Mutagenesis, Vol. 16, No. 5, 431-437,
September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats
Centre for Environmental Health and Department of Biology, University of Victoria, PO Box 3020 STN CSC, Victoria, British Columbia, Canada V8W 3N5
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10–4) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C
T:A and G:CC:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats. |
Introduction |
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a suspected human carcinogen that is formed in cooked meats and fish (Felton et al., 1986
; Wakabayashi et al., 1992; Schut and Snyderwine, 1999). In rats PhIP mainly causes colon and prostate tumors in males and mammary gland tumors in females (Ito et al., 1991; Shirai et al., 1997). PhIP is also potently mutagenic in each of these three tissues (Okonogi et al., 1997; Okochi et al., 1999; Stuart et al., 2000a). PhIP mutagenicity appears to correlate with carcinogenicity in tumor target tissues, since rat liver is neither a target for PhIP-induced DNA adduct formation (Takayama et al., 1989), carcinogenesis (Wakabayashi et al., 1992) nor mutation (Stuart et al., 2000a). Interestingly, colon, breast and prostate are among the most common sites for cancer in humans (Wingo et al., 1999). Therefore, to better evaluate the risk to human health it is particularly important to identify the mechanism by which PhIP contributes to carcinogenesis (Layton et al., 1995; Dingley et al., 1999; Nagao, 1999).
We recently described the mutational specificity of PhIP in the lacI transgene in colon of male and female Big Blue rats (Okonogi et al., 1997). Since male F344 rats predominantly develop colon and prostate cancer while female rats predominantly develop mammary gland tumors after exposure to PhIP (following 52 weeks exposure to 400 p.p.m. PhIP in the diet, 55% of male but only 7% of female rats developed colon tumors, while 47% of the female rats developed mammary gland tumors; see Ito et al., 1991), we predicted that PhIP would induce mutations that would be unique to male colon. In a previous study, initial DNA sequence analysis revealed a sex-specific preference (P = 0.014, 
2 test) for a PhIP `signature' mutation, deletion of a G:C base pair at the 5'-TCCC-3'/5'-GGGA-3' (TCCC/GGGA) sequence at lacI nucleotide positions 89–92. However, this difference disappeared (P = 0.69) as additional mutants were sequenced.
As it is important to understand the basis for the sex-linked specificity of PhIP-induced carcinogenesis in the rat colon, we re-examined the PhIP-induced mutational specificity in this organ using 15 male and 15 female lacI transgenic rats. Since specific regions of the colon are known to differ biochemically, physiologically and also with respect to tumorigenesis in rodents and in humans (see for example Sunter et al., 1979; Gutschmidt et al., 1983; Butler et al., 1992; Hall et al., 1992; Sato and Ahnen, 1992; Breivik et al., 1997), the colon from each animal was trisected into cecum, proximal colon (PC) and distal colon (DC). This enabled us to compare, for the first time, mutant frequency (MF) and mutation spectra (MS) in specific regions of the colon. This strategy additionally tripled the PhIP colon tissue sample sizes, thus increasing the probability of detecting male–female differences should they exist.
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Materials and methods |
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Animal treatments and tissue isolations
Big Blue F344 lacI transgenic rats (Taconic; Germantown, NY) were individually housed and treated following guidelines conforming with the Guide for the Care and Use of Laboratory Animals (Laboratory Animal Resources, National Research Council, Washington, DC, 1996). Food and water were provided ad libitum. The basal (control) diet consisted of powdered AIN-93G (Dyets Inc., Bethlehem, PA) without the t-butylhydroquinone antioxidant, supplemented with 2% (w/w) tocopherol-stripped corn oil (ICN Biomedicals, Aurora, OH). Diets with PhIP (Toronto Research Chemicals, Toronto, Ontario, Canada) consisted of the basal diet supplemented with 200 p.p.m. (w/w) PhIP. Diets were prepared twice weekly and stored at 4°C under argon. The male and female control groups each consisted of five rats, while the PhIP treatment groups each consisted of 15 male and 15 female rats. Upon arrival, the control rats were 4–6 weeks old, while the rats in the PhIP treatment groups were 5 weeks old. After acclimatization to the housing and basal diet for 7 days, the rats received either basal diet (control animals) or basal diet supplemented with PhIP for 61 days, after which all of the animals were returned to the basal diet for 7 days. The amount of diet consumed was recorded biweekly and animal body weights were recorded at least weekly. The rats were killed by CO2 asphyxiation followed by cervical dislocation. Colon tissue, including cecum, was immediately dissected, cut open lengthwise and rinsed with phosphate-buffered saline [0.14 M sodium chloride, 0.0027 M potassium chloride, 0.010 M sodium phosphate (dibasic), 0.0018 M potassium phosphate (monobasic), pH 7.4]. After removing the cecum, the remaining colon was bisected into PC and DC sections of approximately equal length. For reference, the order of the tissues in situ is cecum, PC, DC. The colon mucosal cells were scraped from the tissue sections using a razor blade, flash-frozen in liquid nitrogen and stored at –80°C.
lacI mutational assay
High molecular weight genomic DNA was recovered from the cecal and the colon mucosal cells by a dialysis purification procedure as described previously (Suri et al., 1996). lacI transgenes were recovered from the purified chromosomal DNA by an in vitro 
packaging reaction and the packaged phage were plated on the Escherichia coli SCS-8 host strain following recommended methods (Stratagene, 1997). MF were calculated by dividing the number of lacI mutant plaques by the total number of plaques.
Screening for the PhIP `signature' mutation by oligonucleotide hybridization
To test whether a sex-linked difference exists for the PhIP-induced –1 frameshift mutation at lacI nucleotide positions 90–92 [i.e. –(G:C)90–92] (Okonogi et al., 1997), virtually all of the PhIP-induced lacI mutants that were collected (Table II) were screened for this mutation by hybridization with an oligonucleotide specific for this frameshift deletion. lacI PCR product (1 µl of a standard PCR reaction) (Erfle et al., 1996) was spotted onto a positively charged nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Little Chalfont, UK), air dried and UV cross-linked for 5 min at 312 nm on a UV transilluminator (model FB-TIV-88; Fisher Scientific, Nepean, Ontario, Canada). The membrane was prehybridized at 37°C for 15 min in 1x SSC (1x SSC is 0.015 M sodium citrate, 0.15 M sodium chloride) containing 3 M guanidine thiocyanate (ICN Biomedicals, Aurora, OH). A fluorescein isothiocyanate 5',3'-labeled oligodeoxyribonucleotide probe, 5'-GACCGTTTCCGCGTGGTGAAC-3' (Dalton Chemical Laboratories, North York, Ontario, Canada), specific for the –(G:C)90–92 deoxycytidine deletion (underlined) was then added at a final concentration of 10 µM and allowed to hybridize for 15 min. The membrane was then washed twice with 0.1x SSC containing 2.5 M guanidine thiocyanate (stringent wash) and once with 1x SSC minus guanidine thiocyanate (non-stringent wash); each wash step consisted of 15 min at 37°C. The membrane was blocked for 15 min at room temperature with 2% (w/v) skimmed milk powder in TBS (TBS is 0.15 M sodium chloride, 0.10 M Tris–HCl, pH 7.5) plus 0.5% (v/v) Tween 20. Horseradish peroxidase-conjugated anti-fluorescein antibody (150 U/ml in TBS; Boehringer Mannheim, Mannheim, Germany) was added to the membrane and blocking solution at a final concentration of 0.15 U/ml. After 30 min the membrane was washed four times with TBS plus 0.5% Tween 20 (5 min per wash, at room temperature). The presence of antibody, indicating occurrence of the –(G:C)90–92 deletion, was detected colorimetrically using a 3,3'-diaminobenzidine metal-enhanced kit (ICN Biomedicals, Aurora, OH).
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DNA sequencing and data analyses
Mutations in the lacI transgene were determined using the DNA cycle sequencing method essentially as described previously (Erfle et al., 1996
), using LI-COR automated DNA sequencers. Additionally, all of the –(G:C)90–92 frameshift mutants identified previously by oligonucleotide hybridization were confirmed by DNA sequencing. Mutational data were managed and analyzed using custom software (de Boer, 1995). The lacI gene is numbered according to Farabaugh (1978).
Statistical comparisons (control animal groups compared with sex-matched PhIP-treated animal groups) of average diet consumption rates (following the initial 7 day acclimatization period) and final average animal body weights were conducted by a one-way analysis of variance (ANOVA) using GraphPad InStat v.3.01 (GraphPad Software, San Diego, CA). Statistical comparisons of MS, using the numbers of lacI mutants provided in Table III, were conducted using the algorithm of Adams and Skopek (Adams and Skopek, 1987; Cariello et al., 1994) using a program provided by the authors. This algorithm executes a Monte Carlo approximation to Fisher's exact test and is generally regarded (see Piegorsch and Bailer, 1994) as one of the most robust methods for statistical comparisons of MS. The MS used in the Adams–Skopek analyses consisted of 14 types of mutations: G:CA:T not at 5'-CpG-3' (CpG) dinucleotide sequences; G:CA:T at CpG sequences; A:TG:C; G:CT:A not at CpG sequences; G:CT:A at CpG sequences; G:CC:G; A:TT:A; A:TC:G; +1 frameshifts; –1 frameshifts; deletions; insertions; complex mutations; tandem mutations. Trends (and variance) in the MF data were determined using COCHARM (created by Troy Johnson, Procter & Gamble, Cincinnati, OH), a computer program that executes the Generalized Cochran–Armitage test. Fisher's exact test, used to evaluate specific mutational events, was executed using GraphPad InStat. The 
-level for significance for all tests was set at 0.05.
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Results |
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Diet consumption and animal weights were recorded throughout the study period. Diet consumption rates (g/day) were relatively unaffected by animal age, however, male rats consumed more diet than female rats and control diet rats consumed more diet than PhIP-treated rats (g/day, mean ± SD: male controls, 13.1 ± 0.7; female controls, 9.7 ± 1.1; male PhIP, 11.3 ± 1.0; female PhIP, 7.8 ± 0.7; P < 0.001 for male control rats versus male PhIP-treated rats; P < 0.001 for female control rats versus female PhIP-treated rats). The reduced diet consumption rates in PhIP-treated rats resulted in reduced growth rates, relative to sex-matched controls, throughout the study (growth) period. At completion of the study the average body weights were (mean ± SD): male controls, 331 ± 17 g; female controls, 196 ± 24 g; male PhIP-treated, 275 ± 35 g; female PhIP-treated, 153 ± 14 g. Comparing PhIP-treated rats with sex-matched controls, the final bodyweights were significantly different (males, P < 0.001; females, P < 0.05). With the exception of these growth differences, no other signs of PhIP-induced toxicity were evident in any of the animals.
The recovery of spontaneous (control) and PhIP-induced lacI mutants from cecum, PC and DC of male and female Big Blue rats is summarized in Tables I and II
. The spontaneous MF in the male proximal colon (mean ± SE 4.9 ± 0.3x 10–5) was slightly but significantly lower than that in the remaining male and female control tissues (range 5.4– 7.5x10–5, including SE). Subtracting the spontaneous background, PhIP treatment resulted in 7.0- and 7.2-fold increases, respectively, in the MF of the male and female cecum. Similarly, PhIP-induced MF were elevated 21- and 19-fold, respectively, in the male PC and DC and 14- and 20-fold, respectively, in the female PC and DC. In PhIP-treated males and females MF in the colon were approximately twice as high as in the cecum (P < 10–4) and MF in the PC was slightly but significantly lower than that in the DC for females (P < 10–3) but not for males (P = 0.11). Nevertheless, the overall trend in MF from cecum to PC to DC was significant (P < 10–4) for both male and female rats.
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Randomly selected lacI mutants from each of the tissue sections from control and PhIP-treated male and female rats were sequenced to determine the MS (Table III
). The control MS from cecum, PC and DC of male and female rats principally consisted of 38–47% G:CA:T transitions (1.9–2.9x10–5 MF; mutation-specific MF were calculated by multiplying the prevalence in percent of the mutation in Table III by the appropriate total MF in Table I), with 70–93% (1.6–2.7x10–5) of these occurring at CpG dinucleotide sequences, followed by 29–49% (1.9–2.4x10–5) G:CT:A transversions. Although spontaneous G:CT:A transversions (2.4x10–5) were more prevalent than G:CA:T transitions (1.8x10–5) in the male PC, possibly the result of sampling error due to the relatively small sample size of 37 mutants, overall there were no significant differences (P 
0.06) among any of the spontaneous MS.
The PhIP-induced MS (Table III) consisted predominantly of –1 frameshifts of G:C base pairs (cecum, 21–27%, 11–14x10–5; PC and DC, 27–36%, 27–38x10–5), G:CT:A (cecum, 27–34%, 14–17x10–5; PC and DC, 27–34%, 32–37x10–5) and G:CC:G transversions (cecum, 13–20%, 6.6–9.7x10–5; PC and DC, 9–19%, 9.1–23x10–5) and G:CA:T transitions (cecum, 16%, 7.9–8.0x10–5; PC and DC, 7–14%, 8–15x10–5). Thus, with some exceptions (G:CC:G in female PC; G:CA:T in male DC), MF were generally 2-fold higher in the PC and DC compared with the cecum. Overall, there appeared to be no significant differences or trends between the male and female MS; this was confirmed by the Monte Carlo analyses, which indicated that no significant differences (P 0.08) existed among any of the PhIP-induced MS. As expected, however, the spontaneous and PhIP-induced MS were highly significantly different, with the exception that there was no significant difference (P = 0.13) between the spontaneous female and PhIP-induced male cecal MS, despite the apparent differences (in percentages and in MF) between the various mutational subclasses in each spectrum (Table III).
To determine if a sex-linked difference existed for the –(G:C)90–92 frameshift mutation (Okonogi et al., 1997), PhIP-induced lacI mutants were screened for this mutation by oligonucleotide hybridization and DNA sequencing (Table II). Relatively few (0.9–2% of all mutants, 0.5–2x10–5) PhIP-induced –(G:C)90–92 mutations were recovered and none were recovered from control animals. The frequency of this mutation was similar in males and females in each tissue section, with the exception that –(G:C)90–92 mutations occurred about half as frequently in female cecum (0.9%, 0.5x10–5) compared with the other treatment groups (1.7–2.0%, 2.0–2.2x10–5).
To determine if sex-linked, sequence-specific differences existed between the male and female PhIP-induced mutations that otherwise might not be evident from the overall MS (Table III), a nearest neighbor analysis of the nucleotides flanking mutated sites was conducted [these data are not shown due to limitations of space, but refer to Stuart et al. (2000b) for a description of the method]. While this analysis indicated that some nucleotide sequences (e.g. CGC) were preferentially mutated, there appeared to be no sex-linked component among any of the PhIP-induced mutations, considering the sequence context in which the mutations occurred.
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Discussion |
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We have examined the specificity of PhIP-induced mutagenesis in the lacI transgene from colon of male and female rats. Because of the regional variation in colon physiology and tumorigenesis that is observed in rats and humans, we trisected the colon tissue samples into cecum, PC and DC. This also increased the sample size to 45 tissue sections for the PhIP-treated male and female rats, thus maximizing the power to determine statistical differences in the MS, should they exist. Colon tissue from control rats (five males and five females) was also trisected, thus providing 15 each of matching male and female control tissue samples.
In this study the rats were treated with 200 p.p.m. PhIP for 61 days. Unexpectedly, the colon lacI MF were ~1.6- to 1.7-fold higher than those observed previously using a dose of 400 p.p.m. PhIP for 60 days (Okonogi et al., 1997). The >3-fold net difference in PhIP mutagenic potency between the two studies is likely attributable to one or more of the following factors: the choice of diet; the antioxidant content of the diets; increased toxicity (with reduced dietary consumption) at the higher PhIP dose; possibly, the chemical form of PhIP used in the two studies. The diets used in the two studies differed only by ~10% in their total energy content by weight, making it unlikely that differences in consumption levels could have accounted for the differences in PhIP mutagenic potency. Perhaps more importantly, the lower antioxidant content in the AIN-93G diet might have reduced scavenging of metabolically activated PhIP, consistent with a pathway in which PhIP is metabolized first in the liver by specific cytochrome P450 isozymes and is then transported through the blood to the target tissues, where it is further activated by N-acetyltransferases (Kaderlik et al., 1994; Snyderwine et al., 1994; Kadlubar et al., 1995; Dingley et al., 1999). Alternatively, antioxidants present at higher levels in the CE-2 diet might have suppressed metabolic activation of PhIP (Hirose et al., 1999).
Since rats fed 200 p.p.m. PhIP have depressed growth rates relative to sex-matched controls, it is also possible that increased toxicity at higher doses (e.g. 400 p.p.m. PhIP) also contribute to depressed MF at higher doses of PhIP, due to reduced food consumption. However, male F344 rats fed 50–400 p.p.m. PhIP demonstrate a dose-dependent increase in PhIP–DNA adduct formation (Hasegawa et al., 1992), suggesting that overall consumption of PhIP is higher at the 400 p.p.m. dose, relative to the 200 p.p.m. dose. Additionally, since lacI MF are reported as a ratio (of the number of lacI mutants per 105 lacI sequences analyzed), it is unlikely that PhIP-induced changes in cellular proliferation would significantly affect MF. Furthermore, since high doses (400 p.p.m. for 8 weeks, no effect observed after 4 weeks) of PhIP increase cellular proliferation ~1.5-fold in male (but not female) colon (Ochiai et al., 1996), it seems unlikely that possible differences in cellular proliferation at 200 or 400 p.p.m. PhIP should dramatically affect MF in both male and female colon. Lastly, the hydrochloride form of PhIP (>99.9% pure; Shirai et al., 1997, cited in Okonogi et al., 1997) was used by Okonogi et al. (1997), whereas the free base form of PhIP (>98% pure, assayed by Toronto Research Chemicals) was used in the present study. Therefore, it is possible the free base form of PhIP might be metabolized more efficiently or excreted less efficiently after ingestion, compared with PhIP hydrochloride.
There were no significant differences (Table I) in MF among the control tissue samples, with the exception that the MF in male PC was slightly, but significantly, lower than the MF in the other control tissues. In contrast, PhIP-induced MF were found to increase along the length of the colon, being lowest in the cecum and highest in the DC (Table I). In both PhIP-treated males and females cecal MF were approximately half of those in the PC and DC. The lower cecal MF are consistent with the observation that PhIP–DNA adduct levels in the cecum are approximately half of those in the colon after administration of a single oral dose of 50 mg/kg PhIP in male F344 rats, with similar half-lives in the two tissues (Cummings and Schut, 1994).
Comparisons of PhIP-induced MS from cecum, PC and DC using a Monte Carlo approximation of Fisher's exact test (Adams and Skopek, 1987) indicated that the PhIP-induced MS were significantly different (P 
0.002) from the spontaneous MS, as expected, with the exception that the spontaneous female and PhIP male cecal MS did not differ (P = 0.1). More importantly, there were no significant differences or interpretable trends (P 0.08) among any of the PhIP-induced MS. To determine if any subtle sex-linked or regional differences among the induced MS might nonetheless be found, we examined each PhIP-induced MS in detail. First, with regard to the –(G:C)90–92 mutation reported by Okonogi et al. (1997), virtually all of the PhIP mutants collected in the present study were examined for occurrence of this –1 frameshift mutation (Table II). Combining the –(G:C)90–92 data from PC and DC, there was no difference (P = 1.00) between male (13/684 total mutants screened, 1.9% of all PhIP mutations, 2.2x10–5 MF) and female (9/503, 1.8%, 2.0x10–5) colon, thus confirming the previous report by Okonogi et al. (1997) (males, 8/206, 3.9%, 2.6x10–5; females, 5/161, 3.1%, 2.2x10–5; P = 0.78). For reference, with the exception of PhIP-induced mutations, the –(G:C)90–92 frameshift mutation is otherwise recovered rarely as a Big Blue lacI transgene mutation: 5/5879 (0.08%) spontaneous mutations and 6/12 510 (0.05%) non-PhIP-induced mutations versus 41/1883 (2.2%) PhIP-induced mutations (lacI database; see de Boer, 1995).
–1 frameshift mutations at lacI nucleotide positions 877–879 (males, 6/127 sequenced cecal, 2/99 PC and 5/137 DC mutants; females, 9/132 sequenced cecal, 2/143 PC and 7/116 DC mutants), although occurring at a sequence identical to the –(G:C)90–92 mutation (TCCCGC/GCGGGA), were the most frequent PhIP-induced mutation in the present study, as well as the only frameshift mutational hot spot. The next most frequent mutation was a GC dinucleotide deletion at lacI nucleotides 790–795 (males, 3/127 sequenced cecal, 3/99 PC and 4/137 DC mutants; females, 5/132 sequenced cecal, 5/143 PC and 2/116 DC mutants). Lastly, –1 frameshift mutations of deoxyguanosine at lacI positions 271–272 were the third most frequent mutation (males, 2/127 sequenced cecal, 0/99 PC and 4/137 DC mutants; females, 1/132 sequenced cecal, 3/143 PC and 2/116 DC mutants). The remaining PhIP mutations were more or less randomly distributed among the remaining available lacI nucleotide positions. None of these various mutations demonstrated any obvious sex bias.
The absence of sex-linked differences in PhIP-induced mutational specificity in male and female rat colon raises interesting questions regarding the contribution of PhIP-induced mutations to the development of cancer in colon, and other, tissues. PhIP is potently mutagenic in tumor target tissues (male colon, prostate gland and mammary gland) and the PhIP signature mutation, a –(G:C) frameshift mutation occurring in the sequence 5'-GGGA-3', has been recovered from the Apc tumor suppressor gene of colon tumors from PhIP-treated male rats (Kakiuchi et al., 1995). Thus, PhIP-induced mutations seem likely to contribute to the initiation of carcinogenesis in tumor target tissues. Nevertheless, it is possible that mutational events that contribute to the development of PhIP-induced colon cancer in male rats cannot be detected using the lacI transgene, due to the absence in lacI of nucleotide sequences critical for initiation of carcinogenicity. For example, with the exception of two pentanucleotide deoxyadenosine sequences, the lacI transgene lacks homonucleotide repeat sequences greater than 4 nt in length. Thus, PhIP-induced mutations that target homonucleotide repeat sequences in cancer-associated endogenous genes will likely be missed using the lacI transgene mutagenicity assay. Interestingly, sporadic and hereditary non-polyposis colon cancers often involve frameshift mutations in homonucleotide repeat sequences in genes that are involved in apoptosis, growth regulation, DNA mismatch repair and tumor suppression (see for example Yamamoto et al., 1997, 1998; Duval et al., 1999; Schwartz et al., 1999; Chadwick et al., 2000). PhIP is also known to promote microsatellite instability and loss of heterozygosity (Canzian et al., 1994; Yu et al., 2000).
The contribution of hormonal influences to the development of colon cancer in PhIP-treated male rats is an intriguing possibility. In addition to the well-defined role of sex hormones in the growth, maintenance and pathogenesis of the mammary and prostate glands, sex hormones are also known to influence the growth characteristics of the colon (see for example Fiorelli et al., 1999; Arai et al., 2000; Catalano et al., 2000). Thus, each of the major target tissues for PhIP-induced carcinogenesis in the rat is regulated by sex hormones. Interestingly, it has recently been demonstrated that estrogens reduce, while the withdrawal of estrogens increases the risk of microsatellite instability-positive human colorectal cancer (Slattery et al., 2001). Thus it is possible that PhIP-treated female rats are similarly protected by estrogen against the PhIP-induced microsatellite instability that putatively contributes to colon cancer in male rats.
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Acknowledgments |
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We wish to acknowledge the technical assistance provided at various times by Shulin Zhang (tissue dissections), Catharina de Monyé, Zuzana Gajdosik and Kully Sraw (Big Blue assay), Dr Moyra Brackley (statistics) and the University of Victoria Animal Care Unit (Ralph Scheurle and staff). We also wish to thank Dr J.D.Potter (Fred Hutchinson Cancer Research Center, Seattle, WA) for generously sharing the Slattery et al. manuscript prior to publication.
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Notes |
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1 To whom correspondence should be addressed. Tel: +1 919 541 5424; Fax: +1 919 541 7613; Email: stuart{at}niehs.nih.gov
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References |
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Received on January 2, 2001; accepted on April 19, 2001.
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