Cancer Letters, 1(1976)197--202 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

197

A FURTHER PANCREATIC CARCINOGEN IN SYRIAN GOLDEN HAMSTERS: N-NITROSO-BIS(2-ACETOXYPROPYL)AMINE*

PARVIZ POUR, J U R G E N ALTHOFF, RALPH GINGELL and ROBERT KUPPER

The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.) FRIEDRICH W. K R U G E R t

Institut f~tr Toxicologie und Chemotherapie, Deutsches Krebsforschungzentrum, 6800 Heidelberg, Kirschner Strasse 6 (G.F.R.) ULRICH MOHR

Abteilung far Experimentelle Pathologie, Medizinische Hochschule Hannover, 3000-Hannover Kleefeld, Karl-Wiechert-Allee 9 (G.F.R.) (Received 11 December 1975)

SUMMARY

Weekly subcutaneous injection of N-nitroso-bis(2-acetoxypropyl)amine (BAP) for 20 weeks to Syrian golden hamsters induced pancreatic neoplasms in 53% of the animals, as early as 15 weeks from the start of treatment. The carcinogenic potency and organotropic spectrum of BAP were similar to those of N-nitroso-bis(2-hydroxypropyl)amine (BHP). Metabolism studies indicated that BAP was readily converted in vivo to BHP and that BHP was also the major urinary metabolite.

INTRODUCTION

Investigation of the biological effect of possible metabolites of N-nitrosodi-n-propylamine (DPN)** formed by beta-oxidation showed that N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitroso-bis(2-oxopropyl)amine (BOP) were strong systematic carcinogens which induced a high incidence of pancreatic neoplasms in Syrian golden hamsters [1,2]. However, the effect of BOP differed from that of BHP with regard to toxicity and tumor spectrum. Since acetylation of BHP was thought to be a further step toward either *This study was supported by funds from Public Health Service contract NO1 CP33278 from the National Cancer Institute and from the Fond der Chemischen Industrie. **According to standardized chemical nomenclature; see addendum, this paper. Address correspondence to: Dr. Parviz Pour, The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska

68105, U.S.A.

198

activation or detoxification, the metabolism and potential carcinogenicity of N-nitroso-bis(2-acetoxypropyl)amine (BAP) were examined in Syrian hamsters. MATERIALS AND METHODS

Biological studies: Randomly bred 8-week-old Syrian golden hamsters from the Eppley Colony were housed in groups of 5 by sex under standard conditions in plastic cages and given Wayne pelleted diet (Allied Mills, Chicago, Ill.) and water ad libitum. The LDs0 of BAP (synthesized by Dr. F.W. Krtiger) was calculated b y Weil's method [4] ! after a single subcutaneous (s.c.) injection of the compound in olive oils and an 8-day observation period. In the chronic study, 20 female and 20 male hamsters received weekly s.c. injections of 700 mg/kg b.w. BAP for 20 weeks,* and the survivors were killed 46 weeks after the experiment s began. Control hamsters (20 female and 20 male) received weekly injections of the solvent only. After complete autopsy, organs were prepared using conventional methods for histologic examination. Durations stated are from the beginning of the experiment. Metabolism studies: Pairs of male hamsters were injected intraperitoneally (i.p.) with 100 mg/kg b.w. BAP and maintained in all-glass metabolism cages. Urine was collected frozen. Blood was withdrawn from the orbital venous plexus at various times after i.p. administration of BAP. Blood and urine were extracted with ethyl acetate, and metabolites determined by gas and thin-layer chromatography, using authentic standard compounds. RESULTS

Biological studies: The LDso of BAP was 6967 (5640--8610) mg/kg b.w. (7465 mg/kg b.w. in females and 6500 mg/kg b.w. in males). In the chronic study, 17 females and 13 males died during the first 30 weeks of the experiment and 1 female and 1 male, b e t w e e n 30 and 46 weeks. With the exception of a female hamster which died at 12 weeks, all treated animals developed tumors (Table 1). Three females and 2 males had a single neoplasm, while the rest presented multiple neoplasms in 2--3 organs (7 females a n d 12 males), 4--5 organs (7 females, 5 males) or in 6 or more organs (2 females, i male). The multiplicity of tumors increased during the course of treatment. The earliest neoplasms developed after 6 weeks (males) and 7 weeks (females) in the lungs and liver. Pancreatic neoplasms were found as early as 15 weeks in females and 17 weeks in males. All hamsters with pancreatic lesions had neoplasms of other organs, e.g., the lungs (2 females, 1 male), liver (3 males) or in b o t h the lungs and liver (7 females, 6 males), and gallbladder (1 female). Twelve of the 21 pancreatic tumors were of microscopic size and the rest measured between 0.5--3 mm in diameter; one neoplasm (male, 44 wks) * F u r t h e r t r e a t m e n t was n o t possible due to t h e limited availability of t h e c o m p o u n d .

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200 r e a c h e d a 9 m m diameter. All b u t one of these lesions were localized. T h e largest t u m o r h a d invaded s u r r o u n d i n g tissue, b u t n o d i s t a n t metastases were f o u n d . Neoplasms, o f t e n multiple, were d u c t a l a d e n o m a s , i n t r a d u c t a l c a r c i n o m a s and d u c t a l a d e n o c a r c i n o m a s and were d i s t r i b u t e d in d i f f e r e n t a n a t o m i c a l regions o f t h e pancreas (Table 2). T h e m o r p h o l o g y o f t u m o r s f o u n d in o t h e r organs is given in Table 1. C o n t r o l hamsters had a nasal papillary p o l y p (1 female), a t h y r o i d a d e n o m a (1 female), a t h y r o i d c a r c i n o m a (1 male), an adrenal a d e n o m a (1 male), 2 f o r e s t o m a c h papillomas (2 females), and 7 C o w p e r gland a d e n o m a s . TABLE 2 PATTERNS OF PANCREATIC NEOPLASMS INDUCED BY BAP IN SYRIAN HAMSTERS Survival (wks)

15 20 25 30 35 40 45 46

Number of tumorbearing animals F

M

2 1 3 3 0 1 0 1

0 2 1 6 0 0 1 0

Site of tumors Body

Head

Tail

F

M

F

M

F

M

1A 1C 0 1A 1C 1C 0 0 0 0

0 0 0 5A 2C 0 0 1A 0

IA IA 2C 2A IC 0 IA IC 0 IA IC

0 2C 1A 2A 2C 0 0 1A 1C 0

2A 1A 1A 3A 0 1A 0 1A

0 0 0 3A 1C 0 0 1C 0

1C 2C 1C 1C

F = females M = males A = adenoma C = carcinoma

Metabolism studies: B l o o d collected at various times f r o m animals injected with BAP c o n t a i n e d n o u n c h a n g e d BAP. BHP was the m a j o r m e t a b o l i t e , b u t small a m o u n t s o f t h e m o n o a c e t y l a t e d derivative N - n i t r o s o - ( 2 - h y d r o x y p r o p y l ) ( 2 - a c e t o x y p r o p y l ) a m i n e (HPAP) were d e t e c t e d (Table 3). No BAP was d e t e c t e d in 24-h urine o f hamsters injected w i t h the c o m p o u n d : 19.4+_10.0% o f the dose was e x c r e t e d as BHP, t o g e t h e r with traces o f HPAP. DISCUSSION The t o x i c and carcinogenic effects of BHP and BOP in Syrian h a m s t e r s varied [ 1 , 2 ] . C o m p a r e d to BHP, BOP was m u c h m o r e toxic, a f f e c t e d the pancreas m o r e specifically, i n d u c e d neoplasms in a s h o r t e r time period, and caused fewer liver and lung t u m o r s , while failing t o induce t u m o r s o f t h e nasal cavity, t r a c h e a and kidneys.

201

TABLE 3 BLOOD METABOLITE LEVELS IN HAMSTERS A F T E R BAP Time (min)

10 20 30 60

Blood metabolite conc. (ttg/ml)* BAP

HPAP

BHP

0 0 0 0

88 4 3 2

237 151 69 68

*Blood was taken from the orbital venous plexus of hamsters at various times after i.p. administration of 100 mg/kg BAP. The results are the mean for two animals.

In the present study, BAP which hypothetically could be a further DPN metabolite by beta-oxidation and may form during detoxification of DPN, also showed systematic carcinogenic effects. Its toxicity was similar to that of BHP, as was its carcinogenicity with respect to both the type and site of induced tumors [3]. The lower incidence of pancreatic neoplasms induced by BAP (53%), compared to BHP (100%), may relate to the shorter treatment period of the present study. BHP was the major metabolite in the blood and urine of hamsters administered BAP. The similar carcinogenic potency and organotropic spectrum of BAP and BHP in hamsters are probably due to the facile metabolic conversion of BAP to BHP. These findings indicate that the biological effect of BOP is different from that of analogous hydroxylated and acetylated nitrosamines. ADDENDUM

In an effort to standardize the nomenclature used in referring to the nitroso compounds in this paper and in future studies, the following are suggested: N-nitroso-di-n-propylamine (DPN), formerly di-n-propylnitrosamine (DPN); N-nitroso-bis(2-hydroxypropyl)amine (BHP), formerly 2,2'-dihydroxyn-propylnitrosamine (DHPN): N-nitroso-bis(2-oxopropyl)amine (BOP), formerly 2,2'-dioxopropyl-N-propylnitrosamine (DOPN). ACKNOWLEDGMENTS

The authors are grateful to Kathy Stepan and J. Meehan for technical assistance and Mardelle Susman for editorial advice. REFERENCES Pour, P., Krtiger, F.W., Althoff, J., Cardesa, A. and Mohr, U. (1975) The effect of betaoxidized nitrosamines on Syrian golden hamsters. III. 2,2'-dihydroxy-di-n-propylnitrosamine. J. Natl. Cancer Inst., 54, 145--146.

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2 Pour, P., Althoff, J., Krt~ger, F., Schm~ihl, D. and Mohr, U. (1975) Induction of pancreatic neoplasms by 2,2'-dioxopropyl-N-propylnitrosamine. Cancer Lett., 1, 3--6. 3 Pour, P., Mohr, U., Cardesa, A., Althoff, J. and Kriager, F.W. (1975) Pancreatic neoplasms in an animal model: Morphological, biological and comparative studies. Cancer, 36, 379--389. 4 Weil, C.S. (1952) Tables for convenient calculation of median effective dose (LD~0 or ED~0 ) and instructions in their use. Biometrics, 8 , 2 4 9 - - 2 6 3 .

A further pancreatic carcinogen in Syrian golden hamsters: N-nitroso-bis(2-acetoxypropyl)amine.

Weekly subcutaneous injection of N-nitroso-bis(2-acetoxypropyl)amine (BAP) for 20 weeks to Syrian golden hamsters induced pancreatic neoplasms in 50% ...
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