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Carcinogenesis, Vol. 20, No. 6, 1063-1068, June 1999
© 1999 Oxford University Press

Carcinogenesis

Strain differences of rats in the susceptibility to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine: no implication of Apc and Pla2g2a genetic polymorphisms in differential susceptibility

Yukiko Ishiguro, Masako Ochiai, Takashi Sugimura, Minako Nagao1 and Hitoshi Nakagama2

Biochemistry and
1 Carcinogenesis Divisions, National Cancer Center Research Institute, 1-1 Tsukiji 5, Chuo-ku, Tokyo 104-0045, Japan


   Abstract
Top
 Abstract
Introduction:
 Materials and methods
 Results
 Discussion
 References
 
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine contained in cooked food, induces colon tumors in F344 male rats when administered orally. In the present study, PhIP was introduced to various rat strains, and susceptibility to the induction of aberrant crypt foci (ACFs) was analyzed as a biomarker for colon carcinogenesis. BUF/Nac rats were highly susceptible, giving rise to 12.2 ± 1.7 ACFs per rat. F344 rats were intermediate and ACI/N rats were resistant, giving 3.5 ± 1.8 and 0.9 ± 0.7 ACFs per rat, respectively. In spite of this, the extent of DNA damage by PhIP in F344, in terms of the level of PhIP–DNA adducts, was significantly lower than that in ACI/N. The differences in formation of ACFs could be, in some part, implicated in the differential susceptibility to colon carcinogenesis induced by PhIP, especially in a step later than adduct formation. In an attempt to determine the genetic factors implicated in the susceptibility to formation of ACFs, a possible involvement of the adenomatous polyposis gene (Apc) and its modifier secretory phospholipase A2 (Pla2g2a) was analyzed. No genetic polymorphisms in either Apc or Pla2g2a showed a significant correlation to susceptibility to formation of ACFs among rat strains.

Abbreviations: ACs, aberrant crypts; ACFs, aberrant crypt foci; Apc, adenomatous polyposis coli gene; DMH, 1,2-dimethylhydrazine; HCAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.


   Introduction:
Top
 Abstract
 Introduction:
Materials and methods
 Results
 Discussion
 References
 
Colon cancer used to be less common in the Japanese population. However, paralleling the westernization of the life style of Japanese people, its incidence has been increasing recently (1). Environmental factors are therefore considered to play important roles in human colon carcinogenesis (2,3) in terms of the induction of genetic alterations, such as mutations in the adenomatous polyposis coli gene (Apc), p53, K-ras, DCC and mismatch repair genes (4). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines (HCAs) produced during cooking fish and meat (5,6), and people are exposed to it in daily life. PhIP has been demonstrated to induce colon and prostate tumors in F344 male rats, and mammary tumors in female rats, when fed a diet containing 400 p.p.m. of PhIP for 52 weeks (7,8). As PhIP is the most abundant HCA in cooked food and one of the five HCAs that induce colon tumors in experimental animals (9), the genetic factors of rats implicated in susceptibility to PhIP-induced colon carcinogenesis could also play an essential role in sporadic colon carcinogenesis of humans. Namely, a subset of the general human population could be more sensitive to these environmental chemical insults than other subsets of the population due to differences in genetic polymorphisms.

Genetic alterations in rat colon tumors induced by PhIP have partly been characterized in our previous work (10–12). As for the Apc gene (13,14), PhIP-induced tumors harbored the specific mutation of one guanine base deletion at the 5'-GTGGGAT-3' sites in exons 14 and 15 (11). As there are only two 5'-GTGGGAT-3' sites in the entire Apc gene, the coding sequence of which encompasses >8000 bp, these two sites are considered to be mutational hot spots by PhIP. Thus, polymorphisms at these PhIP target sites could contribute to the differential susceptibility to colon carcinogenesis induced by PhIP.

In a typical adenoma–carcinoma sequence model in human colon carcinogenesis, mutations in Apc are considered to be an initial event followed by the losses of wild-type alleles (4,15,16). A series of experiments using animal models of an Apc mutant mouse strain, Min (17), and various Apc knock-out mice (18–20) also indicated that inactivation of both alleles of Apc seems to be a key genetic event for the induction of intestinal tumors. However, genetic studies have also revealed that the number of intestinal tumors in Min mice is greatly affected by their genetic background (21). As a consequence, quantitative trait loci mapping identified a locus, Mom-1 (modifier of Min 1), which modifies the effects of the Apc mutation on polyp formation (22). Recently, the secretory phospholipase A2 gene (Pla2g2a) was identified as a candidate for Mom-1 (23,24). Thus, Pla2g2a polymorphism could also contribute to the various susceptibilities to PhIP, even with the identical Apc genotype. Genes involved in the metabolic activation of PhIP or the repair capacity of PhIP-induced DNA damage could also be among the candidates.

In the present study, we utilized a rat model to elucidate the genetic factors controlling susceptibility to colon carcinogenesis by PhIP. As a considerable body of data have revealed aberrant crypt foci (ACFs) as potential precancerous lesions in both animal models (25–28) and human colon cancer cases (29–31), ACFs were used as a surrogate biomarker for colon carcinogenesis in this study. PhIP–DNA adduct levels were analyzed as a relevant marker for the extent of DNA damage in different rat strains. Genetic analyses were also employed to identify the role of Apc and Pla2g2a polymorphisms in susceptibility to carcinogenesis induced by PhIP, and the results are discussed.


   Materials and methods
Top
 Abstract
 Introduction:
 Materials and methods
Results
 Discussion
 References
 
Animals and diets
PhIP–HCl (PhIP) was purchased from the Nard Institute (Osaka, Japan) and added to AIN-93G or CE-2 basal diet to give concentrations of 300 or 400 p.p.m. Five-week-old male rats of BUF/Nac (BUF), F344 and ACI/N (ACI) strains were obtained from CLEA Japan (Tokyo, Japan); Wistar/Crj (Wistar) and Brown Norway (BN) rats were from Charles River Japan (Yokohama, Japan). All strains of rats, except Wistar, which is a closed-colony maintained in Charles River Japan, were inbred strains. A genealogic tree for the inbred strains of these rats was reported previously (32). AIN-93G basal diet and a high fat diet (PRIMEX) obtained by adding hydrogenated vegetable oil (23% w/w) to AIN-93G were purchased from Dyets (Bethlehem, PA).

Experimental protocol
The rats were fed 400 p.p.m. of PhIP by the short-exposure protocol for the induction of ACFs as described previously (33). Briefly, for the PhIP-treated group, rats were acclimatized for 1 week to the housing environment and the AIN-93G basal diet, and were fed AIN-93G containing 400 p.p.m. of PhIP for the first 2 experimental weeks followed by PRIMEX feeding for 4 weeks. For the non-treated group, rats were fed AIN-93G without PhIP for the first 2 weeks followed by PRIMEX feeding for 4 weeks.

Detection of ACFs
The rats were killed and the large intestines were removed, flushed out with neutralized 10% formaldehyde and then cut open along the longitudinal median axes. ACFs were detected by staining the colon mucosa as described previously (34), and the number of ACFs and aberrant crypts (ACs) were determined under 40x and 100x magnifications with a light microscope. The size of each ACF was defined as the number of ACs composing each ACF (ACs/ACF).

PhIP–DNA adducts
DNA adduct levels in colon epithelia were measured by the 32P-post-labeling method as described previously (35). Briefly, rats of various strains were fed a CE-2 diet containing 300 p.p.m. of PhIP for 1 week, the animals were then killed and their colon mucosa scraped off with a glass slide, snap-frozen in liquid nitrogen and stored at –80°C until DNA extraction. DNA was extracted and digested with micrococcal nuclease and spleen phosphodiesterase (Worthington Biochemical; Freehold, NJ) at 37°C for 3 h. The DNA digest was 32P-labeled by T4 polynucleotide kinase (Takara; Kyoto, Japan) with [{gamma}-32P]ATP. PhIP–DNA adducts were analyzed by thin-layer chromatography after digesting a labeled product with nuclease P1 (Yamasa shoyu; Choshi, Japan) and venom phosphodiesterase I (Worthington Biochemical).

Statistical analysis
All the statistical results were expressed as means ± SD. Statistical analyses were performed using the Kruskall–Wallis one-way ANOVA and Mann–Whitney U tests, with an SPSS package on a Macintosh computer (SPSS Japan Inc.; Tokyo, Japan).

PCR–SSCP analysis
PCR–SSCP analyses were carried out as described (36) in the presence of [{alpha}-32P]dCTP using the primers listed in Table IGo. All the primers used were designed from rat Apc and Pla2g2a cDNA sequences (11,37). After the PCR, 1 µl aliquots of PCR products were digested with appropriate restriction enzymes in 10 µl 1x reaction mixture at 37°C for 1 h, mixed with the loading buffer (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) and applied to 6% non-denaturing polyacrylamide gels in 0.5x TBE (1x TBE: 89 mM Tris–89mM boric acid, 2 mM EDTA) with or without 5% glycerol. After the electrophoresis, gels were dried and autoradiography was performed with Kodak O-MAT AR film at –80°C for 1–3 days. The annealing temperatures for each primer and restriction enzymes used are also specified in Table IGo.


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  		Table I. PCR primers and restriction enzymes used for PCR–SSCP and PCR–RFLP analyses
 
PCR–RFLP analysis
PCR–restriction fragment length polymorphism (RFLP) analysis was performed as described (38). Briefly, genomic fragments containing polymorphic sites were amplified by PCR, except that [{alpha}-32P]dCTP was omitted in the reaction. Aliquots (5 µl) of PCR products were digested with appropriate enzymes in 10 µl of 1x reaction mixture at 37°C for 1 h, and electrophoresed in 3% agarose gel in 0.5x TBE. The primer sequences for PCR and restriction enzymes used are listed in Table IGo. After the electrophoresis, DNA fragments were made visible by staining the gels with 0.02% ethidium bromide.

Nucleotide sequencing of PCR products
Genomic fragments amplified by PCR were cloned into TA cloning vector, pCR2.1 (Invitrogen, Carlsbad, CA). Plasmid DNA was amplified in XL1-Blue and extracted by the conventional alkaline SDS method, then subjected to the sequencing reaction using an AutoRead Sequencing Kit (Amersham Pharmacia Biotech, Bucks, UK). After the reaction, 2 µl aliquots of the mixture were run and analyzed on an ALF red DNA sequencer (Amersham). Sequences were confirmed by repeating reactions at least twice in both directions.

Northern blot analysis
Large intestines of 6-week-old rats without any treatment were kept frozen at –80°C immediately after resection. Total RNA was extracted by the acid-guanidinium thiocyanate/phenol/chloroform method (39). Samples of 20 µg RNA were separated in a formaldehyde denaturing gel, blotted onto a nylon membrane and subsequently subjected to hybridization. A Pla2g2a cDNA probe for hybridization was prepared as follows. An aliquot of 1 µg of total RNA extracted from rat colon was reverse-transcribed with SuperScript reverse transcriptase (Gibco BRL, Gaithersburg, MD) using oligo(dT)12–18 (Takara) as a primer. Then, the obtained cDNA library was amplified by PCR using primers PL10 and PL18, 5'-GCTAGGAGAGGTGTTAGAGG-3', and 503 bp PCR products including the entire Pla2g2a cDNA were labeled with [{alpha}-32P]dCTP using the Multiprime DNA labeling system (Amersham).


   Results
Top
 Abstract
 Introduction:
 Materials and methods
 Results
Discussion
 References
 
Strain differences of rats in the induction of ACFs by PhIP
The numbers of ACFs induced in five different rat strains by feeding 400 p.p.m. of PhIP were analyzed. As shown in Table IIGo, the average number of ACFs induced was highest in BUF rats (12.2 ± 1.7 per rat), lowest in ACI rats (0.9 ± 0.7 per rat), and Wistar, F344 and BN strains were intermediate, being 5.6 ± 1.7, 3.5 ± 1.8 and 2.8 ± 1.6, respectively, per rat. ACI rats produced a significantly low number of ACFs among all strains examined. Rats without PhIP feeding gave few or no ACFs. The average size of the ACF for each strain varied from 1.4 ACs/ACF in BN to 2.4 ACs/ACF in F344 rats. However, these values were not significantly different from each other (Table IIGo). In Figure 1Go, the average numbers of ACFs per rat in BUF, F344 and ACI strains, were plotted in relation to the sizes of the ACFs. As is clearly demonstrated, none of the ACFs induced in 20 ACI rats was larger than three ACs/ACF. In contrast, the sizes of the ACFs in BUF and F344 strains were more widely distributed ranging from one to seven ACs/ACF, and the numbers of ACFs with four or more ACs/ACF in BUF (1.0 per rat) and F344 strains (0.62 per rat) were significantly high compared with those in ACI rats (P < 0.05).


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  		Table II. Strain differences of rats in the susceptibility to ACF formation
 


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  		Fig. 1. Size distribution of ACFs in various rat strains. ACFs induced in BUF, F344, and ACI rats were subgrouped according to their sizes, from one to six, or seven or more ACs/ACF, and the number of ACFs falling into each subgroup was counted to give an average value of ACFs per rat. The numbers of rats used for this analysis are given in Table IIGo.

 
DNA adduct levels in colon mucosa of various rats
To evaluate the possible contribution of the DNA damage induced by PhIP to the different susceptibilities to ACF formation, the PhIP–DNA adduct level in each rat strain was measured. The results are summarized in Table IIIGo. F344 rats demonstrated the lowest level, 2.8 ± 0.7/107 nucleotides, which was significantly lower than all other strains analyzed (P < 0.05), except BN. The adduct level in ACI rats, which were less susceptible than F344 rats to ACF formation, was significantly higher than that in F344 rats. Therefore, PhIP–DNA adduct levels did not correlate well to the number of ACFs induced by PhIP.


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  		Table III. PhIP–DNA adduct levels in various rat strains
 
Apc gene polymorphism
As the Apc gene was demonstrated to be a target for, and frequently mutated in, PhIP-induced rat colon tumors at three 5'-GGGA-3' sites in exons 14 and 15, including two 5'-GTGGGAT-3' sites as described earlier (11), sequence polymorphisms at those PhIP target sites were analyzed. PCR–SSCP analyses using primer sets 14F/14R, 15B/15D and 15F/15H revealed no genetic polymorphism at the PhIP target sites (Figure 2Go). Another novel polymorphism in exon 15 was identified by SSCP using the same primer set, 15B/15D, and the restriction enzyme, AluI (Figure 3AGo). The nucleotide sequencing revealed the polymorphism without an amino acid change at nucleotide position 2724, that being thymine in BUF, F344 and BN rats, and cytosine in Wistar and ACI rats (data not shown). Since thymine at this position creates a DdeI restriction site, 5'-CTGAG-3', this polymorphism was determined by PCR–RFLP analysis using a primer set, 15BS and 15DS (Table IGo and Figure 3BGo). Another restriction polymorphism at the EcoT21I site in exon 11 (40) was also examined and segregated in Wistar and ACI rats (Figure 3CGo).



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  		Fig. 2. Apc polymorphism at PhIP-target sites in exons 14 and 15. Genomic DNAs (100 ng) from various rat strains were amplified as described in Materials and methods using primer sets 14F/14R, 15B/15D and 15F/15H as indicated in Table IGo. PCR products were labeled with [32P]dCTP, and subjected subsequently to SSCP analyses. DNA samples obtained from PhIP-induced colon tumors in F344 rats (10), T2-1 and T17, were used as positive controls harboring point mutations in exons 14 and 15, respectively. MCR: mutation cluster region. Lane 1, BUF; lane 2, Wistar; lane 3, F344; lane 4, BN; lane 5, ACI.

 


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  		Fig. 3. Two other Apc polymorphisms in exons 11 and 15. (A) An aliquot of 100 ng of each rat genomic DNA was amplified with a primer set, 15B and 15D, digested with AluI and electrophoresed in a 6% SSCP gel. (B) DdeI restriction polymorphism of exon 15. PCR fragments (263 bp) amplified with a primer set, 15BS and 15DS, were digested with DdeI as described in Materials and methods. Both Wistar and ACI strains were missing one DdeI site, giving a long fragment of 215 bp. (C) EcoT22I restriction polymorphism in exon 11. Genomic DNA was amplified using a primer set, 11F and 11R, and PCR products (220 bp) were electrophoresed in a 3% agarose gel without (–) or with (+) EcoT21I digestion. On digestion with EcoT22I, Wistar and ACI alleles gave 126 and 94 bp fragments. {Phi}x174/HaeIII digests were used as a DNA size marker. Lane 1, BUF; lane 2, Wistar; lane 3, F344; lane 4, BN; lane 5, ACI.

 
Pla2g2a gene polymorphism
PCR–SSCP analyses of Pla2g2a gene were performed using both genomic DNA and cDNA fragments. The Pla2g2a cDNA was obtained as described in generating the Pla2g2a cDNA probe in Materials and methods. Three genetic polymorphisms were identified using two primer sets, PL10/PL12 and PL19/PL22, as depicted in Figure 4Go. Two of them were novel and resided in either intron 2 (Figure 4AGo) or the non-coding region of exon 5 (Figure 4CGo), and were demonstrated to be AluI and HinfI RFLPs, respectively. The other one was a C->T substitution at nucleotide position 404 in exon 5 (Figure 4BGo), and accompanied an amino acid substitution from proline (Pro) to leucine (Leu) as reported previously (41), this being Pro in BN and Leu in BUF, F344 and ACI rats. Wistar rats were heterozygous, bearing both alleles for Pro and Leu. All three polymorphisms were segregated in BUF, F344 and ACI strains and did not show any relationship with the differential susceptibility to ACF formation.



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  		Fig. 4. Pla2g2a polymorphism in intron 2, coding and non-coding regions of exon 5. Genomic DNA (100 ng) was amplified by PCR using primer sets, PL10/PL12 (A) and PL19/PL22 (B and C). After the reaction, PCR products of 683 (A) and 540 bp (B and C) in sizes were digested with either AluI (A), RsaI (B) or HinfI (C). In the cases of (A) and (C), samples were electrophoresed in a 3% agarose gel. In the case of (B), the digests were analyzed by SSCP. Lane 1, BUF; lane 2, Wistar; lane 3, F344; lane 4, BN; lane 5, ACI. Wistar rats were heteroalleic (lane 2), and all three polymorphisms were segregated in BUF, F344 and ACI strains (lanes 1, 3 and 5, respectively). {Phi}x174/HaeIII digests were used as a size marker in (A) and (C).

 
Expression of Pla2g2a in various rat strains
We further examined the expression levels of Pla2g2a mRNA to see whether the three polymorphisms described above had any effect on the Pla2g2a gene expression, as in the case of mice (23). Although BUF and F344 strains had a slightly higher level of mRNA expression than Wistar, BN and ACI strains (Figure 5Go), no correlation was observed between sequence polymorphisms and the expression levels.



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  		Fig. 5. Pla2g2a expression in colon tissues from various rat strains. Northern blot analysis for Pla2g2a expression was carried out using 20 µg of total RNA for each sample. All five rat strains expressed 0.8 kb transcripts as indicated (upper panel). To justify the amounts of RNA samples loaded on the gel, ethidium bromide staining of the gel is shown in the lower panel.

 

   Discussion
Top
 Abstract
 Introduction:
 Materials and methods
 Results
 Discussion
References
 
Accumulated data by many researchers so far have characterized and established the biological significance of ACFs as a relevant biomarker for colon carcinogenesis in both human (29–31) and experimental animal cases (25–28). As we expected, obvious strain differences were observed in the susceptibility to ACF formation (Table IIGo). BUF, Wistar and F344 rats were more sensitive to PhIP than were ACI rats, in terms of the induction of ACFs. Moreover, BUF and F344 strains harbored significantly higher numbers of large ACFs than ACI rats, greater than three ACs/ACF. As large ACFs are considered to develop colon adenoma or cancer more readily than small ones (42,43), BUF and F344 rats are therefore considered to be more susceptible to colon carcinogenesis by PhIP than are ACI rats.

As PhIP needs to be metabolically activated to exert its genotoxic effects (44), enzymes involved in this activation pathway could be attributable factors to the strain differences in ACF formation. To assess this point first, the PhIP–DNA adduct levels in colons were analyzed by the 32P-post-labeling method. As shown in Table IIIGo, ACI rats, which are less sensitive to ACF formation, demonstrated significantly higher levels of PhIP–DNA adducts than did F344 rats. Thus, the extent of the overall DNA damage targeted by PhIP in colon epithelial cells was not necessarily the major factor implicated in the differential susceptibility.

We next examined the possible involvement of genetic polymorphism in Apc and Pla2g2a. Apc is considered to be a gate-keeper gene for colon carcinogenesis, and a mutation in Apc could initiate and promote tumor growth (4,45). However, PCR–SSCP analysis of Apc revealed no sequence polymorphism at the PhIP target sites, 5'-GTGGGAT-3', in exon 14 or 15 (11). Two other polymorphisms at exons 11 and 15 were segregated in Wistar and ACI strains. As Wistar rats possessed much higher susceptibility than ACI rats, no relationship was found between Apc polymorphisms and the induction of ACFs by PhIP. As for Pla2g2a, which was first characterized as a modifier gene of Apc mutation in the Min intestinal tumor model, three genetic polymorphisms including two novel ones were identified in intron 2, exon 5 and the 3'-untranslated region of exon 5, and all three polymorphisms were segregated in BUF, F344 and ACI strains. Therefore, allelotypes of Pla2g2a as well as its expression levels in colon epithelium has not dealt with susceptibility to ACF formation in rats. As human PLA2G2A is considered not to be associated with phenotype variations in familial adenomatous polyposis or sporadic colorectal cancer (46,47), the situation is similar in rat colon carcinogenesis by PhIP. Taken together, no genetic polymorphism of either Apc or Pla2g2a has been implicated in the differential susceptibility to ACF formation between ACI and BUF or F344 strains as far as has been examined. We recognize limitations in this work, namely the possibility that undetected polymorphisms in Apc and Pla2g2a might explain the differences in strain susceptibility to ACFs, or that the rate of ACF formation might not reflect the rate of tumor formation. To clarify these points, future studies will use nucleotide sequencing to detect possible polymorphisms implicated in ACF formation and tumor development.

To date, two candidate loci, Scc1 (48) and Ccs 1 (49) were mapped to mouse chromosomes 2 and 12, respectively, as susceptibility loci to 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in mice. In contrast to DMH, which is a synthetic chemical carcinogen, PhIP is produced and present in cooked food, people are exposed to it in daily life and it targets alimentary tracts as well. Therefore, PhIP-induced carcinogenesis in rats seems to be suitable for investigation as a relevant model for human colon carcinogenesis.

At this moment, we do not have any clue as to whether the genes responsible for the susceptibility to ACF formation by PhIP in rats are the same as those identified in mice. Although the Apc or Pla2g2a gene plays major roles in colon carcinogenesis of human and/or animals, genetic polymorphisms of Apc and Pla2g2a do not appear to be associated with differential susceptibility to PhIP-induced ACF formation. A novel genetic factor could be implicated in the susceptibility to PhIP-induced colon carcinogenesis, and a more detailed genetic linkage analysis needs to be applied to extend our knowledge on this point.


   Acknowledgments
 
This study was supported by a Grant-in-Aid for the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research, and by the Research Grant of the Princess Takamatsu Cancer Research Fund.


   Notes
 
2 To whom correspondence should be addressed Email: hnakagam{at}gan2.ncc.go.jp Back


   References
Top
 Abstract
 Introduction:
 Materials and methods
 Results
 Discussion
 References
 

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Received August 25, 1998; revised December 31, 1998; accepted January 25, 1999.


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