (SC) (Figure 2B) are given. Untreated L5178Y cells revealed a mean background MF of 133x10-6 and treatment of cells with FA for 2 h led to a clearly increased MF at concentrations >62.5 µM. FA at 250 µM caused a 7-fold increase in the spontaneous MF. A concentration-related cytotoxic effect was measured which appeared at a concentration of 62.5 µM and reduced the relative cloning efficiency to 30% at 250 µM. 4-NQO was used as a positive control. The distribution of LC and SC is shown in Figure 2B. In untreated controls the frequency of LC is a little higher than that of SC. After treatment with FA (125 and 250 µM) the frequencies of SC are clearly increased, while there is only a marginal increase in the frequency of LC. In contrast, in the positive control cultures treated with 4-NQO, an increase in both SC and LC was measured.
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To determine the types of alterations present in FA-induced mutants, we studied the extent of LOH at five polymorphic markers (D11Agl1, D11Mit67, D11Mit29, D11Mit21 and D11Mit63). The markers used are almost equally distributed along chromosome 11. Independent mutant colonies were isolated for LOH analysis from additionally performed experiments (treatment with 125 µM FA for 2 h resulting in a MF of >700 mutants/106 viable cells at a CE of
70%). A summary of the results of the LOH analysis is given in Figure 3. A total of 47 spontaneous and 37 FA-induced colonies were investigated. LOH at the marker D11Agl1, which is located in the tk gene, occurred in all investigated independent SC mutants from control (25) and FA-treated (18) cultures. In contrast, this type of LOH was only present in 10 of 22 spontaneous LC and in 13 of 19 investigated FA-induced LC. Comparison of LOH patterns at other polymorphic markers revealed extended LOH in six spontaneous SC, five spontaneous LC, one FA-induced SC and seven FA-induced LC. LOH at all investigated markers only occurred in four spontaneous SC, three spontaneous LC and one FA-induced LC.
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Because LOH analysis does not allow distinction between the mechanisms, deletion and recombination, leading to the TK-deficient phenotype, we combined the results from LOH analysis with those from FISH, i.e. whole chromosome 11 painting (Liechty et al., 1998
). The two homologous chromosomes 11 can be distinguished by the size of their centromere; the functional TK+ allele is located on chromosome 11b, with the larger centromere (Hozier et al., 1982). We limited FISH analysis to mutants that showed at least LOH proximal to D11Mit67 (located at 57 cM) because of the limited resolution of the FISH technique used. Mutants caused by a deletion of chromosome 11b material distal to D11Mit67 could not be distinguished by the length of their chromosomes 11 from mutants caused by recombination (Liechty et al., 1998). Translocations and changes in chromosome number are also not detectable. Nineteen mutants fulfilled this requirement: six spontaneous SC, five spontaneous LC, one FA-induced SC and seven FA-induced LC. The results are summarized in Table I. Among the spontaneous mutants only one (Spo25S) revealed a clearly shortened chromosome 11b indicative of a deletion. In five mutants no loss of chromosome 11b material could be detected despite an extended LOH. These were interpreted as a result of a recombination between the two chromosomes 11. One mutant (Spo12S) contained a chromosome 11b with a fluorescence signal in its proximal part only, indicating a translocation with another chromosome. In four mutants the two completely labeled chromosomes 11 were identified as chromosomes 11a due to the small centromeric region. As there was no chromosome 11b, the TFT-resistant phenotype can be explained as a result of an aneugenic event. Among the FA-induced mutants, one (FA9AS) suffered from a chromosomal (11b) deletion. Six colonies did not reveal any aberration of chromosome 11b, suggesting recombination between the homologous chromosomes. One mutant (FA8CL) had a completely labeled prolonged chromosome 11b which could also be due to recombination with chromosome 11a.
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Discussion |
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Previous studies indicated that the Comet assay is well suited for the detection of FA-induced DPC. Induced DPC were measured by the decrease in radiation-induced DNA migration. The results obtained with L5178Y mouse lymphoma cells in the present study exactly correspond with our previous results with V79 cells (Merk and Speit, 1998
, 1999), indicating that there is no difference in the induction of primary DNA damage by FA in the two cell lines. In V79 cells, FA induced DPC, sister chromatid exchanges and micronuclei in the same range of concentrations, in parallel with the induction of cytotoxicity. However, the same treatment did not induce gene mutations at the hprt locus and there is no convincing evidence in the literature for the induction of intragenic mutations/ point mutations by FA (Merk and Speit, 1998). A mutagenic effect of FA was found at the tk locus of TK6 cells, which occurred in parallel with the induction of DPC measured by alkaline elution (Goldmacher and Thilly, 1983; Craft et al., 1987). FA also induced mutations in the mouse lymphoma tk+/– assay with L5178Y cells (Wangenheim and Bolcsfoldi, 1988; Blackburn et al., 1991). Our results are in accordance with these published studies, showing a concentration-related increase in MF. However, beyond the mutagenic effect as such, we found that the increase in MF is mainly due to an increase in SC mutants. The MLA detects a broad spectrum of mutations, including point mutations, partial and total deletions of the gene, chromosomal rearrangements and chromosome loss (Moore and Doerr, 1990; Liechty et al., 1998). It is assumed that the induction of SC mutants is associated with larger chromosomal alterations to chromosome 11b, which carries the active tk gene, while LC mutants can also be due to smaller scale events like intragenic mutations (Hozier et al., 1985; Moore and Doerr, 1990). Therefore, the positive findings in the MLA might be explained by chromosomal alterations and do not disagree with our hypothesis that FA-induced DPC do not represent promutagenic lesions leading to point mutations. Our results rather suggest that the mutagenic effect of FA is based on a clastogenic or recombinogenic mechanism and are in line with the observed strong effects of FA in cytogenetic tests (Merk and Speit, 1998).
One feasible way to elucidate the extent and nature of losses within or around the tk locus is provided by analysis of LOH at polymorphic markers. For mouse chromosome 11, where the tk1 gene is located, several simple sequence repeat polymorphisms (SSRP) have been described (Liechty et al., 1994). LOH of markers within or near the tk allele generally occurs as a consequence of deletion or recombination, while LOH at all loci may be due to total chromosome loss. Because of the almost exclusive induction of SC mutants by FA we focus on these types of mutants for the discussion of the mutation spectrum of FA. LOH analysis of FA-induced SC mutants revealed LOH at the D11Agl1 locus in all mutants investigated, indicating loss of at least parts of the tk gene as the cause for TK deficiency. This loss could have been caused by small-scale deletion or recombination, but because of the limitations of the FISH technique used this could not be further clarified. Interestingly, only one FA-induced SC mutant (FA9AS) was found, which also showed LOH at another marker (D11Mit67). FISH analysis of this mutant revealed a shortened chromosome 11b, indicating a deletion as the cause of the mutant phenotype. In contrast to the FA-induced SC mutants, six spontaneous SC mutants were found which showed LOH at more markers than only D11Agl1. FISH analysis indicated one deletion of chromosome 11b, three recombinations and two numerical aberrations with loss of chromosome 11b. Under the experimental conditions used, no SC mutants with recombinations detectable by FISH or loss of chromosome 11b were found among the FA-induced viable mutants. Although the number of analyzed independent mutants is too limited to draw final conclusions, the present data suggest that the main mechanism involved in FA-induced mutagenesis in the MLA is the production of small-scale chromosomal deletions or recombinations.
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Acknowledgments |
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We thank Dr M.Baumeister (Boehringer Ingelheim Pharma KG, Biberach, Germany) for the gift of L5178Y cells and Dr W.Muster (F.Hoffmann-La Roche Ltd, Basel, Switzerland) for giving advice on the MLA protocol.
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Notes |
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1 To whom correspondence should be addressed. Tel: +49 731 50023429; Fax: +49 731 50023438; Email: guenter.speit{at}medizin.uni-ulm.de
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References |
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Received on October 8, 2001; accepted on December 12, 2001.
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