A Potent Pancreatic Carcinogen in Syrian Hamsters: N-Nitrosobis(2-oxopropyl)amine 1,2 Parviz Pour, 3 Jurgen Althoff,3 F. W. Kruger, 4, 5 and U. Mohr 6
Among the assumed l3-metabolites of DPN, the compounds BHP and BAP were recently found to be pancreatic carcinogens in Syrian hamsters (l -o ). Similar in effect, they also induced neoplasms of the upper and lower respiratory tract, liver, and kidneys (l ,2). Preliminary results from studies of BOP, another postulated 13metabolite of DPN, indicated its higher toxicity and a more specific pancreatic carcinogenicity, in comparison to those of BHP and BAP (5). The present investigation details the biologic effects of BOP in Syrian hamsters treated either once or repeatedly. MATERIALS AND METHODS
Outbred, 8-week-old Syrian hamsters from the Eppley colony were housed in groups of five by sex in plastic cages. They were kept under standard conditions and given Wayne pelleted diet and water ad libitum. BOP was synthesized by Dr. F. W. Kruger. To determine the LD50 by Weil's method (7), a single sc injection of BOP in 0.9% saline was administered to five females and five males at doses of 500,250, 125, and 62.5 mg/kg body weight. Animals that survived the observation period of 8 days were maintained for life (group A). For the chronic study (group B), subgroups of 20 females and 20 males received once weekly sc injections of BOP in 0.9% saline for life in doses of 10 (group B-1), 5 (group B-2), and 2.5 (group B-3) mg/kg body weight. Controls (group C) were treated with the solvent only. Following autopsy, organs were fixed in 10% buffered formalin. Bones were decalcified prior to histologic procedures. Tissues were embedded in Paraplast and sections stained with H & E. The nasal and paranasal cavities, larynx, trachea, lungs, pancreas, extrahepatic bile ducts, kidneys, urinary bladder, and vagina were cut into step sections. The number of tumors per organ was determined by counting the tumors in these sections. RESULTS
The LD50 of BOP was 154 mg/kg body weight for females and 89 rug/kg body weight for males. Cell necrosis of respiratory and intestinal epithelium, liver, pancreas, kidneys, ovaries, and testes, and hemorrhages VOL. 58, NO.5, MAY 1977
of lungs and abdominal organs occurred in animals that died during the 8-day observation period. Among survivors of the LD50 (groups A-I and A-2), all hamsters dying after 24 weeks had tumors (table 1), often simultaneously in several organs. Most affected was the liver, followed by the vagina, pancreas, gallbladder, and other organs. In the chronic experiment (group B), a positive doseresponse relationship relative to survival, body weight, and tumor incidence was observed in both sexes; however, these findings were not unequivocal for the multiplicity of induced neoplasms (table 1). Except for 3 females and 6 males that died between 12 and 19 weeks, all treated hamsters had tumors in one or several organs (table 1). Group B had a high incidence of pancreatic neoplasms, followed by tumors of the lungs, liver, and other sites. Tumors of Pancreas
The incidence of pancreatic neoplasms was 50 and 80%, respectively, in groups A-I and A-2 (single injection) and between 67 and 100% in group B animals (multiple injection) (table 1). No group A males displayed these tumors. In group B hamsters, marked sex differences were observed only in males in subgroup B1,6 of which died between 12 and 17 weeks from the beginning of the experiment. However, proliferative and preneoplastic alterations of the pancreatic ducts were found in these animals, as well as in all other treated animals. All group A hamsters with pancreatic neoplasms simultaneously had tumors of the liver (5 females), gallbladder (5 females), vagina (5 females), and lungs (2 females). In group B hamsters, 108 (92%) had pancreatic neoplasms: Of these, 33% (14 females and 23 males) had pancreatic tumors as their only induced lesion, and these animals died soon after treatment was begun (average survival: 25 wk for females and 22 wk for males). In hamsters surviving longer, tumors also developed in other organs but predominated in the lungs (42%) and liver (10%). Only 50% of all pancreatic lesions were grossly visible, ABBREVIATIONS USED: DPN = dipropylnitrosamine; BHP = N-nitrosobis(2-hydroxypropyl)amine; BAP = N-nitrosobis(2-acetoxypropyl)amine; BOP = N-nitrosobis(2-oxopropyl)amine; LD50 = median lethal dose; H & E = hematoxylin and eosin; HPOP = N-nitroso(2hydroxypropyl) (2-oxopropyl)amine. 1 Received September 9, 1976; accepted November 24, 1976. 2 Supported by Public Health Service contract NOI CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute. 3 Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42d St. and Dewey Ave., Omaha, Nebr. 68105. 41nstitut fur Toxicologie und Chemotherapie, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany. 5 Deceased. 6 Abteilung fur Experimentelle Pathologie der Medizinischen Hochschule, Hannover, Federal Republic of Germany.
1449
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
ABSTRACT -N-Nitrosobis{2-oxopropyl)amine (BOP), a further postulated p-metabolite of di-n-propylnitrosamine, induced a high incidence of pancreatic duct adenomas and adenocarcinomas as early as 13 weeks in Syrian hamsters receiving weekly sc injections for life and a few pancreatic adenomas, after 28 weeks, in those given a single sc dose. Compared to related compounds, N-nitrosobis{2-hydroxypropyl)amine and N-nitrosobis{2-acetoxypropyl)amine which are also pancreatic carcinogens, BOP induced only a few neoplasms of the lung, liver, and kidney and none in the nasal cavity, larynx, and trachea. The results therefore indicate progress in developing a more specific model for pancreatic carcinogenesis studies.-J Natl Cancer Inst 58: 14491453,1977.
1450
POUR, ALTHOFF, KRUGER, AND MOHR TABLE I.-Bioassay results in Syrian hamsters treated with BOP
Group
Average Dose Effective survival mg/kg No. of wk hamsters body wt t;l
A-I A-2 B-1 B-2 B-3 Controls
125 62.5 10 5 2.5 0
0
4 5 20 19 20 19
5 18 20 20 18
t;l
t;l
0
34 47 18 35 42 45
Average body wt g
20 14 22 42 57
t;l
0
78 105 105 86 84 102
No.oftumors per tumorbearing hamsters
Tumor-bearing hamsters No.(%)
4 4 19 18 19 3
59 74 85 71
106
t;l
0
(100) (80) (95) (95) (95) (16)
2 12 20 20 11
2.8 7.0 2.2 2.6 2.1 1.0
(40) (67) (100) (100) (61)
Tumors of the pancreas Ductal adenomas No.(%) t;l
0 1.0 1.6 1.4 2.0 0.6
2 4 17 9 18
(50) (80) (85) (47) (90)
Intraductal carcinomas No.(%)
Adenocarcinomas No.(%)
0
t;l
0
t;l
0
8 (44) 20 (100) 17 (85)
7 (35) 5 (26) 8 (40)
6 (30) 11 (55)
16 (75) 18 (95) 16 (80)
6 (33) 16 (80) 19 (95)
TABLE 2.-Tumor patterns (exclusive of pancreas) in BOP-treated Syrian hamsters
Lungs No.(%)
Group
A-I A-2 B-1 B-2 B-3 Controls
Gallbladder No.(%)
Liver No.(%)
t;l
0
0 3 (60) 6 (30) 14 (70) 13 (65)
0 0 1 (15) 5 (25) 16 (80)
0
0
t;l 4 1 6 4 1
(100) (20) (30) (21) (5) 0
t;l
0 0 2 (50) 1 (16) 0 0 0
1 4 7 6 2
(25) (80) (35) (32) (10) 0
Vagina No.(%)
Cowper's gland No.(%)
Others (sex and age in wk)"
0 0 0 7 (39) 1 (5) 0 0
3 3 1 2
(75) (60) (5) (11) 0
0 0 1 (6) 2 (10) 12 (60)
0
11 (61)
A B C F A
(t;l28) (t;l67), C (t;l67), D (t;l60), E (t;l43) (r;>23), F (n5) (t;l29), G (028) (038), D (t;l62,67), E (t;l55), G (063), H (t;l54), I (t;l67), J (055,64) A (t;l62; 073,73), H (t;l69; 073), K (t;l56), L (053,68), M (035), N (073)
a A=adrenal cortex adenoma; B=forestomach papilloma; C=thyroid adenoma; D=parathyroid adenoma; E=renal adenoma; F=cervical polyp; G=skin papilloma; H=thyroid carcinoma; I=blue nevus; J=basal cell carcinoma; K=vaginal fibroadenoma; L=malignant lymphoma; M=osteogenic sarcoma; N=islet cell adenoma.
and these were often multiple, 2- to 15-mm, grayishwhite nodules (average size, 6 mm), the size of which related proportionately to survival time. These lesions frequently appeared cystic upon sectioning and were distributed mainly in the body and tail regions of the pancreas. The neoplasms appeared as early as 28 weeks in both sexes of group A, and in group B-1 after 13 weeks (males) and 15 weeks (females). Histologically, the tumor incidence reached 100% in some groups; these neoplasms were duct adenomas, intraductal carcinomas, and adenocarcinomas, as described in (2-4). Foci of squamous cell metaplasia were observed in the margin of a 1.5-cm tumor invading the gastric pylorus of a B-1 female at the 28th treatment week (figs. 1, 2). Mixed ductal and islet cell-like neoplasms were found in two B3 males at 36 and 55 weeks, respectively. No acinar cell neoplasms were seen. Invasion of surrounding tissue by tumors (e.g., the peritoneum and pelvic cavity) occurred in 25 instances (16 females and 9 males) in B-2 and B-3 animals. Metastatic foci were seen in the regionallymph nodes of a female (44 wk) and male (51 wk) and in the lungs of a male (46 wk). Three B-2 females exhibited jaundice (at 38, 40, and 44 wk) due to tumor invasion of the common duct. Hemorrhagic ascites was observed in 3 females, diarrhea in 2 males, portal vein thrombosis in 2 females and 2 males, and lung thrombosis in 2 females with pancreatic carcinomas. Fat necrosis was not seen. Controls showed focal proliferation and
J
NATL CANCER INST
distension of pancreatic ducts (2 females) and portal vein thrombosis (2 females). Lung Tumors
The incidence of lung neoplasms was 0% in group AI, 60% in group A-2 (all females), and between 25 and 80% in group B-1, B-2, and B-3 hamsters, respectively. The frequency and multiplicity of tumors increased by decreasing the dose level (i.e., No. of tumors per animal by group: B-1, 1.2 in females; B-2, 8.8 in females, 0.3 in males; B-3, 5.5 in females, 6.3 in males). The first neoplasms were found at 43 weeks in group A females and at 15 and 17 weeks in group B males and females, respectively. Histologically, the tumors were mostly adenomas of an apparent alveologenic origin and less frequently were adenocarcinomas (5 females, 1 male, group B-1; 6 females, group B-2; 4 males, group B-3) and squamous cell carcinomas (1 female, group B-2; 2 females, group B-3) (table 2). Lung neoplasms always occurred simultaneously with tumors of the pancreas and other organs. Proliferative and dysplastic alterations of the epithelial lining were found in many treated hamsters, with or without lung tumors, and in a male control hamster (59 wk). Liver Tumors
Liver neoplasms were induced in groups A and B in VOL. 58, NO.5, MAY 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
Tumor sites
1451
BOP-A POTENT PANCREATIC CARCINOGEN
Gallbladder Tumors
Only group A females and 16 females and 7 males of group B had papillary polyps of the gallbladder, often of multiple origin. The tumor incidence decreased with lowered dose levels (table 2). The first tumor was found at 32 weeks in group A females and at 15 weeks in group B females. These neoplasms always occurred in hamsters with pancreatic tumors. In all instances, hepatic and common bile ducts were free of neoplastic growth. Focal proliferation of gallbladder epithelium was observed in 4 females (group B-2), in 7 males (1 in group B-1 and 6 in group B-2), and in 1 female control. Gallstones were found in 2 female controls. Tumors of Urogenital Organs
Renal adenomas were found in 1 group A and 1 group B female. However, regenerative and degenerative alterations, unrelated to dose level, were evident in the tubular epithelium of 64% (38 females and 37 males) of group B animals. Atypical cell proliferation of proximal tubules was observed in 2 males at 23 and 64 weeks, respectively. Acute inflammation or local proliferation of urinary bladder or urethral epithelium was seen in 5 females and 6 males (3 females and all males were from group B-1). The incidence of vaginal papillomas was higher in group B (table 2). All papillomas were close to the vaginal orifice and extended to the labiae. Papillary adenomas of Cowper's gland were found in incidences up to 60% in group B males (table 2), and hyperplasia of Cowper's gland in 3 group B-1 males (between 16 and 21 wk). Nearly all male controls older than 53 weeks had Cowper's gland adenomas (61 % incidence). Tumors of Other Sites
Epidermal tumors were found at injection sites in 1 female and 4 males (table 1). In males, 2 of these lesions were squamous cell papillomas; the others were basal cell carcinomas displaying focal cornification. The female had a small blue nevus. Lesions in other tissues (table 2) were within the range of spontaneously occurring neoplasms in Eppley colony hamsters. VOL. 58, NO.5, MAY 1977
DISCUSSION
The present investigation indicated that BOP is a potent pancreatic carcinogen in the Syrian hamster. Following weekly sc treatment of hamsters with this compound, a high incidence of pancreatic duct adenomas and adenocarcinomas was induced as early as 13 weeks. Animals without pancreatic neoplasms exhibited no other tumors; since these hamsters (all of which died early in the course of treatment) showed proliferative and preneoplastic alterations of the pancreas, the pancreatic tumor incidence may have reached 100% if animals had survived longer. The specific affinity of BOP for the hamster pancreas was also evidenced by the marked multiplicity of adenocarcinomas, their large size, and their potential for invasion, leading to mechanical obstruction of bile ducts and subsequent jaundice in 3 hamsters. Furthermore, a single dose of BOP led to development of pancreatic neoplasms; however, compared to chronic treatment, the tumor incidence was lower, the latency longer (32 wk), and all tumors benign. Conversely, the single BOP application yielded a significantly higher incidence of liver neoplasms, the frequency of which was twice as great in animals receiving 125 mg BOP/kg body weight than in those treated with half that dose. Also, in animals treated weekly for life, despite an increased survival rate, the incidence of liver neoplasms was decreased by lowering the dose. In the group receiving the lowest dose, only those hamsters surviving longer than 62 weeks developed hepatic neoplasms. These data may indicate that induction of liver neoplasms depends on administration of an adequate BOP dose (above 150 mg/kg body wt), regardless of treatment frequency. In the case of lung neoplasms, which showed similar latencies among the different subgroups but occurred in higher incidences at lower doses, the frequency of BOP application, rather than the quantity, may be important in tumorigenesis. Consequently, it may be possible to suppress or even to eliminate tumor induction in the liver and lungs by an appropriate dose regimen. Further studies in this regard are underway in this laboratory. BOP's toxicity and the tumor spectrum induced differed markedly from those of the related compounds, BHP and BAP. BOP induced pancreatic neoplasms with shorter latencies in almost all hamsters and at doses considerably lower than those used in BHP and BAP studies (1-6). Moreover, in contrast to the latter two compounds, BOP produced fewer liver and lung tumors and no tumors in the nasal cavity, larynx, and trachea. The similar biologic effects of BHP and BAP were assumed to be mediated by metabolic conversion of BAP to BHP (8). The different toxicity and tumorigenicity of BHP (and consequently also BAP) and BOP seem related to their metabolic fate, since BOP in hamsters gives HPOP as a major metabolite with traces of BHP. After BHP administration, however, unchanged BHP with only traces of HPOP was excreted (8). Direct bioassay of HPOP as a possible proximate pancreatic carcinogen is currently underway. Development of vaginal tumors after BOP treatment may be an effect of BHP, formed by metabolism from
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
an incidence directly related to dose; i.e., a higher dose induced a higher tumor incidence (table 2). The first neoplasms were observed at 28 weeks in groups A-I females and A-2 males, at 15 and 20 weeks in B-1 females and males, respectively, at 32 weeks in group B-2 females, and at 62 weeks in group B-3 females. Most neoplasms were cholangiomas, whereas hemangioendotheliomas (in 2 females of group A-2), cholangiocarcinomas (in 1 female and 1 male, group B-1), and hyperplastic nodules (in 2 females of group A-I) were found only occasionally (table 2). Animals with liver neoplasms had simultaneous tumors of other organs. Focal proliferation and distension of bile ducts, often with acute inflammation, were found in 81 hamsters (19 females and 15 males of group B-1, 16 females and 8 males of group B-2, and 13 females and 10 males of group B-3). Similar proliferative lesions were also seen in controls (5 females, 12 males).
1452
POUR, ALTHOFF, KRUGER, AND MOHR
BOP, since these neoplasms were also found in a high incidence and attributed to BHP formation when BAP was administered to hamsters (8). BOP-induced pancreatic neoplasms were morphologically similar to those found in man, and the presence of other conditions associated with pancreatic cancer, such as obstructive icterus, diarrhea, ascites, weight loss, and vascular thrombosis (all known to occur in human pancreatic cancer victims), indicated that these experiments represent a further step in developing an appropriate model for investigation of pancreatic carcinogenicity. The findings also make evident the significant advantages of the present model, compared to the few other equivalent studies on pancreatic tumor induction (913 ).
(5)
(6) (7) (8)
(9)
(10)
(1) POUR P, ALTHOFF ], GINGELL R, et al: A further pancreatic carcinogen in Syrian golden hamsters: N-Nitroso-bis(2-acetoxypropyl)amine. Cancer Lett 1:197-202, 1976 (2) POUR P, MOHR U, CARDESA A, et a1: Pancreatic neoplasms in an animal model: Morphological, biological and comparative studies. Cancer 36:379-389, 1975 (3) POUR P, KRUGER FW, ALTHOFF]: Effect of beta-oxidized nitrosamines on Syrian golden hamsters. III. 2,2'-Dihydroxy-di-npropylnitrosamine.] Nat! Cancer lnst 54:141-146,1975 (4) POUR P, KRUGER FW, ALTHOFF ], et al: A new approach for
J NATL CANCER INST
(11) (12) (13)
VOL. 58, NO.5, MAY 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
REFERENCES
induction of pancreatic neoplasms. Cancer Res 35:2259-2266, 1975 POUR P, ALTHOFF], KRUGER FW, et al: Induction of pancreatic neoplasms by 2,2'-dioxopropyl-N-propylnitrosamine . Cancer Lett 1:3-6, 1975 POUR P, ALTHOFF ], GINGELL R, et al: N-Nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters. Cancer Res 36:2877-2884, 1976 WElL CS: Tables for convenient calculation of median effective dose (LD50 or ED50) and instructions in their use. Biometrics 8:249-263, 1952 GINGELL R, WALLCAVE L, NAGEL D, et al: Metabolism of the pancreatic carcinogens N -nitroso-bis(2-oxopropyl)amine and N-nitroso-bis(2-hydroxypropyl)amine in the Syrian hamster. ] Nat! Cancer lnst 57:1175-1178,1976 DISSIN], MILLS LR, MAINS DL, et al: Experimental induction of pancreatic adenocarcinoma in rats. J Nat! Cancer lnst 55:857864, 1975 ELKORT R], HANDLER AH, WILLIAMS DL: Early neoplasia of rabbit pancreatic ductal cells induced by dimethylhydrazine. Cancer Res 35:2292-2294, 1975 HAYASHI Y, HASEGAWA T: Experimental pancreatic tumor in rats after intravenous injection of 4-hydroxyaminoquinoline 1oxide. Gann 62:329-330, 1971 LONGNECKER DS, CRAWFORD BG: Hyperplastic nodules and adenomas of exocrine pancreas in azaserine-treated rats. ] Nat! Cancer lnst 53:573-577, 1974 REDDY ]K, SVOBODA D], RAO MS: Susceptibility of an inbred strain of guinea pig to the induction of pancreatic adenocarcinoma by N-methyl-N-nitrosourea. ] Nat! Cancer lnst 52:991993, 1974
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
I.-Pancreatic adenocarcinoma showing local squamous cell metaplasia with keratinization. Larger nests of metaplastic squaous cells were found in periphery of tumor invading gastric pylorus. H & E. X 100 FIGURE 2.-Another area of same tumor showing transition of cylindric epithelium to squamous cells. H & E. x 100
FIGURE
POUR, ALTHOFF, KRUGER, AND MOHR
1453
Among the assumed l3-metabolites of DPN, the compounds BHP and BAP were recently found to be pancreatic carcinogens in Syrian hamsters (l -o ). Similar in effect, they also induced neoplasms of the upper and lower respiratory tract, liver, and kidneys (l ,2). Preliminary results from studies of BOP, another postulated 13metabolite of DPN, indicated its higher toxicity and a more specific pancreatic carcinogenicity, in comparison to those of BHP and BAP (5). The present investigation details the biologic effects of BOP in Syrian hamsters treated either once or repeatedly. MATERIALS AND METHODS
Outbred, 8-week-old Syrian hamsters from the Eppley colony were housed in groups of five by sex in plastic cages. They were kept under standard conditions and given Wayne pelleted diet and water ad libitum. BOP was synthesized by Dr. F. W. Kruger. To determine the LD50 by Weil's method (7), a single sc injection of BOP in 0.9% saline was administered to five females and five males at doses of 500,250, 125, and 62.5 mg/kg body weight. Animals that survived the observation period of 8 days were maintained for life (group A). For the chronic study (group B), subgroups of 20 females and 20 males received once weekly sc injections of BOP in 0.9% saline for life in doses of 10 (group B-1), 5 (group B-2), and 2.5 (group B-3) mg/kg body weight. Controls (group C) were treated with the solvent only. Following autopsy, organs were fixed in 10% buffered formalin. Bones were decalcified prior to histologic procedures. Tissues were embedded in Paraplast and sections stained with H & E. The nasal and paranasal cavities, larynx, trachea, lungs, pancreas, extrahepatic bile ducts, kidneys, urinary bladder, and vagina were cut into step sections. The number of tumors per organ was determined by counting the tumors in these sections. RESULTS
The LD50 of BOP was 154 mg/kg body weight for females and 89 rug/kg body weight for males. Cell necrosis of respiratory and intestinal epithelium, liver, pancreas, kidneys, ovaries, and testes, and hemorrhages VOL. 58, NO.5, MAY 1977
of lungs and abdominal organs occurred in animals that died during the 8-day observation period. Among survivors of the LD50 (groups A-I and A-2), all hamsters dying after 24 weeks had tumors (table 1), often simultaneously in several organs. Most affected was the liver, followed by the vagina, pancreas, gallbladder, and other organs. In the chronic experiment (group B), a positive doseresponse relationship relative to survival, body weight, and tumor incidence was observed in both sexes; however, these findings were not unequivocal for the multiplicity of induced neoplasms (table 1). Except for 3 females and 6 males that died between 12 and 19 weeks, all treated hamsters had tumors in one or several organs (table 1). Group B had a high incidence of pancreatic neoplasms, followed by tumors of the lungs, liver, and other sites. Tumors of Pancreas
The incidence of pancreatic neoplasms was 50 and 80%, respectively, in groups A-I and A-2 (single injection) and between 67 and 100% in group B animals (multiple injection) (table 1). No group A males displayed these tumors. In group B hamsters, marked sex differences were observed only in males in subgroup B1,6 of which died between 12 and 17 weeks from the beginning of the experiment. However, proliferative and preneoplastic alterations of the pancreatic ducts were found in these animals, as well as in all other treated animals. All group A hamsters with pancreatic neoplasms simultaneously had tumors of the liver (5 females), gallbladder (5 females), vagina (5 females), and lungs (2 females). In group B hamsters, 108 (92%) had pancreatic neoplasms: Of these, 33% (14 females and 23 males) had pancreatic tumors as their only induced lesion, and these animals died soon after treatment was begun (average survival: 25 wk for females and 22 wk for males). In hamsters surviving longer, tumors also developed in other organs but predominated in the lungs (42%) and liver (10%). Only 50% of all pancreatic lesions were grossly visible, ABBREVIATIONS USED: DPN = dipropylnitrosamine; BHP = N-nitrosobis(2-hydroxypropyl)amine; BAP = N-nitrosobis(2-acetoxypropyl)amine; BOP = N-nitrosobis(2-oxopropyl)amine; LD50 = median lethal dose; H & E = hematoxylin and eosin; HPOP = N-nitroso(2hydroxypropyl) (2-oxopropyl)amine. 1 Received September 9, 1976; accepted November 24, 1976. 2 Supported by Public Health Service contract NOI CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute. 3 Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42d St. and Dewey Ave., Omaha, Nebr. 68105. 41nstitut fur Toxicologie und Chemotherapie, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany. 5 Deceased. 6 Abteilung fur Experimentelle Pathologie der Medizinischen Hochschule, Hannover, Federal Republic of Germany.
1449
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
ABSTRACT -N-Nitrosobis{2-oxopropyl)amine (BOP), a further postulated p-metabolite of di-n-propylnitrosamine, induced a high incidence of pancreatic duct adenomas and adenocarcinomas as early as 13 weeks in Syrian hamsters receiving weekly sc injections for life and a few pancreatic adenomas, after 28 weeks, in those given a single sc dose. Compared to related compounds, N-nitrosobis{2-hydroxypropyl)amine and N-nitrosobis{2-acetoxypropyl)amine which are also pancreatic carcinogens, BOP induced only a few neoplasms of the lung, liver, and kidney and none in the nasal cavity, larynx, and trachea. The results therefore indicate progress in developing a more specific model for pancreatic carcinogenesis studies.-J Natl Cancer Inst 58: 14491453,1977.
1450
POUR, ALTHOFF, KRUGER, AND MOHR TABLE I.-Bioassay results in Syrian hamsters treated with BOP
Group
Average Dose Effective survival mg/kg No. of wk hamsters body wt t;l
A-I A-2 B-1 B-2 B-3 Controls
125 62.5 10 5 2.5 0
0
4 5 20 19 20 19
5 18 20 20 18
t;l
t;l
0
34 47 18 35 42 45
Average body wt g
20 14 22 42 57
t;l
0
78 105 105 86 84 102
No.oftumors per tumorbearing hamsters
Tumor-bearing hamsters No.(%)
4 4 19 18 19 3
59 74 85 71
106
t;l
0
(100) (80) (95) (95) (95) (16)
2 12 20 20 11
2.8 7.0 2.2 2.6 2.1 1.0
(40) (67) (100) (100) (61)
Tumors of the pancreas Ductal adenomas No.(%) t;l
0 1.0 1.6 1.4 2.0 0.6
2 4 17 9 18
(50) (80) (85) (47) (90)
Intraductal carcinomas No.(%)
Adenocarcinomas No.(%)
0
t;l
0
t;l
0
8 (44) 20 (100) 17 (85)
7 (35) 5 (26) 8 (40)
6 (30) 11 (55)
16 (75) 18 (95) 16 (80)
6 (33) 16 (80) 19 (95)
TABLE 2.-Tumor patterns (exclusive of pancreas) in BOP-treated Syrian hamsters
Lungs No.(%)
Group
A-I A-2 B-1 B-2 B-3 Controls
Gallbladder No.(%)
Liver No.(%)
t;l
0
0 3 (60) 6 (30) 14 (70) 13 (65)
0 0 1 (15) 5 (25) 16 (80)
0
0
t;l 4 1 6 4 1
(100) (20) (30) (21) (5) 0
t;l
0 0 2 (50) 1 (16) 0 0 0
1 4 7 6 2
(25) (80) (35) (32) (10) 0
Vagina No.(%)
Cowper's gland No.(%)
Others (sex and age in wk)"
0 0 0 7 (39) 1 (5) 0 0
3 3 1 2
(75) (60) (5) (11) 0
0 0 1 (6) 2 (10) 12 (60)
0
11 (61)
A B C F A
(t;l28) (t;l67), C (t;l67), D (t;l60), E (t;l43) (r;>23), F (n5) (t;l29), G (028) (038), D (t;l62,67), E (t;l55), G (063), H (t;l54), I (t;l67), J (055,64) A (t;l62; 073,73), H (t;l69; 073), K (t;l56), L (053,68), M (035), N (073)
a A=adrenal cortex adenoma; B=forestomach papilloma; C=thyroid adenoma; D=parathyroid adenoma; E=renal adenoma; F=cervical polyp; G=skin papilloma; H=thyroid carcinoma; I=blue nevus; J=basal cell carcinoma; K=vaginal fibroadenoma; L=malignant lymphoma; M=osteogenic sarcoma; N=islet cell adenoma.
and these were often multiple, 2- to 15-mm, grayishwhite nodules (average size, 6 mm), the size of which related proportionately to survival time. These lesions frequently appeared cystic upon sectioning and were distributed mainly in the body and tail regions of the pancreas. The neoplasms appeared as early as 28 weeks in both sexes of group A, and in group B-1 after 13 weeks (males) and 15 weeks (females). Histologically, the tumor incidence reached 100% in some groups; these neoplasms were duct adenomas, intraductal carcinomas, and adenocarcinomas, as described in (2-4). Foci of squamous cell metaplasia were observed in the margin of a 1.5-cm tumor invading the gastric pylorus of a B-1 female at the 28th treatment week (figs. 1, 2). Mixed ductal and islet cell-like neoplasms were found in two B3 males at 36 and 55 weeks, respectively. No acinar cell neoplasms were seen. Invasion of surrounding tissue by tumors (e.g., the peritoneum and pelvic cavity) occurred in 25 instances (16 females and 9 males) in B-2 and B-3 animals. Metastatic foci were seen in the regionallymph nodes of a female (44 wk) and male (51 wk) and in the lungs of a male (46 wk). Three B-2 females exhibited jaundice (at 38, 40, and 44 wk) due to tumor invasion of the common duct. Hemorrhagic ascites was observed in 3 females, diarrhea in 2 males, portal vein thrombosis in 2 females and 2 males, and lung thrombosis in 2 females with pancreatic carcinomas. Fat necrosis was not seen. Controls showed focal proliferation and
J
NATL CANCER INST
distension of pancreatic ducts (2 females) and portal vein thrombosis (2 females). Lung Tumors
The incidence of lung neoplasms was 0% in group AI, 60% in group A-2 (all females), and between 25 and 80% in group B-1, B-2, and B-3 hamsters, respectively. The frequency and multiplicity of tumors increased by decreasing the dose level (i.e., No. of tumors per animal by group: B-1, 1.2 in females; B-2, 8.8 in females, 0.3 in males; B-3, 5.5 in females, 6.3 in males). The first neoplasms were found at 43 weeks in group A females and at 15 and 17 weeks in group B males and females, respectively. Histologically, the tumors were mostly adenomas of an apparent alveologenic origin and less frequently were adenocarcinomas (5 females, 1 male, group B-1; 6 females, group B-2; 4 males, group B-3) and squamous cell carcinomas (1 female, group B-2; 2 females, group B-3) (table 2). Lung neoplasms always occurred simultaneously with tumors of the pancreas and other organs. Proliferative and dysplastic alterations of the epithelial lining were found in many treated hamsters, with or without lung tumors, and in a male control hamster (59 wk). Liver Tumors
Liver neoplasms were induced in groups A and B in VOL. 58, NO.5, MAY 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
Tumor sites
1451
BOP-A POTENT PANCREATIC CARCINOGEN
Gallbladder Tumors
Only group A females and 16 females and 7 males of group B had papillary polyps of the gallbladder, often of multiple origin. The tumor incidence decreased with lowered dose levels (table 2). The first tumor was found at 32 weeks in group A females and at 15 weeks in group B females. These neoplasms always occurred in hamsters with pancreatic tumors. In all instances, hepatic and common bile ducts were free of neoplastic growth. Focal proliferation of gallbladder epithelium was observed in 4 females (group B-2), in 7 males (1 in group B-1 and 6 in group B-2), and in 1 female control. Gallstones were found in 2 female controls. Tumors of Urogenital Organs
Renal adenomas were found in 1 group A and 1 group B female. However, regenerative and degenerative alterations, unrelated to dose level, were evident in the tubular epithelium of 64% (38 females and 37 males) of group B animals. Atypical cell proliferation of proximal tubules was observed in 2 males at 23 and 64 weeks, respectively. Acute inflammation or local proliferation of urinary bladder or urethral epithelium was seen in 5 females and 6 males (3 females and all males were from group B-1). The incidence of vaginal papillomas was higher in group B (table 2). All papillomas were close to the vaginal orifice and extended to the labiae. Papillary adenomas of Cowper's gland were found in incidences up to 60% in group B males (table 2), and hyperplasia of Cowper's gland in 3 group B-1 males (between 16 and 21 wk). Nearly all male controls older than 53 weeks had Cowper's gland adenomas (61 % incidence). Tumors of Other Sites
Epidermal tumors were found at injection sites in 1 female and 4 males (table 1). In males, 2 of these lesions were squamous cell papillomas; the others were basal cell carcinomas displaying focal cornification. The female had a small blue nevus. Lesions in other tissues (table 2) were within the range of spontaneously occurring neoplasms in Eppley colony hamsters. VOL. 58, NO.5, MAY 1977
DISCUSSION
The present investigation indicated that BOP is a potent pancreatic carcinogen in the Syrian hamster. Following weekly sc treatment of hamsters with this compound, a high incidence of pancreatic duct adenomas and adenocarcinomas was induced as early as 13 weeks. Animals without pancreatic neoplasms exhibited no other tumors; since these hamsters (all of which died early in the course of treatment) showed proliferative and preneoplastic alterations of the pancreas, the pancreatic tumor incidence may have reached 100% if animals had survived longer. The specific affinity of BOP for the hamster pancreas was also evidenced by the marked multiplicity of adenocarcinomas, their large size, and their potential for invasion, leading to mechanical obstruction of bile ducts and subsequent jaundice in 3 hamsters. Furthermore, a single dose of BOP led to development of pancreatic neoplasms; however, compared to chronic treatment, the tumor incidence was lower, the latency longer (32 wk), and all tumors benign. Conversely, the single BOP application yielded a significantly higher incidence of liver neoplasms, the frequency of which was twice as great in animals receiving 125 mg BOP/kg body weight than in those treated with half that dose. Also, in animals treated weekly for life, despite an increased survival rate, the incidence of liver neoplasms was decreased by lowering the dose. In the group receiving the lowest dose, only those hamsters surviving longer than 62 weeks developed hepatic neoplasms. These data may indicate that induction of liver neoplasms depends on administration of an adequate BOP dose (above 150 mg/kg body wt), regardless of treatment frequency. In the case of lung neoplasms, which showed similar latencies among the different subgroups but occurred in higher incidences at lower doses, the frequency of BOP application, rather than the quantity, may be important in tumorigenesis. Consequently, it may be possible to suppress or even to eliminate tumor induction in the liver and lungs by an appropriate dose regimen. Further studies in this regard are underway in this laboratory. BOP's toxicity and the tumor spectrum induced differed markedly from those of the related compounds, BHP and BAP. BOP induced pancreatic neoplasms with shorter latencies in almost all hamsters and at doses considerably lower than those used in BHP and BAP studies (1-6). Moreover, in contrast to the latter two compounds, BOP produced fewer liver and lung tumors and no tumors in the nasal cavity, larynx, and trachea. The similar biologic effects of BHP and BAP were assumed to be mediated by metabolic conversion of BAP to BHP (8). The different toxicity and tumorigenicity of BHP (and consequently also BAP) and BOP seem related to their metabolic fate, since BOP in hamsters gives HPOP as a major metabolite with traces of BHP. After BHP administration, however, unchanged BHP with only traces of HPOP was excreted (8). Direct bioassay of HPOP as a possible proximate pancreatic carcinogen is currently underway. Development of vaginal tumors after BOP treatment may be an effect of BHP, formed by metabolism from
J
NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
an incidence directly related to dose; i.e., a higher dose induced a higher tumor incidence (table 2). The first neoplasms were observed at 28 weeks in groups A-I females and A-2 males, at 15 and 20 weeks in B-1 females and males, respectively, at 32 weeks in group B-2 females, and at 62 weeks in group B-3 females. Most neoplasms were cholangiomas, whereas hemangioendotheliomas (in 2 females of group A-2), cholangiocarcinomas (in 1 female and 1 male, group B-1), and hyperplastic nodules (in 2 females of group A-I) were found only occasionally (table 2). Animals with liver neoplasms had simultaneous tumors of other organs. Focal proliferation and distension of bile ducts, often with acute inflammation, were found in 81 hamsters (19 females and 15 males of group B-1, 16 females and 8 males of group B-2, and 13 females and 10 males of group B-3). Similar proliferative lesions were also seen in controls (5 females, 12 males).
1452
POUR, ALTHOFF, KRUGER, AND MOHR
BOP, since these neoplasms were also found in a high incidence and attributed to BHP formation when BAP was administered to hamsters (8). BOP-induced pancreatic neoplasms were morphologically similar to those found in man, and the presence of other conditions associated with pancreatic cancer, such as obstructive icterus, diarrhea, ascites, weight loss, and vascular thrombosis (all known to occur in human pancreatic cancer victims), indicated that these experiments represent a further step in developing an appropriate model for investigation of pancreatic carcinogenicity. The findings also make evident the significant advantages of the present model, compared to the few other equivalent studies on pancreatic tumor induction (913 ).
(5)
(6) (7) (8)
(9)
(10)
(1) POUR P, ALTHOFF ], GINGELL R, et al: A further pancreatic carcinogen in Syrian golden hamsters: N-Nitroso-bis(2-acetoxypropyl)amine. Cancer Lett 1:197-202, 1976 (2) POUR P, MOHR U, CARDESA A, et a1: Pancreatic neoplasms in an animal model: Morphological, biological and comparative studies. Cancer 36:379-389, 1975 (3) POUR P, KRUGER FW, ALTHOFF]: Effect of beta-oxidized nitrosamines on Syrian golden hamsters. III. 2,2'-Dihydroxy-di-npropylnitrosamine.] Nat! Cancer lnst 54:141-146,1975 (4) POUR P, KRUGER FW, ALTHOFF ], et al: A new approach for
J NATL CANCER INST
(11) (12) (13)
VOL. 58, NO.5, MAY 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
REFERENCES
induction of pancreatic neoplasms. Cancer Res 35:2259-2266, 1975 POUR P, ALTHOFF], KRUGER FW, et al: Induction of pancreatic neoplasms by 2,2'-dioxopropyl-N-propylnitrosamine . Cancer Lett 1:3-6, 1975 POUR P, ALTHOFF ], GINGELL R, et al: N-Nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters. Cancer Res 36:2877-2884, 1976 WElL CS: Tables for convenient calculation of median effective dose (LD50 or ED50) and instructions in their use. Biometrics 8:249-263, 1952 GINGELL R, WALLCAVE L, NAGEL D, et al: Metabolism of the pancreatic carcinogens N -nitroso-bis(2-oxopropyl)amine and N-nitroso-bis(2-hydroxypropyl)amine in the Syrian hamster. ] Nat! Cancer lnst 57:1175-1178,1976 DISSIN], MILLS LR, MAINS DL, et al: Experimental induction of pancreatic adenocarcinoma in rats. J Nat! Cancer lnst 55:857864, 1975 ELKORT R], HANDLER AH, WILLIAMS DL: Early neoplasia of rabbit pancreatic ductal cells induced by dimethylhydrazine. Cancer Res 35:2292-2294, 1975 HAYASHI Y, HASEGAWA T: Experimental pancreatic tumor in rats after intravenous injection of 4-hydroxyaminoquinoline 1oxide. Gann 62:329-330, 1971 LONGNECKER DS, CRAWFORD BG: Hyperplastic nodules and adenomas of exocrine pancreas in azaserine-treated rats. ] Nat! Cancer lnst 53:573-577, 1974 REDDY ]K, SVOBODA D], RAO MS: Susceptibility of an inbred strain of guinea pig to the induction of pancreatic adenocarcinoma by N-methyl-N-nitrosourea. ] Nat! Cancer lnst 52:991993, 1974
Downloaded from http://jnci.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 2, 2016
I.-Pancreatic adenocarcinoma showing local squamous cell metaplasia with keratinization. Larger nests of metaplastic squaous cells were found in periphery of tumor invading gastric pylorus. H & E. X 100 FIGURE 2.-Another area of same tumor showing transition of cylindric epithelium to squamous cells. H & E. x 100
FIGURE
POUR, ALTHOFF, KRUGER, AND MOHR
1453
