Carcinogenicity in rats of a mutagenic compound, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, from tryptophan pyrolysate

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a mutagenic principle isolated from a tryptophan pyrolysate. When Trp-P-1 was given to male and female F344 rats at concentrations of 0.015% and 0.02%, respectively, in the diet, it induced hepatocellular carcinomas in high incidence within a year. No tumor was found in control rats.     

Generation of intracellular active oxygens in mouse FM3A cells by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, the activated Trp-P-2

Mouse FM3A cells in culture were treated with a reactive metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2-(NHOH]. When the treated cells, which were judged as viable on the basis of trypan-blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp-P-2. Single-strand breaks in the cellular DNA took place during this treatment with Trp-P-2(NHOH). Since Trp-P-2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp-P-2(NHOH) treatment produces intracellular active oxygens that can damage DNA.     

Inhibition of DNA Adduct Formation and Mutagenic Action of 3-Amino-l-methyl-5H-pyrido[4,3-b]indole by Chlorophyllin-chitosan in rpsL Transgenic Mice

We have studied the inhibitory effect of chlorophyllin-chitosan (Chl-Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen-carcinogen, 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp-P-2, DNA adducts were formed in various tissues in a dose-dependent manner, with the maximum level observed in the liver. Addition of 3% Chl-Chi to the diet reduced the Trp-P-2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp-P-2 diet. Addition of Chl-Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl-Chi was detected during these experiments. The results show that Chl-Chi may be a candidate chemopreventive agent against the genotoxic action of Trp-P-2, and possibly also other aromatic carcinogens in the diet.     

Metabolic activation of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole(TRP-P-2) by isolated rat liver nuclei

The metabolic activation of a tryptophan pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2), by rat liver nuclei was studied. Nuclei from the livers of rats treated with polychlorinated biphenyl (PCB) or 3-methylcholanthrene (MC) showed high mutagenic activity with Trp-P-2 in the Ames test, but activities with nuclei of untreated or phenobarbital (PB)-treated rat livers were quite low. The formation of N-hydroxy-Trp-P-2 by nuclei of PCB- or MC-treated rat livers was greater than that by nuclei of untreated or PB-treated rat livers. Similar results were observed with microsomes, which suggests that Trp-P-2 is metabolized by the same type of monoxygenase system in nuclei as in microsomes.     

Inhibition of DNA adduct formation and mutagenic action of 3-amino-1-methyl-5h-pyrido[4,3-b]indole by chlorophyllin-chitosan in rpsL transgenic mice.

We have studied the inhibitory effect of chlorophyllin-chitosan (Chl-Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen-carcinogen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp-P-2, DNA adducts were formed in various tissues in a dose-dependent manner, with the maximum level observed in the liver. Addition of 3% Chl-Chi to the diet reduced the Trp-P-2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp-P-2 diet. Addition of Chl-Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl-Chi was detected during these experiments. The results show that Chl-Chi may be a candidate chemopreventive agent against the genotoxic action of Trp-P-2, and possibly also other aromatic carcinogens in the diet.     

Absorption of 3-amino-1-methyl-5H-pyrido[4,3-b]indole, a mutagen-carcinogen present in tryptophan pyrolysate, from the gastro-intestinal tract in the rat

The absorption characteristics of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a mutagen-carcinogen present in tryptophan pyrolysate, in the gastrointestinal tract was investigated in Wistar male rats by an in situ loop technique. Trp-P-2, a weak base with a pKa of 8.2, was poorly lipophilic below pH 5 and therefore the gastric absorption was negligible at pH 1.1 and 3.0. On the other hand, this compound was rapidly absorbed from the small intestine even at pH 4.0 and the absorption was even faster at higher pH where the unionized fraction increases. The absorption from the large intestine was also rapid. Although the intestine is one of the major metabolic organs for ingested materials, metabolism of Trp-P-2 by the scraped mucosa of the small intestine was slow in comparison with that in the liver. Formation of direct-acting mutagens (towards S. typhimurium TA98) associated with the metabolism was also much slower in the small-intestinal mucosa than in the liver. These results suggest that this mutagen is activated mainly after being incorporated into the liver, and are consistent with the liver-specific carcinogenicity of Trp-P-2 in rats.     

3-Amino-1-methyl-5H-pyrido[4,3-b]-indole initiates two-stage carcinogenesis in mouse skin but is not a complete carcinogen

In two separate experiments, 2.0 mg of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was applied to the back of female CD-1 mice twice weekly for 5 weeks followed by application of 2.5 micrograms of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) twice weekly for 47 weeks. Skin squamous cell papillomas and carcinomas developed in 6 of 20 mice (30%) in Experiment 1 and in 3 of 19 mice (16%) in Experiment 2. In contrast, application of 2.0 mg of Trp-P-2 twice weekly for 52 weeks did not produce any skin tumors. These data suggest that Trp-P-2 acts primarily as an initiator, not as a complete carcinogen, for mouse skin.     

Enzyme immunoassay for (3-amino-1-methyl-5H-pyrido[4,3-b]indole), a tryptophan pyrolysate

For evaluation of the cancer risk by the tryptophan pyrolysates a sensitive enzyme immunoassay for 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), a mutagenic and carcinogenic substance, found in foods and biological fluids has been developed. The dose-response curve obtained was suitable for the determination of Trp-P-2 in the range of 20-800 pg. The immunoassay system was characterized to be satisfactorily specific as judged by cross-reactivity to gamma-carboline derivatives and their precursors, and comparison of immunoreactive Trp-P-2 levels in the basic and neutral fraction derived from pyrolyzed amino acids and related compounds.     

The Rat Urinary Bladder as a New Target of Heterocyclic Amine Carcinogenicity: Tumor Induction by 3-Amino-l-methyl-5H-pyrido[4,3-β]indole Acetate

In order to examine the carcinogenicity of 3-amino-l-methyl-5 H -pyrido[4,3-β]indole acetate (Trp-P-2), 30 male and 30 female F344 rats were maintained on diet containing 0, 30, or 100 ppm Trp-P-2 for 112 weeks. The overall mean chemical intakes in the 100 ppm and 30 ppm groups were 3.84 and 1.14 mg/kg/day in males, and 4.57 and 1.34 ing/kg/day in females, respectively. Females of the 100 ppm group showed increased mortality in the late period of the study. In the 100 ppm group, significant increases in the incidences of neoplastic lesions were found in the liver, urinary bladder and mammary gland in males, and in the mammary gland, hematopoietic system and clitoral gland in females. Histologically, tumors induced by Trp-P-2 were hepatocellular adenomas, transitional cell tumors (papillomas and carcinomas) of the urinary bladder, fibroadenomas/fibromas of the mammary gland, malignant lymphomas and clitoral gland tumors (adenomas and adenocarcinomas). These results indicate multi-target carcinogenicity of Trp-P-2 in F344 rats and provide evidence that the urinary bladder is also a target for heterocyclic amine action.     

Determination of human exposure to the dietary carcinogen 3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) from hemoglobin adduct: the relationship to DNA adducts.

A quantitative method for estimation of the exposure of the food-borne carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), was developed by the analysis of its hemoglobin binding in rats. This method was then applied to show the presence of Trp-P-1-hemoglobin adducts in human blood. In rat experiments, 0.2 and 0.07% of the administered [14C]Trp-P-1 formed stable covalent adducts with blood hemoglobin and plasma proteins respectively. Subsequent strong acidic treatment (6 N HCl, 110 degrees C, 24 h) of the Trp-P-1-hemoglobin adducts cleaved peptide bonds of globin, and yielded mainly three derivatives of Trp-P-1. One of them (TPHB) represented approximately 50% of the total Trp-P-1-hemoglobin adducts and was suitable for detection through its strong fluorescence. TPHB was used as a surrogate marker of the Trp-P-1-hemoglobin adducts. Linear dose dependency of Trp-P-1 binding to liver DNA and hemoglobin in rats was confirmed by 32P-postlabeling analysis and TPHB assay. The absence of Trp-P-1-DNA adducts and TPHB in nontreated rats was also confirmed. Using TPHB as a tool for human dosimetry of Trp-P-1, human blood samples from four healthy individuals were examined. TPHB was detected in all samples ranging from 0.23 to 4.33 pmol/g hemoglobin. These results suggest human exposure to Trp-P-1, probably from cooked foods or cigarette smoke, and its possible relationship to human carcinogenesis.     

DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces apoptosis via caspase-9 in primary cultured rat hepatocytes

The mechanism of cytotoxicity induced by the DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1) was investigated in primary cultured rat hepatocytes. Cytotoxicity was caused by intact Trp-P-1 and not by metabolically activated derivatives prepared using a recombinant yeast strain AH22/pAMR2 expressing rat cytochrome P450 1A1, and not by metabolically activated derivatives. We also found internucleosomal DNA fragmentation 6 h after treatment with 30 microM Trp-P-1, indicating that the cytotoxicity was due to the induction of apoptosis. After treatment with Trp-P-1, c-Myc protein level increased in a time-dependent manner and p53 protein also increased transiently with a subsequent increase in Bax protein level. This apoptotic pathway required the activation of caspase-9 as an initiator after leakage of cytochrome c into the cytosol from mitochondria and the activation of caspase-3 and -7 as executioners, but not caspase-1, -6 or -8 as measured using the corresponding peptide inhibitors and substrates or western blotting. The activated caspases in turn cleaved poly(ADP-ribose) polymerase as an intracellular substrate. Furthermore, we detected NUC18-like endonuclease activity during apoptosis induced by Trp-P-1. These findings suggest that this apoptosis may have a role against heterocyclic amine-type carcinogens in normal cells.     

Induction of DNA Recombination by Activated 3-Amino-1-methyl-5H-pyrido[4,3-b]indole

To investigate the genotoxic properties of a food-derived carcinogen, 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), we have tested whether Trp-P-2 and its metabolically transformed products can induce DNA recombinations. Trp-P-2 is a strong mutagen and its activated form, the N-hydroxylated derivative, Trp-P-2(NHOH), is known to form DNA adducts and cause DNA chain cleavage. Using a system in which phage λ undergoes recombination inside host Escherichia coli, we have found that Trp-P-2(NHOH), but not Trp-P-2 itself, can induce recombination. A nitroso derivative of Trp-P-2, Trp-P-2(NO), which can be reduced intracellularly to form Trp-P-2(NHOH), also induced recombination. Active oxygens are implicated in this recombinogenic action, since Trp-P-2(NHOH) is known to undergo spontaneous oxidative degradation, generating active oxygen radicals which can cause DNA chain cleavages. 4-Hydroxyaminoquinoline N-oxide and phenyl-hydroxylamine also showed recombinogenic actions in this assay system; hence, it is suspected that aromatic amine-type carcinogens have this property in common.     

Carcinogenic effect of the simultaneous administration of five heterocyclic amines to F344 rats

F344 rats were given five mutagenic and carcinogenic heterocyclic amines, i.e., 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AaC), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), mixed together in the diet each at one-fifth of the concentration used in the previous single-compound carcinogenesis experiment. Liver, colon and Zymbal gland tumors in both sexes, skin tumors in males and clitoral gland tumors in females were induced at significantly higher incidences than in control groups. Among them, the incidences of liver tumors in both sexes, skin tumors in males and clitoral gland tumors in females were significantly higher than those expected from the simple assumptions that the incidence of tumors induced by each compound would be one-fifth of that in the corresponding previous experiment and that the combined effect of the five chemicals would be additive.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) triggers apoptosis by DNA double-strand breaks caused by inhibition of topoisomerase I

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is one of the dietary carcinogens. At the initial step in the carcinogenic process, its exocyclic amino group is metabolically activated to the hydroxyamino derivative by the cytochrome P450 (CYP) 1A and 1B subfamily and then form DNA adducts, which are considered to be the main cause of DNA damage during the carcinogenic process. On the other hand, our previous study has shown that Trp-P-1 exhibits cytotoxicity to primary cultured rat hepatocytes, via induction of caspase-9-dependent apoptosis without being metabolized by CYP 1A1. In the present study, we investigated what type of DNA damage would be involved in the induction of apoptosis induced by Trp-P-1. When RL-34 cells derived from normal rat liver were treated with a high (30 microM) concentration of Trp-P-1, apoptotic events such as the loss of cell viability, nuclear condensation and the activation of caspase-3 were observed. In these apoptotic cells, intracellular topoisomerase I activity was inhibited and histone H2AX phosphorylation, which occurs after introduction of DNA double-strand breaks (DSBs), was observed in the early phase of the apoptosis. On the other hand, treatment with a non-apoptotic concentration (1 microM) of Trp-P-1 increased the formation of 8-hydroxy-2'-deoxyguanosine. The formation of DNA adducts was detected at almost the same level in both cells exposed to the apoptotic and non-apoptotic concentrations of Trp-P-1. These results indicate that Trp-P-1-induced apoptosis was triggered by DNA DSBs through the inhibition of topoisomerase I but not the formation of DNA adducts.     

Inhibitory effect of chlorophyll on the genotoxicity of 3-amino-1-methyl-5H-pyrido[4,3-b indole (Trp-P-2)

Genotoxicity of 3-amino-1-methyl-5H-pyrido [4,3-b indole(Trp-P-2)on Drosophila was suppressed by chlorophyll. Using the winghair spot test, we found that the formation of mutant hairsin adult files as a result of feeding them with Trp-P-2 in theirlarval stage was efficiently inhibited by coadininistrationof chlorophyll. The decrease in the spot frequencies was dependenton the dose of chlorophyll, and at the highest dose used, wherethe ratio in weight of Trp-P-2 to chlorophyll was 1:80, a completeprevention of the small single-spot formation was observed.A similar inhibitory effect was detected for chlorophyllin,the chromophore of chlorophyll. In the studies to investigatethe mechanism of inhibition, we observed that the mutagenicityof 3-hydroxyamino-1-methyl-5H-[4,3-bpyrido [Trp-P-2(NHOH)],the metabolically activated form of Trp-P-2, in Salmonella typhimuriumTA98 was suppressed effectively with chlorophyll and chlorophyllin.We also found that chlorophyll and chlorophyllin can producecomplexes with Trp-P-2 and Trp-P-2(NHOH). A straightforwardmechanism of these inhibitions is that Trp-P-2 [and Trp-P-2(NHOH)]becomes no longer available to organisms on forming the chlorophyllcomplex.     

The food mutagen 2-amino-9H-pyrido[2,3-b]indole (AalphaC) but not its methylated form (MeAalphaC) increases intestinal tumorigenesis in neonatally exposed multiple intestinal neoplasia mice

The heterocyclic amines 2-amino-9H-pyrido[2,3-b]indole (AalphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) are carcinogenic in several organs in rodents, but not in the intestinal tract. However, AalphaC induces DNA adducts, mutations and preneoplastic aberrant crypt foci (ACF) in rodent colons. The purpose of this study was to examine whether AalphaC and MeAalphaC could affect intestinal tumorigenesis in C57BL/6J-Min/+ (multiple intestinal neoplasia) mice. These mice are heterozygous for a germline nonsense mutation in codon 850 of the tumor suppressor gene adenomatous polyposis coli (Apc), producing a truncated non-functional Apc protein. They develop multiple intestinal adenomas, and are particularly susceptible to intestinal carcinogens that affect the Apc gene, especially when exposed neonatally. Whole litters consisting of Min/+ and +/+ (wild-type) mice of both sexes were given a single s.c. injection of 0.22 mmol/kg AalphaC (40.3 mg/kg) or MeAalphaC (43.4 mg/kg) or the vehicle 1:1 dimethylsulfoxide:0.9% NaCl on days 3-6 after birth, and were terminated at 11 weeks. AalphaC increased the number and diameter of small intestinal tumors, but not the number of colonic tumors or dysplastic ACF, in female and male Min/+ mice separately. In pooled data from females and males, colonic tumors and ACF found after AalphaC exposure appeared to be smaller than the spontaneous lesions, indicating later induction, slower growth or both. In contrast to AalphaC, MeAalphaC did not affect intestinal tumorigenesis in Min/+ mice. No effects were found by any of the amino-alpha-carbolines in the +/+ mice. AalphaC was less potent than the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.     

Catalysis of the covalent binding of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole to DNA by a L-proline- and adenosine triphosphate-dependent enzyme in rat hepatic cytosol

An enzymatic mechanism involved in the activation of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), a mutagenic intermediate of a tryptophan pyrolysate, was studied in vitro. In hepatic cytosol supplemented with adenosine triphosphate and L-proline, N-hydroxy-Trp-P-2 was converted to a form which reacts readily with DNA. The enzyme responsible for the activation was partially purified and identified as prolyl transfer RNA synthetase as judged by their cofactor requirements, inhibition by pyrophosphate or adenosine monophosphate, and copurification of their activities. The prolyl transfer RNA-dependent covalent binding of N-hydroxy-Trp-P-2 to DNA of hepatic cytosol was highest in rats, followed by mice, hamsters, rabbits, and guinea pigs in that order. The capacity for the binding of N-hydroxy-Trp-P-2 was largely consistent with their prolyl transfer RNA synthetase activity. With regard to the ultimate form of N-hydroxy-Trp-P-2 for the covalent binding, a possible formation of N,O-prolyl-3-amino-1-methyl-5H-pyrido[4,3-b]indole was proposed.     

Sex-dependent induction of hepatic enzymes for mutagenic activation of a tryptophan pyrolysate component, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole, by feeding in mice

Male and female BALB/c X DBA/2 F1 mice were treated with a diet containing 0.02% 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp P-1), a hepatocarcinogenic tryptophan pyrolysate component, and the capacities of subcellular fractions of the liver to catalyze the mutagenic activation of Trp P-1 and its analogue Trp P-2 (4-demethylated Trp P-1) were examined by the in vitro Salmonella test with strain TA 98. In mice on control diet, both 9000 X g supernatant (S-9) and microsomal fractions from female mice livers displayed only 1.1- to 1.3-fold higher capacities for the mutagenic activation of either Trp P-1 or Trp P-2 than did those from male mice livers. When mice were treated with the Trp P-1 diet for 1 week, the S-9 activity in male mice for the Trp P-1 mutagenesis did not change, but that in females was increased to 2.5-fold of the female control. Treatment of mice with the dietary Trp P-1 for 2 weeks increased the S-9 activities to 2.8-fold in males and 4.9-fold in females of the same sex controls and the increased S-9 activities were not significantly changed by additional Trp P-1 feeding for 2 weeks. Similar changes in the S-9 activity were observed for the Trp P-2 mutagenesis. The overall changes in the S-9 activities induced by feeding Trp P-1 were reflected in the isolated microsomes. However, microsomes derived from the same volume of S-9 used exhibited only about one-half (Trp P-1) or one-third (Trp P-2) of the activity of the respective complete S-9 mixtures. Addition of liver cytosolic fractions (105,000 X g supernatants) from untreated or Trp P-1-treated mice to microsomes resulted in enhanced activities. Cytosols alone did not activate the compounds to mutagens. The microsome-mediated mutagenicity of either Trp P-1 or Trp P-2 was diminished by removal of NADPH from the assay system. It was also inhibited by addition of 7,8-benzoflavone and to a lesser extent by SKF 525A. Enzyme(s) for the mutagenic activation of Trp P-1 was induced by an i.p. injection of 3-methylcholanthrene to mice and to a lesser extent by an injection of phenobarbital, but no sex differences were observed in these enzyme inductions as opposed to the Trp P-1 feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutational specificity of the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]-indole in mammalian cells

We have used the pZipHprtNeo shuttle vector to determine the types of DNA sequence alterations induced by a potent carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2). The shuttle vector contains a human cDNA hprt as the target gene and is stably integrated into a chromosome of the mouse cell line VH12. After Trp-P2 treatment, 59 independent HPRT- mutant clones of VH12 were isolated and altered sequences of the mutant hprt- cDNA genes were determined. Mutations induced by Trp-P2 comprised a variety of events; base substitutions, frameshifts, deletions and complex. Frameshifts were the most frequent mutational events (51%), and base substitutions were the next most frequent (30%) followed by deletions (14%). Examination of the DNA sequence context in the mutant genes revealed that approximately 70% of mutations induced by Trp-P2 occurred at G:C sites and thymine residues were the suggested target for the remainder of mutations. The results seem consistent with the previously reported finding that in vivo, metabolically activated Trp-P2 specifically binds to the C8 position of guanine residues in DNA to form C8G-Trp-P2 adducts (Hashimoto et al., Mutat, Res., 105, 9-13, 1982). As for molecular mechanisms, we showed that slippage and slippage misalignment could predict the generation of a large portion of Trp-P2-induced mutations found in the cDNA gene.     

Carcinogenicity of heterocyclic amines for the pancreatic duct epithelium in hamsters.

Effects of eight heterocyclic amines (HCAs) on pancreatic duct carcinogenesis were investigated in a rapid production model in hamsters. N-Nitrosobis(2-oxopropyl)amine (BOP) was given to effect initiation, followed by augmentation pressure consisting of four daily i.p. injections of 500 mg/kg DL-ethionine, a choline-deficient (CD) diet, a single i.p. injection of 800 mg/kg L-methionine and a s.c. injection of 20 mg/kg BOP. After two cycles of augmentation pressure, the HCAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) or 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [4,8-DiMeIQx) were administered in the diet for 50 or 70 days. The numbers of pancreatic ductal hyperplasias (H) and a total lesions including, atypical hyperplasia (AH), carcinomas in situ (CIS) and invasive carcinomas, were increased in hamsters given the diet containing 0.02% Trp-P-1 for 50 days. This result was confirmed and extended by the finding of increased numbers of invasive carcinomas in hamsters given 0.02% Trp-P-1 for 70 days. The number and incidence of invasive carcinomas were also elevated in hamsters given the diet containing 0.06% 4,8-DiMeIQx for 50 days. These results suggest a possible involvement of Trp-P-1 and 4,8-DiMeIQx in pancreatic duct carcinogenesis.