Genetic analysis of the susceptibility in rats to aberrant crypt foci formation by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced while cooking fish and meat, induces aberrant crypt foci (ACF) and colon cancers in rats. We previously reported that F344 rats were sensitive and ACI rats resistant to ACF formation by PhIP, and that the genetic susceptibility in F344 rats to ACF formation by PhIP was autosomally dominant over ACI rats. To identify candidate susceptibility genes in F344 rats, a preliminary genome-wide linkage analysis was employed using a subset of 170 progeny of (F344 x ACI)F1 x ACI backcross rats with either high or low sensitivity to ACF formation by PhIP. Three chromosomes, 1, 6 and 16, demonstrated the presence of loci with a logarithm of the odds (lod) scores of more than 1.0, and a susceptible gene for ACF formation by PhIP was suggested to reside on chromosomes 16.     

Differential gene expression profiles in colon epithelium of two rat strains with distinct susceptibility to colon carcinogenesis after exposure to PhIP in combination with dietary high fat

Colon cancers develop through accumulation of multiple genetic and epigenetic alterations in colon epithelial cells, and the environment of the genetically altered epithelial cells may also have a substantial impact on their further development to cancer. In the present study, groups of 6-week-old F344 and ACI male rats, the former strain being susceptible to colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the latter being relatively resistant, were subjected to a long-term carcinogenesis experiment using our intermittent feeding protocol of PhIP in combination with a high-fat diet, which serves as a relevant risk factor that promotes the development of colon cancers. Animals were sacrificed at 60 weeks, and global gene expression analyses of normal parts of colon epithelial tissues were conducted using a high-density oligonucleotide microarray to elucidate the differential gene expression profile (environment) in normal colonic regions between F344 and ACI strains. Of 8799 entries on the RatU34A array, 74 genes exhibited 3-fold or greater variation. A subset of genes encoding ribosomal RNAs and proteins were highly preferentially expressed in the F344 strain. In addition, genes encoding fatty acid binding proteins and the peroxisome membrane protein 70 appeared up-regulated in the susceptible F344 strain. In the ACI strain, a mismatch repair gene, Msh2 , was preferentially expressed, at approximately 20-fold the F344 level, along with a gene encoding a detoxification enzyme, catechol-O-methyltransferase. The combined effects of the repertoire of these differentially expressed genes in normal colon epithelial tissues may account for the distinct susceptibilities of F344 and ACI strains to colon carcinogenesis.     

Efficient Method for Rapid Induction of Aberrant Crypt Foci in Rats with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) induces aberrant crypt foci (ACFs) as well as colon cancer in F344 male rats. Conditions allowing rapid development of ACFs over a short period were investigated. F344 male rats were fed 400 ppm of PhIP-HCl in a low-fat diet (LF) for 2 weeks and then given a PhIP-free, high-fat diet containing PRIMEX (HF-PR) or safflower oil, or PhIP-free LF for 4 or 12 weeks. Rats fed HF-PR for 4 weeks gave the highest number of ACFs/rat (3.3) and their size in terms of aberrant crypts/ACF(2.7) was much larger than that obtained with conventional continuous feeding of PhIP for 25 weeks in the CE-2 diet. Therefore, 2 weeks of dietary exposure to 400 ppm of PhIP-HCl, followed by HF-PR for 4 weeks, is a practical and convenient method for obtaining ACFs. This protocol should be useful for studies of the early phase of colon carcinogenesis.     

High susceptibility of the ACI and spontaneously hypertensive rat (SHR) strains to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) prostate carcinogenesis

Carcinogenic responses in the prostate to 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) were compared among seven rat strains (F344, ACI, Spontaneously Hypertensive Rat (SHR), Sprague-Dawley (SD), Wistar, Lewis and Brown Norway (BN)). Ten-week-old animals of each strain were given PhIP at 400 ppm in the diet for 20 weeks then maintained until week 54. The final survival rates were 92, 92, 83, 75, 67, 42 and 42%, respectively, and the SHR strain showed the highest sensitivity with regard to development of prostatic intraepithelial neoplasias (PINs) in the ventral prostate. With regard to the induction of adenocarcinomas of the ventral prostate, the ACI strain was most sensitive, whereas Lewis and F344 rats were relatively resistant. No adenocarcinomas were found in the dorsolateral or anterior prostate or seminal vesicles in any of the strains. The levels of serum testosterone and estrogen, PhIP-DNA adducts and cell kinetics did not correlate with the development of ventral prostatic lesions and thus other factors are presumably responsible for the variations in susceptibility. The present data indicate that ACI and SHR rats are appropriate strains for experimental investigation of PhIP-induced prostate carcinogenesis.     

Experimental chemical carcinogenesis in the stomach and colon

Experimental chemical carcinogenesis in the digestive tract is reviewed, mainly on the basis of information obtained in the laboratories of the National Cancer Center Research Institute. It is generally accepted that cancer is the outcome of DNA damage, resulting in mutation, loss, amplification and recombination of genes. Gastric cancer is no exception. It was shown very early that cancer of the glandular stomach can be produced in rats by administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a widely used mutagen. However, this depends on the genotype. Whereas the ACI rat is susceptible to MNNG, the Buffalo rat is resistant and this is a dominantly inherited trait. Genes responsible for the sensitivity to gastric cancer induction are at present under investigation by linkage analysis of rat genome markers. With regard to cancer in humans, our finding that cooked proteinaceous foods can give rise to a series of heterocyclic amines (HCAs) is of major significance. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most abundant, causes colon cancers in male rats, whereas in females it induces breast cancers. The colon cancers induced by PhIP feature a deletion of G as represented by 5-GGGA-3-->5-GGA-3 in the Apc gene, resulting in a truncated Apc molecule. Microsatellite mutations have also been found in PhIP-induced colon tumors, as in human hereditary non-polyposis colorectal cancer cases. Similarly to the case of gastric cancer production by MNNG, there is a genetic component and F344 rats are more susceptible to PhIP colon carcinogenesis than the ACI/N strain and the gene responsible is being sought. Since carcinogenesis proceeds with accumulation of genetic alteration, often involving genomic instability, exposure to any kind of carcinogenic substances, either xeno- or autobiotics, needs to be reduced as far as possible, taking account of inconvenience at the individual and socio-economical levels.      

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine-induced colon carcinogenesis by 1-O-hexyl-2,3,5-trimethylhydroquinone after initiation with 1,2-dimethylhydrazine in F344 rats

The aim of this study is to investigate the chemopreventive effects of the synthetic phenolic antioxidant 1-O-hexyl-2,3, 5-trimethylhydroquinone (HTHQ) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colon carcinogenesis in rats after initiation with 1,2-dimethylhydrazine (DMH) in male F344 rats. Groups of 20-22, 6-week-old male F344 rats were given four subcutaneous injections of 40 mg/kg body wt of DMH during the initial 4 weeks. They were then maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, 0.5 or 0.125% HTHQ alone or basal diet for 32 weeks. A small number (1.1 +/- 1.1/rat) of colon tumors were induced by DMH treatment alone. After initiation with DMH, the number of colon tumors was greatly increased to 8.3 +/- 5.6 by the administration of PhIP. Additional treatment with HTHQ dose-dependently decreased the multiplicity of colon adenocarcinomas to 4.9 +/- 2.8 and 2.6 +/- 1.4 with 0.125 and 0.5%, respectively. This treatment similarly reduced atypical hyperplasias of the ventral prostate. Furthermore, HTHQ significantly reduced the multiplicity of duodenal adenocarcinomas induced by DMH + PhIP or DMH alone. Immunohistochemically, HTHQ was revealed to suppress PhIP-DNA adduct formation in the epithelial cells of the colon and prostate in a separate 2 weeks experiment. The present results clearly showed that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis. The observed inhibition may largely be due to interference with PhIP-DNA adduct formation. In addition, HTHQ has been demonstrated to inhibit duodenal carcinogenesis in the post-initiation stage.     

DNA adduct formation, cell proliferation and aberrant crypt focus formation induced by PhIP in male and female rat colon with relevance to carcinogenesis

2-Amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) inducescolon tumors in male, but not female, F344 rats, We investigatedthe mechanisms leading to this difference by measuring the levelof PhlP-DNA adducts, the enhancement of cell proliferation andaberrant crypt focus (ACF) formation in colon mucosa. PhIP wasadministered in the diet at a level of 0.04% to both male andfemale F344 rats for 1–8 weeks. The level of DNA adductsin the colon mucosa was measured using the ³²P-postlabelingmethod. Four major PhlP-DNA adducts were detected in fairlyconstant proportions in all the animals examined. The levelof PhlP-DNA adducts in male and female rats was the same, indicatingno direct correlation between adduct levels and carcinogenesis.Labeling indices (LIs) were determined by measuring BrdU incorporationin rats after feeding with a PhIP diet for 4, 8 and 12 weeks.After 8 weeks administration the LI had increased 1.5-fold inthe colon of the male rats, but no increase was observed inthe female rats. ACF formation was examined after feeding witha PhIP diet for 14 weeks. The number of aberrant crypt fociwas 6.6 ± 1.5 per rat in males and 1.9 ± 0.5 perrat in females. Thus differences in colon tumor developmentin male and female rats takes place at an early stage(s). Ourresults suggest that, in addition to DNA adduct formation, enhancedproliferation contribites to the formation of ACFs, which arepremalignant lesions of the colon.     

Mechanistic approach of contrasting modifying effects of caffeine on carcinogenesis in the rat colon and mammary gland induced with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Caffeine exerts potent chemopreventive action against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland carcinogenesis, but acts as a co-carcinogen in the colon. The present work was performed to clarify mechanisms underling these organ dependent actions. Female F344 rats were given PhIP and caffeine, PhIP alone, caffeine alone or no treatment for 4 weeks. PhIP-DNA adduct formation in the colon was significantly higher in the PhIP+caffeine than in the PhIP group, but levels in the mammary glands showed no inter-group differences. CYP1A2 mRNA expression in the livers of the PhIP+caffeine group tended to be higher than in either the PhIP or the caffeine alone groups. High mRNA expression for both N-acetyltransferase (NAT) 1 and NAT2 was observed in the colon, with less expression in the mammary gland. The levels of four DNA-repair enzymes were not influenced by the caffeine treatment. In conclusion, only increased level of DNA adducts in the colon partially related to the modifying effects of caffeine on PhIP-induced rat carcinogenesis. Thus, other unknown factors must be contributory.     

Protection by chlorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat

The most abundant heterocyclic amine in fried ground beef, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP), induces colon carcinomas in the male F344 rat. The potentialchemopreventive effects of two compounds, namely, the ‘interceptormolecule’ chlorophyllin (CHL) and a modulator of carcinogenactivation, indole-3-carbinol (I3C), were examined in a PhIPcolon carcinogenesis model. During weeks 3 and 4 of a 16-weekstudy, F344 rats were given PhIP by oral gavage (50 mg/kg bodyweight, alternating days). Inhibitors were given either beforeand during PhIP exposure, after PhIP treatment, or continuouslyfor 16 weeks. Treatment of rats with 0.1% CHL in the drinkingwater inhibited the formation of aberrant crypt foci (ACF) with     

Reduction in formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP)-induced aberrant crypt foci in the rat colon by docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2- dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.      

Association between acetylator genotype and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) DNA adduct formation in colon and prostate of inbred Fischer 344 and Wistar Kyoto rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine (HCA) found in cooked meats, causes colon and prostate tumors in male rats. Polymorphic N-acetyltransferase metabolizes N-hydroxy-PhIP to a DNA-reactive form. Liver, colon, and prostate PhIP-DNA adduct levels were compared in male rapid-acetylator Fischer 344 (F344) and slow-acetylator Wistar-Kyoto (WKY) rats fed 0.01 or 0.04% PhIP. Liver PhIP-DNA adduct levels at both PhIP doses, and colon PhIP-DNA adduct levels at the 0.01% PhIP dose were unaffected by acetylator genotype. However, in rats fed 0.04% PhIP, colon PhIP-DNA adduct levels were higher in rapid acetylator F344 rats (P < 0.05). Similarly, prostate PhIP-DNA adduct levels were higher in rapid acetylator F344 rats at both PhIP doses (P < 0.05). The combination of the high-PhIP dose and rapid-acetylator genotype resulted in the highest level of PhIP-DNA adducts in rat colon and prostate.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine induces a higher number of aberrant crypt foci in Fischer 344 (rapid) than in Wistar Kyoto (slow) acetylator inbred rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine carcinogen in the human diet and is a colon carcinogen in the rat. N-Acetyltransferase-2 (NAT2) catalyzes the conversion of PhIP and other heterocyclic amines to a DNA-reactive form. NAT2 has a polymorphic distribution in humans and other mammals, including rats. The rapid NAT2 genotype has been shown to be associated with increased colorectal cancer risk in some, but not all, human epidemiological studies. This investigation was designed to study the role of acetylator genotype in PhIP-induced colon carcinogenesis using aberrant crypt foci (ACF) as an intermediate biomarker. Five-week-old male, rapid-acetylator Fischer 344 (F344) rats and slow-acetylator Wistar-Kyoto (WKY) rats were fed the semipurified AIN76A diet with 0.01% PhIP, 0.04% PhIP, or no PhIP (control) for 8 weeks. PhIP induced ACF in both rapid- and slow-acetylator rats; 0.04% PhIP induced more ACF than 0.01% PhIP. There was no difference in the number of ACF between rapid- and slow-acetylator rats that were fed 0.01% PhIP. However, 0.04% PhIP induced 2-fold higher ACF and a greater dose-dependent increase in PhIP-induced ACF in the rapid-acetylator F344 rats compared with the slow-acetylator WKY rats. The results support human epidemiological studies showing higher risk for colorectal cancer in rapid acetylators who frequently consume meat that is very well done.     

Organ dependent enhancement of rat 3,2'-dimethyl-4-aminobiphenyl (DMAB) carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): positive effects on the intestine but not the prostate

In order to evaluate tumor enhancing effects of the heterocyclic carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), doses of 100 and 300 p.p.m. PhIP were given for 40 weeks to male F344 rats, which initially received 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB shows a similar carcinogenic organ spectrum to that of PhIP, including the prostate and colon. PhIP alone at a dose of 300 p.p.m. resulted in the development of prostate and intestine cancers. Furthermore, among the DMAB-treated group, enhancement of intestinal carcinogenesis by 300 p.p.m. PhIP was observed. However, no prostate enhancement was demonstrated in the DMAB + PhIP group. Since PhIP-DNA adduct formation in the prostate epithelial cells in a satellite experiment was not affected by pre-treatment with DMAB, it is speculated that the contradictory findings between the intestine and prostate may be due to the specific biological effects of PhIP. Taking into account previous data, that PhIP clearly enhanced rat 1,2-dimethylhydrazine-initiated colon tumorigenesis, the potential of PhIP to enhance colon carcinogenesis may be initiator dependent.     

Heterocyclic amine mixture carcinogenesis and its enhancement by caffeine in F344 rats.

In order to elucidate whether mixed exposure to environmental carcinogens and caffeine increases the risk of cancer induction, we investigated the relationship between preneoplastic lesion development in the liver and colon and drug metabolizing enzyme induction and DNA adduct formation, in rats treated with a mixture of heterocyclic amines (HCAs) and caffeine. In Experiment 1, male F344 rats were administered 3 different HCAs, the food carcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in combinations of 2 or 3 at 50 ppm in the diet for 16 weeks. The numbers of hepatic glutathione-S-transferase P form positive (GST-P+) foci and colonic aberrant crypt foci (ACF) were greater in the IQ + MeIQx group than expected from simple summation and increased levels of HCA-DNA adducts were noted. However, no summation was obtained when combined with PhIP, which rather caused inhibition. In Experiment 2, the effects of concurrent caffeine administration on the PhIP carcinogenicity were assessed. Caffeine at 1000 and 500 ppm in the drinking water for 2 weeks significantly increased levels of CYP1A2. Ten weeks concurrent administration of caffeine (1000 ppm) and PhIP (400 ppm) resulted in significant increase of colon ACFs and CYP1A2 expression. Thus, concurrent administration of IQ and MeIQx caused elevation of their carcinogenicity but other mixtures with PhIP did not enhance carcinogenicity. However, a non-carcinogen, caffeine, enhanced PhIP colon carcinogenesis, possibly due to induction of CYP1A2.     

Enhanced colitis-associated colon carcinogenesis in a novel Apc mutant rat

To establish an efficient rat model for colitis-associated colorectal cancer, azoxymethane and dextran sodium sulfate (AOM/DSS)-induced colon carcinogenesis was applied to a novel adenomatous polyposis coli ( Apc ) mutant, the Kyoto Apc Delta (KAD) rat. The KAD rat was derived from ethylnitrosourea mutagenesis and harbors a nonsense mutation in the Apc gene (S2523X). The truncated APC of the KAD rat was deduced to lack part of the basic domain, an EB1-binding domain, and a PDZ domain, but retained an intact β-catenin binding region. KAD rats, homozygous for the Apc mutation on a genetic background of the F344 rat, showed no spontaneous tumors in the gastrointestinal tract. At 5 weeks of age, male KAD rats were given a single subcutaneous administration of AOM (20 mg/kg, bodyweight). One week later, they were given DSS (2% in drinking water) for 1 week. At week 15, the incidence and multiplicity of colon tumors developed in the KAD rat were remarkably severe compared with those in the F344 rat: 100 versus 50% in incidence and 10.7 ± 3.5 versus 0.8 ± 1.0 in multiplicity. KAD tumors were dominantly distributed in the rectum and distal colon, resembling human colorectal cancer. Accumulation of β-catenin protein and frequent β-catenin mutations were prominent features of KAD colon tumors. To our knowledge, AOM/DSS-induced colon carcinogenesis using the KAD rat is the most efficient to induce colon tumors in the rat, and therefore would be available as an excellent model for human colitis-associated CRC.     

Inhibitory effects of mofezolac,a cyclooxygenase-1 selective inhibitor,on intestinal carcinogenesis

Cyclooxygenase (COX)-2, one enzyme isoform responsible for producing prostanoids from arachidonic acid, contributes to colon carcinogenesis. Recently, genetic disruption of COX-1, the other isoform, was shown to decrease the number of intestinal polyps and prostaglandin E(2) levels in intestinal mucosa, like the case with COX-2 gene disruption, in Min mice. We therefore investigated whether a COX-1 selective inhibitor, mofezolac, suppresses intestinal carcinogenesis in rodents. F344 male rats, receiving azoxymethane (AOM, 15 mg/kg body wt) s.c. injections at 5 and 6 weeks of age, were fed a diet containing 600 or 1200 p.p.m. mofezolac for 4 weeks. The number of aberrant crypt foci (ACFs) per rat and the bromodeoxyuridine labeling index of the crypt epithelium were dose-dependently decreased by administration of mofezolac, the value for the former at 1200 p.p.m. being 60% of control value. When Apc gene knockout mice (APC1309 mice) were given 600 or 1200 p.p.m. mofezolac in their diet for 8 weeks, the numbers of intestinal polyps were also dose-dependently decreased, with reduction to 59% of that in the control diet group at the higher dose. Nimesulide, a COX-2 selective inhibitor used as positive control, showed similar suppressive effects on the development of ACFs in AOM-treated rats and polyps in Apc gene knockout mice. The data indicate that both COX-1 and COX-2 can contribute to intestinal tumorigenesis.     

Suppression of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced DNA damage in rat colon after grapefruit juice intake

The influence of grapefruit juice intake on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colon DNA damage was examined using comet assay in F344 rats given 60 mg/kg of PhIP by gavage. F344 rats allowed free access to grapefruit juice for 5 days experienced clearly reduced DNA damage in the colon to a 40% level of control rats. The suppression of PhIP-induced colon DNA damage depended on the grapefruit juice concentrations. The serum concentration of PhIP was compared between grapefruit juice-pretreated and non-pretreated rats, but showed no significant difference in the areas under their concentration-time curves, peak values and half lives of PhIP. Furthermore, no obvious difference was found in the liver capacity for mutagenic activation of PhIP in Ames assay between grapefruit juice-pretreated and non-pretreated rats. These results suggest that grapefruit juice suppresses PhIP-induced colon DNA damage by a mechanism independent of PhIP absorption in the intestine.     

Formation of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in male Fischer-344 rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) is known to induce colon tumors in male Fischer-344 rats. Using 32P-postlabeling assays, we have examined PhIP-DNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer-344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhIP/kg. Three PhIP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed as relative adduct labeling values (RAL x 10(7), or adducts per 10(7) nucleotides assuming complete labeling) were highest in the large intestine (5.66), followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhIP forms high levels of specific DNA adducts in the large intestine, the target organ in PhIP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form of PhIP are present in the circulation.