Mutagenesis, Vol. 17, No. 2, 105-109,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Mutant frequencies and loss of heterozygosity induced by N-ethyl-N-nitrosourea in the thymidine kinase gene of L5178Y/TK+/–-3.7.2C mouse lymphoma cells
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA and 1 National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27709, USA
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Abstract |
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N-ethyl-N-nitrosourea (ENU) is a potent monofunctional ethylating agent that has been found to be mutagenic in a wide variety of organisms from viruses to mammalian germ cells. To elucidate the mutagenicity of ENU at the Tk+/– locus of mouse lymphoma cells and to confirm the ability of the mouse lymphoma assay (MLA) to detect both point mutations and large DNA alterations, Tk+/– L5178Y cells were exposed to different doses of ENU. Treatment of the cells with ENU resulted in a linear dose response with mutant frequencies of up to 16-fold over control. Evaluation of mutant clone size showed that 36% of the 100 µg/ml ENU-induced clones (66% in control) were small colony mutants and 64% (34% in control) were large colony mutants. DNA isolated from mutants in the control culture and the 100 µg/ml ENU treatment group was analyzed for loss of heterozygosity (LOH) using allele-specific PCR. The majority of the small colony mutants, both ENU-treated (97%) and spontaneous (91%), lost the Tk1b allele. The percentage of allele loss in ENU-induced large colony mutants was distinctly different from that of the control. Twenty-three percent of ENU-induced large colony mutants lost their Tk1b alleles, whereas 73% of the large colony mutants from the control culture lost the allele (P < 0.001). Overall, 50% of the Tk mutants from the 100 µg/ml ENU-treated cultures (86% in control) showed LOH. Our data indicate that ENU is a potent mutagen in mouse lymphoma cells and that 100 µg/ml ENU induces equal numbers of point mutations and chromosomal mutations. This study serves to verify that the MLA detects both point mutations and chromosomal mutations.
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Introduction |
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The mouse lymphoma assay (MLA) detects a broad spectrum of genetic damage, including both point and chromosomal mutations (Hozier et al., 1985
; Moore et al., 1985; Blazak et al., 1986; Applegate et al., 1990). This assay, therefore, is the most widely used mammalian cell gene mutation assay and is included in the core battery of regulatory agency tests world wide (Dearfield et al., 1991; Department of Health and Human Services, 1997). In this assay, thymidine kinase-deficient (Tk-/–) mutants of L5178Y/Tk+/– cells are selected with the pyrimidine analog trifluorothymidine (TFT). Unlike the X-linked functionally hemizygous Hprt locus, the Tk locus is located on mouse chromosome 11 and is present in two copies, one of which is functional in the heterozygote. Because of its autosomal location, the Tk locus can recover types of mutants not detected at the Hprt locus, i.e. the Tk+ allele may be lost due to mitotic recombination (which cannot occur with the X-linked Hprt locus) or the mutant cells can survive large DNA deletions that are lethal to such Hprt mutants. Hence, MLA detects not only intragenic events, mainly point mutations, but also large genetic damage ranging from entire gene loss to karyotypically visible deletions and rearrangements of the Tk+-bearing chromosome 11b. The Tk mutant colonies fall into a bimodal size distribution, with the larger clones growing at a faster rate and the smaller clones at a slower rate (Moore et al., 1985). It has been suggested that the small mutant colony mutants result from large scale damage to chromosome 11 on which the Tk1 gene resides (Hozier et al., 1985; Moore et al., 1985; Applegate et al., 1990).
For assessing this large scale alteration to chromosome 11, two assays for detecting loss of heterozygosity (LOH) have been developed. An NcoI heteromorphism immediately adjacent to Tk1 that distinguishes the non-functional Tk1a allele from the functional Tk1b allele was identified and Southern blot analysis of NcoI-digested DNA was utilized to screen for loss of the Tk1b allele (Applegate et al., 1990; Clive et al., 1990). Later, Liechty et al. (1996) identified a heteromorphic microsatellite within the Tk1 gene in mouse lymphoma cells and developed a PCR-based analysis to identify Tk1b loss. This new assay is perfectly correlated with Southern analysis of a NcoI digest, but is much faster, simpler and more reliable. To date, hundreds of mutants have been analyzed and it has been demonstrated that a large proportion of mutants isolated from L5178Y mouse cells (typically 70–75% of spontaneous mutants and varying proportions of induced mutants, depending on the mutagen) have lost the Tk1b allele (Applegate et al., 1990; Clive et al., 1991; Liechty et al., 1996).
N-ethyl-N-nitrosourea (ENU) is one of the most potent mutagens in a wide variety of mutational test systems. It has been used as a positive control substance for genotoxicity studies and as a means of inducing new mutations in various organisms. This compound, however, has not been evaluated in the MLA. In most mutagenic test systems, ENU induces point mutations because of its characteristics as a monofunctional alkylating agent (Shibuya and Morimoto, 1993). ENU is also mutagenic at the chromosomal level. ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations (Sanger and Eisen, 1976). ENU also induced DNA strand breaks in mouse lymphoma cells at concentrations of >5 mM (Garberg et al., 1988) and increased the frequency of micronuclei in mice (Suzuki et al., 1997). In the present study, mouse lymphoma cells were treated with different doses of ENU. Then, both large and small colony Tk-deficient mutants from the control and ENU-treated cells were isolated to evaluate them for LOH at the Tk1 locus. In addition, we evaluated and report the sequence of the heteromorphic microsatellite within the Tk1 gene in Tk+/–-3.7.2C mouse lymphoma cells. The possible relationship of LOH, clone size and mechanisms of mutation induction by ENU at the Tk+/– locus of mouse lymphoma cells are also discussed.
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Materials and methods |
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Chemicals
ENU, TFT, Tris–HCl, MgCl2, Triton X-100 and Tween 20 were obtained from Sigma (St Louis, MO). Fischer's Medium for Leukemic Cells of Mice, horse serum, penicillin/streptomycin, sodium bicarbonate, sodium pyruvate, pluronic, phosphate-buffered saline (PBS) and proteinase K were purchased from Gibco (Grand Island, NY). The QiaAmp Blood Kit was purchased from Qiagen (Chatsworth, CA).
Cells and chemical treatment
The Tk+/–-3.7.2C heterozygote of the L5178Y mouse lymphoma cell line was utilized for the mutation assay. Growth conditions, treatment and expression were performed according to the procedures described by Harrington-Brock et al. (1998). Cells were maintained in logarithmic growth in culture using Fischer's Medium for Leukemic Cells of Mice supplemented with 10% horse serum, sodium pyruvate, pluronic, penicillin and streptomycin. For treatment, cells were centrifuged and suspended at a concentration of 0.6x106 cells in 10 ml of medium in 50 ml polystyrene tubes. ENU was added (0, 75, 100, 125 and 150 µg/ml) and these tubes were gassed with 5% CO2 in air and sealed. The cell culture tubes were placed on a roller drum and incubated at 37°C for 4 h. At the end of the treatment period, the cell cultures were centrifuged and washed twice with fresh medium and resuspended in fresh medium. The culture tubes were placed on a roller drum in a 37°C incubator and maintained in log phase growth for a 2 day expression period. Relative survival values were calculated according to the method described by Clive and Spector (1975).
Tk microwell mutation assay
Mutant selection was performed using the microwell version of the assay as described by Cole et al. (1986). Simply, the treated cells in medium containing 1 µg TFT/ml for selection or without TFT for cloning efficiency were distributed at 200 µl/well into 96-well flat-bottom microtiter plates. For mutant selection, four plates were seeded with ~2000 cells/well. For cloning efficiency, three plates were seeded with ~1 cell/well. All plates were incubated in 5% CO2 in air in a humidified incubator at 37°C. After 14 days incubation, clones were counted and the colony size distribution was determined. Mutant frequencies were calculated according to the methods reported by Oberly et al. (1997).
Detection of LOH at the Tk1 locus
Mutant cells were directly isolated from the TFT selection plates and washed in PBS buffer once by centrifugation. The cell pellets were frozen quickly and stored at –80°C. Genomic DNA was extracted from the cell pellets as purified DNA or cell lysate. Purified DNA was isolated using the QiaAmp Blood Kit according to the manufacturer's protocol for blood and body fluids. Cell lysate was produced by digesting the cells in lysis buffer (10 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 1% v/v Triton X-100, 1% v/v Tween 20) with 200 µg/ml proteinase K at 60°C for 90 min, the proteinase K being inactivated at 95°C for 10 min. The procedure for PCR analysis was modified from the method described by Liechty et al. (1996). The amplification reactions were carried out in a total volume of 30 µl using reagents purchased from the Applied Biosystems Division of Perkin-Elmer (Foster City, CA). Each reaction mixture contained genomic DNA from ~5000 cells, 0.15 µl each of primers Agl2.frw and Agl2.rev (Liechty et al., 1996), 1.5 U AmpliTaq DNA polymerase, 100 µM each dNTP and 1x AmpliTaq PCR buffer with 1.5 mM MgCl2. The PCR was carried out in 96-well plates using a GeneAmp PCR System 9600 thermocycler (Perkin-Elmer). The thermal cycling conditions consisted of an incubation at 94°C for 3 min, followed by two cycles of 94°C denaturation for 30 s, 72°C annealing for 30 s and 72°C extension for 30 s. The annealing temperature was decreased by 1°C for each additional two cycle set until 65°C was reached. Then 19 additional cycles were performed at the 65°C annealing temperature, followed by a 72°C extension for 7 min. The reaction products (10 µl) were resolved by electrophoresis using a 2% agarose gel that contained 0.5 µg/ml ethidium bromide and visualized on a UV light box. An example of the PCR analysis of LOH at the Tk1 locus of ENU-induced mouse lymphoma mutants is shown in Figure 1.
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Determination of the nucleotide sequences of the heteromorphic microsatellite within the Tk gene
Genomic DNA was isolated from mouse lymphoma cells. The heteromorphic microsatellite within the Tk1 gene (Liechty et al., 1996
) was amplified. The PCR products were used as template to determine the nucleotide sequences of the microsatellite loci in Tk1a and Tk1b. The sequencing reactions were conducted with an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit and a 377 DNA Sequencing System (Applied Biosystems). The nucleotide sequence of the heteromorphic microsatellite within Tk1a was compared with those within Tk1b of mouse lymphoma cells and with the wild-type sequence (Gudas et al., 1992).
Statistics
The one way ANOVA Bonferroni t-test was used to evaluate the significance of the difference between the control and ENU-treated cultures. Induced mutant frequencies, cloning efficiency and cell growth were evaluated. The Fisher exact test was utilized to analyze the proportions of small and large colony mutants and the percentage of mutants showing LOH.
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Results |
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ENU was shown to induce a large number of mutants in mouse lymphoma cells. A dose-related cytotoxic and mutagenic effect was observed (Figure 2
). The survival and cloning efficiency of the cells decreased with increasing ENU concentration while the Tk mutant frequency increased with dose. The mutant frequency in every treatment group was significantly different from that in the control (P < 0.05).
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Evaluation of the proportion of small colony Tk mutants showed a significant difference (P < 0.05) between the 100 µg/ml ENU-induced (36%) and spontaneous mutants (66%) (Table I
). DNA isolated from 602 Tk-/– mutants (both 100 µg/ml ENU treated and controls) was analyzed for LOH caused by allele loss at the Tk1 locus using allele-specific PCR. This analysis revealed that allele loss occurred in both large and small clones from both treatment and control groups. The percentage allele losses in both ENU-induced and spontaneous small colony mutants were similar, 97% in ENU-induced and 92% in controls. The percentage allele loss in ENU-induced large colony mutants, however, was distinctly less than that of the controls. Only 23% of ENU-induced large colony mutants lost their Tk1b allele, whereas 73% of large colony mutants in the controls lost the allele (P < 0.001). The total percentage LOH in mutants from ENU-treated and control groups were 50 and 86%, respectively (Table I).
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The nucleotide sequence of the heteromorphic microsatellite within Tk1 from L5178Y mouse lymphoma cells was determined by sequencing and compared with the wild-type sequence from liver of C57BL/10J mouse in Figure 3
. Tk1b (the wild-type allele) has 95 more base pairs than Tk1a (the mutated allele), which is the basis for PCR analysis of LOH of Tk mutants from mouse lymphoma cells. The length of the PCR product is 525 bp for Tk1b and 430 bp for Tk1a and, as shown in Figure 1
, LOH is determined by the absence of the larger (525 bp) band. There are also base pair differences between the sequences of Tk1b in mouse lymphoma cells and in normal mouse cells. We have placed the sequences in GenBank (accession nos AF149051 and AF149052).
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Discussion |
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As expected, ENU appears to be a potent mutagen in mouse lymphoma cells. Mutant induction showed a linear dose–response relationship. Treatment of cells with ENU resulted in a very high mutant frequency (1255.3 ± 42.5x10-6) in the 150 µg/ml treatment group, a 16-fold increase in mutant frequency over the controls (Figure 2
). Point mutation and LOH contributed equally to this high mutant frequency, with 50% LOH and 50% intragenic mutation induced by ENU in the Tk gene of mouse lymphoma cells (Table I).
In a separate experiment, we found that treatment of mouse lymphoma cells with 100 µg/ml ENU resulted in an ~2-fold higher induced frequency in the Tk gene than in the Hprt gene, 680x10-6 in Tk and 304x10-6 in Hprt (Chen et al., 2000). Thus, it is apparent that the MLA recovers mutants with large DNA alterations induced by ENU that are not detected in the X-linked Hprt gene.
ENU ethylates nucleic acids directly and the reactive sites have been identified, including N3, N7 and N1 of adenine, N7, O6 and N3 of guanine, O2, O4 and N3 of thymine, O2 and N3 of cytosine and the phosphate groups in the DNA backbone (Shibuya and Morimoto, 1993). It has been suggested that point mutations correlate best with O6-ethylguanine and O2- and O4-ethylthymine, whereas O6-ethylguanine, 3-ethylguanine and phosphotriesters relate best to large DNA changes (Heflich et al., 1982; Natarajan et al., 1984; Asita et al., 1992; Bronstein et al., 1992; Galloway et al., 1995; Suzuki et al., 1997). Although the production of O6-ethylguanine has been thought to be the most important parameter for the prediction of mutation induction by ethylating agents such as ENU (van Zeeland et al., 1985), the predominant mutation induced by ENU in mammalian cells is T:A
A:T transversion. This difference is caused by efficient repair of O6-ethylguanine in most mammalian cells by p53-activated O6-alkylguanine-DNA-alkyltransferase (Russell et al., 1995; Rafferty et al., 1996; Reese et al., 2001) in comparison with the persistence of O2- and O4-ethylthymine adducts (Liem et al., 1994; Jansen et al., 1995). We have previously found, however, that in L5178-3.7.2C cells G:CA:T transition is the major point mutation induced by ENU (Chen et al., 2000), probably because these p53-deficient cells (Clark et al., 1998) do not repair O6-ethylguanine adducts efficiently.
The majority of mutants due to ENU treatment were large colony mutants (64% in ENU-treated versus 34% in controls) while the average proportion of large colony mutants induced by most mutagens is <50% (Clive et al., 1990). This ENU-induced proportion of large colony mutants induced is close to that induced by ethyl methanesulfonate, another known point mutagen (Clive et al., 1990).
This study confirms other analyses showing that the majority of small colony mutants (both spontaneous and induced) have lost heterozygosity. The proportion of large colony mutants showing allele loss was distinctly different between the ENU-treated and controls (23% in ENU-induced versus 73% in controls). Recently, in other studies we have found that potassium bromate, a potent clastogen, induces 94% LOH among large colony mutants (Chen et al., 2001). These data are consistent with a previous report that most small Tk-/– mutants result from large DNA damage, while the frequency of mutants with intragenic mutation in the large Tk-/– clones are mutagen dependent (Applegate et al., 1990). Chemicals that mainly induce point mutations induce a high proportion of large colony mutants retaining the Tk allele, while clastogens show a high proportion of LOH in large colony mutants.
In conclusion, ENU is a potent mutagen to mouse lymphoma cells and the types of mutations induced by ENU at the Tk locus include both point mutations and LOH. Our results further confirm that the MLA can be used to detect chemicals that cause both point mutations and large DNA alterations. These events can be distinguished by LOH analysis of mutants. Therefore, the MLA can be used to provide valuable mode of action information that can be used for the hazard characterization portion of cancer risk assessment and can contribute to elucidating the types of mutations induced by the test chemical.
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Acknowledgments |
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This work was funded in part by the Duke/EPA Toxicology Training Program, Training Agreement CT826514 with the Integrated Toxicology Program of Duke University. This manuscript has been reviewed by National Center for Toxicological Research, Food and Drug Administration and the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agencies nor does mention of trade names of commercial products constitute endorsement or recommendation for use.
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Notes |
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2 To whom correspondence should be addressed. Tel: +1 870 543 7954; Fax +1 870 543 7682; Email: tchen{at}nctr.fda.gov
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References |
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Received on October 8, 2000; accepted on March 10, 2001.
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