Preferential induction of guanine deletion at 5'-GGGA-3' in rat mammary glands by 2-amino- 1-methyl-6-phenylimidazo[4,5-b]pyridine

Eriko Okochi, Naoko Watanabe, Yoshiya Shimada1, Satoru Takahashi2, Kuniko Wakazono, Tomoyuki Shirai2, Takashi Sugimura, Minako Nagao and Toshikazu Ushijima3

Carcinogenesis Division, National Cancer Center Research Institute,1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045,
1 Low Dose Radiation Risk and Carcinogenesis Research Group, National Institute of Radiological Sciences, Anakawa 4-9-1, Inage-ku, Chiba 263-8555 and
2 First Department of Pathology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan


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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is known to induce a characteristic mutation, G deletion at the 5'-GGGA-3' site, preferentially in the lacI transgene of the colonic mucosa of Big Blue® rats (BBR) and mice and specifically in the Apc gene of rat colon tumors. In this study, lacI mutations of the mammary glands in PhIP-treated rats were investigated. Six-week-old female (BBRxSprague–Dawley)F1 rats were administered 10 gavages of 65 mg/kg/day PhIP. Mammary ducts were collected from the macroscopically normal mammary tissue of PhIP-treated and untreated rats at 56–69 weeks of age by collagenase treatment. The mutant frequencies were 25 ± 2.1x10–6 in control rats and 323 ± 44x10–6 in the PhIP-treated rats. By sequencing 40 and 177 mutants in the control and PhIP-treated groups, respectively, 34 and 149 mutations were considered independent mutations. In the control group, G:C->A:T transitions at CpG sites dominated and no G:C deletions were detected. In the PhIP-treated group, G:C->T:A transversions were most frequent (43%), followed by single base pair deletions of G:C (21%). A total of nine deletions were at 5'-GGGA-3' sites, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations at one nucleotide position were observed at 12 positions and two were G deletions at 5'-GGGA-3' sites. Comparison of the PhIP-induced mutations in the mammary glands with those previously reported in the colon revealed that G:C->T:A transversions occurred at a significantly higher frequency in the mammary glands and that G:C deletions occurred at a significantly lower frequency. However, the signature mutation, G deletion at the 5'-GGGA-3' site, was commonly observed in both tissues.

Abbreviations: BBM, Big Blue® mouse; BBR, Big Blue® rat; DMEM, Dulbecco's modified Eagle's medium; F344, Fischer 344; MF, mutant frequency; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; SD, Sprague–Dawley; SSCP, single-strand conformation polymorphism.


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Characteristic mutations of the p53 gene in human cancers have potential as molecular markers of past exposure to specific carcinogens. A few examples of such `signature mutations' have been identified by analysis of human cancers whose etiology can be inferred and the usefulness of the signature mutations in risk assessment has been proposed (1). For example, the G->T transversion at the third position of codon 249 of the p53 gene indicates past exposure to aflatoxin B1 in human hepatocellular carcinomas (24) and the CC->TT double base change in the p53 gene indicates involvement of UV exposure (5). However, it is generally difficult to identify signature mutations of human carcinogens directly in human samples. This is because the cause of a human cancer may not be a single component, only a limited number of human populations have elevated exposure to a specific carcinogen and only a limited number of mutations can be recovered in the tumor-related genes of the cancers in these populations.

To identify such signature mutations, the use of transgenic rodent models with marker genes, such as Big Blue® mice (BBM) and rats (BBR) with the lacI gene and MutaTM mice with the lacZ gene, is advantageous, because a large number of mutations induced by a specific carcinogen can be analyzed in the marker transgenes. With this approach, the same mutational spectra were observed with UV exposure as in human skin cancer, including CC->TT mutations (6). Further, a high rate of G->T transversions at the 5'-CGT-3' sequence were found to be induced in the mouse colon by a food-borne carcinogen, 2-amino-9H-pyrido[2,3-b]indole (7). Characteristic mutations identified in the transgenes were commonly observed in endogenous marker genes such as Hprt (8) and tumor-related genes of the rodent (7,9). It is reasonable to expect that some of these characteristic mutations will also be found in the tumor-related genes of human cancers and that they will then be useful as signature mutations.

Dietary factors play important roles in human cancers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the mutagenic heterocyclic amines present in cooked meat and fish (10). PhIP induces mammary carcinomas in female and colon carcinomas, prostate microcarcinomas and lymphomas in male Fischer 344 (F344) rats (1113). Previously, we found a characteristic mutation, G deletion at the 5'-GGGA-3' site, in the Apc gene of colon carcinomas induced by PhIP (9). We also demonstrated that PhIP induced this type of mutation in the lacI gene of the colon mucosa of BBM and BBR and that this mutation accounted for 7 and 10% of the total mutations of this gene in each of these animals, respectively (7,14). Based on these results, we assume that PhIP induces various types of mutations in the Apc gene of normal colonic mucosa, with G deletions at 5'-GGGA-3' sites at a frequency of ~7–10%, and that cells having this deletion mutation acquire a growth advantage and result in tumor formation. Therefore, the G deletion at the 5'-GGGA-3' site is considered to have potential as a molecular marker of past exposure to PhIP, i.e. as a signature mutation of PhIP.

To date, however, there have been no investigations into mutations in the mammary gland, the target of PhIP-induced carcinogenesis, in female rats. In this study, we analyzed mutant frequencies (MFs) and mutational spectra of the lacI gene in the macroscopically normal mammary glands of female rats that had been treated with a carcinogenic dose of PhIP.


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Animals
Female BBR with the genetic background of the F344 strain were purchased from Stratagene (La Jolla, CA) and male Sprague–Dawley (SD) rats were purchased from CLEA Japan Inc. (Tokyo, Japan). A total of 102 (BBRxSD)F1 rats were produced by mating six pairs of female BBR and male SD rats. After confirming the presence of the lacI transgene by Southern blot analysis, 47 female F1 rats were fed a CE-2 basal diet (CLEA Japan, Tokyo, Japan) and used for this study.

PhIP treatment
PhIP–HCl was purchased from the Nard Institute (Osaka, Japan) and dissolved in distilled water at a concentration of 13 mg/ml PhIP.

In the experimental group, 33 F1 rats (6 weeks old) were administered 65 mg/kg/day of PhIP by gavage five times a week for 2 weeks. In the control group, 14 F1 rats were administered distilled water alone. After the tenth dose, both experimental and control rats were placed on a diet containing 23.5% corn oil (15), Dyets no. 111140 (Dyets Inc., Bethlehem, PA). Both groups were killed at 56–69 weeks of age. Mammary carcinomas were observed in 15 of 33 PhIP-treated rats and in three of 14 control rats. PhIP-treated and untreated rats without macroscopic mammary tumors were used for this study.

Isolation of mammary ducts
Mammary tissues that appeared macroscopically normal were removed en bloc. Mammary ducts were collected by a modification of the method previously reported (16). Mammary glands in the inguinal areas were removed with fat tissues, avoiding contamination of lymph nodes, and then placed in a Petri dish with serum-free Dulbecco's modified Eagle's medium (DMEM) without serum or phenol red (serum-free DMEM). The tissue was minced with scissors to yield fragments <1 mm3. The minced tissue was digested at 37°C for 3 h with collagenase type III (Gibco BRL, New York, NY) dissolved in 90 ml of serum-free DMEM (2 mg/ml) with vigorous shaking. During the last 15 min, 3 ml of 0.04% DNase type I (Sigma-Aldrich, St Louis, MO) was added. The suspension was centrifuged at 480 g for 3–6 min and the pellet was suspended in 30 ml of DMEM supplemented with 10% fetal bovine serum. The centrifugation was repeated once at 480 g for 3–6 min and twice at 30 g for 2 min and the pellet was suspended in 10 ml of DMEM with serum. The suspension was passed through a nylon filter of 180 µm mesh to trap mammary ducts and the collected mammary ducts were suspended in 10–40 ml of DMEM with serum. The suspension was centrifuged once at 480 g for 3–6 min. The final pellet was examined under a stereomicroscope to confirm that it consisted mainly of mammary ducts and was then kept frozen at –80°C.

DNA extraction and packaging
High molecular weight genomic DNA was extracted by the phenol/chloroform extraction method, and {lambda} phages were recovered using Transpack® Packaging Extract (Stratagene). MFs of lacI were analyzed by the method recommended by Stratagene with a modification (17). Phages of each fully blue plaque were stored in 500 µl of SM buffer (0.1 M NaCl, 8 mM MgSO4, 50 mM Tris–HCl, pH 7.5, and 0.01% gelatin) with 50 µl of chloroform at 4°C.

Analysis and classification of mutations
To avoid sequencing of the entire lacI gene, the region containing the mutation was first identified by PCR–restriction–single-strand conformation polymorphism (SSCP) analysis (18). The region covering the lacI coding region and its promoter was amplified by PCR with primers 5'-GACACCATCGAATGGTGCAA-3' (5'-position –50) and 5'-TTCCACACAACATACGAGCC-3' (5'-position 1197) in the presence of [{alpha}-32P]dCTP and the PCR product was digested with HaeIII, EcoRV and BclI (Toyobo, Tokyo, Japan). The digestion solution was then subjected to SSCP analysis under four conditions (20°C with or without 5% glycerol and 10°C with or without 5% glycerol).

Then, sequencing was performed on the fragment that showed a mobility shift in the SSCP analysis (14). Initial PCR was performed for the entire lacI gene and the PCR product was purified using a MicroSpinTM S-300 HR column (Amersham Pharmacia Biotech, Uppsala, Sweden). Using the purified PCR product as a template, fluoro-cycle sequencing was performed for both strands with Cy5TM-labeled primers specific for the fragment and Thermo Sequenase (Amersham Pharmacia Biotech). After the cycle sequencing reaction, the product was mixed with the loading dye and sequenced using an ALFexpress automatic DNA sequencer (Amersham Pharmacia Biotech).

When a mutation was observed twice or more in a rat, only the first mutation was considered as independent and used for the following analysis. Mutations were classified into three categories: base substitutions, single base pair deletions and `other mutations'. The `other mutations' category included deletions of two or more base pairs, insertions and other complex mutations. When a base pair deletion mutation occurred in a consecutive run of the same nucleotide, the nucleotide with the lowest number was assigned as the mutation site.

Statistical analysis
To analyze the difference in MFs, the non-parametric Cochran–Armitage test was used (COCHARM). Mutational spectra were analyzed by the {chi}2 test.


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Mutant frequencies
MFs in the mammary ducts were measured in five rats from the control group and in six rats from the PhIP-treated group (Table IGo). The MF in the control group was 25 ± 2.1x10–6 (mean ± SE), which was in the same range as the MFs reported for the colon, liver and lymphocytes in BBR and BBM (14,1923). In the PhIP-treated group, the MF was 323 ± 44x10–6, a value 13-fold higher than that in the control group (P = 0.0017, Cochran–Armitage test).


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Table I. MF of lacI in the mammary glands of (BBRxSD)F1 rats
 
Mutational spectra of lacI
All 40 mutants obtained in the five control rats were analyzed for the sequence of the lacI gene by PCR–restriction–SSCP and sequencing. From the six PhIP-treated rats, 177 mutants (30 mutants randomly selected from each of five rats and all 27 mutants from rat 4) were analyzed. After removal of mutants that were suspected to have clonally expanded, 34 and 149 mutants were considered as independent mutations in the control and PhIP-treated groups, respectively. All the mutations are listed in Table IIGo.


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Table II. List of mutations in thelacI gene in the mammary glands of PhIP-treated Big Blue® rats
 
The mutations detected were classified according to our previous reports (7,24) and are summarized in III. In the control group, the predominant mutation was a G:C->A:T transition at CpG sites (44%). In the PhIP-treated group, G:C->T:A transversions (43%) and single base pair deletions of G:C (21%) occurred at much higher rates, compared with the control group (P = 0.02 and 0.004, respectively). Nine deletions were observed at 5'-GGGA-3' sequences, nt 90, 513, 675, 743 and 877, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations were observed at 12 positions (Table IIGo) and two of the 12 clusters were G deletions at 5'-GGGA-3' sequences (nt 675 and 877).


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PhIP at a carcinogenic dose was shown to induce a 13-fold increase in MF in the mammary gland, the target tissue for carcinogenicity in female F344 and (BBRxSD)F1 rats. This is the first report on MF in the mammary gland induced by PhIP or other mammary carcinogens. PhIP was administered at 6–8 weeks of age and the MFs were measured at 56–69 weeks. The manifestation time for genotoxic insult (25) is estimated to be 4 weeks in the mammary gland (26) and the MFs in this study can be considered to reflect MFs in stem cells. The 13-fold elevation in MF corresponded with the cancer incidence of 45% (15/33) in the case of PhIP-induced rat mammary carcinogenesis. However, we still cannot exclude the possibility that the F1 rats with mammary tumors had a somewhat different genetic background than those without tumors, since the SD rat is an outbred strain. As for contamination with neoplastic lesions in the samples, we histologically examined the entire mammary tissue in two PhIP-treated rats without any macroscopic lesions and confirmed the absence of any neoplastic lesions. DNA was extracted from mammary ducts of six other PhIP-treated rats without any macroscopic lesions.

The mutational spectra of PhIP in the mammary glands were somewhat different from those in the colon. Since the spectra of the lacI gene have been shown to be the same in male and female rat colons (14), the mutational spectra of the female rat mammary glands and those of the male and female rat colons were compared. G:C->T:A transversions were observed at a significantly higher frequency in the mammary gland than in the colon (P = 0.0003) and G:C deletions were found at a significantly lower frequency (P = 0.002). The mutational clusters at nt 92 and 877 were common between the mammary gland and the colon (14), but those at nt 56 and 82 were detected only in the mammary gland. One possible mechanism is the difference in the species of DNA adducts formed in the two tissues. Although PhIP is known to yield almost the same adduct levels in the two tissues (27,28), the structures of the adducts in the mammary gland have not been analyzed in detail. A second possible mechanism is the higher rate of cell proliferation in the colon (27,28). The mammary gland has a better chance of DNA repair before cell replication and the DNA repair might involve specific sequence contexts. A third possible mechanism is the difference in the local chromatin structure, which can affect mutational types and clusters. Finally, we used (BBRxSD)F1 rats in this study but BBR in our previous study for the mutational spectra in the colon (7).

The signature mutation of PhIP, G deletion at the 5'-GGGA-3' site, was shown to be commonly observed in the mammary gland and colon. The signature mutation was previously detected in the lacI gene of the mouse colon and in the lacI and Apc genes of the rat colon. It is also known that the signature mutation is preferentially induced in the lacZ transgene of the intestine of male MutaTM mice (5%) (29), the supF gene of human fibroblasts in vitro (3%) (30) and the Chinese hamster endogenous Hprt gene (10%) (31). A search of a human p53 mutation database (32) identified 33 cases of G deletions at the 5'-GGGA-3' site among 9959 somatic p53 mutations. If we assume that G deletions at the 5'-GGGA-3' site are not induced by any other genotoxic agents to which humans are ubiquitously exposed and also that the deletion mutation accounts for 3–10% of the total PhIP-induced mutations in the human p53 gene, 330–1100 mutations in the database (3.3–11% of the total 9959 mutations) can be estimated to have been induced by PhIP. This is an approach to assess the risk of an environmental carcinogen and, of course, these assumptions must be refined by clarifying whether there are other human carcinogens that induce G deletions at the 5'-GGGA-3' site. The use of the signature mutation of PhIP as a molecular marker seems to be a promising approach for evaluating the role of PhIP in human cancers.


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Table III. Types of PhIP-induced lacI mutations in the mammary glands
 

    Acknowledgments
 
The authors are grateful to Ms A. Ohtake for her technical help. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.


    Notes
 
3 To whom correspondence should be addressed Email: tushijim{at}ncc.go.jp Back


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 References
 

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Received February 16, 1999; revised June 22, 1999; accepted June 22, 1999.