EXPERIMENTAL AND MOLECULAR PATHOLOGY 53, 203-210 (1%)

Genotoxicity of Pancreatic Chemical Carcinogens to Propagable Cultured Normal Pancreatic Epithelial Cells

JESSICA SHEPHERD, MING-SOUND TSAO,’ AND WILLIAM P. DUGUID

Department of Pathology, Montreal General Hospital, and McGill University, Montreal, Quebec, Canada H3G lA4

Received April 23, 1990, and in revised form August 14, 1990

The cytotoxic and DNA-damaging effects of the pancreatic carcinogens azaserine, strep- tozotocin, and N-nitrosobis(2-oxyopropyl)amine (BOP) on propagable cultured normal rat pancreatic epithelial cells have been studied. All three chemicals in micromolar concentra- tions produced cytotoxicity to these cells. The concentrations that caused 50% reduction in colony formation (LD,) were approximately 0.3 mM for azaserine, 0.4 mM for BOP, and 2 m&f for streptozotocin. Comparatively, the LD,, for N-methyl-N’-nitro-N-nitrosoguanidine was 3 PM. The toxicity of both azaserine and BOP did not require exogeneously added S9 microsomal enzymes, indicating that the cells were capable of metabolic activation of these carcinogens. All three compounds induced unscheduled DNA synthesis, thus suggesting their mutagenic and carcinogenic potential in these cultured cells. 6 1990 Academic PRSS, hc.

INTRODUCTION

The histogenetic origin of pancreatic carcinomas induced by chemical carcin- ogens in experimental animals is controversial (Rao, 1987). In Syrian golden ham- sters, N-nitrosobis(2-oxopropyl)amine (BOP) and its analogues typically produce ductal adenocarcinomas (Pour et al., 1975, 1977). In rats, the majority of carcin- ogens tested including azaserine have produced predominantly acinar cell carci- nomas, although a smaller percentage were undifferentiated and ductal carcino- mas (Longnecker et al., 1981; Longnecker and Curphey, 1975; Riverson et al., 1988). The diabetogenic substance streptozotocin has also been shown to induce islet cell tumors in rat pancreas (Kazumi et al., 1978; Rakieten er al., 1971; Yoshino et al., 1977). In guinea pigs, N-methyl-N-nitrourea (MNU) has been reported to produce adenocarcinomas of varying differentiation (Reddy and Rao, 1975). While Pour strongly defends the concept that ductal carcinomas in the hamster model originated from the ductal or centro-acinar cells (Pour, 1984, 1988), others believe that all carcinomas, whether ductal or acinar, may arise from “metaplastic transformation/de-differentiation” of acinar cells (Scarpelli et al., 1984; Flaks, 1984; Bockman, 1981). Both hypotheses may be correct in experi- mental models but are dependent on the species used (Longnecker, 1983, 1984). Based on the premise that chemical carcinogens are mutagens and also induce cellular toxicity and DNA damage on their target cells, we have studied the effect of BOP, azaserine, streptozotocin, and iV-methyl-N’-nitro-N-nitrosoguanidine (MNNG) on propagable cultured epithelial cells derived from duct/ductular cells of normal rat pancreas, in order to assess their carcinogenic potential on these cells.

’ To whom reprint requests should be addressed at Department of Pathology, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G lA4.

203

0014-4800190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

204 SHEPHERD, TSAO, AND DUGUID

MATERIALS AND METHODS

Materials

Corning’s tissue culture plates were used in all experiments. Ham’s F-12 me- dium, trypsin, Hanks’ balanced salt solution (HBSS), and fetal bovine serum (FBS) were obtained from Gibco Laboratories (Grand Island, NY). Azaserine and MNNG were from Sigma Chemical (St. Louis, MO), BOP was from Ash Stevens (Detroit, MI). Streptozotocin, [methyl-3H]thymidine (sp act 60 Cilmole) and Eco- lume liquid scintillation fluid were from ICN Canada (St. Laurent, Quebec). The Aroclor-S9 rat liver microsomal enzyme preparation was obtained from Organon Teknika (Durham, NC).

Cell Lines

Most experiments were performed on the RP-F344-1 normal pancreatic epithe- lial cell line derived from an adult male Fischer-344 rat, as previously reported (Tsao and Duguid, 1987). Some experiments were also repeated on another but identically derived normal pancreatic epithelial cell line (RP-J). Cells were rou- tinely cultured in Ham’s F-12 medium containing 10% FBS and were subcultured regularly as soon as they reached confluence. Cells between passages 10 to 18 (RP-F344-1) and 5 to 10 (RP-J) were used for these experiments.

Assays for Cellular Toxicity

The toxic effect of chemical carcinogens on these normal cultured pancreatic epithelial cells was assessed by measuring the inhibition of growth or colony formation in monolayer cultures. Measurements of growth inhibition were per- formed on cells plated in six-well tissue culture plates with approximately 2 x lo4 cells seeded into each of the replicate wells. When the logarithmic growth phase had commenced, 2-3 days later, the medium was changed to fresh medium con- taining various concentrations of carcinogens to be tested. Cells in triplicate wells were trypsinized and counted during the following 3 to 7 days or when the un- treated control plates had reached 7O-gO% confluence. Cells were counted using a ZM Coulter cell counter (Hialeah, FL). For colony-forming efficiency, 1000 cells were plated on lOO-mm tissue culture plates. Treatment with chemicals was also performed 2-3 days after plating and colonies were allowed to form in 2 to 3 weeks before they were fixed and stained with 4% Giemsa. When S9 microsomal enzyme preparation was used, it was added to a final concentration of 250 l&ml at the same time the chemical carcinogens were added.

Unscheduled DNA Synthesis

DNA synthesis was performed according to a modified method of Williams (1977). Cells were grown to confluence in two-chamber Labtek’s tissue culture chamber-slides (Nunc Inc., Naperville, IL). One hour prior to chemicai treat- ments, cells were preincubated in medium containing 5 mM hydroxyurea to in- hibit replicative DNA synthesis. Cells were then treated with fresh medium con- taining 5 mM hydroxyurea and various concentrations of the chemicals to be tested. After 1-hr incubation, 10 &i of [3H]thymidine was added to each well and further incubated for 18 hr. Cells were then washed three times with HBSS and incubated in 1% sodium citrate for 10 min. Subsequently they were fixed in acetic acid:ethanol (1: 1) mixture and were air-dried. After removal of the chamber walls,

GENOTOXICITY OF PANCREATIC CARCINOGENS 205

the slides were coated with NTB photographic emulsion and developed 2 weeks later. They were then counterstained with hematoxylin and examined under a microscope with a grided eyepiece. Nuclei covered with heavy grains (more than 100) were considered to represent replicative DNA synthesis, while those with light grains (less than 25-30) represented unscheduled (repair) DNA synthesis. For each treatment regimen, approximately 4000 to 6000 nuclei in 8 to 12 micro- scopic fields of duplicate chambers were counted.

RESULTS

Since BOP and possibly azaserine are indirect carcinogens which require en- zymatic activation for their methylating action (Longnecker, 1983), initial exper- iments were performed to test if the effect of these chemicals on the proliferation of normal cultured rat pancreatic epithelial cells requires the addition of exoge- nous activating microsomal enzyme preparation. Figure 1 showed that millimolar concentrations of both azaserine and BOP inhibited the proliferation of these cells, and the levels of inhibition were unaffected by the presence of exogenously added S9 rat microsomal enzyme preparation. Streptozotocin alone also signifi- cantly and dose-dependently inhibited the growth of these cells at a concentration greater than 1 mM (data not shown). These results suggest that normal propagable

0 CONTROL 6Sl MICROSOME

AZASERINE CONCENTRATION (mM)

10

7

0 CONTROL m MICROSOME

BOP CONCENTRATION (mM)

FIG. 1. The effect of adding exogenous S9 rat liver microsomai enzymes on the growth inhibitory effect of azaserine and BOP in normal cultured rat pancreatic epithelial cells. At the time these chemicals were added, there were approximately 20,000 cells in each well of six-welt tissue culture plates. Values represent the means and SD of the number of cells in each of triplicate wells 4 days later. S9 microsomal enzymes were added to a final concentration of 250 &ml at the same time azaserine and BOP were added.

206 SHEPHERD, TSAO, AND DUGUID

cultured rat pancreatic epithelial cells possess the necessary metabolic capacity to activate these pancreatic carcinogens into their toxic forms. Therefore, all sub- sequent experiments were performed without the addition of microsomal enzyme preparation.

The dose-response effect of azaserine, streptozotocin, BOP, and MNNG on these cultured pancreatic epithelial cells were measured by their effects on colony formation. The LD,, respectively were approximately 0.3 mit4 for azaserine, 0.4 mi$4 for BOP, and 2 mM for streptozotocin (Fig. 2). In contrast, the direct-acting carcinogen N-methyl-N’-nitro-N-nitrosoguanidine was toxic when present in mi- cromolar concentrations.

Treatment of these normal cultured pancreatic duct/ductular epithelial cells by azaserine, BOP, streptozotocin, and MNNG resulted in significant unscheduled DNA synthesis in their nuclei (Table 1). The highest UDS was noted in cells treated with MNNG and streptozotocin. The numbers of heavily grained nuclei were similar in control untreated cells and cells treated with various chemicals, and consistently represented approximately 3.05 + 0.73% of the nuclei.

DISCUSSION

Based on serial ultrastructural observations of cell aggregates isolated from enzymatic digest of adult rat pancreas that became attached to the plastic surface and subsequently proliferated as a monolayer culture, we have previously con- cluded that these propagable cells originated from the duct/ductular epithelium (Tsao and Duguid, 1987). The ductiductular origin of these rat pancreatic epithe- ha1 cells is also supported by reports that monolayer cultures of pancreatic epi- thelial cells have also been established from isolated fragments of guinea pig pancreatic ducts (Hootman and Longsdon, 1988) and enzymatically dissociated bovine pancreatic duct epithelial cells (Stoner et al., 1978). In contrast, although prolonged maintenance of function can be achieved in cultured pancreatic acinar or islet cells (Oliver, 1980; Ono et al., 1979; Hegre er al., 1983; Jain et al., 1985; Brannon et al., 1985), sustained cell proliferation to form propagable cell lines by normal acinar or islet cells of adult mammalian pancreases has never been re- ported.

The lack of available proliferating cultured pancreatic epithelial cells has re- sulted in the paucity of in vitro chemical carcinogenesis studies of adult mamma- lian pancreases (Jones et al., 1981). Studies using explanted pancreases have been reported (Parsa et al., 1980a,b, 1984), but the results have not provided further significant insight than the knowledge already obtained from the in viva studies of pancreatic chemical carcinogenesis models. The availability of propagable cul- tured rat pancreatic epithelial cells will provide novel approaches to the in vitro studies of pancreatic chemical carcinogenesis, especially in view of the fact that the majority of human pancreatic carcinomas are of ductal origin.

Our results show that the three major prototypes of chemical carcinogens used in the in vivo pancreatic carcinogenesis models, namely azaserine, BOP and strep- tozotocin, are cytotoxic to the propagable cultured normal rat pancreatic epithe- lial cells. On an equimolar basis, azaserine and BOP are more toxic than strep- tozotocin but all three are lOOO-fold less toxic than the direct-acting methylating carcinogen MNNG. Although comparative toxicity studies on cell proliferation for cultured rat acinar and islet cells are unavailable, Zucker et al. (1986) have previously reported that azaserine at 0.2 mM also caused 50% inhibition (I,,) in

GENOTOXICITY OF PANCREATIC CARCINOGENS 207

CONCENTRATION (pM)

CONCENTRATION (mu)

BOP

CONCENTRATION (mu)

FIG. 2. The effect of MNNG, streptozotocin (STZ), azaserine (AZA), and BOP on the colony- forming efficiency of normal cultured rat pancreatic epithelial cells. Values for STZ, AZA, and BOP represent the means f SEM of two or four experiments, and for MNNG represent the mean t SD.

protein synthesis by primary cultured rat pancreatic acinar cells. I,,-, for primary cultured islet cells was 1 m&Z for azaserine and approximately 2.5 mM for strep- tozotocin. Streptozotocin did not inhibit protein synthesis in rat acinar cells. BOP and HPOP were ineffective on both the rat acinar and islet cells. Although it is difficult to make a direct comparison between our results and the data of Zucker et al. (1986), it appears that azaserine is toxic to all three types of epithelial cells in rat pancreas, whereas streptozotocin is mainly toxic for duct and islet cells. In

208 SHEPHERD, TSAO, AND DUGUID

TABLE 1 Unscheduled DNA Synthesis in Normal Cultured Rat Pancreatic Epithelial Cells Treated with

Chemical Carcinogens

Carcinogen Concentration % Nuclei with UDS”

None (control) - 0 Azaserine 0.1 mM 4.0%

0.3 mM 3.3% BOP 2mM 3.8%

5mM 3.3% MNNG 34 PM (5 t&ml) 15.0% Streptozotocin 2mM 1.1%

5mM 11.3%

0 Values represent the percentage of light-grained nuclei among 4000 to 6000 nuclei counted.

contrast, BOP appears to be toxic mainly for rat pancreatic duct/ductular epithe- lial cells. We have also demonstrated that all three chemicals caused DNA dam- age and induced unscheduled (repair) DNA synthesis in these cultured pancreatic epithelial cells, and all of them have been recognized to methylate DNA and induce DNA repair (Lilja et al., 1977; Zurlo et al., 1982; Bennett and Pegg, 1981; Lawson et al., 1981).

The cytotoxic effect of BOP, azaserine, and streptozotocin occurs without requiring the addition of exogenous activating microsomal enzymes. While aza- serine requires at least a simple enzymatic (Y$ elimination of diazoacetic acid catalyzed by pyridoxal to produce its DNA alkylating effect, it is directly muta- genic in the Ames assay and hence independent of the mixed-function oxidase system for its activation (Zurlo et al., 1982). Streptozotocin is a naturally occur- ring glucose derivative of methylnitrosourea (Herr et al., 1967) with potent DNA alkylating activity, but the mechanism for its activation is unclear (Bennett and Pegg, 1981). Of the three carcinogens, only BOP is definitely known to require microsomal activation for its mutagenic and carcinogenic activity (Scarpelli et al., 1984; Mangino et al., 1987; Kokkinakis and Scarpelli, 1989). Our results strongly suggest that propagable cultured rat pancreatic duct epithelial cells not only are susceptible to the toxic effect of BOP, they also have the metabolic enzymes to activate BOP to its toxic form. Lawson et al. (1989) have also reported recently that rat pancreatic duct epithelial cells isolated as duct tissue fragments main- tained in agarose were capable of activating 3H-labeled BOP to its DNA- interacting active forms, and incubation of these isolated duct tissues with BOP resulted in a similar level of alkylation as that produced in isolated hamster duct tissue. The reason for the poor carcinogenicity of BOP in rat pancreas is unclear. Although rat duct and hamster duct tissues are alkylated to the same extent when incubated with BOP, the loss of alkylation from rat ducts is almost twice that from hamster duct cells, suggesting a more efficient “repair” system in the former.

The morphological and growth properties of propagable cultures of normal pancreatic epithelial cells that we established are very similar phenotypically to those of propagable cultured normal rat liver epithelial cells that are posited to originate from intrahepatic bile ductular cells (Grisham, 1980; Tsao et al., 1984). Tumors derived from these liver epithelial cells following repeated treatments with MNNG demonstrated diverse histological phenotypic expression including resemblance to hepatoblastoma, cholangiocarcinoma, and hepatocellular carci-

GENOTOXICITY OF PANCREATIC CARCINOGENS 209

noma (Tsao and Grisham, 1987). Based on the results that propagable cultured pancreatic duct/ductular epithelial cells are susceptible to the DNA-damaging effect of these pancreatic carcinogens, a novel approach to the in vitro studies of pathogenesis and lineage in pancreatic chemical carcinogenesis becomes feasible.

ACKNOWLEDGMENTS

This work was supported by a grant from the Cancer Research Society Inc. of Montreal, Canada. Dr. Tsao is a scholar of the Medical Research Council of Canada.

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