Mutagenesis, Vol. 14, No. 1, 113-119,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
A comparison of the effects of diverse mutagens at the lacZ transgene and Dlb-1 locus in vivo
Department of Biology, York University, Toronto, Canada M3J 1P3
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Abstract |
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Transgenic assays permit the detection of mutations in any tissue, whereas endogenous mutations can be measured in very few. For this reason comparisons between these loci when both can be measured in the same cells are of considerable interest. Previous comparisons have been inconsistent: usually these loci have responded alike, however, in some cases the endogenous locus has been more sensitive and at other times the transgenic locus has been more sensitive. Here we report a comparison of the lacZ transgene of the MutaTMMouse and the endogenous Dlb-1 gene in the epithelium of the small intestine after acute exposure to seven mutagens. Benzo[a]pyrene, 5-bromo-2'-deoxyuridine, methyl methane sulphonate, ethyl methane sulphonate, N-ethyl-N-nitrosourea, mitomycin C and N-methyl-N-nitrosourea were all given by gavage to F1 (MutaMousexSWR) mice. Mutations were quantified 2 weeks after the end of treatment. The data shows that all of the agents induced similar mutant frequencies at the Dlb-1 locus and at the lacZ transgene. The acute treatments generally produced only modest increases in mutant frequency at both loci. The higher background frequency observed at the lacZ transgene reduces the ability of the transgenic assay to detect the same absolute increase in mutant frequency.
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Introduction |
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An assumption in the development and use of transgenic assays is that mutations at these loci accurately reflect mutations at endogenous loci. Nevertheless, these targets differ in several ways. First, their sequences and location in the genome differ. Secondly, the prokaryotic DNA is heavily methylated, is non-transcribed and is embedded in viral DNA. Third, the transgenes are usually present in multiple tandem copies. Although some differences observed between the loci would not be surprising, as all endogenous genes are not identical, comparisons of mutations in endogenous genes and transgenes in the same tissue are valuable. Previous studies have demonstrated that the lacI transgene and the endogenous Dlb-1 locus respond similarly in vivo after acute i.p. treatment with N-ethyl-N-nitrosourea (ENU) but respond differently to X-rays in the mouse small intestine (Tao et al., 1993
; Tao and Heddle, 1994). A significant increase in the mutant frequency was detected at the Dlb-1 locus after X-ray treatment, but at lacI the increase in mutant frequency was only barely detectable. This difference is probably due to the fact that X-rays produce mostly deletions and deletions that extend into the 
vector will not produce viable phage and will not be recovered. This indicates that some classes of mutations or some classes of mutagens may not produce detectable increases in mutant frequency at this transgene. The plasmid mouse was developed, in part, to overcome this difficulty (Gossen et al., 1995). Comparisons between the endogenous hprt locus and the lacI transgene in splenocytes after acute exposure have given mixed results depending on the mutagenic agent. The induction of mutations by ENU is very similar (Skopek et al., 1995), whereas benzo[a]pyrene [B(a)P] produces many more lacI mutations than hprt mutations (Skopek et al., 1996). The hprt data is complicated by the fact that the mutant frequency varies with time after treatment and this time response is age dependent (Jones et al., 1987; Walker et al., personal communication). It is not known if the lacI mutant frequency reflects the changing pattern with time found for hprt in these cells. In this respect the comparisons in the small intestine are simpler, since Dlb-1, lacI and lacZ frequencies each reach a stable plateau soon after induction (Tao et al., 1993; Cosentino and Heddle, 1996).
Previous studies have suggested that the response of endogenous genes and transgenes to different mutagenic agents differ, thus we investigated the effects of various agents at the Dlb-1 locus and lacZ transgene. Here we report comparisons of the mutant frequencies at these two loci after acute exposure, by gavage, to seven agents. The agents were selected according to the different DNA alterations they create; the presumptive repair pathways involved and whether the compound is a direct acting or a metabolically activated agent (Table I). With the use of various compounds, each inducing a different spectrum of adducts, it is possible to investigate the mutagenic effects of the various adducts in the DNA. N-Methyl-N-nitrosourea (MNU) was chosen because it is a strong mutagen and carcinogen and it reacts directly with DNA producing methylated bases. MNU methylates DNA at the N7 position of guanine and at the O6 position of guanine residues (IARC, 1978). These adducts, after two rounds of replication, produce GC
AT transitions. The responses of Dlb-1 and lacI were comparable after treatment with ENU, thus it was of interest to observe the effect of another ethylating agent. Ethyl methane sulphonate (EMS) is also a direct alkylating agent which generates a high level of nitrogen adducts compared with ENU (IARC, 1974). Thus, an effect specific to O6-ethylguanine would be observed with ENU but not with EMS. Methyl methane sulphonate (MMS) was selected because it produces primarily methylation at N7 of guanine (81–85% of total DNA methylation), producing GCAT transitions (Lawley, 1976). In contrast, B(a)P and mitomycin C (MMC) were selected because, unlike the other agents, they require metabolic activation. B(a)P is a potent mutagen and carcinogen in animals. Its carcinogenic effects depend on host metabolic activation to produce chemically reactive products such as B(a)P diol epoxide, which is capable of forming bulky DNA adducts and ultimately inducing GCTA transversions (IARC, 1973). MMC, on the other hand, is a bifunctional alkylating agent that can crosslink DNA and is a potent clastogen (IARC, 1976). Interstrand crosslinks represent an important class of chemical damage, as they block DNA replication and transcription.
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Materials and methods |
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Animals
All homozygous lacZ (Dlb-1b/Dlb-1b) MutaTMMouse were obtained from Hazleton Research Products Inc. (Denver, PA) and bred with non-transgenic SWR (Dlb-1a/Dlb-1a) mice, obtained from The Jackson Laboratory (Bar Harbor, ME) to produce the animals used here. The F1 animals, therefore, were hemizygous for the lacZ transgene and heterozygous at the Dlb-1 locus (Dlb-1b/Dlb-1a). The F1 animals were ~3 months of age and weighed between 20 and 31 g. Two females and three males were randomly assigned to each treatment group and four males and four females to each control group. Mice were housed in standard plastic cages with wood chip bedding. Mouse chow and water was supplied ad libitum. An independent Animal Care Committee approved all experimental protocols in advance.
Chemical treatment
All animals were treated by gavage. One group of animals were treated with 10, 50 or 100 mg/kg B(a)P. The second group was treated with either 2500 or 5000 mg/kg bromodeoxyuridine (BrdU). A third group was treated with 50, 100 or 250 mg/kg EMS. Another group was treated with 100 mg/kg ENU. The MMC group received 1, 2 or 4 mg/kg and the MMS group was given an acute treatment of 50, 100 or 150 mg/kg. The last group of animals was treated with 50, 75 or 100 mg/kg MNU. Animals were sacrificed 2 weeks after treatment.
Chemicals
B(a)P (CAS no. 50-32-8), ENU (CAS no. 759-73-9) and MNU (CAS no. 000684935) were purchased from Sigma Chemical Co. (St Louis, MO). BrdU (CAS no. 59-14-3) and MMC (CAS no. 50-07-7) were purchased from Boehringer Mannheim (Laval, Quebec, Canada). EMS (CAS no. 000062500) was purchased from Aldrich (Milwaukee, WI) and MMS (CAS no. 000063273) was purchased from Eastman Kodak Co. (Rochester, NY). After test solutions were freshly prepared, all animals were treated with the appropriate dose of the agent. Control animals were treated with a phosphate-buffered saline (PBS) solution, also by gavage.
Tissue collection
All animals were sacrificed by cervical dislocation. The jejunal section of the small intestine was reserved for the Dlb-1 assay, while the remainder of the tissue was used for the lacZ assay. After the intestine was flushed with PBS and inverted, it was placed in 3 ml KCl/EDTA solution and forced in and out of a 5 ml needleless syringe. The cell suspension was stored at –70°C for future use.
DNA isolation
Genomic DNA was purified from the cell suspension with a proteinase K solution (2 mg/ml) for 3 h at 55°C, followed by phenol–chloroform (1:1) extraction and precipitation with ethanol as described by Kohler et al. (1990). The precipitated DNA was spooled onto a hooked glass Pasteur pipette, air dried and dissolved in Tris–EDTA buffer. The concentration of DNA was determined spectrophotometrically at 260 nm.
DNA packaging of lacZ mutations
The phage shuttle vector, which contains the entire lacZ target gene, was recovered by in vitro packaging with TranspackTM packaging extract (Strategene, La Jolla, CA), under conditions recommended by Hazleton Research Products Inc. (Denver, PA). Briefly, 8 µl genomic DNA (1–2 mg/ml) was used in each packaging reaction and incubated for a total of 3 h at 30°C. The reaction was terminated by dilution with 470 µl phage buffer. Approximately 500 µl packaged phage were incubated in 2 ml bacterial suspension at room temperature for 20–30 min. A 5 µl aliquot was diluted in 100 µl LB broth containing 10 mM MgSO4 for a concurrent titer on non-selective agar plates. The remaining phage/bacteria mixture was mixed with ~8 ml freshly prepared top selection agar supplemented with 0.3% phenyl-ß-D-galactopyranoside (P-gal) (Sigma). P-gal is used as a selective agent for bacteria infected with phage containing non-functional lacZ genes. P-gal in the selection plates prevents the formation of wild-type plaques and thus the plaques observed represent lacZ mutants (Gossen et al., 1992). After incubation overnight at 37°C, mutant and non-mutant plaques were scored. The number of plaques recovered from each animal varied, with a mean of ~200 000 plaques and a range of 47 000–1 575 000 plaques.
Dlb-1 assay
Whole mounts of the small intestine were prepared as described by Winton et al. (1988). Briefly, the small intestine was divided into its three sections (duodenum, jejunum and ileum) and flushed clean with PBS. The jejunum was used for this assay while the remaining sections of the intestine were used for the transgenic assay. After the jejunum was flushed with 10% formal saline, one end was sealed between two microscope slides and clipped. The intestine was inflated with 10% formal saline using a blunt end needle and fixed for ~3 min. It was cut along the mesenteric side, placed on a microscope slide, villi side up, stretched and held in place by plastic-coated paper clips under which a small piece of coverslip was placed. The slides were placed in 10% formal saline to fix for at least 1 h, at which time they were then rinsed with PBS and incubated overnight in 20 mM DL-dithiothreitol (Sigma) dissolved in 20% ethanol, 150 mM 80% Tris (pH 8.2). Mucus was removed by pipetting the solution over the intestinal tissue. Before staining, slides were rinsed three times with PBS and incubated in 0.1% phenylhydrazine hydrocholoride (Sigma) in PBS for 30 min to block endogenous peroxidases. After three washes with PBS and a 10 min incubation in PBS containing 0.5% albumin (fraction V; Boehringer), the slides were stained with the Dolichos biflorus agglutinin–peroxidase conjugate (Sigma) at 5 µg/ml in PBS/albumin (fraction V). The peroxidase was developed using 3,3'-diaminobenzidine (Sigma) solution for 45 min. The slides were rinsed twice with PBS and stored in 10% formal saline until analyzed. The slides were scored with a dissecting microscope at 50x magnification. The Dlb-1b/Dlb-1a epithelial cells stain dark brown; mutant cells, which have no lectin-binding ability, appear as unstained white vertical ribbons on the villus. The number of villi scored per animal was estimated from duplicate counts of the number of villi in the first and last field. Each field contained ~200 villi, to yield an average of 9000 villi/animal. Since there are ~10 stem cells/villus (Cosentino et al., 1996), ~90 000 stem cells were analyzed per animal.
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Results |
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Individual animal data and mutant frequencies are presented in Table II
. The majority of agents produced only a marginal increase in mutant frequencies at either locus. MMC proved to be the weakest mutagen, whereas MNU was the most mutagenic agent, inducing a 200- to 400-fold increase in both Dlb-1– and lacZ– mutations.
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The dose–response data were tested for linearity using the SAS statistical test for regression analysis. Only treatment with B(a)P, MMS and MNU resulted in a dose-related increase in mutant frequency for both loci (Table III
). Treatment with BrdU resulted in a dose-related increase in Dlb-1– mutants but not lacZ– mutants (Table III).
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Because not all dose–response curves are linear, the mutant frequencies of the highest dose was tested for significance against controls by one-way analysis of variance (ANOVA) using Microsoft® Excel 97. EMS (F = 64.41, P < 0.05) and MMC (F = 8.74, P = 0.02) induced a significant increase in Dlb-1 mutations at the highest dose, whereas a significant increase in lacZ mutations was not detected. The higher background mutant frequency observed at the lacZ locus (3.1 ± 1.2) compared with that of the Dlb-1 locus (1.2 ± 0.5) reduces the ability of the transgenic assay to detect small increases, as discussed later. Despite the different spontaneous mutation frequencies, the induced mutant frequencies at the Dlb-1 locus and lacZ transgene were not significantly different and were nearly identical after treatment with B(a)P (P = 0.93), BrdU (P = 0.97), EMS (P = 0.85), MNU (P = 0.87), MMC (P = 0.86) and MMS (P = 0.74) (Figure 1
). It is interesting to note that although each mutagen produces a distinct spectrum of mutations, resulting from the specificity of DNA binding and the type of DNA repair involved, all of the agents induced similar mutant frequencies at both loci. The large error bars seen for the 100 mg/kg EMS treatment group are partly a result of a small sample size, as two males died, leaving only one male and two females for that treatment group (Figure 1c).
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) the Dlb-1 locus (–SEM) and (
) lacZ transgene (+SEM). |
Discussion |
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The similarity in the response of the lacZ transgene and the Dlb-1 gene is surprising (Figure 1
). The induced mutant frequencies detected at the Dlb-1 locus and the lacZ transgene for all the mutagens tested are highly correlated (Figure 2). The correlation derived here implies that for an increase in lacZ– mutants, the number of Dlb-1– mutants also increases. The almost perfect quantitative agreement must be regarded as a matter of chance, for other endogenous loci and other transgenes can show different mutant frequencies. That there are exceptions is shown by the differential response of lacI and Dlb-1 to X-rays, with Dlb-1 being the more sensitive locus (Tao et al., 1993). The sensitivity of any locus to mutation depends on a number of factors. Clearly the larger the mutable target, the more likely mutations are to arise, although the mutable target may not be in proportion to the size of the locus. The 
X174 transgenic mouse, for example, has a mutable target of 1 bp within the 5 kb gene sequence (Burkhart et al., 1993). Secondly, the nature of the mutation screen influences the size of the mutable target and thus the mutation frequency. If the selection is very rigorous, then only the more extreme mutants will be detected. lacI mutants, for example, vary in the intensity of the blue color on selection plates with X-gal. The nature of the agar and the amount of X-gal in the plates will influence the fraction of lacI mutations detectable and thus the mutation frequency. This is as true of endogenous loci as transgenic ones. Ouabain resistance in mammalian cells is much less frequent than thioguanine resistance (Baker et al., 1979). In the case of ouabain, the enzyme is essential and the mutation is dominant. Probably the mutational target is a few base pairs at most and only base pair substitutions are recoverable. Thioguanine resistance can arise by deletion of the whole gene, by single base pair substitutions and by frameshift mutations at numerous sites in the hprt gene. This provides a satisfactory explanation for the ~100-fold difference in mutation rates at these loci.
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Differences between loci are much more likely than similarities. Nevertheless, the lacI transgene (1 kb), about one third the length of the lacZ transgene (3 kb), the hprt endogenous gene (~1 kb of coding sequence) and Dlb-1 (of unknown size) all show very similar mutant frequencies after ENU treatment (Tao et al., 1993
; Skopek et al., 1995; Cosentino and Heddle, 1996). Other comparisons are few and involve lacI and hprt. One chemical, B(a)P, produces many more lacI mutations than hprt mutations (Skopek et al., 1996). It is noteworthy that, unlike Dlb-1, the hprt mutant frequency changes with time, rising to a maximum and falling thereafter. This makes comparisons difficult, especially as the lacI time course has not always been determined and might differ. These comparisons indicate that age, manifestation time and the kinetics of the lymphoid system, in addition to treatment effects of various chemical agents, influence the frequency of hprt– mutations. Clearly, more studies are needed, but the Dlb-1 comparisons suggest that the transgene does not usually behave anomalously.
The data reported here shows that MNU, B(a)P and MMS induced a significant dose-related increase in Dlb-1– and lacZ– mutant frequencies (Table II). In addition to EMS (250 mg/kg), MMC (4 mg/kg) and BrdU (5000 mg/kg) induced a significant increase in Dlb-1– mutations (P < 0.05, P = 0.02 and P = 0.003, respectively) but not in lacZ– mutations. The lack of significant increases in lacZ– mutations is probably due to a higher background mutant frequency observed at the lacZ locus (3.1 ± 1.2) compared with that at the Dlb-1 locus (1.2 ± 0.5). Thus, the induced mutant frequency (fold increase) was significantly lower for lacZ than for Dlb-1. The lower spontaneous mutant frequency in the Dlb-1 assay allowed this test to detect smaller absolute numbers of mutations.
The spontaneous frequency influences the sensitivity of these assays, as pointed out by Skopek et al. (1995). Induced mutations arise as an absolute increase in mutant frequency, so the lower the spontaneous frequency, the more sensitive the assay is to the same increase. This is apparent in our studies with these mutagens, most of which produced rather small increases in mutant frequency. The increases are remarkably similar at the lacZ and the Dlb-1 loci, but the Dlb-1 data are much more significant because of the lower spontaneous mutant frequency at this locus. This was also observed in a study when animals were treated with cyclophosamide (CP). CP-induced mutations were detected at the hprt locus but not in the lacI transgene in splenic T cells (N.J.Gorlick, personal communication). This fully confirms the prediction of Skopek et al. (1995). Thus the high spontaneous frequency reduces the sensitivity of the transgenic assays. The solution that has been proposed is to increase the exposure by using subacute and chronic protocols as recommended by Shephard et al. (1994), Tao et al. (1994) and Heddle et al. (1995). Evidence supporting this solution is accumulating rapidly. Suzuki et al. (1993), for example, have shown that multiple treatments with MMC resulted in a 2-fold increase in lacZ mutations in the bone marrow, whereas a single acute dose did not induce a significant increase in lacZ mutations.
It should be mentioned that the Dlb-1 locus and the lacI transgene respond differently to chronic treatment, i.e. when animals are treated chronically with ENU there is an initial deficiency of Dlb-1 mutations relative to lacI mutations. With continued treatment, lacI mutations accumulate linearly for 90 days. In contrast, the Dlb-1 mutants accumulate more slowly at first and then at an accelerated rate (Shaver-Walker et al., 1995). This is also true for the lacZ transgene (Cosentino and Heddle, in preparation). In addition to treatment protocols, we have observed that the induced mutant frequency is greatly influenced by the route of mutagen administration. When animals are treated with ENU (100 mg/kg) by gavage, a lower mutant frequency is induced at the Dlb-1 locus when compared with animals treated i.p. (50x105 versus 80x105, respectively).
It is evident that there are many factors that can affect the induction of mutations in animals. Although Dlb-1 and lacZ respond similarly to acute doses, the exposure given may not accurately reflect all of the biological phenomena occurring in vivo, as observed by the differential mutation frequencies obtained during a chronic dosing regime. Like Shephard et al. (1994), we believe that a subacute or chronic treatment protocol would likely maximize the sensitivity of the assays (Heddle et al., 1995). It is evident from this study that in addition to treatment protocol, background mutant frequency, mode of mutagen administration, selection of test substances and dose level influence the detection limit of these mutational assays. Although the majority of agents induced similar mutant frequencies at the two loci, they were not very mutagenic in the small intestine. It is possible that each agent may be tissue-specific, species-specific or strain-specific. Obviously, one must consider all influential factors when designing experiments in order to maximize the power of the assay.
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Acknowledgments |
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This work was supported by a grant from the National Cancer Institute of Canada. We thank HRP Inc. for permission to cross the MutaTMMouse to produce the F1 necessary for this study. We also thank Cesare Urlando and Jennifer Moody who helped with the experiments. We are also grateful to Jason Belas for his helpful comments and suggestions.
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Notes |
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1 To whom correspondence should be addressed. Tel: +1 416 736 2100, ext. 33053; Fax: +1 416 736 5698; Email: lidiac{at}yorku.ca and jheddle@yorku.ca
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