Male lacI transgenic rats were fed a diet containing 200 ppm of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP), a heterocyclic amine present in cooked meats. PhIP was found to be a powerful prostate mutagen. After 61 days of treatment, the lacI mutant frequency was 71 × 10−5, >20-fold higher than the spontaneous mutant frequency of 3.2 × 10−5. The predominant PhIP-induced mutations were G:C→T:A transversions and deletions of G:C bp. The results directly link PhIP-induced mutations with the earlier observation of PhIP-induced prostate cancer in rats and suggest that exposure to dietary PhIP could be a risk factor in the incidence of human prostate cancer.

Prostate cancer is the most prevalent cancer affecting United States males and, after lung cancer, is the most prevalent cause of cancer mortality in males (1). Although the specific factors contributing to the initiation and progression of prostate cancer remain undefined, genetic and environmental factors are likely to be involved. Epidemiological studies indicate that populations that consume more saturated fat and meats have a greater incidence of prostate cancer (2). Of particular interest are the heterocyclic amines, an important class of food mutagens and animal carcinogens (3, 4). One of the more prevalent heterocyclic amines,PhIP2(5), is a carcinogen in rat colon, mammary gland, and prostate (6, 7); interestingly, these three organs are among the sites of the most prevalent human cancers (1). Using the Big Blue lacI mutational assay (8),we show for the first time that PhIP can induce a large increase in MF in the rat prostate. The results suggest that PhIP could be a potential factor in the incidence of human prostate cancer.

Animal Treatments.

Big Blue F344 lacI transgenic rats (Taconic, Germantown, NY)were housed individually and treated following guidelines conforming to the Guide for the Care and Use of Laboratory Animals (Laboratory Animal Resources, National Research Council). Food and water were provided ad libitum. The basal (control) diet consisted of powdered AIN-93G (Dyets Inc., Bethlehem, PA) minus the tert-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 ppm (w/w) of PhIP. Diets were prepared twice weekly and stored at 4°C under argon. The control and PhIP study groups each consisted of five male rats. The control rats were 4–6 weeks old, whereas the rats in the PhIP treatment group were 5 weeks old. After acclimatization to the housing and basal diet for 7 days, rats received either basal diet (control animals) or basal diets supplemented with PhIP for 61 days, after which all of the animals were returned to the basal diet for 7 days. The rats were sacrificed by CO2 asphyxiation, followed by cervical dislocation. Tissues, including the prostate, were immediately dissected, flash-frozen in liquid nitrogen, and then stored at−80°C.

lacI Mutational Assay.

High molecular weight DNA was recovered from prostate tissue by a dialysis purification procedure described previously (9). lacI transgenes were recovered from purified rat chromosomal DNA by an in vitro λ packaging reaction, and packaged phage were plated on the Escherichia coli host strain following recommended methods (10). MFs were calculated by dividing the total number of lacI mutant plaques by the total number of mutant plus wild-type plaques.

DNA Sequencing and Data Analyses.

Mutations in the lacI transgene were determined by the PCR cycle sequencing method described previously (11). All 12 spontaneous prostate mutants were sequenced, as were 178 mutants from PhIP-treated rats. The lacI gene is numbered according to Farabaugh (12).

Previous studies using the Big Blue lacI transgenic rodent assay have shown that PhIP causes mutations in rat colon (13) and mammary gland (14), which are also sites for PhIP-induced carcinogenesis (6). In the present study, we examined the frequency and specificity of PhIP-induced mutation in the prostate from Big Blue rats. We found PhIP to be highly mutagenic in rat prostatic tissue, inducing a MF of 71 ± 4 × 10−5, >20-fold higher than the spontaneous MF of 3.2 ± 1.5 × 10−5 in control animals (Table 1).

PhIP is unique among heterocyclic amines in that rat liver is a target for neither PhIP-induced DNA adduct formation (15) nor carcinogenesis (3). We therefore assessed the MF from the liver of PhIP-treated male and female Big Blue rats (data not shown). The MF of 4 × 10−5 in either the PhIP-treated male or female liver was not significantly different from the spontaneous MF of 3 × 10−5observed previously in male rat liver (16). These data indicate that PhIP-induced MFs are elevated in tissues that are actual targets for PhIP-induced carcinogenesis.

We then determined the mutational specificity of spontaneous and PhIP-induced mutations in rat prostate tissue. Twelve mutants were recovered and sequenced from the prostates of the untreated animals,and 11 independent mutations were identified (Tables 1,2). Despite the small sample size, the prostate spontaneous MS was consistent with those previously determined in lacItransgenes recovered from a variety of tissues (16, 17). Specifically, the spontaneous prostate MS was characterized by 55%G:C→A:T transition mutations (all of which occurred at 5′-CpG-3′dinucleotide sites), 18% transversions, and 27% non-base substitution mutations (Table 2).

One hundred and seventy-eight PhIP prostate mutants were sequenced,yielding 155 independent mutations. The PhIP MS consisted of 12%transitions, 42% transversions, and 46% other mutations (mostly deletions of G:C bp). The predominant PhIP-induced mutations included 39% minus one (−1) frameshifts [of which 59 of 60 (98%) involved deletions of G:C bp], 32% G:C→T:A transversions, and 10% each of G:C→A:T transitions and G:C→T:A transversions. (In the following sentence, the numbers in parentheses indicate the numbers of independent mutations.) With regard to sequence specificity, the PhIP mutations included five (four) −1 frameshifts of G:C at lacI nucleotide positions 90–92, five (four) base substitution mutations at position 92, and 11 (four) −1 frameshifts of G:C at position 877. Of the six independent deletion mutations, five were dinucleotide deletions involving cytosine and guanine:(a) CC; (b) CG; (c) CG; (d)GC; and (e) GC.

The PhIP-induced MS from prostate was consistent with those determined previously in the rat colon (13) and mammary gland (14). The large increase in the proportion of −1 frameshifts involving G:C bp and the increase in the proportion of G:C→T:A transversions in PhIP-treated prostate (Table 2) are particularly characteristic of PhIP mutational spectra recovered from colon and mammary gland.

To our knowledge, this is the first report of spontaneous and induced mutation frequencies and spectra from prostate. The PhIP mutational data from prostate, combined with the previous observation that PhIP(mixed into the diet at a dose of 400 ppm for 52 weeks) causes prostate tumors in the rat (7), provide convincing evidence that PhIP is a genotoxic carcinogen in the case of rat prostate cancer.

The rats in our mutagenicity study received 200 ppm of PhIP mixed into the diet for 61 days, an exposure sufficient to dramatically elevate the mutation frequency in this organ. During this period, the average daily food consumption and body weights were approximately 13 and 180 g, respectively, giving an estimated daily consumption of PhIP of 14 μg of PhIP per gram of body weight (i.e., 14 ppm PhIP, adjusted for body weight). It has been estimated that daily human dietary intake of heterocyclic amines approaches microgram quantities (3, 18). Therefore, assuming that the average human consumes 1 μg of PhIP daily at a body weight of 70 kg [calculated using the data provided in Table 3 in the article by Layton et al.(18)], the estimated daily human consumption is approximately 1.4 × 10−5 μg of PhIP per gram of body weight(i.e., 1.4 × 10−5ppm, adjusted for body weight), a difference of approximately 106-fold compared to the rats. However, this fold difference in exposures is approximately 103-fold once the durations of exposures are considered (61 days for the rat mutagenicity study; assumed exposure of 60 years for humans).

Wakabayashi et al.(3) have noted that the carcinogenic effects of various heterocyclic amines appear to be additive or synergistic, whereas Felton et al.(5) have found that the binding of heterocyclic amines to DNA is linear at doses well in excess of the average daily intakes of heterocyclic amines (18). Felton and colleagues have estimated that the overall risk of cancer due to dietary exposure to heterocyclic amines is approximately 1 × 10−4, with the incremental risk of cancer due to exposure to PhIP being approximately 5 × 10−5(18).

Thus the mutagenicity data presented here, combined with the previous demonstration that PhIP is a rat prostate carcinogen, provide additional evidence that humans who consume excessive amounts of PhIP may risk developing prostate cancer. This conclusion is also consistent with the recent demonstration that PhIP appears to be a substrate for N-acetyltransferase activity present in human prostate epithelial cells in vitro(19) and in human prostate tissue implanted into nude mice (20).

The transgenic rodent model provides a practical opportunity to investigate the mechanisms contributing to cancer in the prostate. Studies including the role of diet, as well as the efficacy of potential chemopreventive therapies, can be undertaken using the approach described.

We acknowledge the excellent technical assistance provided at various times by Ralph Scheurle (University of Victoria Animal Care Unit) and Glickman laboratory members Amanda Thornton-Glickman,Jana Kangas, Paul Kotturi, Ken Sojonky, Erika Thorleifson, David Walsh,Haiyan Yang, and Shulin Zhang. We also thank Drs. T. Ushijima, E. Okochi, and colleagues (National Cancer Center Research Institute,Tokyo, Japan) for generously sharing PhIP-treated mammary gland data prior to publication.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: PhIP,2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; MF,mutant frequency; MS, mutational spectrum.

Table 1

Mutant frequencies in the lacI transgene from prostate of Big Blue rats

Male PhIP-treated Big Blue lacI transgenic rats received 200 ppm of PhIP mixed into the diet for 61 days. After isolation of chromosomal DNA from prostate glands, lacI transgenes were recovered by an in vitro λ packaging reaction and plating on the E. coli host strain on media containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. Mutant frequencies were calculated by dividing the number of blue(lacI mutant) plaques by the total number of plaques. Data for each animal are shown, as well as the average mutant frequency for each group.

TreatmentAnimalNo. of plaquesMF
TotalMutant
Control MC1 14,000 <7 × 10 
 MC2 136,000 7 × 10 
 MC3 164,000 1 × 10 
 MC4 30,000 <3 × 10 
 MC5 31,000 3 × 10 
 Total 375,000 12 (3.2± 1.5) × 10a 
PhIP MP1 14,000 11 79 × 10 
 MP2 47,000 26 55 × 10 
 MP3 134,000 117 87 × 10 
 MP4 222,000 140 63 × 10 
 MP5 283,000 206 73 × 10 
 Total 700,000 500 (71± 4) × 10a 
TreatmentAnimalNo. of plaquesMF
TotalMutant
Control MC1 14,000 <7 × 10 
 MC2 136,000 7 × 10 
 MC3 164,000 1 × 10 
 MC4 30,000 <3 × 10 
 MC5 31,000 3 × 10 
 Total 375,000 12 (3.2± 1.5) × 10a 
PhIP MP1 14,000 11 79 × 10 
 MP2 47,000 26 55 × 10 
 MP3 134,000 117 87 × 10 
 MP4 222,000 140 63 × 10 
 MP5 283,000 206 73 × 10 
 Total 700,000 500 (71± 4) × 10a 
a

Average MF ± SE of the mean.

Table 2

PhIP-induced lacI mutations from rat prostate

Blue (lacI mutant) plaques from the Big Blue assay were purified, and the mutations in the lacI transgene were identified by PCR cycle DNA sequencing. To ensure that only independent(nonclonal) mutations were analyzed, identical mutations recovered more than once from the same animal were counted as one mutation.

UntreatedPhIP-treated
No.%No.%
Transitions     
G:C → A:T 55 16 10 
At CpG sequences 55 
A:T → G:C 
Transversions     
G:C → T:A 49 32 
At CpG sequences 12 
G:C → C:G 16 10 
A:T → T:A 
A:T → C:G <1 
Other mutations     
+1 frameshift <1 
−1 frameshift 60 39 
Deletions 
Insertions 
Complex changes <1 
Tandem mutants 
Total independenta 11 100 155 100 
Clonal mutations  19  
lacI nonmutantb   
UntreatedPhIP-treated
No.%No.%
Transitions     
G:C → A:T 55 16 10 
At CpG sequences 55 
A:T → G:C 
Transversions     
G:C → T:A 49 32 
At CpG sequences 12 
G:C → C:G 16 10 
A:T → T:A 
A:T → C:G <1 
Other mutations     
+1 frameshift <1 
−1 frameshift 60 39 
Deletions 
Insertions 
Complex changes <1 
Tandem mutants 
Total independenta 11 100 155 100 
Clonal mutations  19  
lacI nonmutantb   
a

The total numbers of mutants after correction for clonal expansions.

b

lacI nonmutants were blue mutant plaques that did not contain mutations in the lacI gene.

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