Early detection of 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine(PhIP)-induced mutations within the Apc gene of rat colon
Dominique Y. Burnouf3,
Roman Miturski2,2,
Minako Nagao1,
Hitoshi Nakagama1,
Marc Nothisen,
Jérome Wagner and
Robert P.P. Fuchs
1 Groupe d'Epidémiologie Moléculaire du Cancer, UPR 9003, Centre National de la Recherche Scientifique, Institut de Recherche sur les Cancers de l'Appareil Digestif, 1 Place de l'Hopital, 67097 Strasbourg,France and
2 National Cancer Center Research Institute, Tsukiji 5, Chuoku, Tokyo 104-0045, Japan
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Abstract
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A large proportion of human cancers result from exposure of individuals to environmental or occupational carcinogens. The early detection of carcinogen-induced mutations is a prerequisite for the identification of individuals at risk for developing cancer. Short G-rich repetitive sequences have been previously identified as hot-spots for frameshift mutagenesis induced by a large variety of carcinogens belonging to several families of widespread environmental pollutants. In order to test if these sequences, when mutated, might serve as biomarkers for carcinogen exposure, we designed a sensitive PCR-based strategy that allows the detection of rare mutational events within a whole genome. 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), the most abundant carcinogenic heterocyclic amine generated in cooked meat, induces mammary and colon carcinoma in F344 rats. About 25% of male rats exposed to 400 p.p.m. PhIP in the diet for >43 weeks present colon tumors with specific 1G mutations within 5'-GGGA-3' sequences of the Apc gene. Using our PCR assay we have assessed the occurrence of such specific events in rats exposed to PhIP for only 1, 2, 4 and 6 weeks. A specific amplification signal was already observed in the 1 week-treated population and increases in a treatment time-dependent manner. These data validate this approach for the early detection of mutations and demonstrate its usefulness for molecular epidemiology and early diagnosis.
Abbreviations: Apc, adenomatous polyposis coli gene; gDNA, genomic DNA; HCA, heterocyclic amine; IS, internal standard; MAMA, mismatch amplification mutation assay; PhIP, 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine; SML, somatic mutational load; UDG, uracil-DNA glycosylase.
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Introduction
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Living organisms are continuously exposed to a broad range of endogenous or exogenous genotoxics, which, after metabolic activation, may form premutagenic DNA lesions. If left unrepaired, these lesions interfere with DNA replication and eventually give rise to mutations that may lead to cancer development through activation of oncogenes and/or inactivation of tumor suppressor genes (1). The adenomatous polyposis coli gene (Apc) is an 8.5 kb tumor suppressor gene which encodes a 2834 amino acid multi-functional protein involved in cell signaling pathways (2). This locus has been linked to familial adenomatous polyposis, an inherited form of colorectal cancer (3) characterized by the development of multiple colorectal adenomatous polyps. Mutations of the Apc gene, which have been found in ~85% of all sporadic and hereditary colorectal tumors (4), are considered an early event in the colon carcinogenic pathway (5). The large majority of mutations are frameshifts and nonsense mutations that result ultimately in truncated proteins (6). Heterocyclic amines (HCAs) are widespread environmental genotoxics found particularly in cooking procedure of ordinary cooked meat and fish and also in cigarette smoke (7). 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is the most prevalent food-derived HCA (8) and has been shown to induce mammary and colon cancers in rodents (9). This compound undergoes metabolic activation in vivo and the resulting ultimate carcinogens (10) bind to DNA to form N-(deoxyguanosin-8-yl)-PhIP as a major adduct (11). PhIP-induced mutational spectra reveal that G:C base pair deletion is the most frequent mutation in the Big Blue rat lacI gene (12), while GC
TA transversions are prevalent in both the Big Blue mouse lacI and Chinese hamster fibroblasts hprt genes (13,14). However, in all three studies G:C base pair deletions occur preferentially within the sequence 5'-GGGA-3', which was previously identified as a PhIP mutational hot-spot in the Apc gene of rat adenocarcinoma (15). The surrounding sequence appears to be critical for induction of guanine deletion mutations by PhIP: while the rat Apc gene contains 26 5'-GGGA-3' sequences, four of the five mutations isolated from PhIP-induced colon tumors were detected within the sequence 5'-GTGGGAT-3', which occurs twice in the gene (15). We used the specificity and sensitivity of mismatch amplification mutation assay (MAMA)PCR (16) to probe one of these two sequences for the occurrence of G deletions in the normal colon mucosa of rats exposed to PhIP for a short period (16 weeks). A specific amplification signal which indicates the presence of this mutation is detected in PhIP-treated rats and increases in both intensity and frequency with time of exposure. These results demonstrate the usefulness of such a strategy in sorting very rare molecular events, allowing discrimination between unexposed and mutagen-exposed organisms, even after a short period of treatment.
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Materials and methods
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Enzymes, reagents and oligonucleotides
BamHI, HindIII, NdeI and ScaI restriction enzymes, T4 polynucleotide kinase, DNA ligase and uracil-DNA glycosylase (UDG) were purchased from New England Biolabs (Beverley, MA) and used as suggested by the supplier. Gold Star DNA polymerase `Red' and oligonucleotides were from Eurogentec (Seraing, Belgium). dNTPs, [
32P]ATP, the ThermoSequenase radiolabeled terminator cycle sequencing kit and T7 DNA polymerase were from Amersham Pharmacia Biotech (Saclay, France). PhIP was purchased from the Nard Institute (Osaka, Japan) and added to the basal CE-2 diet. Ampicilin was from Boehringer. Oligonucleotides were designed using the Oligo v.4.0 software (MedProbe, Oslo, Norway).
Animals and genomic DNA (gDNA) purification
F344 male rats were purchased from Clea Japan (Tokyo, Japan) at the age of 5 weeks. After acclimatization to the housing environment and the CE-2 basal diet for 1 week, rats were divided into five groups (IV). Groups IIV were fed a PhIP-containing CE-2 diet, at a concentration of 400 p.p.m., continuously for 1, 2, 4 and 6 weeks, respectively. Group I was the control group. Rats in each group were killed and dissected according to the protocols. The colons were then removed, flushed out with phosphate-buffered saline and cut open along the longitudinal median axis. Colonic mucosal tissues (~0.5 g) from each rat were scraped off using the edge of a slide glass and incubated in 5 ml of lysis buffer (20 mM TrisHCl, 10 mM EDTA, pH 8.0, 0.4% SDS, 500 mg/ml proteinase K) at 37°C overnight. gDNA was then extracted using the conventional phenol/chloroform extraction method and was finally resuspended in 0.01x SSC, 0.1 mM EDTA, pH 8.0, to give a concentration of 0.22.0 mg/ml. DNA from untreated rat liver, used as an external untreated control DNA, was purified according to a published protocol (17). All DNAs were subjected to NdeI digestion before PCR analysis, so that the target sequence is located within a 1.2 kb linear fragment. The final DNA concentration was determined by UV measurement at 260 nm.
Construction of an internal standard (IS)
An IS that shares identical priming sites with the mutant gDNA sequence was used in all reactions to allow quantification of each PCR amplification (18). To construct the IS we amplified a gDNA fragment, located in exon 14 of the rat Apc gene, from nucleotide position 1856 to 1922 of the cDNA (15), which encompasses the target sequence to be analyzed (5'-GTGGGAT-3'). One of the primers, 5'-CGCGGATCCTTACCGGAGCCAGACAAGAATTCTAGAATACGTTAGC-3', contains an extra 10 nt sequence (underlined), allowing the IS amplification products to be distinguished from the gDNA amplification products by their length. The other primer, 5'-CGCGGATCCGGACACATTCCGTAATATCCACCTCCAC-3', overlaps the target sequence (underlined) and introduces the desired 1G mutation. PCR reactions were done on a MJ Research Minicycler in 30 µl of 1x Pfu buffer supplemented with 100 µM each dNTP, 1.25 mM MgCl2, 225 ng rat liver DNA, 15 pmol each primer and 1 U Pfu DNA polymerase. After 3 min at 94°C, cycling conditions were 1.5 min at 92°C, 45 s at 52°C and 45 s at 72°C for 35 cycles, with a final extension at 72°C for 5 min. The amplification product was purified by 20% non-denaturing PAGE, eluted overnight in a 0.16 M ACONa, 1 mM EDTA solution and ethanol precipitated. After digestion for 1 h at 37°C by BamHI, it was gel purified as described above and cloned in the BamHI site of vector pUC19. Selected recombinants plasmids were sequenced with T7 DNA polymerase and analyzed on an 8% denaturing (8 M urea) polyacrylamide gel. The selected IS plasmid was produced on a large scale by the usual procedures. Before being used in PCR experiments it was digested with ScaI and HindIII so that the target sequence is located within a 1.7 kb linear fragment. The concentration of IS was determined by UV measurement at 260 nm.
MAMAPCR
The general strategy of MAMAPCR has been described elsewhere (16). Both primers, 5'-GGACACATTCCGTAATATCAA-3' (M) and 5'-TTA- CCGGAGCCAGACAA-3' (W), were purified on a 15% (8 M urea) polyacrylamide gel and eluted as described above. M was radiolabeled using [
32P]ATP at a sp. act. of 60 Ci/mmol in order to detect the PCR products. MAMAPCR experiments were carried out in 10 µl containing 1x polymerase buffer supplemented with 3.3 mM MgCl2, 40 µM each dNTP, but using dUTP in place of dTTP, 0.6 pmol each primer, 565 ng gDNA (170 000 copies), 29 ag IS (10 copies), 0.6 U Gold Star polymerase. An aliquot of 0.1 U UDG was added to each PCR assay and let react for 10 min at room temperature in order to eliminate any carryover contamination. PCR amplifications were performed on a Perkin Elmer 9600 thermocycler. After 10 min at 94°C, cycling conditions were 50 s at 92°C and 20 s at 56°C for 39 cycles, followed by a denaturation step at 92°C for 5 min. Samples of 3 µl of the amplification mixture were analyzed on a 12% denaturing (8 M urea) polyacrylamide gel. Analysis was done using a PhosphorImager 445SI and ImageQuaNT software (Molecular Dynamics). Gel-purified PCR products (0.52 fmol) were sequenced using a ThermoSequenase radiolabeled terminator cycle sequencing kit and analyzed on 15% denaturing (8 M urea) polyacrylamide gels.
Statistics
Data are reported as means ± SEM. Statistical differences between groups were evaluated by the MannWhitney test, using the Instat v.2.00 software (GraphPad Software).
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Results and discussion
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Aim of the project
Cancer prevention requires the identification of carcinogens present in the external or cellular environment and the evaluation of susceptibility factors (19). For these purposes the detection of biomarkers such as DNA or protein adducts or intermediate metabolites has been developed (20). Alternatively, the measurement of accumulation of mutations in non-tumorous tissues, also referred to as somatic mutational load (SML), integrates both exposure to carcinogens and various genetic predisposition factors of each individual, such as metabolic activation and DNA repair. Consequently, measuring accumulation of mutations within a genome may allow identification of individuals at increased cancer risk. Spontaneous mutation frequency per base pair in normal human cells is estimated to be in the range 1081010 and, despite an increase upon exposure to a mutagen, highly specific and sensitive techniques are required in order to detect a mutation at any given position in the genome. Short repetitive sequences are spontaneous hot-spots for frameshift mutagenesis, within which the mutation frequency is increased by two to five orders of the magnitude upon treatment with mutagens (21,22). The structural basis for this induction relies on formation of a slipped mutagenic intermediate stabilized by presence of the adduct (22). This mechanistic model probably accounts for the PhIP-induced frameshift mutations at 5'-GGGA-3' hot-spot sequences in the rat (15). Repetitive sequences thus represent ideal molecular targets for the detection of induced mutations. In order to validate the early detection of mutations as ultimate biomarkers of exposure we have combined the use of such a reporter sequence along with a sensitive PCR-based assay to detect, in a whole genome, PhIP-induced frameshift mutations at a specific position within the Apc gene of rats exposed for a short period to the carcinogen.
Sensitivity of the MAMAPCR
The MAMAPCR procedure (16) is based on the observation that a PCR primer carrying a 3'-terminal double mismatch is amplified less efficiently by Taq DNA polymerase than a primer carrying a single mismatch at the penultimate 3'-position. The use of two instead of one 3'-terminal mismatches, as in usual allele-specific PCR, allows the sensitivity of detection to be increased by two to three orders of magnitude (16). Consequently, the mutation-specific primer M, which hybridizes from nucleotide position 1902 to 1922 of the rat Apc wild-type cDNA, was designed to present two 3'-terminal G:A mismatches when hybridized to the wild-type target sequence. G:A mispairs were shown to be among the less efficiently extended mismatches (23). When paired with the expected 1 frameshift mutant sequence this primer presents a canonical A:T 3'-terminal base pair and a single internal G:A mismatch (Figure 1
). The second primer, W, perfectly matches the sequence to be amplified, from nucleotide position 1856 to 1872. The expected lengths of the mutant and wild-type gDNA PCR products are 66 and 67 nt, respectively.
Preliminary experiments were conducted to assess the conditions for efficient MAMAPCR amplification and to determine the absolute and relative sensitivities of the detection assay. The overall amplification efficiency (
) was measured according to the equation logY = logX + nlog(1 +
), where Y is the amount of PCR products, X the amount of initial target sequences and n the number of cycles. The average value of
is derived from the slope of the regression line obtained when plotting log(Y) versus n.
was found to be equal to 0.671 when amplifying 10 copies of IS alone and to 0.662 when 10 copies of IS were amplified in the presence of 170 000 copies (565 ng) of rat liver DNA (Figure 2
). In order to avoid the reaction mixture being too viscous, we did not analyze a larger amount of gDNA in a single PCR experiment.

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Fig. 2. Determination of the amplification efficiencies of the internal standard. Ten copies of IS were amplified either alone (dark squares) or in the presence of 170 000 copies of rat liver DNA (open squares). After 42 cycles, amplification of IS alone was no longer exponential. Results are means of five independent experiments.
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Absolute sensitivity of the test
The absolute sensitivity of our test was estimated by amplifying a solution of IS containing an average of one DNA molecule per assay, in the absence or presence of 170 000 genome equivalent copies of liver DNA (Table I
). In each experiment ~30 independent amplifications were performed and we determined the fraction of samples that failed to display any IS-specific amplification products. This value was used to calculate the initial number of targets present in the PCR reaction, according to Poisson's law. Despite slight variations between experiments, the mean estimated number of targets was close to one, thus indicating that a single molecule can be detected under these experimental conditions (Table I
).
Relative sensitivity of the test
The relative sensitivity of our test represents the lowest number of mutant copies that can be detected among 170 000 copies of wild-type DNA under our experimental conditions and is expressed as the ratio between the number of mutant and wild-type copies that yield the same amount of PCR products. In order to estimate its value, we performed reconstruction experiments where 10 copies of IS DNA were co-amplified with 170 000 copies of liver DNA and we measured the ratio of the signal intensities of each PCR product (gDNA/IS). From 18 independent amplifications the mean ratio value was calculated to be 0.1 ± 0.02. In other words, one copy of IS would have given a signal intensity equivalent to that resulting from amplification of 170 000 copies of untreated wild-type gDNA, which actually represents the noise of gDNA amplification. This background was kept at a low level after 39 cycles of amplification. Moreover, as these gDNA PCR products migrate at the same position as the expected mutant products, we considered that a specific signal due to the mutant should be at least twice that of the noise generated by amplification of the wild-type gDNA. This yields a relative sensitivity of 1.1x105 [= {(10x0.1)x2}/170 000], which is in accordance with previous work using the MAMA technique (16,24,25).
Sequence analysis of the PCR products
As noted above, amplification of untreated control DNAs from liver or colon produced small amounts of products (Figure 3
, lanes 47) with an expected size of 67 nt. However, gel analysis showed that these products migrate at the same position as the stronger signals obtained upon amplification of carcinogen-treated DNAs (Figure 3
) which result from amplification of the mutant targets. Indeed, direct sequencing of all these PCR products confirmed a length of 66 nt and the presence of the mutant sequence 5'-GTTGAT-3' (Figure 1
). These unexpected products from wild-type untreated DNA may result from elongation of the M primer after a one base slippage has occurred in the three G wild-type sequence. Indeed, repetitive sequences are prone to slippage (26) and amplification of M in the absence of template slippage would give a 67 nt product bearing the sequence 5'-GTTTGAT-3'. This last event was observed in <5% of all the PCR assays performed.

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Fig. 3. Gel analysis of the MAMAPCR amplification products. Migration of the amplification products of the genomic and internal standard templates (gDNA and IS products, respectively) is indicated on the left. IS tracks (lanes 13) correspond to samples containing 10 copies of internal standard. H2O samples (lanes 2931) contained no DNA. L tracks correspond to samples containing 10 copies of IS and 170 000 copies of genomic DNA purified from liver of unexposed rats. Groups II, IV and V correspond to samples also containing 10 copies of IS and 170 000 copies of genomic DNA extracted from colon mucosa of rats exposed to PhIP for 1, 4 or 6 weeks, respectively. Two different animals from groups IV and V were analyzed. The individual amplification factor A is given for each PCR experiment. The mean A value is also indicated for each animal analyzed and for each group.
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MAMAPCR analysis of PhIP-treated rat gDNAs
The colon DNA from four groups of rats treated with PhIP for 1, 2, 4 and 6 weeks (4, 3, 5 and 3 animals, respectively) were analyzed by MAMAPCR for the occurrence of a 1G mutation within a sequence 5'-GTGGGAT-3' of the Apc gene. Liver or colon gDNA of two untreated rats were used as controls (Table I
). For each MAMAPCR experiment three tubes containing the reaction mix but no added DNA were amplified in order to assess potential external contamination. A typical analysis of the PCR products is presented in Figure 3
. All signals appear as doublets: the lower band corresponds to the full-length PCR product while the higher band results from incorporation of an extra non-templated nucleotide by the polymerase, which is devoid of 3'
5' proofreading activity. IS and gDNA amplification products migrate at positions 7677 and 6667, respectively. Little variability in the IS signal intensity is observed for a given gDNA (Figure 3
). However, variations in the amplification efficiency of the IS is observed between the various DNA samples and is attributed to different purity of each preparation (Figure 3
). Sequencing analysis of the gDNA amplification products showed that they all present the mutant sequence (5'-GTTGAT), however, in <5% of the cases mixed sequences of mutant and wild-type products were observed in the 67 nt band. In some cases triplets were observed (lane 9) with an additional band corresponding to a fragment of 68 nt. Such a band may partly result from a background product which is also observed when amplifying the IS alone (lanes 13). Alternatively, it may also result from addition of an extra nucleotide to a wild-type PCR product migrating at position 67. Strong signals migrating at positions 6768 were observed in 2% of all cases analyzed and were attributed to amplification of a mutated sequence bearing a GC
TA transversion (GTGGGAT
GTTGGAT). Indeed, G
T transversion is the most common base substitution induced by PhIP in rat, hamster, mouse and human and the resulting mutated sequence is readily amplified in our assay, giving a 67 nt PCR product.
For each PCR reaction the relative amplification (Ra) of the DNA mutant targets is measured as the ratio of the signal intensities (I) of the DNA and IS amplification products (Ra = IDNA/IIS). Several samples containing control liver DNA (L DNA) were always amplified in each experiment, serving as internal controls to normalize the results between different PCR reactions or gels. We thus define the amplification factor as A = Ra/Rc, where Rc is the mean relative amplification measured for this control liver DNA (Rc = mean Icontrol L DNA/mean IIS).
By definition, the individual A values for control DNAs are close to 1 (Figures 3 and 4
). In contrast, stronger signals are observed when amplifying PhIP-treated DNAs (Figure 3
). These signals do not appear in all amplification experiments performed with the DNA from a given rat (Figures 3 and 4
), possibly because the expected mutations are rare events. We thus performed several independent amplifications for each DNA (Table I
) and calculated an average A factor, which clearly indicates that the mutant signal increases as a function of PhIP exposure (Figures 3 and 4
). Indeed, the mean A value for liver and colon controls were 1.16 and 0.66, while it rises to 1.98, 3.09, 5.49 and 13.46 for PhIP-treated groups II, III, IV and V, respectively (Table II
). Statistical comparison of the A values shows that group II (1 week PhIP treatment) already differs significantly from control group I (P = 0.022) (Figure 5
). This statistical difference increases for groups III, IV and V relative to control group I (P < 0.0001). However, no statistical difference is observed between groups II and III and groups III and IV, although groups II and IV are significantly different (P = 0.012). The 6 weeks-treated group (V) is significantly different from all other groups (P < 0.0001) (Figure 5
). The statistical analysis is still valid when omitting the highest A values observed for group III (A = 53 and A = 49; P = 0.0001) and group IV (A = 81 and A = 62; P = 0,0001) (Figure 4
). In summary, statistical analysis of the data reveals that the different groups exposed to PhIP differ significantly from the control group and that a significant difference can be observed between groups exposed for a short or a longer period to the carcinogen. Indeed, we were able to identify in a blind study samples from unexposed, 1 week- and 6 weeks-treated rats (data not shown).

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Fig. 4. This graph represents the individual amplification factor A calculated for each MAMAPCR experiment. L and I correspond to amplifications performed with control gDNA (liver and colon, respectively) from unexposed rats. Groups II, III, IV and V correspond to samples containing genomic DNA purified from colon mucosa of rats exposed to PhIP for 1, 2, 4 or 6 weeks, respectively. Each data point corresponds to the different amplifications performed for each animal (see Table II ). The mean amplification factor (± SEM) for each group of rats is indicated. The number of independent PCRs performed for each animal is indicated on the left.
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Table II. Characteristics and results of the MAMAPCR analysis of colon genomic DNA from rats exposed (or not) to PhIP
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Fig. 5. Statistical analysis of the amplification factor A. Values are means ± SEM. Columns not sharing the same superscript differ significantly. The P values relative to the control group I are indicated.
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Conclusion
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This study describes the early detection of a specific mutation induced in rats exposed for a short period to a food-borne carcinogen. Because the mutation frequency is expected to be low and in order to increase the chances of their early detection, a sensitive and selective PCR-based assay was used to detect PhIP-induced mutations within a hot-spot sequence, i.e. a short run of G residues. Such mutational hot-spots, where the mutation frequency is increased by two to five orders of magnitude over background, are specific targets for carcinogen-induced 1 frameshift mutations. Indeed, several families of widespread chemical carcinogens, such as aromatic amines and amides such as acetylaminofluorene (21), HCAs including PhIP (15) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) (27), polycyclic hydrocarbons like benzo[a]pyrene (Bintz and Samuel, unpublished results) and some oxidative lesions (28), have been shown to trigger 1 frameshift mutations within such sequences. All these compounds form covalent adducts either at position C8 or N2 of guanine residues. A common property of all these adducts is their capacity to drastically slow down DNA polymerase progression, which in turn may explain their common mutation specificity within repetitive sequences. Indeed, a model mechanism that accounts for acetylaminofluorene-induced mutations within short runs of G residues has been proposed (21). It suggests that a DNA polymerase incorporates a C opposite the modified base but is delayed in elongation past the lesion. This delay allows slippage to occur so that the incoming C will pair with the G located 5' of the modified base. The slipped mutagenic intermediate is stabilized by the lesion (22) and presents a correct GC 3'-terminal base pair that can be elongated by the polymerase, thus generating a targeted 1 frameshift mutation. This mechanism, which has been observed in both Escherichia coli and yeast, may similarly occur in mammals, as exemplified by the pattern of PhIP-induced mutations in the Apc gene of rat colon (15). Thus, any lesion that would disturb the replication process is susceptible to induce such a type of mutation within repetitive sequences.
Several genotypic selection strategies have been developed for the detection of rare sequences (reviewed in ref. 29). Among the most sensitive assays, the restriction fragment length polymorphism/PCR approach has allowed the detection of mutations induced by carcinogens such as N-ethyl-N-nitrosourea with a sensitivity of 107106 (30). The same sensitivity has been obtained by combining a MutS-based enrichment of the target sequence and a PCR amplification similar to the MAMAPCR assay (31). Although the sensitivity of the MAMAPCR assay is lower and has been determined to be ~105 (16, 24, 25; this work), it seems to be sufficient for the detection of point mutations, provided that the target sequence is a hot-spot for carcinogen-induced mutagenesis. Indeed, our results show that upon carcinogenic treatment the specific mutant signal is increased by a factor of 210 above background. In other words, the fraction of mutated sequences reaches 104 after 6 weeks PhIP exposure. This result is in agreement with previous results obtained in LacI transgenic rats fed with PhIP for 60 days and where the fraction of mutant sequences was measured to be ~7x104 (12,32)
Consequently, we suggest that these mutated sequences might be considered as a new class of biomarker for carcinogen exposure. This approach could thus represent a general strategy to assess the SML in a genome, independently of the nature of the carcinogens or their specificity of mutation or target sequence. Because SML results from both exposure and the genetic predisposition of each individual, its assessment is of great interest in epidemiology, either for identifying people at higher risk for cancer or for determining the genotoxic potency of carcinogens in humans.
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Notes
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2 Present address: 2nd Department of Gynecological Surgery, Medical Academy, Jaczewski Street 8, 20-290 Lublin, Poland 
3 To whom correspondence should be addressed. Email; dominique.burnouf{at}ircad.u-strasbg.fr 
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Acknowledgments
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We thank J.M. Clément for helpful discussions and P. Krupnik for technical assistance. This work was partly supported by grants from the Comités Départementaux du Bas-Rhin et du Haut-Rhin de la Ligue Nationale Franciaise Contre le Cancer, which are gratefully acknowledged. R.M. was supported by grants from the French government and Polish State Committee for scientific research (#4P05E00714).
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Received April 27, 2000;
revised August 23, 2000;
accepted October 10, 2000.