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Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.

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c-K-ras is activated by mutation at codon 12 in the majority of human pancreatic carcinomas of ductal but not acinar phenotype. The Ela-1-myc transgene when expressed in transgenic mice induces pancreatic carcinomas of both acinar and mixed acinar/ductal phenotype. The histopathology of 110 pancreatic carcinomas were characterized in this model. A high percentage of the low to moderately differentiated acinar cell carcinomas contain areas of ductal metaplasia. The latter tumors and several well-differentiated acinar tumors were evaluated for c-K-ras mutation to determine whether there is a relationship between the ductal phenotype and c-K-ras mutation. The polymerase chain reaction and allele-specific oligomer hybridization were used to determine whether the c-K-ras gene was mutated at codons 12, 13, or 61. Amplified DNA products from these tumors were also evaluated by single strand conformation polymorphism analysis. Only wild-type c-K-ras was found in these tissues. Not finding c-K-ras mutation in tumors containing ductal morphology indicates that c-K-ras mutation is not a required factor for acinar to ductal metaplasia or a factor in the tumorigenesis of pancreatic tumors that arise in acinar tissue.

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The objective of this study was to determine whether small human pancreatic adenocarcinomas contain activated c-K-ras as an approach to answering the question of whether c-K-ras mutation is an early change in this disease. Eight pancreatic adenocarcinomas in the range 1.2-3 cm were analyzed for c-K-ras mutation at codon 12 by amplifying the c-K-ras gene around codon 12 out of paraffin-embedded tissue sections using the polymerase chain reaction. c-K-ras mutations were detected by allele-specific oligonucleotide hybridization. Six of the eight small pancreatic adenocarcinomas contained mutated c-K-ras at codon 12, position 2, and two of the six tumors had an additional mutation at position 1 of codon 12. Our results indicate that small pancreatic adenocarcinomas are similar to large, late-stage pancreatic adenocarcinomas in that 75% of the tumors analyzed contain mutated c-K-ras at codon 12, position 2. These data suggest that c-K-ras mutation occurs early and may therefore have a role in initiation of human pancreatic adenocarcinoma.

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Several heterocyclic amines, found in cooked food, are powerful mutagens in the Ames Salmonella mutagenicity test system. One of these, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is one of the most mutagenic chemicals tested in this assay. In primary cultures of chick and rat hepatocytes, MeIQ, by itself, induced cytochrome P450 from the IA subfamily but was a weak inducer compared to 3-methylcholanthrene. However, in both chick and rat hepatocytes in culture, MeIQ decreased the amount of 3-methylcholanthrene-induced ethoxyresorufin deethylase activity, which is catalyzed by cytochrome P450 IA. The protein moiety of cytochrome P450 IA was decreased at MeIQ concentrations of 2.5 micrograms/ml or greater in chick hepatocytes and 25 micrograms/ml in rat hepatocytes. In hepatic microsomes from methylcholanthrene-treated chicks and rats, MeIQ was a competitive inhibitor of both ethoxyresorufin deethylase activity, a reaction catalyzed mainly by rodent cytochrome P450 IA1, and uroporphyrinogen oxidation, a reaction catalyzed by rodent P450 IA2. In cultured chick hepatocytes, MeIQ also decreased cytochrome P450-mediated oxidation of uroporphyrinogen by intact cells. The ability of MeIQ to inhibit as well as to induce cytochrome P450s of the IA subfamily may be important in assessing the mutagenic and carcinogenic effects of MeIQ in mammals.

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Nodules of acinar cells with increased proliferative potential develop in the pancreas of carcinogen-treated rats and in untreated aged rats. Large nodules are classed as adenomas. Phenotypic and genotypic characteristics of nodule cells were compared with normal pancreas and transplantable acinar cell carcinomas by several methods. Nuclei of acinar cells from normal pancreas, adenomas, and three carcinomas in situ had normal diploid DNA content as determined by flow cytometry. One of two primary carcinomas had a hypodiploid DNA content. Two of three transplantable carcinomas were aneuploid with a DNA content in the tetraploid range. Explants from nodules and adenomas failed to grow in soft agar, whereas several carcinomas were positive in this assay. A primary carcinoma was serially transplanted, but transplantation of nodules or adenomas failed. Transfection of DNA from carcinomas in situ yielded a higher frequency of NIH 3T3 transformants than DNA from adenomas. DNAs from the transformants did not contain ras sequences. These studies indicate that cells from nodules and adenomas have low growth potential and lack critical phenotypic and genotypic characteristics of transformed malignant cells that were present in some primary and transplanted carcinomas.

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The objective of this study was to determine whether activation of c-Ki-ras occurs in carcinogen-induced rat pancreatic tumors. DNAs from 27 samples, which included adenomas, carcinomas in situ, and adenocarcinomas arising in azaserine-treated rats and corn oil-gavaged rats along with a nafenopin-induced rat pancreatic adenocarcinoma were examined for mutation of c-Ki-ras at codons 12, 13, and 61 by using the polymerase chain reaction. Our results indicate that activation of c-Ki-ras is not a common event during pancreatic carcinogenesis in the rat.

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Using the technique of alkaline elution analysis, the ability of 11 known or suspected pancreatic carcinogens to damage the DNA of pancreatic acinar cells when administered to rats and hamsters was examined. The two species respond differently to several agents. In selected instances, DNA damage was also assessed in cultured pancreatic acinar cells exposed in vitro to the agents. Comparisons of DNA damage produced in vivo with that produced in vitro gave useful information on the role of pancreatic metabolism in activating pancreatic carcinogens. Finally, information germane to the question of the cell of origin for pancreatic cancer was obtained.

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The mutagenicity of azaserine was determined in a pancreatic acinar cell-mediated mutagenesis assay using V79 cells as the responder cell line. The mutation frequency of V79 cells was increased in direct culture with azaserine as well as in coculture with rat and hamster pancreatic acinar cells. Although slightly higher mutation frequencies were seen with coculture, the mutation frequency induced by azaserine in coculture was not significantly enhanced over that observed in direct culture. Thus, azaserine cannot be used as a positive control to monitor the level of acinar cell metabolism in such cell-mediated mutagenesis assays. Statistical analysis suggested that hamster acinar cell cocultures were more effective at increasing the mutation frequency of azaserine as compared to rat acinar cell cocultures. Hamster acinar cell cocultures, but not rat acinar cell cocultures, increased the mutagenicity of azaserine in a dose-response fashion. These results suggest that azaserine may be a pancreatic carcinogen for the hamster as well as the rat.

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Acinar cells were isolated by enzymatic digestion from normal pancreas and from azaserine-induced atypical acinar cell nodules and a transplantable acinar cell carcinoma. Normal acinar cells were predominately binuclear, with abundant cytoplasm. They were 10-24 micron in diameter, with a size distribution skewed toward smaller sizes but with a peak at 18 micron. Cells were isolated from 41 enucleated nodules varying in weight from 1.1 to 200 mg. These cells were predominantly mononuclear, with a more uniform size than normal cells and a peak at 9 micron diameter. Cells from all nodules studied were grossly similar, and there was no relation between nodule size and the degree of mononucleation. Cells from the transplantable tumor were small, with little cytoplasm, and were almost exclusively mononuclear. The extent of binucleation in normal and microscopic atypical acinar cell nodules was also studied in sections from the pancreas of rats injected with azaserine 4 months before killing. Nuclear and cell counts in these sections confirmed that binucleation is more frequent in normal than in nodule tissue. These studies emphasize the high degree of binucleation found in normal pancreatic acinar cells. They demonstrate the feasibility of using cell separation techniques to obtain preparations of acinar cells from normal and neoplastic pancreatic tissue for studies of functional and morphological differences in these cells.

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Isolated rat and hamster acinar cell suspensions possess the ability to carry out the cytochrome P-450-dependent O-deethylation of 7-ethoxycoumarin, 2-,3-, and 4-hydroxylation of biphenyl and 3-hydroxylation of benzo(a)pyrene. Rat and hamster acinar cells isolated from 5,6-benzoflavone-pretreated animals oxidize all three substrates at measurable rates. These rates are considerably lower (16-210-fold in the rat and 290-2670-fold in the hamster) than those in incubations using hepatocytes isolated from 5,6-benzoflavone-pretreated animals. Hydroxylation of biphenyl at the 2-, 3-, and 4-positions proceeds at similar rates in rat acinar cells. The rate of 3-hydroxybiphenyl formation is barely detectable in hamster acinar cells where the rates of 2- and 4-hydroxybiphenyl formation are slower than in the rat. No detectable oxidation products of 7-ethoxycoumarin, biphenyl, or benzo(a)pyrene are found with acinar cells of either species isolated from untreated and phenobarbital-pretreated animals. The O-deethylation of 7-ethoxycoumarin in rat and hamster acinar cells is decreased in the presence of inhibitors of the cytochrome P-450-dependent monooxygenase system, 7,8-benzoflavone being much more effective than metyrapone. The deethylation product of 7-ethoxycoumarin, 7-hydroxycoumarin, is conjugated with sulfate and glucuronic acid moieties at appreciable rates by acinar cells isolated from both rat and hamster. Pretreatment of rats and hamsters with either 5,6-benzoflavone or phenobarbital has little effect on the rates of conjugation in isolated acinar cell preparations.

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Utilizing the technique of alkaline elution analysis, the ability of N-nitrosomethyl(2-oxopropyl)amine (MOP), a potent pancreatic carcinogen, to damage pancreatic DNA in rats and hamsters was examined. Pancreatic DNA isolated from hamsters exposed for 1 h to MOP given i.p. at doses of 7-60 mg/kg showed dose-related DNA damage. A similar dose-response was observed in the pancreas of rats receiving 20-180 mg MOP/kg, suggesting that hamsters were 2-3 times more sensitive than rats. In contrast to the results obtained in vivo, functionally viable acinar cells from both rat and hamster pancreas, when exposed in vitro to levels of MOP comparable to those in vivo (20-180 micrograms/ml), failed to show dose-related DNA damage. Acinar cells from hamsters pretreated with 5,6-benzoflavone, an inducer of cytochrome P-450 activity, showed greatly enhanced drug-metabolizing capability, but again no DNA damage was observed upon exposure to MOP. Minced hamster or rat pancreas also failed to show DNA damage in response to MOP treatment. When hamsters in which hepatic blood supply was interrupted by ligation were given 60 mg/kg MOP i.v. and sacrificed 15 min later, damage to pancreatic and liver DNA was comparable to that observed in ligated controls which had received saline only. Administration of MOP to sham-operated animals led to extensive DNA damage in both pancreas and liver at 15 min. Analysis by h.p.l.c. showed an almost 2-fold increase in the amount of MOP present in the pancreases of the liver-ligated animals as compared to the sham-operated unligated animals. MOP was absent from the liver of the ligated animals. These experiments strongly suggest that DNA damage by MOP to the pancreatic acinar cells and probably to other pancreatic cell types, as well, requires metabolic activation by the liver.

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Carcinomas of the pancreas, stomach, and breast, as well as mesotheliomas and ovarian stromal tumors, were induced in Syrian golden hamsters treated with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine (MNCO), which has previously been shown to cause pancreatic acinar cell carcinomas in rats. The pancreatic carcinomas in hamsters appeared ductlike. The nonneoplastic and preneoplastic lesions induced in the hamster pancreas included cystic ductal complexes, tubular complexes, intraductal hyperplasia and atypical hyperplasia, focal eosinophilic metaplasia, and foci of atypical acinar cells. High doses of 654 mg MNCO/kg body weight were cytotoxic for acinar cells and caused atrophy of the pancreas. Alkaline elution analysis of DNA from acinar cells treated in culture with MNCO showed an increased rate of elution characteristic of single-strand breaks. A group of hamsters treated with a low dose of N-nitrosobis(2-oxopropyl)amine (BOP) developed pancreatic lesions similar to those seen when a subcarcinogenic dose of MNCO was given. The results suggest that MNCO affects both acinar and ductal cells in the hamster and that the response of the hamster pancreas to MNCO and BOP is similar in many respects.

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