Hyperplastic Nodules and Adenomas of Exocrine Pancreas in Azaserine-Treated Rats 1, 2

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1 Hyperplastic Nodules and Adenomas of Exocrine Pancreas in Azaserine-Treated Rats 1, 2 Daniel S. Longnecker 3 and Barbara G. Crawford 3.4 SUMMARY-Long-term studies of the carcinogenicity of azaserine in rats were undertaken to develop a model of adenocarcinoma of the pancreas. Rats were treated for 6 weeks to 6 months with once or twice weekly intraperitoneal azaserine injections. Single doseswere 5-25 mgjkg; total doses were mgjkg. All of 18 rats autopsied 31/2, 6, or 8 months after initiation of treatment had nodular groups of atypical exocrine cells which seemed to represent hyperplastic foci, and 3 rats had small exocrine adenomas. No malignant tumors were found. One of 6 control rats contained 1 hyperplastic nodule in exocrine pancreas. J Natl Cancer Inst 53: , SPONTANEOUS and experimentally induced carcinomas are rare in the exocrine pancreas of common experimental animals (1, 2). Thus no satisfactory an imal model of carcinoma of the pancreas is available for study. Development of such an animal model is our current goal. We tried to induce malignant change in the pancreas of rats with a chemical which 1) is a known or suspected carcinogen, and 2) has a natural affinity for pancreas. Since natural alpha-amino acids and several of their analogs have a high affinity for pancreas, we gave special attention to amino acid derivatives. We found that azaserine (NSC-742), a potential alkylating agent, behaved as a mutagen in 2 bacterial screening systems designed to detect mutagenic activity of chemicals (3-5). We also found in preliminary studies with 3H-azaserine that the pancreas was second only to kidney in concentration of trichloroacetic acid-soluble radioactivity after intravenous injection; it attained the highest specific radioactivity in a trichloroacetic acidinsoluble fraction when compared with kidney, liver, intestine, spleen, testis, thymus, and skeletal muscle (unpublished). Acinar cell injury has been reported in the pancreas of rats treated with azaserine (6). With this background, we undertook long-term studies in rats to evaluate the carcinogenicity of azaserine, with special attention to the exocrine pancreas. Preliminary results revealed that all 17 animals killed at the end of 6 months contained nodular groups of atypical exocrine cells which we believe qualify as hyperplastic nodules; we also found 3 lesions which have the characteristics of "adenomas" of exocrine origin. These lesions and the details of their induction are described here. MATERIALS AND METHODS We used Charles River Wistar strain rats fed Purina rat chow ad libitum. Azaserine (Calbiochem, La Jolla, Calif.) dissolved in 0.9% NaCl was injected intraperitoneally (ip) at doses of 5, 10, 15, and 25 mg/kg according to schedules shown in table 1. Sixteen rats were killed and autopsied 6 months after the first injection, 1 was killed after 17 weeks, and 1 died 8 months after the first treatment, apparently as the result of suppurative meningitis. Those animals treated for 6 weeks and examined at 6 months were survivors of preliminary toxicity studies and were not part of long-term studies of a larger group of rats. Rats treated for 6 months were part of a long-term (2-year) study involving 180 rats still in progress. Six control animals were given equivalent volumes of saline according to the same schedule as azaserinetreated rats. Four were treated like groups 3, 4, and 5, and 1 each was treated like groups 1 and 2. All pancreatic tissue taken at autopsy was embedded as slices 1-2 mm thick. Representative sections were taken of lungs, liver, kidneys, intestine, stomach, salivary glands, and spleen. Thymus and prostate were taken from selected animals. Tissues were fixed in Susa's solution, paraffin-embedded, and stained with hematoxylin and eosin. Aldehyde fuchsin and toluidine blue stains were used on selected blocks. RESULTS The pancreases of all 18 rats examined to date have small masses of histologically atypical cells which we call hyperplastic nodules (figs. 1-4). A few larger lesions were visible grossly as pale areas measuring up to 1 mm in diameter; however, most such foci have been detected only microscopically. These nodules consist of groups of cells that vary in size from a few dozen cells to masses several times as large as an islet (i.e., = 1 mm in diameter). Cells of the nodules are distinguished from the surrounding pancreas by altered differentiation, increased nuclear size, and increased mitotic activity (figs. 2, 4). None of these features is constant; i.e., the degree of differentiation varies from nodule to nodule but tends to be consistent within a nodule, not all nodules show increased nuclear size, and mitotic activity of nodulevaries. The nodules appear "clonal" in nature, inasmuch as they tend to be circumscribed masses of cells-often spherical unless they are subserosal or at the periphery of a lobule, when they conform to the surface or septal limit (figs. 1, 3). They are not encapsulated. Subserosal nodules often bulge above the surface of adjacent pancreas (fig. 1). More than 300 such foci have been encountered with a frequency of 1 Received March 21, 1974; accepted May 10, Supported by Public Health Service contracts NIH NCI-E and NOl-CP from the National Cancer Institute. 3 Department of Pathology, Dartmouth Medical School, Hanover, N.H We acknowledge the technical assistance of Susan T. James, Maureen Devine, and Richard Markley. JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 53, NO.2, AUGUST

2 574 LONGNECKER AND CRAWFORD Group* la B C 2A B TABLE I.-Summary of azaserine treatment for 18 rats Dose/injection Injections/week Duration of Number of rats (mg/kg) treatment mo 1 1 " 1 2 " 1 " 16 wk wk " " 8 Sex All rats were autopsied after 6 months except 2B, which was examined at 17weeks, and TC. examined at 8 months. 4-90/rat in the routinely examined single section/ block. The true frequency is unknown, since we have not made serial or step sections to search for additional nodules which could be present in deep areas. Only 1 similar focus was encountered in 6 control rats. Three pancreases, 1 each from groups 1, 2, and 5, contained encapsulated spherical masses of acinar cells less differentiated than hyperplastic nodules (figs. 5, 6, 7). Zymogen granules were present in some cells of each lesion indicating acinar cell origin. Mitotic activity was increased in 2 lesions. We regard these lesions as acinar cell adenomas. No similar lesion was encountered among controls. The group 2B (5 mg/kg twice weekly) rat was a replacement for one that died early in the experiment, and it was under treatment for only 16 weeks before autopsy. The pancreas contained fewer nodules and the nodules were smaller than those typical of rats killed at 6 months. No consistent abnormalities have been found in sections from other organs, though many rats had focal bronchopneumonia or intrapulmonary hemorrhage. One azaserine-treated rat exhibited a 3-mm subcutaneous fibroma on a hind limb (not an injection site). This was the only other neoplasm found to date. It should be noted that the doses of azaserine used in these experiments were lower than those reported to cause acute cytotoxic effects in the pancreas (6). DISCUSSION Lesions of the exocrine pancreas in rats similar to those we described have been reported previously. Some have apparently arisen spontaneously (1), and some have followed treatment with chemical carcinogens (7-9). The "hyperplastic adenoma-like" lesions reported by Morris et al. (8) induced by feeding N,N' 2,7-fluorenylenebisacetamide and N-2-fluorenylacetamide seem similar to the lesions we described as hyperplastic nodules. The spontaneously arising exocrine adenomas described by Rowlatt and Roe (1) seem more like the lesions we called adenomas, though those illustrated in their report seem more highly differentiated and were larger than ours. The pancreatic tumors induced by Hayashi et al. (7) by 4-nitroquinoline-l-oxide and 4-hydroxyaminoquino Iine-Ivoxide are much like those we observed in azaserine-treated rats. The largest tumors reported by Hayashi are larger than any adenoma we found. His observations were made after 14 months, i.e., a longer interval than ours. The foci of atypical acini described by Hayashi appeared similar to our hyperplastic nodules, and similar nodules reported by Shinozuka and Konishi (9, and personal communication), appeared after a single injection of 4-hydroxyaminoquinoline-I-oxide. The azaserine-induced lesions are of interest because they may represent early stages in carcinogenesis. At this time, the absence of hyperplastic or neoplastic changes in other tissues suggests a specific effect in the pancreas. These lesions are clearly of acinar cell origin, whereas human adenocarcinoma of the pancreas is generally interpreted as ductlike. Hopefully, continued observation of azaserine-treated rats will indicate whether the lesions reported here represent a stage in the histogenesis of a neoplasm comparable to that in the majority of human pancreatic malignancies. REFERENCES (1) ROWLATT U, ROE FJ: Epithelial tumors of the rat pancreas. J Natl Cancer lnst 39: 17-32, 1967 (2) SNELL KC: Spontaneous lesions of the rat. In The Pathology of Laboratory Animals (Ribelin WE, McCoy JR, eds.). Springfield, Ill., Charles C Thomas, 1965, pp (3) AMES BN LEE FD DURSTON WE: An improved bacterial test system for' the detection and c1assificat!on of mutagens and carcinogens. Proc Nat! Acad SCI USA 70: , 1973 (4) SLATER EE, ANDERSON MD, ROSENKRANZ, HS: Rapid detection of mutagens and carcinogens. Cancer Res 31 : , (5) LONGNECKER DS, CUR~HEY TJ, JAMES S!, et al:. Trial of a bacterial screemng system for rapid detection of mutagens and carcinogens. Cancer Res. In press (6) HRUBAN Z, SWIFT H, SLESERS A: Effect of ~zaserine on the fine structure of the liver and pancreatic acinar cells. Cancer Res 25: , 1965 (7) HAYASHI Y FURUKAWA H, HASEGAWA T: Pancreatic tumors i~ rats induced by 4-nitroquinoline-l-oxide derivatives. In Topics in Chemical Carcinogenesis (Nakahara W, Takayama S, Sugimura T, et al, eds.). Baltimore, University Park Press, 1972, pp 53-:65 (8) MORRIS HP WAGNER BP RAY FE, et al: Comparative study of c~ncer and oth~r lesions of rats fed N,N'_~, 7 fluorenylenebisacetamide or N-2-fluorenylacetamlde. Nat! Cancer lnst Monogr 5: 1-53, 1961 (9) SHINOZUKA H, KONISHI Y: Degenerative and hyperplastic changes in rat pancreatic acinar cells induced by 4-hydroxyaminoquinoline-l-oxide. Am J Pathol 74:59a, 1974

3 FIGURE I.-Portion of exocrine pancreas from a rat killed 6 months after the first of 12 biweekly lo-mg injections of azaserine/kg. A subserosal nodule of hyperplastic exocrine cells occupies major portion of the field. Nonhyperplastic cells are at bottom and right. Cells in hyperplastic area are on the average larger, have larger nuclei, and have paler-staining cytoplasm. Hematoxylin and eosin (H & E). X284 FIGURE 2.-Portion of pancreas from rat killed 6 months after the first of 12 biweekly 25-mg ip injections of azaserine/kg. All cells in the field are from a hyperplastic nodule except for a few in lower left corner which have smaller nuclei. This nodule displayed an especiallyhigh rate of mitotic activity (arrows). H & E. X 254 FIGURE 3.-Portion of exocrine pancreas from a rat treated for 6 months with biweekly 5-mg ip injections of azaserine/kg. An interlobular connective-tissue septum comprises the top border. Upper central portion shows a focus of pale-staining acinar cells with enlarged nuclei. This group of cells is poorly delineated from the surrounding acinarcells with smaller nuclei. H & E. X 262 LONGNECKER AND CRAWFORD 575

4 FIGURE 4.-Portion of pancreas from same rat as in figure 2. A hyperplastic nodule of acinar cells occupies center and left side of the field, and normal acinar cells are at top right and bottom. Cells in the nodule contain abundant zymogen and have nuclei slightly larger than those of the surrounding pancreas. Several mitotic figures are present (arrows). H & E. X 315 FIGURE 5.-Portion of exocrine pancreas from rat killed 5 months after receiving the first of 12 biweekly 25-mg ip doses of azaserine/ kg. Note encapsulated mass of acinar cells with little zymogen in the centerof the picture. These are surrounded by normal-appearing acinar cells. An aldehyde fuchsin stain, used because of the islet-like appearance of the nodule, failed to demonstrate granulation of the cells. Lesion was interpreted as a small adenoma. H & E. X 190 FIGURE 5.-Portion of exocrine pancreas from rat killed after 5 months of treatment with 5 mg azaserine/kg ip I X/week. Central portion shows an encapsulated area of acinar cells, some of which contain significant zymogen. A small islet is at right adjacent to wall of a duct. H & E. X97 FIGURE 7.-Portion of exocrine pancreas from rat which died spontaneously 8 months after initiation of treatment with biweekly ip injections of 5 mg azaserine/kg, continued for 5 months. Central portion of field contains an encapsulated nodule of acinar cells lying at the edge of a lobule. Most of these cells contain less zymogen than the adjacent pancreas; however, a few appear to contain numerous zymogen granules. Lesion was interpreted as a small exocrine adenoma. H & E. X

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