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Response of the Syrian golden hamster to a nitrosourea amino acid carcinogen
Cancer Letters, 8 (1979) 163-168 0 Elsevier/North-Holland Scientific Publishers Ltd.
163
RESPONSE OF THE SYRIAN GOLDEN HAMSTER TO A NITROSOUREA AMINO ACID CARCINOGEN*
DANIEL S. LONGNECKER, HERMAN S. LILJA** Department
of Pathology,
THOMAS J. CURPHEY, JANICE I. FRENCH and
Dartmouth
Medical School,
Hanover,
NH 03755
(U.S.A.)
(Received 4 June 1979) (Accepted 7 August 1979)
SUMMARY
Syrian golden hamsters were treated with j@ -(N-methyl-l\r-nitrosocarbamoyl)-L-ornithine (MNCO), a nitrosourea amino acid, which has induced a high incidence of breast, kidney and skin neoplasms and a low incidence of pancreatic carcinoma in rats. MNCO induced a few breast and skin carcinomas, and a high incidence of foci of atypical acinar cells and of focal ductular abnormalities in the exocrine pancreas. The latter were similar to lesions observed in rats treated with MNCO. MNCO was less toxic and less effective as a carcinogen in hamster than in the rat.
INTRODUCTION
The nitrosourea amino acid N6 -(N-methyl-N-nitrosocarbamoyl)-Lornithine (MNCO), has been shown to induce neoplasms in breast, kidney, skin and pancreas of the Wistar rat [ 31. MNCO was synthesized for evaluation as a pancreatic carcinogen with the rationale that the N-methyl-Nnitrosourea moiety was likely to confer carcinogenic activity while the intact a-amino acid configuration of the diamino acid would provide a
Address all correspondence to: Daniel S. Longnecker, M.D., Department of Pathology, Dartmouth Medical School, Hanover, NH 03755, U.S.A. * Supported by Contract NIH-NCI-E-72-3274 and 3296, NIH-NOl-CP-55708, and NOl-CP-33378 from the National Cancer Institute. Preparation of this manuscript was completed while Dr. Longnecker was a Guest Worker in the Laboratory of Toxicology, National Cancer Institute, National Institutes of Health, Bethesda, MD, U.S.A. ** Present address: MA 01608, U.S.A.
Dr. H.S. Lilja, E.G. and G. Mason Research Institute, Worcester,
164
basis for uptake by the pancreas. A simiiar rationale had led to evaluation of azaserine as a pancreatic carcinogen [ 21. MNCO has been shown to induce DNA damage in the rat pancreas using alkaline sucrose gradient analysis [l] and to induce atypical acinar cell nodules, adenomas and carcinomas in rat pancreas [3, and in preparation]. Dramatic differences have been noted in the response of different species to specific carcinogens. Although several chemicals have induced carcinomas of the pancreas in rats or hamsters, none has been shown to be effective in both species. Notably, azaserine has induced pancreatic cancers in rats but failed in hamsters [2,7], and derivatives of dipropyl-nitrosamine have induced carcinomas in hamster pancreas but failed in the rat and guinea pig [4- -61. We have studied the effect of MNCO in hamster using doses and regimens which can be compared to previous studies in rats and found that MNCO is less effective as a carcinogen in hamsters than in Wistar rats. We did observe a low incidence of breast and skin cancers and a high incidence of foci of atypical acinar cells and ductular abnormalities in the pancreas of MNCOtreated hamsters. MATERIALS
AND METHODS
Syrian golden hamsters obtained from Charles River Breeding Laboratories, Wilmington, MA, were housed in groups of 4 in plastic cages with wood shavings as bedding and fed commercial laboratory chow (Ralston Purina Co., St. Louis, MO) and water ad lib. MNCO was prepared from commercially obtained chemicals. Its synthesis will be described elsewhere (Curphey, T.J., in preparation). MNCO was dissolved in 0.9% NaCl and injected i.p. at doses of 218 and 70 mg/kg. The formula weight is 218. Group 1 received 6 injections of 218 mg/kg at weekly intervals. Group 2 received 6 weekly injections of 70 mg/kg. Group 3 received saline. Hamsters were 22. days old when first injected. All survivors were killed for autopsy 6 months after initial injection. The animals were killed by decapitation and skin, breast and all thoracic and abdominal viscera were examined for tumors or other abnormalities. All grossly observed tumors were sampled and, in the absence of such lesions, all of the pancreas and sections of kidney, liver and testis or ovary were routinely fixed in Susa’s solution, embedded in paraffin, and sections stained with hematoxylin and eosin. RESULTS
Carcinomas were present in breast and skin of several group 1 female hamsters, but not in males or in low dose or control groups (Table 1). Two females had epithelial breast neoplasms which were histologically
165 TABLE
1
MNCO-INDUCED Group
LESIONS
MNCO”
IN THE Sex
SYRIAN nb
(mg/kg)
1
218
2
70
3
0
GOLDEN
HAMSTER
Pancreatic
lesionsC
AC
DC
Renal cysts
Other
F M F
11 8 6
10 6 2
11 7 0
7 5 0
5* -
M F M
10 6 8
6 0 0
4 0 0
1 0 0
le -
A MNCO was dissolved in 0.9% NaCl and 6 weekly i.p. injections were given at the dose indicated. Group 3 was given 0.9% NaCI. ’ The number of hamsters surviving until 6 months is given. There was 1 early death in group 1, 2 in group 2, and 1 in group 3, i.e. the original group sizes were 20, 18 and 1.5, respectively. ” Pancreatic lesions are (AC) foci of atypical acinar cells and (DC) ductal complexes. !$ee the text for descriptions. Two hamsters had adenocarcinoma of the breast and 3 had squamous carcinomas of the skin Another had an apparently benign epidermal inclusion cyst of skin, and there was 1 granulosa cell tumor of ovary. e One hamster had a focus of anaplastic stromal cells in the kidney consistent with a microscopic mesenchymal tumor.
consistent with a diagnosis of adenocarcinoma although there was no evidence of invasion or metastasis. The tumors included both ductal and solid epithellal areas. Grossly, they measured 0.5-0.8 cm in diameter. A third hamster had intraductal epithelial hyperplasia in the breast. Three female hamsters had squamous cell carcinomas of the skin. The largest measured 1 cm in diameter. None had metastasized. Although no neoplasms were present in pancreas or kidney of MNCOtreated hamsters, MNCO-induced lesions were prevalent in these tissues. Pancreas Sixteen of 19 hamsters in group 1 and 10 of 16 hamsters in group 2 had foci of dysplastic acinar cells in the pancreas (Table 1). None of the group 3 control hamsters had such foci. These foci consisted of circumscribed groups of phenotypically altered cells which differed from surrounding acinar cells by virtue of one of the following changes: (1) nuclear enlargement (Fig, 1); (2) diminished zymogen content; (3) increased zymogen content; or (4) decreased cytoplasmic mass. These foci were generally smaller than islets and ranged up to about 250 pm in diameter. The average number of such lesions/pancreas was greater in group 1 than in group 2. As many as 11 foci/pancreas were counted. Males appeared to be slightly more responsive at the lower dose.
166
A second type of lesion was encountered in 18 of 19 group 1 hamsters and in 4 of 16 group 2 hamsters, but was absent in group 3. These appeared as groups of small duct-like structures lined by cuboidal epithelium which did not display anaplasia or hyperplastic (papillary) change. Some of these ductular complexes were surrounded by fibrous tissue while others had minimal fibrous stroma. A few were intimately associated with islets, sometimes engulfing islet tissue (Fig. 2). These lesions were not associated with evidence of inflammation. The number of focal ductular complexes/pancreas was greater in group 1 than in group 2 as was the average size of the lesions. The epithelium of the main pancreatic duct and interlobular ducts appeared normal in all groups. There was histologic evidence of mild increase in stromal fat in the pancreases of group 1 hamsters in comparison with groups 2 and 3, but the wet weight of group 1 pancreases was not decreased below that of controls. Kidney Twelve of 19 group 1 hamsters had small cysts in the renal cortex lined by cuboidal or columnar epithelium similar to that of the renal tubules. The diameter of the cysts averaged about 1 mm. The epithelial cells in some cysts had enlarged nuclei compared with surrounding tubules, but there were not marked anaplastic or hyperplastic changes.
Fig. 1. A subserosal group of atypical acinar cells in the center of this field has enlarged nuclei and altered cytoplasmic staining. From the pancreas of an MNCO-treated hamster. H and E x 360.
167
Fig. 2. The pancreas of an MNCO-treated hamster contains a group of dilated duct-like structures which surround an islet. Adjacent acinar cells have normal appearance. H and E. x 145. DISCUSSION
The low incidence of neoplasms of all sites among MNCO-treated hamsters is in striking contrast to the high incidence of breast, kidney, and skin neoplasms previously noted in MNCO-treated rats [3]. MNCO seems to be less effective as a carcinogen in hamster than in rat and it provides another example of differing sensitivity between species to a specific carcinogen. The pancreatic lesions are of interest although none was regarded as a neoplasm. The foci of atypical acinar cells in the pancreases of MNCOtreated hamsters were similar in appearance to the smaller variants of the focal acinar cell lesions induced in rats by azaserine [ 2,7], MNCO [ 31, and 4-hydroxyaminoquinoline-l-oxide [ 81. All of these compounds have induced pancreatic carcinomas in rats. In rats, a small fraction of atypical acinar cell nodules appear to progress to cancers. A few small atypical acinar cell nodules have been encountered in pancerases of azaserine treated hamsters [ 71, although the incidence and number was very low. The focal ductular complexes are of interest because they seem to overlap the spectrum of ductular lesions induced in hamster pancreas by N-nitroso bis(2-oxopropyl)amine and related compounds which also induce hyperplastic and dysplastic ductular lesions as well as pancreatic’ductal carcinomas in this species 151. MNCO has induced cystic ductural lesions but not dysplastic ductular changes in the rat pancreas. Thus, MNCO has induced morphologic
168
lesions of acinar cells and ductular cells which are qualitatively similar in rat and hamster pancreas. Both lesions have been produced by other chemicals which are effective pancreatic carcinogens in one or the other species. MNCO and related nitrosourea amino acids are highly effective renal carcinogens in rats inducing both epithelial and mesenchymal tumors (in preparation). Although no renal neoplasms developed in hamsters, it seems likely that the small epithelial lined cysts noted in groups 1 and 2 reflect a renal effect of the carcinogen. Similar small renal cysts were seen in MNCO-treated rats. The low mortality in the high dose group suggests that hamsters may tolerate larger doses of MNCO than rats which have shown a high mortality when treated with similar regimens (in preparation). Higher doses, longer treatment or a longer period of observation following treatment seem likely to yield a higher incidence of skin and breast cancers in hamsters and might also cause progression of pancreatic or renal lesions such as were noted in the present study to form neoplasms. No animals were observed for longer than 6 months in the present study. Such studies may be of special interest in the hamster because MNCO induces focal atypical acinar cell lesions which have been previously noted primarily in rat models of pancreatic carcinogenesis. This provides an approach for evaluation of the relative importance of species and chemical agent in determining the histogenesis of pancreatic cancer. ACKNOWLEDGEMENTS
Joan Johnson (Histology) technical assistance.
and Richard Markley
(Photography)
provided
REFERENCES 1 Lilja, H.S., Curphey, T.J., Yager, J.D., Jr. and Longnecker, D.S. (1978) Persistence of DNA damage in rat pancreas following administration of three carcinogens and/or mutagens. Chem.-Biol. Interact., 22, 287-295. 2 Lopgnecker, D.S. and Curphey, T.J. (1975) Adenocarcinoma of the pancreas in azaserine-treated rats. Cancer Res., 35, 2249-2258. 3 Longnecker, D.S., Curphey, T.J., Lilja, H.S., French, J.I. and Daniel, D.S. (1978) Carcinogenicity of a methylnitrosourea amino acid in rats. Fed. Proc., 37, 231. 4 Pour, P. (1978) A new and advantageous model for colorectal cancer: its comparison with previous models for a common human disease. Cancer Letters, 4, 293-298. 5 Pour, P., Althoff, .J. and Takahashi, M. (1977) Early lesions of pancreatic ductal carcinoma in the hamster model. Am. J. Pathol., 88, 291-308. 6 Rao, M.S. and Pour, P. (1978) Development of biliary and hepatic neoplasms in guinea pigs treated with N-nitrosobis(2-oxopropyl)amine. Cancer Letters, 5, 31-34. 7 Roebuck, B.D. and Longnecker, D.S. (1977) Species and rat strain variation in pancreatic nodule induction by azaserine. J. Natl. Cancer Inst., 59, 1273-1277. 8 Shinozuka, H., Popp, J.A. and Konishi, Y. (1976) Ultrastructures of atypical acinar cell nodules in rat pancreas induced by 4-hydroxyaminoquinoline-l-oxide. Lab Invest., 34, 501-509.
163
RESPONSE OF THE SYRIAN GOLDEN HAMSTER TO A NITROSOUREA AMINO ACID CARCINOGEN*
DANIEL S. LONGNECKER, HERMAN S. LILJA** Department
of Pathology,
THOMAS J. CURPHEY, JANICE I. FRENCH and
Dartmouth
Medical School,
Hanover,
NH 03755
(U.S.A.)
(Received 4 June 1979) (Accepted 7 August 1979)
SUMMARY
Syrian golden hamsters were treated with j@ -(N-methyl-l\r-nitrosocarbamoyl)-L-ornithine (MNCO), a nitrosourea amino acid, which has induced a high incidence of breast, kidney and skin neoplasms and a low incidence of pancreatic carcinoma in rats. MNCO induced a few breast and skin carcinomas, and a high incidence of foci of atypical acinar cells and of focal ductular abnormalities in the exocrine pancreas. The latter were similar to lesions observed in rats treated with MNCO. MNCO was less toxic and less effective as a carcinogen in hamster than in the rat.
INTRODUCTION
The nitrosourea amino acid N6 -(N-methyl-N-nitrosocarbamoyl)-Lornithine (MNCO), has been shown to induce neoplasms in breast, kidney, skin and pancreas of the Wistar rat [ 31. MNCO was synthesized for evaluation as a pancreatic carcinogen with the rationale that the N-methyl-Nnitrosourea moiety was likely to confer carcinogenic activity while the intact a-amino acid configuration of the diamino acid would provide a
Address all correspondence to: Daniel S. Longnecker, M.D., Department of Pathology, Dartmouth Medical School, Hanover, NH 03755, U.S.A. * Supported by Contract NIH-NCI-E-72-3274 and 3296, NIH-NOl-CP-55708, and NOl-CP-33378 from the National Cancer Institute. Preparation of this manuscript was completed while Dr. Longnecker was a Guest Worker in the Laboratory of Toxicology, National Cancer Institute, National Institutes of Health, Bethesda, MD, U.S.A. ** Present address: MA 01608, U.S.A.
Dr. H.S. Lilja, E.G. and G. Mason Research Institute, Worcester,
164
basis for uptake by the pancreas. A simiiar rationale had led to evaluation of azaserine as a pancreatic carcinogen [ 21. MNCO has been shown to induce DNA damage in the rat pancreas using alkaline sucrose gradient analysis [l] and to induce atypical acinar cell nodules, adenomas and carcinomas in rat pancreas [3, and in preparation]. Dramatic differences have been noted in the response of different species to specific carcinogens. Although several chemicals have induced carcinomas of the pancreas in rats or hamsters, none has been shown to be effective in both species. Notably, azaserine has induced pancreatic cancers in rats but failed in hamsters [2,7], and derivatives of dipropyl-nitrosamine have induced carcinomas in hamster pancreas but failed in the rat and guinea pig [4- -61. We have studied the effect of MNCO in hamster using doses and regimens which can be compared to previous studies in rats and found that MNCO is less effective as a carcinogen in hamsters than in Wistar rats. We did observe a low incidence of breast and skin cancers and a high incidence of foci of atypical acinar cells and ductular abnormalities in the pancreas of MNCOtreated hamsters. MATERIALS
AND METHODS
Syrian golden hamsters obtained from Charles River Breeding Laboratories, Wilmington, MA, were housed in groups of 4 in plastic cages with wood shavings as bedding and fed commercial laboratory chow (Ralston Purina Co., St. Louis, MO) and water ad lib. MNCO was prepared from commercially obtained chemicals. Its synthesis will be described elsewhere (Curphey, T.J., in preparation). MNCO was dissolved in 0.9% NaCl and injected i.p. at doses of 218 and 70 mg/kg. The formula weight is 218. Group 1 received 6 injections of 218 mg/kg at weekly intervals. Group 2 received 6 weekly injections of 70 mg/kg. Group 3 received saline. Hamsters were 22. days old when first injected. All survivors were killed for autopsy 6 months after initial injection. The animals were killed by decapitation and skin, breast and all thoracic and abdominal viscera were examined for tumors or other abnormalities. All grossly observed tumors were sampled and, in the absence of such lesions, all of the pancreas and sections of kidney, liver and testis or ovary were routinely fixed in Susa’s solution, embedded in paraffin, and sections stained with hematoxylin and eosin. RESULTS
Carcinomas were present in breast and skin of several group 1 female hamsters, but not in males or in low dose or control groups (Table 1). Two females had epithelial breast neoplasms which were histologically
165 TABLE
1
MNCO-INDUCED Group
LESIONS
MNCO”
IN THE Sex
SYRIAN nb
(mg/kg)
1
218
2
70
3
0
GOLDEN
HAMSTER
Pancreatic
lesionsC
AC
DC
Renal cysts
Other
F M F
11 8 6
10 6 2
11 7 0
7 5 0
5* -
M F M
10 6 8
6 0 0
4 0 0
1 0 0
le -
A MNCO was dissolved in 0.9% NaCl and 6 weekly i.p. injections were given at the dose indicated. Group 3 was given 0.9% NaCI. ’ The number of hamsters surviving until 6 months is given. There was 1 early death in group 1, 2 in group 2, and 1 in group 3, i.e. the original group sizes were 20, 18 and 1.5, respectively. ” Pancreatic lesions are (AC) foci of atypical acinar cells and (DC) ductal complexes. !$ee the text for descriptions. Two hamsters had adenocarcinoma of the breast and 3 had squamous carcinomas of the skin Another had an apparently benign epidermal inclusion cyst of skin, and there was 1 granulosa cell tumor of ovary. e One hamster had a focus of anaplastic stromal cells in the kidney consistent with a microscopic mesenchymal tumor.
consistent with a diagnosis of adenocarcinoma although there was no evidence of invasion or metastasis. The tumors included both ductal and solid epithellal areas. Grossly, they measured 0.5-0.8 cm in diameter. A third hamster had intraductal epithelial hyperplasia in the breast. Three female hamsters had squamous cell carcinomas of the skin. The largest measured 1 cm in diameter. None had metastasized. Although no neoplasms were present in pancreas or kidney of MNCOtreated hamsters, MNCO-induced lesions were prevalent in these tissues. Pancreas Sixteen of 19 hamsters in group 1 and 10 of 16 hamsters in group 2 had foci of dysplastic acinar cells in the pancreas (Table 1). None of the group 3 control hamsters had such foci. These foci consisted of circumscribed groups of phenotypically altered cells which differed from surrounding acinar cells by virtue of one of the following changes: (1) nuclear enlargement (Fig, 1); (2) diminished zymogen content; (3) increased zymogen content; or (4) decreased cytoplasmic mass. These foci were generally smaller than islets and ranged up to about 250 pm in diameter. The average number of such lesions/pancreas was greater in group 1 than in group 2. As many as 11 foci/pancreas were counted. Males appeared to be slightly more responsive at the lower dose.
166
A second type of lesion was encountered in 18 of 19 group 1 hamsters and in 4 of 16 group 2 hamsters, but was absent in group 3. These appeared as groups of small duct-like structures lined by cuboidal epithelium which did not display anaplasia or hyperplastic (papillary) change. Some of these ductular complexes were surrounded by fibrous tissue while others had minimal fibrous stroma. A few were intimately associated with islets, sometimes engulfing islet tissue (Fig. 2). These lesions were not associated with evidence of inflammation. The number of focal ductular complexes/pancreas was greater in group 1 than in group 2 as was the average size of the lesions. The epithelium of the main pancreatic duct and interlobular ducts appeared normal in all groups. There was histologic evidence of mild increase in stromal fat in the pancreases of group 1 hamsters in comparison with groups 2 and 3, but the wet weight of group 1 pancreases was not decreased below that of controls. Kidney Twelve of 19 group 1 hamsters had small cysts in the renal cortex lined by cuboidal or columnar epithelium similar to that of the renal tubules. The diameter of the cysts averaged about 1 mm. The epithelial cells in some cysts had enlarged nuclei compared with surrounding tubules, but there were not marked anaplastic or hyperplastic changes.
Fig. 1. A subserosal group of atypical acinar cells in the center of this field has enlarged nuclei and altered cytoplasmic staining. From the pancreas of an MNCO-treated hamster. H and E x 360.
167
Fig. 2. The pancreas of an MNCO-treated hamster contains a group of dilated duct-like structures which surround an islet. Adjacent acinar cells have normal appearance. H and E. x 145. DISCUSSION
The low incidence of neoplasms of all sites among MNCO-treated hamsters is in striking contrast to the high incidence of breast, kidney, and skin neoplasms previously noted in MNCO-treated rats [3]. MNCO seems to be less effective as a carcinogen in hamster than in rat and it provides another example of differing sensitivity between species to a specific carcinogen. The pancreatic lesions are of interest although none was regarded as a neoplasm. The foci of atypical acinar cells in the pancreases of MNCOtreated hamsters were similar in appearance to the smaller variants of the focal acinar cell lesions induced in rats by azaserine [ 2,7], MNCO [ 31, and 4-hydroxyaminoquinoline-l-oxide [ 81. All of these compounds have induced pancreatic carcinomas in rats. In rats, a small fraction of atypical acinar cell nodules appear to progress to cancers. A few small atypical acinar cell nodules have been encountered in pancerases of azaserine treated hamsters [ 71, although the incidence and number was very low. The focal ductular complexes are of interest because they seem to overlap the spectrum of ductular lesions induced in hamster pancreas by N-nitroso bis(2-oxopropyl)amine and related compounds which also induce hyperplastic and dysplastic ductular lesions as well as pancreatic’ductal carcinomas in this species 151. MNCO has induced cystic ductural lesions but not dysplastic ductular changes in the rat pancreas. Thus, MNCO has induced morphologic
168
lesions of acinar cells and ductular cells which are qualitatively similar in rat and hamster pancreas. Both lesions have been produced by other chemicals which are effective pancreatic carcinogens in one or the other species. MNCO and related nitrosourea amino acids are highly effective renal carcinogens in rats inducing both epithelial and mesenchymal tumors (in preparation). Although no renal neoplasms developed in hamsters, it seems likely that the small epithelial lined cysts noted in groups 1 and 2 reflect a renal effect of the carcinogen. Similar small renal cysts were seen in MNCO-treated rats. The low mortality in the high dose group suggests that hamsters may tolerate larger doses of MNCO than rats which have shown a high mortality when treated with similar regimens (in preparation). Higher doses, longer treatment or a longer period of observation following treatment seem likely to yield a higher incidence of skin and breast cancers in hamsters and might also cause progression of pancreatic or renal lesions such as were noted in the present study to form neoplasms. No animals were observed for longer than 6 months in the present study. Such studies may be of special interest in the hamster because MNCO induces focal atypical acinar cell lesions which have been previously noted primarily in rat models of pancreatic carcinogenesis. This provides an approach for evaluation of the relative importance of species and chemical agent in determining the histogenesis of pancreatic cancer. ACKNOWLEDGEMENTS
Joan Johnson (Histology) technical assistance.
and Richard Markley
(Photography)
provided
REFERENCES 1 Lilja, H.S., Curphey, T.J., Yager, J.D., Jr. and Longnecker, D.S. (1978) Persistence of DNA damage in rat pancreas following administration of three carcinogens and/or mutagens. Chem.-Biol. Interact., 22, 287-295. 2 Lopgnecker, D.S. and Curphey, T.J. (1975) Adenocarcinoma of the pancreas in azaserine-treated rats. Cancer Res., 35, 2249-2258. 3 Longnecker, D.S., Curphey, T.J., Lilja, H.S., French, J.I. and Daniel, D.S. (1978) Carcinogenicity of a methylnitrosourea amino acid in rats. Fed. Proc., 37, 231. 4 Pour, P. (1978) A new and advantageous model for colorectal cancer: its comparison with previous models for a common human disease. Cancer Letters, 4, 293-298. 5 Pour, P., Althoff, .J. and Takahashi, M. (1977) Early lesions of pancreatic ductal carcinoma in the hamster model. Am. J. Pathol., 88, 291-308. 6 Rao, M.S. and Pour, P. (1978) Development of biliary and hepatic neoplasms in guinea pigs treated with N-nitrosobis(2-oxopropyl)amine. Cancer Letters, 5, 31-34. 7 Roebuck, B.D. and Longnecker, D.S. (1977) Species and rat strain variation in pancreatic nodule induction by azaserine. J. Natl. Cancer Inst., 59, 1273-1277. 8 Shinozuka, H., Popp, J.A. and Konishi, Y. (1976) Ultrastructures of atypical acinar cell nodules in rat pancreas induced by 4-hydroxyaminoquinoline-l-oxide. Lab Invest., 34, 501-509.