In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations.

Neoplastic transformation of Syrian golden hamster (SGH) pancreatic duct cells was induced by in vitro treatment with the direct-acting carcinogens N-methylnitrosourea (MNU) and N-(2-hydroxypropyl)nitrosourea (HPNU), with subsequent selection by sustained culture in serum- and epidermal growth factor (EGF)-deprived medium. The present study examines the efficacy of serum and EGF deprivation as a selection pressure and the effect of the carcinogen dose, frequency and interval of exposure on tumorigenesis and K-ras mutation. Selection of carcinogen-initiated duct cells by serum and EGF deprivation is highly reproducible and effective, increasing the incidence of tumors from 26 to 93% for MNU or from 0 to 100% for HPNU. SGH pancreatic duct cells exposed to 0.5 mM MNU for 13 weeks (long-treatment schedule) produced K-ras mutations at codon 12 in six of six tumors. However, when cells were exposed to 0.125, 0.25 or 0.5 mM MNU daily for 5 days (short-treatment schedule), mutations of K-ras at codon 13 were identified in four of 16 tumors, the remaining 12 showing no mutations. Duct cells exposed to 0.5 mM HPNU by the short-treatment schedule produced K-ras mutations in codon 13 in six of six tumors, as contrasted to 12 tumors that developed from cells exposed to 0.125 or 0.25 mM HPNU, which all contained K-ras codon 12 mutations. The current experiments demonstrate that K-ras mutation in pancreatic carcinogenesis in vitro by MNU or HPNU can be modified by the nature and dose of the carcinogen as well as the frequency and duration of exposure.

Stimulation of DNA synthesis in pancreatic duct cells by gastrointestinal hormones: interaction with other growth factors.

Pancreatic duct cells of the Syrian hamster were grown as monolayers on thin layers of type I collagen coated onto microporous membranes. The effects of a number of potential trophic factors were tested by their ability to increase [3H]thymidine incorporation into cellular DNA. To measure the effect of growth factors, cells were subjected to a period of growth factor depletion to induce a state of partial quiescence in DNA synthesis. Cells responded with a significant increase in thymidine incorporation after the addition of epidermal growth factor (EGF) alone or a growth factor mixture containing EGF plus insulin, transferrin, selenium, linoleic acid, bovine pituitary extract, triiodothyronine, and dexamethasone. When the serum substitute, Nu Serum IV (5%, vol/vol), was added to this mixture, addition of several gastrointestinal (GI) hormones including secretin, vasoactive intestinal polypeptide (VIP), bombesin, and gastrin caused significant increases in thymidine incorporation at concentrations of 0.01-1 microM. At 1 microM, these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) greater than bombesin (153%) greater than VIP (138%) greater than gastrin (126%). Cholecystokinin octapeptide, a known trophic factor for pancreatic acinar cells, did not cause significant increases in thymidine incorporation in cultured duct cells. These results suggest that pancreatic duct cells possess receptors for a number of GI hormones and respond to the trophic effects of hormones known to stimulate pancreatic growth in vivo.

Characterization of differentiated Syrian golden hamster pancreatic duct cells maintained in extended monolayer culture.

Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium supplemented with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal growth factor, 3,3',5-triiodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to bovine serum albumin (ITS+), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous elongated mitochondria, and both free and membrane-bound ribosomes. Cells grown as monolayers for 3 mo. were more flattened and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic AMP (cAMP) after stimulation by 1 X 10(-6) M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold. At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels of cAMP after exposure to secretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic studies.

Metabolism and activation of the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine by isolated hepatocytes and pancreatic cells of the Syrian hamster.

The metabolism of the pancreatic carcinogen N-nitroso-bis(2-oxopropyl)amine (BOP) was studied using primary hepatocytes and acinar and duct cells isolated from Syrian hamsters. Metabolic activation of BOP was verified by detecting its conversion to CO2 covalently bound metabolites and soluble products containing the alpha-carbon of the nitrosamine. At concentrations below 0.2 mM, BOP was completely activated by hepatocytes within 60 min. At high substrate concentration (1 mM) or high cell density (5 x 10(6) cells/ml), reduction of BOP to N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine and N-nitrosobis(2-hydroxypropyl)amine contributed significantly to the metabolic profile. The conditions which favored metabolic activation of BOP were used to compare metabolism by hepatocytes and pancreatic cells. Under such conditions, the ratio of activation products formed by hepatocytes versus those formed by acinar cells was 14.5:1; the corresponding ratio for covalently bound metabolites was 19:1. Hepatocytes activated BOP 106 times more rapidly than duct cells as determined from yields of activation products or 152 times more rapidly as determined from labeling of cellular macromolecules. Acinar cells showed a higher capacity for metabolic activation than duct cells. The ratio for the yield of activation products from acinar versus duct cells was 4.3:1; the corresponding ratio for covalent binding was 5.8:1. The relatively low capacity of pancreatic cells for activation of BOP compared to hepatocytes is in agreement with the low levels of DNA binding in the pancreas compared to other organs after administration of BOP to the hamster in vivo. The observation that the ratio for total covalent binding of BOP in hepatocytes versus acinar cells was higher than that seen previously for the liver versus the pancreas in vivo is consistent with the hypothesis that alkylating agents derived from BOP reach the pancreas after formation in other organs. The liver would be the prime source for such alkylating agents.

Uptake of N-nitrosobis(2-oxopropyl)amine by isolated rat and hamster hepatocytes: species differences and evidence for an active carrier-mediated transport process.

The rates of uptake of the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) by hepatocytes isolated from Fischer rats and Syrian hamsters were determined in order to investigate species differences in cellular transport of the carcinogen. Initial rates of uptake of [1-14C]BOP by hepatocytes were measured using a rapid centrifugation technique. At cell densities from 1.5 to 6 x 10(6) cells/ml, initial rates of uptake were as much as 4-fold more rapid in hamster hepatocytes than in those of the rat. The cell/medium distribution ratio for hamster hepatocytes reached a value of 9.0 after a 20-min incubation with an extracellular BOP concentration of 20 microM. Under the same conditions, the cell/medium distribution ratio for rat hepatocytes was only 2.4. These results indicated that BOP uptake proceeded against a concentration gradient and was more rapid in hamster hepatocytes. In both species, the rates of uptake were saturable with increasing concentration (2-685 microM) and displayed biphasic kinetics characteristic of high-affinity (Km less than 20 microM) and low-affinity (Km greater than 30 microM) processes for the uptake of BOP. Evidence for the involvement of an ATP-dependent active carrier-mediated transport process was obtained from experiments in which hepatocytes were preincubated with metabolic inhibitors. Significant inhibition of uptake was observed in the presence of KCN, carbonyl cyanide-3-chlorophenylhydrazone, antimycin A, oligomycin and other agents which interfere with electron transport or ATP generation. Based on the reduction in uptake rates, rat hepatocytes were more sensitive to the effects of these inhibitors. These results suggest that the entry of BOP into hepatocytes is under cellular regulation and that the more rapid rate of uptake in liver cells of the hamster may be one factor responsible for the observation that BOP is a more potent hepatotoxin and carcinogen in this species.

Species specificity in the metabolism of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine to mutagens by isolated rat and hamster hepatocytes.

The metabolic activation of the carcinogens N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) by Fischer rat and Syrian hamster hepatocytes was investigated in order to determine the existence of species differences in the induction of cell mutation. The conversion of BOP and HPOP into forms mutagenic to V79 cells was studied by using the hepatocyte-mediated mutagenicity assay. Mutations at the hypoxanthine:guanine phosphoribosyltransferase locus and the Na-K-ATPase locus were scored by the induction of 6-thioguanine resistance (TGr) or ouabain resistance (Ouar), respectively. Hepatocytes of both species were capable of converting BOP and HPOP to mutagens for V79 cells in a dose-dependent manner. Metabolism of BOP by rat hepatocytes resulted in higher mutation frequencies than that by hamster hepatocytes. At a BOP concentration of 240 microM, rat hepatocyte metabolism yielded 90.7 TGr mutants and 19.5 Ouar mutants per 10(5) V79 cells. At the same concentration, hamster hepatocyte metabolism of BOP yielded 54.1 TGr mutants and 13.0 Ouar mutants per 10(5) V79 cells. These results did not correlate with the known carcinogenic potency of BOP in the hamster as compared to the rat. Hamster hepatocytes carried out the catabolism of BOP to CO2 at faster rates than rat hepatocytes; therefore, the species difference in mutagenic activation was not due to a defect in BOP uptake or metabolism by hamster hepatocytes. In contrast, metabolism of HPOP by hamster hepatocytes resulted in significantly higher mutation frequencies than that by rat hepatocytes. At an HPOP concentration of 240 microM, hamster hepatocyte metabolism yielded 83.5 TGr mutants per 10(5) V79 cells; rat hepatocyte metabolism yielded only 19.8 TGr mutants per 10(5) V79 cells. This species difference in mutagenic activation correlated well with the known potency of HPOP as a carcinogen for the hamster as compared to the rat. Since hamster pancreatic cells and subcellular fractions are known to have very limited capacity to perform the metabolic activation of HPOP, the results of this study imply that liver metabolism plays an important role in the conversion of HPOP to an agent(s) which subsequently affects the hamster pancreas. The mutagenic potency of BOP versus HPOP was compared after metabolism by hepatocytes from both species. Following their metabolism by hamster hepatocytes, the two compounds were nearly equivalent in mutagenic potency. After metabolism by rat hepatocytes, BOP was significantly more potent mutagen than HPOP.(ABSTRACT TRUNCATED AT 400 WORDS)

Tannic acid-induced nucleolar changes in hepatocytes transplanted into syngeneic or xenogeneic host and in hepatocytes maintained in primary culture.

In this study we have investigated the effect of a single dose of tannic acid, administered s.c., on the nucleolar ultrastructure of hepatocytes transplanted into a syngeneic or xenogeneic host in order to evaluate the validity of our hepatocyte transplantation system as an in vivo alternative to the use of whole animals to test for species and strain differences to the effects of hepatotoxins. Within 4-6 h following tannic acid injection, rat hepatocytes transplanted into the anterior chamber of eye and inguinal fat pads of rat and athymic nude mouse, showed changes of nucleolar components, with separation of ribonucleoprotein containing granules into discrete dark zones. These dark areas were surrounded by light areas consisting of granular and fibrillar components of the nucleolus. These changes were identical to tannic acid-induced nucleolar alterations in the homotopic liver. Hamster and rat hepatocytes xenotransplanted into athymic nude mice also displayed prominent nucleolar alterations in response to tannic acid. The similarity and extent of nucleolar alterations observed in transplanted hepatocytes and the in situ homotopic liver cells attest to the usefulness of the hepatocyte transplantation system for the evaluation of species differences in biological response to toxic/carcinogenic effects of xenobiotics.

Activation of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)-(2-oxopropyl)amine to mutagens for V79 cells by isolated hamster and rat pancreatic acinar cells.

A pancreatic acinar cell-mediated mutagenicity assay was developed as an in vitro model system to study the metabolism of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amino (HPOP) into forms mutagenic for Chinese hamster V79 cells. Mutations at the hypoxanthine:guanine phosphoribosyltransferase locus and the Na/K ATPase locus were scored by resistance to 6-thioguanine and ouabain, respectively. The ability of both Syrian golden hamster and Fischer rat pancreatic acinar cells to convert BOP and HPOP to mutagens for V79 cells was investigated in order to examine the basis for species specificity. Acinar cells of both species were capable of activating BOP and HPOP to mutagens for V79 cells in a dose-dependent manner. In the 6-thioguanine resistance assay, rat acinar cells induced higher mutation frequencies than hamster acinar cells with both BOP and HPOP. In the ouabain resistance assay, both cell types induced equivalent levels of mutation with the respective nitrosamines. BOP was a considerably more potent mutagen than HPOP after activation by either cell type. This is consistent with the known in vivo specificity of BOP versus HPOP in the hamster pancreas and suggests that BOP may be activated to mutagenic metabolites by a pathway(s) independent from its enzymatic reduction to HPOP. The comparable abilities of rat and hamster acinar cells to convert BOP or HPOP to mutagenic forms imply that pancreatic metabolic activation alone cannot explain the difference in organotropism of BOP and HPOP in the two species.

The effect of 3-methyl substitution on the carcinogenicity of nitroso-4-piperidone.

The carcinogenic activity of the 3-methyl derivative of nitroso-4-piperidone was compared with that of the compound without the methyl group, by chronic administration to rats at equimolar doses in drinking water. Nitrosopiperidone induced both liver and esophageal tumors, whereas the 3-methyl derivative induced only tumors of the esophagus in a much shorter time.

Rapid formation of N-nitrosodimethylamine from gramine, a naturally occurring precursor in barley malt.

The two tertiary amine alkaloids, hordenine and gramine, which are biosynthesized in malt during germination, were subjected to nitrosation under conditions typical for the study of tertiary amine nitrosation. At 65 degrees C in dilute aqueous acid (pH 4.4 or pH 6.4), nitrosation of both amines resulted in formation of N-nitrosodimethylamine (NDMA). At 24 degrees C in dilute acid (pH 3.4), the initial rate of NDMA formation from gramine was nearly equal to the initial rate of NDMA formation from dimethylamine. At the same temperature, the ratio of initial rates of formation of NDMA from gramine and trimethylamine was 6250:1. At 23 degrees C, the ratio of initial rates of formation of NDMA from gramine and hordenine was 5200:1. The rapid reaction of gramine with nitrous acid and the nature of the gramine nitrosation reaction products both indicated that gramine did not undergo nitrosation by the expected mechanism of nitrosative dealkylation. A new mechanism if proposed to explain the labile nature of the dimethylamino group of gramine and to account for the fact that NDMA is the only N-nitrosamine formed during the nitrosation of gramine.

Nitrosation of N-methyltyramine and N-methyl-3-aminomethylindole, two barley malt alkaloids.

The secondary amine barley malt alkaloids N-methyltyramine and N-methyl-3-aminomethylindole were synthesized, nitrosated in dilute acetic acid and the products characterized by mass spectrometry and NMR spectroscopy. The nitrosation products of N-methyltyramine were p-hydroxy-m-nitro-N-nitroso-N-methyl-2-phenylethylamine and p-hydroxy-N-nitroso-N-methyl-2-phenylethylamine. Upon nitrosation, N-methyl-3-aminomethylindole formed N-nitroso-N-methyl-3-aminomethylindole and the dinitrosated product N1-nitroso-N-nitroso-N-methyl-3-aminomethylindole.

Plant anticancer agents V: new bisindole alkaloids from Tabernaemontana johnstonii stem bark.

The isolation and structure elucidation of the three new bisindole alkaloids, gabunamine, tabernamine, and 19,20-epoxyconoduramine, from Tabernaemontana johnstonii stem bark are described. The isolation of the seven known alkaloids, conodurine, conoduramine, gabunine, isovacangine, ibogamine, pericyclivine, and perivine, from the same source also is noted. The alkaloids gabunamine, gabunine, and tabernamine showed significant cytotoxicity against the P-388 cell culture system.