Response of two rodents, Mastomys natalensis and Mystromys albicaudatus, to the pancreatic carcinogen azaserine.

The response of two rodents to azaserine carcinogenicity for the pancreas was evaluated. Mystromys albicaudatus was not responsive; however, Mastomys natalensis developed large numbers of atypical acinar cell nodules and several adenomas in a 6-month study. Mastomys is the most responsive of several animals in which azaserine has been studied as a pancreatic carcinogen.

Species and rat strain variation in pancreatic nodule induction by azaserine.

Subtoxic doses of azaserine induced atypical acinar cell nodules (AACN) in the pancreases of outbred Wistar rats, inbred W/LEW and F344 rats, and outbred Charles River CD-1 albino mice 4-6 months after initiation of treatment in the growing animal. These AACN apparently represented preneoplastic lesions, some of which have the potential to develop into adenomas or adenocarcinomas. Wistar and W/LEW rats were highly responsive to nodule induction; AACN developed in about 90% of the outbred Wistar rats and in all of the W/LEW rats tested. F344 rats were less susceptible and developed about 10% as many AACN as the Wistar rats. Female rats developed approximately half as many AACN as males. The mouse was intermediate in response between the F344 and the two Wistar rats. Syrian golden hamsters and strain 13 guinea pigs were relatively unresponsive. These studies of azaserine-induced AACN provided a basis for selection of carcinogenic azaserine regimens and suggested that the young male W/LEW rat was the most sensitive of the animals studied.

Positive correlation between pancreatic DNA damage and species specificity in response to N-nitrosobis(2-oxopropyl)amine.

N-nitrosobis(2-oxopropyl)amine (BOP), a potent pancreatic carcinogen in hamsters that has failed to induce pancreatic tumors in rats, was studied for its effects on the DNA of both rat and hamster pancreas in order to relate DNA damage (as measured by alkaline elution) to carcinogenicity in vivo. At doses of 10, 20, and 40 mg BOP/kg, extensive DNA damage was detected in male Syrian golden hamster pancreas but Lewis rat pancreatic DNA was not affected. Only at doses of 100 mg BOP/kg or greater could pancreatic DNA damage in the rat be detected. DNA damage was also observed in both rat and hamster livers at 10, 20, and 40 mg BOP/kg. Alkaline elution analysis of DNA from isolated rat and hamster acinar cells treated in vitro with BOP revealed that only hamster acinar cell DNA was damaged. Rat acinar cell DNA was unaffected at all doses examined, up to 200 micrograms BOP/ml medium. Unscheduled DNA synthesis studies in cultured acinar cells confirmed the observations that BOP is genotoxic to hamster but not to rat acinar cells. The results strongly suggested that rat pancreas did not have the ability to metabolically activate BOP, which accounted for lack of both BOP-induced DNA damage and carcinogenicity in the rat.

Relationship of age to prevalence of focal acinar cell dysplasia in the human pancreas.

The prevalence of focal dysplastic lesions of acinar cells in the pancreata of autopsied children and adults was compared. The lesions were recognized in sections stained with hematoxylin and eosin because acinar cells forming islet-sized foci or larger nodules contained one or more of the following cytologic abnormalities: reduced cytoplasmic basophilia, reduced cytoplasm, reduced zymogen, cytoplasmic vacuoles, or nuclear abnormalities. Lesions were found in only 1 patient (age, 7 yr) of 170 patients whose ages ranged from birth to 9 years, whereas 7 of 49 patients 10-19 years old had focal acinar cell dysplasia. The prevalence of such lesions among adults was comparable to that encountered in individuals during the second decade of life and distinctly higher than that found among children during the first decade. Six of the 8 children in whom dysplastic acinar cell foci were found had cancers in other tissues that had been treated by chemotherapy. The data are consistent with the interpretation that dysplastic acinar cell lesions in the pancreas are acquired.

Enhancement of pancreatic carcinogenesis by a dietary unsaturated fat in rats treated with saline or N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine.

The effect of diets high in an unsaturated fat on the enhancement of pancreatic carcinogenesis in saline-treated rats and in rats treated with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) was examined. Young male LEW rats were treated with a single dose of HPOP (160 mg/kg body wt) or saline, fed diets containing 5 or 20% corn oil, and then autopsied 12 months later. The pancreata of HPOP-treated rats fed the diet with 5% fat contained multiple foci and nodules of atypical acinar cells (AACN), acinar cell adenomas, and localized carcinomas. Rats fed the diet with 20% fat developed a similar spectrum of pancreatic lesions and also developed carcinomas that showed local invasion or metastasis to regional lymph nodes. The incidence and multiplicity of localized carcinomas was significantly higher in the group that was fed the high-fat diet. HPOP also induced neoplasms in the liver, lungs, and kidneys, but none of these had a higher incidence in the group fed the high-fat diet. Among rats that received no carcinogen, the incidence of AACN was high, but the multiplicity of these lesions was low, an average of three per pancreas in groups fed both levels of fat; however, the average area of AACN transections was larger in the high-fat diet group. One acinar cell adenoma and 1 carcinoma developed in the group of 11 rats fed the 20% corn oil diet, whereas no neoplasms developed in the group of 12 rats fed the 5% corn oil diet. Although the incidence of pancreatic neoplasms is not significantly different in these 2 groups, the data are consistent with the hypothesis that initiated foci are promoted to grow and become neoplasms in the pancreas of rats that are fed diets with a high content of unsaturated fat--as was demonstrated in the HPOP-treated rats.

Pathologic and biochemical effects of azaserine in inbred Wistar/Lewis rats and noninbred CD-1 mice.

Inbred W/LEW rats and noninbred CD-1 mice were compared for responsiveness to induction of pancreatic adenocarcinomas by azaserine. At 1 year following the first of 15 weekly ip injections of 10 mg azaserine/kg body weight, the rats had a pancreatic adenoma incidence of 71% (12/17) and a pancreatic adenocarcinoma incidence of 35% (6/17), with 1 invasive adenocarcinoma. The mice showed none of these advanced lesions, although numerous pancreatic atypical acinar cell nodules (AACN) were present. An examination of the number and size of the AACN showed the rats to hve more and larger AACN thatn did the mice. The concentration of [14C]azaserine and/or its metabolites was greater in rat pancreas than in mouse pancreas. Alkaline sucrose gradients were used to compare azaserine-induced pancrewtic and liver DNA damage in W/LEW and F344 rats, the CD-1 mouse, the Syrian golden hamster, and the strain 13 guinea pig. DNA damage was detected 1 hour following azaserine administration in both pancreata and livers of all animals tested and persisted for at least 1 week in the pancreata of all animals except the CD-1 mouse; however, neither the degree nor persistence of DNA damage accurately reflected the differing responsiveness of these species to induction of pancreatic AACN or neoplasms by azaserine.

Induction of pancreatic carcinomas in rats with N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine: histopathology.

The carcinogenicity of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) for rat pancreas was evaluated. Two-week-old male LEW rats were given a single ip injection of HPOP, 160 mg/kg body weight; the rats were autopsied 4, 6, or 12 months later. Histologic examination showed that the pancreata contained multiple foci of atypical acinar cells and nodules of atypical acinar cells (AACN), acinar cell adenomas, localized carcinomas, and carcinomas. The incidence of carcinomas was 77%. The carcinomas were composed of poorly differentiated acinar cells and ductlike structures. Pancreatic ducts were unaffected. The prominence of AACN, the histologic type of the neoplasms, and the absence of hyperplastic changes in ductal epithelium suggest that the pancreatic carcinomas were derived from acinar cells. The incidence of liver cell carcinomas and pulmonary adenomas was similar to that of localized pancreatic carcinomas. Neoplasms of other organs were less frequent. HPOP has been shown to induce pancreatic carcinomas in hamsters but has not previously been reported to be a pancreatic carcinogen in rats.

Experimental induction of pancreatic carcinomas in the hamster with N delta-(N-methyl-N-nitrosocarbamoyl)-L-ornithine.

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.

Inhibition by retinoids of the growth of azaserine-induced foci in the rat pancreas.

The usefulness of a short-term azaserine [CAS: 115-02-6; diazoacetate serine (ester)]-rat model for the screening of retinoids (known chemopreventive agents) and the effect of two retinoids on the growth of azaserine-induced, presumptive preneoplastic foci of acinar cells were examined. At 14 days of age, male Lewis rats were each given injections of a single dose of 30 mg azaserine/kg body weight. These rats were weaned to test diets to which retinoids were added. At 4 months post initiation, pancreata were examined by quantitative stereologic methods to determine number and mean size of foci. Two phenotypically different populations of foci were observed and characterized as acidophilic or basophilic. Retinylidene dimedone and N-2-hydroxyethylretinamide decreased the number and size of the acidophilic foci but not the basophilic foci. The inhibition of growth of the acidophilic foci correlates well with the known effects of these retinoids in long-term carcinogenicity studies.

Effect of age on nodule induction by azaserine and DNA synthesis in rat pancreas.

The efficacy of various azaserine treatment durations was evaluated with respect to induction of atypical acinar cell nodules in Wistar rat pancreas and was related to animal age and rate of pancreatic DNA synthesis during growth. The sensitivity to nodule induction was maximal in postnatal rats when the rate of pancreatic DNA synthesis was high, whereas treatment of weanlings was less effective and treatment of mature rats was least effective. When weaned growing rats were given 1, 3, or 5 weekly injections of 30 mg azaserine/kg, the number of nodules induced was proportional to the number of injections. A single dose at this level did not induce detectable pancreatic necrosis or inflammation; therefore, DNA synthesis due to regeneration was probably not a major factor in the initiation of nodules. We concluded that multiple daily injections of [3H]thymidine during the first or second postnatal week provided DNA of sufficiently high specific activity for use in DNA repair and biochemical toxicity studies.

Adenocarcinoma of the pancreas in azaserine-treated rats.

Development of a model of carcinoma of the pancreas in rats was approached by attempting to identify chemicals that (a) behave as mutagens and (b) localize in the pancreas following systemic administration; and then to study the effects of long-term administration. Azaserine was selected because it behaves as a direct-acting mutagen in two bacterial test systems and because tissue distribution studies showed concentration especially in kidney and pancreas. Groups of rats have been given i.p. injections once or twice weekly for 6 months, and rats have been autopsied after 6 to 18 months. During the first year pancreases developed (a) nodules of atypical exocrine cells which seem to represent hyperplastic foci and (b) encapsulated adenomas. After 1 year most pancreases from treated rats are diffusely abnormal and contain many hyperplastic nodules and adenomas, while more than one-quarter have had pancreatic adenocarcimona. Metastases have been observed in lymph nodes, liver, and lung. No carcinomas or adenomas have been observed in control rats. No other organ shows as high an incidence of involvement as pancreas, but renal neoplasms were frequent. Studies with another chemical O-(N-methyl-N-nitroso-beta-alanyl)-L-serine, are at an earlier stage. The tissue distribution of radioactivity following injection of a 14C-labeled sample is similar to that of azaserine; however, this compound is not a direct-acting bacterial mutagen. Rats treated for 6 months twice weekly i.p. have a higher incidence of nodules of atypical acinar cells than did controls, although the number of nodules per rat is few. No adenomas or carcinomas have been found during 13 months of the study. We conclude that azaserine is a carcinogen in rats and causes major abnormalities of growth and differentiation of the exocrine pancreas, including adenocarcinoma in some rats. O-(N-Methyl-N-mitroso-beta-alanyl)-L-serine had less effect than azaserine on pancreatic growth and differentiation.

Effect of bombesin and caerulein on early stages of carcinogenesis induced by azaserine in the rat pancreas.

This study was designed to analyze the effect of two pancreaticotrophic peptides on pancreatic carcinogenesis in the azaserine-rat model. The rats were treated with bombesin or caerulein for 16 weeks after initiation with azaserine. Two-week-old Lewis rats were given injections of a single dose of azaserine (30 mg/kg) and the control pups received an injection of saline. They were divided into ten groups for peptide treatment as follows: Group 1, azaserine-saline; Group 2, azaserine-bombesin, 10 micrograms/kg; Group 3, azaserine-bombesin, 30 micrograms/kg; Group 4, azaserine-caerulein, 5 micrograms/kg; Group 5, azaserine-caerulein, 15 micrograms/kg; Group 6, control-saline; Group 7, control-bombesin, 10 micrograms/kg; Group 8, control-bombesin, 30 micrograms/kg; Group 9, control-caerulein, 5 micrograms/kg; and Group 10, control-caerulein, 15 micrograms/kg. At 3 weeks of age, they were weaned. Peptides or saline were injected 3 consecutive days a week for 16 weeks. Rats were autopsied 4 months after the administration of azaserine. Pancreatic weight was increased by bombesin and decreased by caerulein treatment. Quantitative histological analysis of azaserine-induced atypical acinar cell nodules in the pancreas showed that the size and number of atypical acinar cell nodules were increased in both bombesin- and caerulein-treated groups. Thus, these peptides appear to stimulate the growth of preneoplastic acinar cell lesions.

Effects of four retinoids in N-nitrosobis(2-oxopropyl)amine-treated hamsters.

Four synthetic retinoids were evaluated with regard to chemo-prevention of pancreatic carcinomas in carcinogen-treated hamsters. Syrian golden hamsters were given two injections of N-nitrosobis(2-oxopropyl)amine (20 mg/kg) and then were fed retinoid-supplemented diets for 1 year. The incidence of pancreatic carcinomas was lower in six of eight retinoid-fed groups than in the control group, although the differences were not statistically significant. The lowest incidence was observed in groups fed N-(4-pivaloyloxyphenyl)retinamide and N-(2-hydroxypropyl)retinamide. Testicular atrophy with decreased spermatogenesis was noted in males fed N-(2-hydroxypropyl)retinamide, N-(3-hydroxypropyl)retinamide, and N-(2,3-dihydroxypropyl)retinamide.

Dietary modulation of azaserine-induced pancreatic carcinogenesis in the rat.

Because diet has been shown to modulate the incidence of a wide variety of chemically induced cancers in experimental animals, various dietary constituents were evaluated for their ability to modulate the incidence of pancreatic exocrine cancer in male Wistar/Lewis rats given injections of the pancreatic carcinogen, azaserine. Ten different diet regimens were fed. The incidence of pancreatic cancers in rats fed a control diet was compared to that in groups fed diets formulated to evaluate the effect of caloric restriction, high protein, low protein, low fat, cyclopropenoid fatty acids, lipotrope deficiency, high unsaturated fat, and high saturated fat. The incidence of pancreatic adenomas and carcinomas was evaluated by light microscopy. The number of pancreatic neoplasms was reduced in carcinogen-treated groups which were underfed the control diet or fed the diet high in protein. Pancreatic carcinogenesis appeared to be enhanced in two groups which were fed diets containing 20% corn oil, i.e., high in unsaturated fat; whereas, the group fed a diet high in saturated fat had the same incidence of neoplasms as did the group fed the control diet. The pancreatic neoplasms from groups in which the incidence was enhanced by diet showed less evidence of acinar cell differentiation and displayed diverse histological types. In the lipotrope-deficient group, there was a significantly increased incidence of hepatocellular carcinoma; however, a low incidence of liver tumors was encountered in all other dietary groups.

Characterization of the elastase 1-simian virus 40 T-antigen mouse model of pancreatic carcinoma: effects of sex and diet.

Elastase 1-simian virus transgenic mice, strain Tg(Ela-1, SV40E) Bri18, were studied to characterize the development of pancreatic neoplasms. The incidence of pancreatic carcinomas was compared in groups of male and female mice fed one of three diets chosen because of their effect on the development of pancreatic carcinomas in other animal models. Male mice developed more exocrine carcinomas than female mice and their tumors were larger. Groups fed chow had fewer exocrine carcinomas than groups fed purified diets. The level of fat in the latter diets, 5 versus 20% corn oil, did not alter tumor incidence. An unexpectedly high incidence of islet cell tumors was found in all dietary groups, with a higher incidence in females than in males.

Inhibition of a transplantable pancreatic carcinoma by castration and estradiol administration in rats.

Influence of sex steroids on the growth of an azaserine-induced transplantable rat pancreatic carcinoma, DSL-2, was studied. This established transplantable tumor has been maintained in syngeneic rats. Inbred male Lewis rats were pretreated with castration and s.c. implantation of 1.0-mg 17 beta-estradiol (CAS: 50-28-2; estradiol) pellets at 7 weeks of age. Tumor cells were inoculated s.c. on the back of intact male, castrated male, or 17 beta-estradiol-treated castrated male rats. Additional male rats served as non-tumor-bearing controls. There was no difference in the body weight between tumor-bearing and non-tumor-bearing male rats. A distinct difference in the tumor growth was observed in variously conditioned recipients. In castrated male hosts, the serum testosterone levels and the epididymis weights were significantly decreased, and the tumor weights were significantly less as compared to intact control hosts. Additional pretreatment with 17 beta-estradiol caused a markedly slower growth of tumors and increases of the serum 17 beta-estradiol levels and the pituitary weights in castrated male recipients. The remarkable response of tumor growth to castration was also observed in a fast-growing tumor derived from DSL-2. Moreover, close positive relationships between tumor weights and the activities of both serum amylase and lipase were observed. Results showed that the pretreatment with castration alone or in combination with 17 beta-estradiol treatment was able to inhibit the growth of the transplantable tumor. In addition, tumor cells had an ability to produce amylase and lipase, and the amount of enzymic activity was related to the tumor volume. Thus, these data indicate that the transplantable rat pancreatic carcinoma retains physiological function. Our previous study has shown the modulation by sex steroids of azaserine-induced preneoplastic lesions of pancreas in rats. Therefore, androgens and estrogens may play key roles as promoters and inhibitors during the process of pancreatic carcinogenesis.

Divergent effects of retinoids on pancreatic and liver carcinogenesis in azaserine-treated rats.

Chemoprevention by synthetic retinoids of the progression of carcinomas of the pancreas induced in rats by azaserine was evaluated. Lewis rats were given five weekly injections of azaserine, 30 mg/kg, while being fed a chow diet. Two weeks after completion of carcinogen treatment, groups of rats were fed the chow diet supplemented with four different retinoids at the level of 0.5 to 2 mmol/kg of diet for 1 year. The incidence of pancreatic and other neoplasms was determined by autopsy and histological study. The incidence of localized pancreatic carcinoma among male and female non-retinoid-treated controls was 25 and 17%, respectively. No invasive or metastatic carcinomas were found in the control group. The combined incidence of localized and invasive pancreatic carcinomas among male and female rats treated with retinoids was: N-(4-pivaloyloxyphenyl)retinamide, 4 and 0%; N-(2-hydroxypropyl)retinamide, 14 and 6%; N-(3-hydroxypropyl)retinamide, 16 and 4%; and N-(2,3-dihydroxypropyl)retinamide, 12 and 6%. High- and low-dose groups are combined in this summary of data. Thus, there was a trend towards fewer pancreatic carcinomas among all retinoid-treated groups. The reduction in incidence was significant in both male and female rat groups given N-(4-pivaloyloxyphenyl)retinamide and N-(2,3-dihydroxypropyl)retinamide. The principal evidence of retinoid toxicity was growth failure, which was most severe in animals treated with N-(4-pivaloyloxyphenyl)retinamide, and testicular atrophy, which was most severe among male animals treated with N-(3-hydroxypropyl)retinamide. Among the females, groups treated with three of the four retinoids showed a dose-related increase in incidence of hepatocellular carcinomas. Since the retinoids were fed after the completion of exposure to the carcinogen, the effects on both pancreatic and liver carcinogenesis were exerted during the postinitiation phase of carcinogenesis.

Identification of 7-carboxymethylguanine in DNA from pancreatic acinar cells exposed to azaserine.

Studies were undertaken to determine the identity of an azaserine:DNA adduct. The most probable adduct, 7-carboxymethylguanine, was synthesized. DNA isolated from pancreatic acinar cells treated in culture with [14C]azaserine was hydrolyzed under neutral conditions to liberate N-alkylated purines. The neutral hydrolysate was subjected to high-performance liquid chromatography along with the synthetic standard. One of the radioactive peaks from the treated DNA was found to cochromatograph with 7-carboxymethylguanine in three systems: reverse phase; anion exchange; and ion pair reverse phase. These results suggest that azaserine metabolism in acinar cells results in carboxymethylation of DNA, supporting previously proposed models of azaserine degradation.

Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterization.

Many reports have emphasized the role of gastrin as a growth factor for normal gastrointestinal mucosa and gastrointestinal cancers. Recent studies have pointed out that this peptide acts also as a growth factor for the pancreatic cancer cell line AR42J. This effect is mediated by gastrin [cholecystokinin (CCK)-B] receptors. In the present study, we investigated gastrin (CCK-B) receptor expression in the azaserine-induced rat pancreatic carcinoma DSL-6, comparing it to normal rat pancreas, and we also characterized CCK receptor subtypes in this tumor. The results showed that there is extensive gastrin binding to the DSL-6 pancreatic carcinoma. No evidence of specific gastrin binding to normal pancreas was found. Analysis of the ability of gastrin-17-I to inhibit 125I-gastrin-I binding demonstrated that gastrin bound to a single class of receptors with a Kd of 0.21 +/- 0.04 nM and a binding capacity of 184 +/- 29 fmol/mg protein. 125I-Gastrin-I binding was inhibited by the specific CCK-B receptor antagonist L365,260 approximately 40 times more effectively than by the specific CCK-A receptor antagonist L364,718. Analysis of the ability of cholecystokinin octapeptide (CCK-8) to inhibit 125I-Bolton-Hunter-CCK-8 binding revealed two CCK binding sites, i.e., a high affinity site and a low affinity site. The observed binding affinities of CCK-8 were then introduced into the computer analysis of the dose-inhibition curve of the ability of gastrin-17-I to inhibit binding of 125I-Bolton-Hunter-CCK-8, which was significantly better fit by a three-site model than by a two-site model. The three sites meet the criteria for CCK-B, high affinity CCK-A, and low affinity CCK-A receptors. The binding capacity of CCK-B receptors constitutes 34% of the total high affinity CCK binding sites. This study demonstrated that DSL-6 pancreatic carcinoma expresses three subtypes of CCK receptors. Gastrin (CCK-B) receptors, which were not detected in normal rat pancreas, constitute about one third of the total high affinity CCK receptors. We suggest that novel expression of gastrin (CCK-B) receptors may be generated by gene mutation or amplification during carcinogenesis and may play an important role in promoting tumor growth.

Carcinogen-induced lesions in the rat pancreas: effects of varying levels of essential fatty acid.

The ingestion of high levels of fats, especially unsaturated fats, has been shown to enhance carcinogenesis in a variety of experimental model systems. Recently attention has focused upon the unsaturated linoleic fatty acid (18:2 omega 6) as a key component for this postinitiation enhancement. We have investigated the dose-effect relationship of this essential fatty acid (EFA), in a well-characterized experimental model of pancreatic cancer. Male Lewis rats were given injections i.p. of azaserine (30 mg/ kg) at 14 days of age. The pups were weaned to test diets that contained 20% total dietary fat with EFA compositions varying from 0.5 to 11.5% of the diet. After 4 months of feeding these 20% fat diets, the pancreases were evaluated in situ for grossly visible tumors and microscopically for the number and size of the azaserine-induced, putative preneoplastic lesions (foci). Grossly visible tumors increased significantly in number as the EFA content of the diet increased. Two populations of microscopic foci were observed in these azaserine-initiated rats; namely, acidophilic foci and basophilic foci. Quantitative stereological analyses of these foci revealed that the acidophilic population of foci increased in both number and size as the EFA content of the diet increased. This increase was particularly apparent from 4.4 to 8.5% dietary EFA content. The basophilic population showed no similar response to increasing dietary EFA. These results indicate that the minimum dietary EFA required for enhancement of azaserine-induced, pancreatic carcinogenesis by a high fat diet lies in the range of 4 to 8%.