[CANCERRESEARCH36, 2877-2884,August1976]

SUMMARY

N-Nitroso-bis(2-acetoxypropyl)amine , a possible f3 metabolite of N-nitroso-di-n-propylamine, was shown to be apotent carcinogen in the Syrian golden hamster. After asingle s.c. treatment, the pancreas was the most affectedorgan, followed by the liver, respiratory tract, and kidneys.However, repeated application resulted in a higher mcidence of neoplasms of the respiratory tract than of thepancreas and kidneys. The effect of N-nitroso-bis(2-acetoxypropyl)amine on toxicity, target tissues, and carcinogenicity was similar to that of N-nitroso-bis(2-hydroxypropyl)amine. The assumption that these two compounds mayhave similar metabolic pathways was confirmed; N-nitrosobis(2-acetoxypropyl)amine was readily deesterified to N-nitroso-bis(2-hydroxypropyl)amine in vivo and in vitro.

INTRODUCTION

The postulated metabolites of DPN3 by f3oxidation (1-3),BHP and N-nitroso-bis(2-oxopropyl)amine , were potentpancreatic carcinogens in Syrian golden hamsters (7, 9, 10,14), although the biological effect of these 2 compoundsdiffered with regard to toxicity and the organotropic spectrum. To obtain additional information on the carcinogenicity of DPN, we examined the effect of BAP, which hypothetically could be a further DPN metabolite by /3oxidation andacetylation in vivo. Since preliminary results indicated asimilar carcinogenic effect of BAP and BHP (6), a parallelstudy of the metabolism of both compounds was appropriate.

MATERIALS AND METHODS

Chemicals. For preparation of BAP, 16.6 g (0.21 mole)anhydrous pyridine and 16.2 g (0.1 mole) BHP were placedin a 3-necked, 500-mi round-bottom flask with stirrer, add ition funnel, condenser, and drying tube. To this solution

I Supported by USPHS Contract NO1 CP33278 from the National Cancer

Institute, NIH.2 To whom requests for reprints should be addressed, at Eppley Institute

for Research in Cancer, University of Nebraska Medical Center, 42nd Streetand Dewey Avenue, Omaha, Ncbr. 68105.

3 The abbreviations used are: DPN, N-nitroso(di-n-propyl)amine; BHP, N-

nitroso-bis(2-hydroxypropyl)amine; BAP, N-nitroso-bis(2-acctoxypropyl)amine; HPAP, N-nitroso-(2-hydroxypropyl) (2-acctoxypropyl)aminc; LD50,acute lethal dose.

Received February 5, 1976; accepted April 29, 1976.

were added 250 ml anhydrous ether, followed by dropwiseaddition of 16.4 g (0.21 mole) acetyl chloride at a rate thatmaintained a gentle reflux. The resulting solution was allowed to stir for 1 hr. It was then filtered, washed with 100ml 10% HCI,100 ml 5% NaCO3, and 2 x 100 ml H2O, and the

organic phase was dried over Na2SO4.The ether was removed on a Buchi Aoto-vap, leaving 23.8 g pure BAP as alight yellow oil; the yield was 97%.

C10H18N2O@

Calculated: C 48.76, H 7.37, N 11.38Found: C 48.53, H 7.52, N 11.34

To prepare HPAP, 8.2 g (0.11 mole) anhydrous pyridineand 16.2 g (0.1 mole) BHP were placed in a 3-necked, 500-ml round-bottom flask with stirrer, reflux condenser, andaddition funnel. To this solution were added 250 ml anhydrous ether, followed by the dropwise addition of 8.6 g (0.11mole) acetyl chloride over 2 hr. The solution was stirred for1 hr. The ether was then removed on a Roto-vap, and thecrude orange oil was chromatographed on a 6- x 50-cmcolumn of silica gel with methylene chloride:ethyl acetate,4:1 , as elutant. The 2nd main band eluting gave 17.5 g pureHPAP as a light yellow viscous oil: yield 86%.

C8H1.N2O4

Calculated: C 47.03, H 7.90, N 13.72Found: C 46.85, H 8.07, N 13.94

Elemental analyses were performed by Micro-Tech Laboratories, Inc., Skokie, Ill.

Nuclear magnetic resonance spectra were obtained foreach compound on Varian HA-100 and CFT-20 spectrometers. Spectra were complex but were consistent with theproposed structures. Mass spectral data were obtained withan AEI-MS9 gas chromatographer-mass spectrometer. Nomolecular ions were observed, but major fragments wereidentified.

Biological Studies. Random-bred, 8-week-old Syriangolden hamsters from the Eppley colony were housed bysex in plastic cages in groups of 5. They were kept understandard conditions and given Wayne pelleted diet andwater ad !ibitum.

For determination of the LD@,,,according to Weil's methods (15), hamsters (5 females and 5 males/dose level) received a single s.c. injection of BAP dissolved in olive oil.Animals that survived for 8 days were maintained for life

AUGUST 1976 2877

N-Nitroso-bis(2-acetoxypropyl)amine as a Further PancreaticCarcinogen in Syrian Golden Hamsters1

Parviz Pour,2 Jurgen Althoff, Ralph Gingell, Robert Kupper, Friedrich Kruger, and Ulrich Mohr

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 (P. P., J. A. , R. G. , R. K.]; Institut fürToxikologie undChemotherapie, Deutsches Krebsforschungszentrum, Heidelberg, West Germany (F. K.]; and Abteilung fürExperimentelle Pathologic, MedizinischeHochschule Hannover, Hannover, Germany (U. M.J

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CompoundThin

layer(RF X 100)Gas-liquid retention time

(mm)A°BCDBAP7550806518HPAP5015755012BHP30565408Silylated

HPAP19SilylatedBHP20

Blood metabolite levels in hamstersafter BAPadministrationBloodwas taken from the orbital venous plexus of hamstersat

various times after i.p. administration of 100mg BAP per kg. Theresults representvaluesobtained from 2animals.Blood

metabolite concentration(@g/ml)Time(min)

BAP HPAPBHP100 94,82 332,142

20 0 3,5 228,7530 0 2,4 81,5760 0 3,1 90,46

Deesterification of BAP.bybloodControl

blood (0.2ml) was incubatedfor varioustimeswith 1 mgBAP in 0.1 M sodium phosphate-potassiumphosphate buffer, pH7.4, with 2.5%ethanol in a final volume of 1 ml. Metaboliteswereextracted

and estimatedas described in thetext.Metabolitesformed by blood(.tmobes)Time(hr)

BAP HPAPBHP0

4.0 000.53.7 0.201.02.6 1.201.51.9 1.90180 1.91.8600 03.9Nobbood,603.5 0.4 0

P. Pour et a!.

(Group A). For the chronic study (Group B), 20 females and20 males were used per dose level and received weekly s.c.injections of BAP in olive oil for 20 weeks in doses (mg/kgbody weight) corresponding to 0.1 (Group B-i), 0.05 (GroupB-2), and 0.025 (Group B-3) of the LD@.Controls (Group C)were treated with solvent only. Surviving animals (includingcontrols) were killed at 46 weeks from the beginning of theexperiment. Following complete autopsies, organs werefixed in 10% bufferedformalin. Bonesweredecalcified priorto histological procedures. Tissues were embedded in Paraplast and sections were stained with hematoxybin and eosin.Data are stated from the beginning of the experiment. Thenumber of tumors per organ was determined by countingthe tumors in representative histological sections. The critenon used for diagnosis of the induced lesions was basedon our previous investigations (8, 11—13).

Metabolic Studies. Six male Syrian golden hamsters weregiven i.p. injections of 100 mg BAP per kg body weight inolive oil (5 mg/mI). Animals were maintained in pairs in allglass metabolism cages with no food, but with water ad!ibitum. Urine was collected frozen for 24 hr and stored at—20°before extraction.

Aliquots of urine were saturated with solid NaCI, thenextracted 3 times with an equal volume of ethyl acetate.Extracts were pooled, dried with anhydrous magnesiumsulfate, and evaporated to a small volume under nitrogen.Extracted metabolites were identified and estimated chromatographically by comparison with authentic standards(Table 1).

Blood was taken from the optic vein of hamsters at various times after i.p. injection of BAP. It was first extractedwith an equal volume of hexane, which was discarded, toremove soluble lipids, and then with ethyl acetate as forurine.

Control hamster blood (0.2 ml) was incubated at 37°in ashaking water bath for various times with 1 mg BAP in afinal volume of 1 ml 0.1 M sodium phosphate-potassiumphosphate buffer, pH 7.4, containing 2.5% ethanol. Nitrosamines were extracted and estimated as described above.

RESULTS

MetabolicStudies

Recovery of BAP, BHP, and HPAP from blood and urine atconcentrations of 50 to 250 pg/mb was between 50 and 60%,and the blood and urine concentrations reported are corrected by this factor. Twenty-four-hr urine from hamstersgiven i.p. injections of BAP contained 19.4 ±10.0% of thedose as BHP. No unchanged BAP was detected, but tracesof an unidentified metabolite with a gas chromatographyretention time of 5 mm were observed. Preliminary experiments indicated that 23.1 ±9.7% of a BHP dose of 100 mgIkg body weight was excreted unchanged in 24-hr hamsterurine.

Blood from animals given i.p. injections of BAP containedno unchanged BAP; only BHP and traces of HPAP weredetected (Table 2). Levels of metabolites were highest 10mm after injection. When incubated for various times withcontrol blood, BAP was quantitatively deacetylated to HPAP

and, eventually, to BHP. Incubation of BAP with bufferalone yielded only small amounts of HPAP (Table 3).

Biological Studies

The LD@of BAP was calculated to be 6967 (5640 to 8610)mg/kg body weight (7465 mg/kg body weight in femalesand 6500 mg/kg body weight in males). Necrosis of thesensory cells and mucoid degeneration of Bowman'sglands in the nasal olfactory epithelium were found, as wascell degeneration and necrosis of the liver, proximal renaltubules, and germinal epithelium of the testis in animalsthat died during the 1st 8 days of the experiment. Hemorrhages of lungs and abdominal organs were also seen. Alltreated hamsters surviving the acute phase (Group A) hadbower body weights compared with controls (Table 4). Of 26

Table 1Chromatographyof nitrosamines

Thin-layer chromatography was performed on Kodak Chromagramsilica gel sheetswith fluorescent indicator. Gaschromatographywasperformedon a PackardModel7820chromatograph usinga 6-foot, all-glass column containing 10%OV-1at 140°with flameionization detection.

(I A, methylene chboride:ethyl acetate, 4:1 ; B, methylene chloride;

C, acetonitrile:gbacialacetic acid, 99:1; D, toluene:ethanol, 9:1.

Table 2

Table 3

I

2878 CANCERRESEARCHVOL. 36

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A Further Pancreatic Carcinogen

hamsters in this group, 23 developed neoplasms in variousorgans as early as 22 weeks into the experiment. (Of 3hamsters without neoplasms, 2 died after 2 weeks and 1 at37 weeks of the experiment.) The main sites of tumor devebopment were the pancreas (in 50% of the animals), respiratory tract (38%), liver (35%), kidneys (31%), and vagina(23%).

In the chronic study (Group B), all but 9 hamsters developed neoplasms. The hamsters without neoplasms were 3females (which died at 12 weeks, Group B-i ; 7 weeks, GroupB-2; and 23 weeks, Group B-3) and 6 males (1 died at 19weeks in Group B-2, 4 died at 30 weeks, and the other diedat 41 weeks in Group B-3). A dose-dependent effect wasindicated, since the overall tumor incidence increased andthe latency and/or average survival decreased with risingdose levels (Table 4). This effect did not influence the multiplicity of neoplasms, since, on the average, 2.5, 3.6, and 2.9tumors were found per animal in the respective dosagegroups (Table 4). The most affected organs were the respiratory tract (tumor incidence, 73%), followed by the liver(59%), pancreas (58%), vagina (37%), and kidneys (13%).Controls showed a tumor incidence of 30% with a multiplicity of 0.3 tumor/animal.

Tumors of Pancreas. Pancreatic neoplasms were foundafter a single dose of BAP in 77% of the females and 23% ofthe males (Table 5). The frequency of pancreas tumors didnot rise significantly after chronic treatment, occurring in62% of the females and 53% of the males. The 1st neoplasms were found at 14 weeks (females) and at 17 weeks(males). A dose-dependent effect could not be established.Of a total 82 hamsters with pancreatic neoplasms, 4 (1female and 3 males) had only pancreatic neoplasms, whilethe remaining 78 hamsters presented tumors simultaneously in other organs, but mainly in the lungs and liver.

Only 40 (about 50%) of the pancreatic neoplasms wereseen macroscopically; of these, 9 were from Group A animals (in 7 females and 2 males) and the rest were fromGroup B (in 20 females and ii males). The anatomicallocations ofthese tumors by sex were as follows: tail, 16 (10females, 6 males); body, 3 (2 females, 1 male); head, 3 (1female, 2 males); body and tail, 6 (4 females, 2 males); headand body, 1 (female); head and tail, 1 (female); all portions,10 (8 females, 2 males). The neoplasms ranged from 2-mmglassy, cystic lesions to 12-mm grayish white nodules. Table5 demonstrates in detail the anatomical locations of histologically observed pancreatic neoplasms, according to theirmorphological patterns. A relationship could not be foundbetween tumor type, location, and duration of treatment.The neoplasms were ductal adenomas (Figs. 1 and 2), intraductalpapillomas(Fig.3),intraductalcarcinomas (Fig.4), and adenocarcinomas (Fig. 5). One carcinoma in a female (at 41 weeks, Group A) showed a mixture of ductab andacinarcell-likepatterns(Fig.6). Pure acinarcell carcinomaswere not seen. Adenomas (8 in Group A and 59 in GroCip B)were found as early as 14 weeks after the beginning oftreatment; their size usually did not exceed 2 mm. Theywere multiple lesions that developed more frequently in thetail of the pancreas than in the body and head (Table 5). In40 animals(21 femalesand 19 males),adenomaswere theonly pancreatic tumors, while 9 hamsters (6 females, 3males) had simultaneous intraductal carcinomas, 10 (7 fe

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Distribution ofpancreatic neoplasms in pancreasof hamsterstreatedwithBAPTumor

bearingTumortypes―hamstersGroupFb

MFAdenoma

Head Body

M FMTailIntraductal

carcinomaFHead

MAdenocarcinoma

TailBody

F MFMFMFHead

Body Tail

M F MFMA-i2000 102000 1 0 00000 000A-22010 000000 0 0 00200 010A-33010 203010 1 0 20001 010A-43300 011000 0 0 00021 1 11

Segmentaldistribution of tumorsinduced by BAP in the respiratorysystemof SyriangoldenhamstersTumor

bearinghamstersNasal

cavitiesLaryngo trachealtract

PapillarypolypLungsAdenomaAdenocarcinoma

.Squamous cell carcinomaAdenomaPapillary

polyps papilbomaAdenocarcinomaGroupF

MF MF MF MF MF MF MFMA-i

A-2A-3A-42

01 05 01 10

10 00 00 00

10 00 00 01

00 00 01 10

00 00 00 12

01 05 00 00

00 00 0i 00

00 00 000B-i

B-2B-316

1416 1217 130

02 01 01

11 14 13

13 44 18

57 54 713

1217 iO16 94

21 31 20

10 001Control1

01 00 00 00 00 00 00 0

P. Pour et a!.

males, 3 males) had adenocarcinomas, and 8 (5 females, 3 located in the body and tail, respectively. Both animals andmales) had all 3 types of neoplasms. Intraductal carcinomas another group B male hamster with a large adenocarcinoma(3 in Group A and 18 in Group B) were seen, with 1 excep- of the head presented hemorrhagic ascites; the male withtion (in a Group B male), simultaneously with adenomas (6 lung metastases also had multiple lung thrombosis. Focalfemales, 3 males), adenocarcinomas (1 female, 2 males), or fat necrosis was found at 24 weeks in a Group B female withwith both adenomas and carcinomas (5 females, 3 males), adenocarcinomas of both the head and tail regions.as early as 24 weeks. The often multiple intraductal carcino- Tumors of Respiratory Tract. The incidence of nasal caymas occurred more frequently in the body of the pancreas ity neoplasms was 12% (2 females, 1 male) in Group A andthan in the tail or head. Adenocarcinomas (7 in Group A and 22% (17 females, 9 males) in Group B (Table 6). Most of the25 in Group B), often multifocal, were the only pancreatic tumors in both groups were adenocarcinomas (in 16 hamtumor in 11 hamsters (5 from Group A and 6 from Group B), sters), all of which originated in the olfactory epithelium,whereas the rest were found simultaneously with adenomas followed by papillary polyps and squamous cell papibbomas(7 females, 3 males), with intraductal carcinomas (1 female, (in 10 hamsters), adenomas (in 4), and squamous cell carci2 males), or with both adenomas and intraductal carcino- nomas (in 1), which arose in the respiratory epithelium ofmas (5 females, 3 males). The carcinomas originated more the anterior portion. The benign tumors were observed asfrequently in the body of the pancreas than in the tail and early as 33 weeks from the beginning of treatment, andhead (Table 5). In 4 animals, the adenocarcinomas invaded malignant neoplasms were first seen 7 weeks later. Proliferthe extrapancreatic tissues and, in 2 instances, metasta- ative and hyperplastic changes of the respiratory epitheliumsized to distant organs, such as the regional lymph nodes, were evident in 15% (Group A) and 24% (Group B) of thekidneys, adrenal glands, genital organs, or lungs. The me- animals; 1 animal treated with a single dose also showedtastasizing carcinomas (in a Group B male and female) were glandular proliferation and hyperplasia of the mucosa in the

Table 5

B-i 12 9 3 6 6 3 10 3 2 0 3 2 4 1 2 3 2 5 4 2B-2 12 16 6 4 7 10 10 13 1 1 2 2 1 1 1 0 3 3 4 2B-3 13 7 2 1 5 2 12 5 1 0 1 3 0 1 0 0 2 1 0 1C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

a By location in pancreas segment.b F, female; M, male.

Table 6

2880 CANCERRESEARCHVOL. 36

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Tumors induced by BAP in the gallbladder and liver of Syrian goldenhamstersTumor-bear-

Gallbladdering hamsters polyp

— CholangiomaLiver

tumorsCholangiocar-

Hemangioencinoma dothebiomaNodular

livercell hyperpla

siaGroupF

M F M F MF M F MFMA-i

A-2A-3A-42

0 1― 0 2 01 3 0 0 1 32 2 1 1 0 22 0 1 0 1 01

0 1 01 1 0 00 0 1 00 0 1 02

00 01 000B-i

B-2B-313

18 2 3 12 1614 14 4 3 12 26 6 3 0 3 15

7 0 10 1 4 ii1 0 1 33

102 542Control0

0 0 0 0 00 0 0 000aAdditionaI papillary adenocarcinoma of gallbladder (A-i , 31weeks; B-i , 30 weeks).

A Further Pancreatic Carcinogen

olfactory region. All animals with nasal cavity tumors hadsimultaneous neoplasms of other sites. A small adenomatous polyp in the anterior nasal region was found in a femalecontrol.

One hamster in Group A (4%) developed papillary polypsof the larynx. In Group B animals, the majority of papillarypobyps found in 36 hamsters (30%) developed in the trachea(14 females, 11 males), less frequently in the larynx (2 females, 5 males), and least in stem bronchi (2 females, 2males). The anatomical distribution of tracheal tumors wasas follows: upper portion (in 14 animals), middle portion (in7), lower portion (in 4). These were solitary tumors in 28animals; multiple papillary polyps occurred in the larynxand trachea (in 4 animals), or in the different tracheal segments (in 4 animals). The first of these neoplasms wasobserved earlier in females (at 9 weeks) than in males (at 17weeks). Three animals (1 female at 21 weeks and 2 males at28 and 29 weeks) had a tracheal tumor as their only neoplasm . Focal epithelial alterations (hyperplasia, metaplasia,and dysplasia) occurred frequently in animals with or without respiratory tract tumors.

Lung neoplasms developed as early as 31 weeks after asingle injection in females only (in a 35% incidence) andwere bronchiogenic adenomas, with 1 exception (a bronchiogenic adenocarcinoma at 60 weeks). In Group B, thelung tumor incidence was higher (66%), and these neoplasms were found as early as 9 weeks in females and at 14weeks in males. Also in this group, more tumors wereobserved in females than in males (Table 6). These consisted mainly of adenomas (46 females, 31 males) and adenocarcinomas (6 females, 7 males). Squamous cell carcinomas were seen in 2 males only (at 17 and 30 weeks). Oftenneoplasms were of multiple origin in one or all lobes (3 to 20tumors/animal). Only 3 animals had the lungs as their soletumor site (females: 16 weeks, Group B-i , and 18 weeks,Group B-2; male, 46 weeks, Group B-3), whereas the resthad simultaneous neoplasms in other organs, primarily ofthe liver (26 females, 21 males).

Tumors of Gallbladder and Liver. Fifteen % (3 females, 1male) of Group A hamster and 13% (9 females, 6 males) ofthose in Group B developed gallbladder neoplasms (Table7). Except for 2 invasive papillary carcinomas (in a female,

31 weeks, Group A; male, 30 weeks, Group B-i), the neoplasms were papillary polyps, often of multiple origin, andobserved as early as 15 weeks in females and 18 weeks inmales. Simultaneously occurring with the gallbladder tumors were cholangiogenic neoplasms (in 6 females, 4males), pancreatic duct tumors (in 7 females, 2 males) andlung neoplasms (in 2 females). Focal proliferation of gallbladder epithelium was observed in 5 females and 2 males(23%) of Group A and in ii females and 12 males (19%) ofGroup B.

Most liver neoplasms were of bile duct origin and consisted of cholangiomas (Group A, 35%; Group B, 38%) andcholangiocarcinomas (Group A, 12%; Group B, 12%). Thefirst of these neoplasms was found after 31 weeks in afemale and at 22 weeks in a male in Group A and after 9 and7 weeks in Group B, respectively. Proliferative changes ofbile ducts, often mubtifocal, along with cystic distention andgoblet cell metaplasia, occurred before tumors developed(for diagnostic criteria, see Ref. 13).

Hemangioendothebiomas were found in 12% of Group A(females only) after 22 weeks and in 17% of Group B hamsters after 7 weeks, predominantly in males (15:5). In animals that died earlier, peliosis and focal proliferation of thevascular endothebium were seen. Hyperplastic liver cellnodules occurred after a single dose only in females (in a12% incidence) as early as 31 weeks, whereas in the chronicstudy they were found more frequently in males than infemales by a ratio of 17:9 (a 22% incidence) and as early as 9weeks (female) and 7 weeks (male). Hepatocellular carcinomas were not observed.

Tumors of Urogenital Organs. A single dose and weeklyinjections of BAP bedto development of renal neoplasms inincidences of 31% and 13% for Groups A and B, respectively(Table 8). A clear dose-dependent effect could not be established. Twenty-one tubular adenomas and 3 tubular adenocarcinomas were found. Among these renal neoplasms,only 1 adenoma (1 mm in size) and the 3 carcinomas (6 to 8mm) could be observed grossly. The first adenoma wasfound in Group A after 29 weeks in males and after 31 weeksin females, and in Group B after 28 and 29 weeks, respectively. Adenocarcinomas occurred at 30 weeks from thebeginning of the experiment in a male and at 27 weeks in a

Table 7

AUGUST1976 2881

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Tumorsof urogenital tract, forestomach,and other sites in Syriangoldenhamsters treated withBAPKidneysVagina

PapilbomaCow

per'sgland

AdenomaForestomach

Pa ‘Iboma

Female MaleOther

sites(wk)GroupTumor-bear

ing hamsters

Female MaleAd

n me o a

Female MaleAdenocarci

noma

Female MaleFemaleMaleA-i

A-2A-3A-4B-iB-2

B-3C1

02 12 20 01 23 7

1 20 01

01 12 20 01 12 7

1 20 00

01 00 00 00 11 0

0 00 01

02069

700

01113

250

00 10 02 31 44 2

0 22 0A@(50)

00 B (35)C(49) 0A (44), D (60) E (58)F (19) B (24)D (27),G(44),H(46), H(46),L (46),M (46),

I (30),J (27),K (46) N (46), P (46)0 B (36)G (46),0 (46) L (46), N (46)

P. Pour et a!.

Table 8

a A, Parathyroid adenoma; B, Harderian gland adenoma; C, rectal adenocarcinoma; 0, splenic hemangioendothelioma; E, flank organepithelioma; F, colon polyp; G, thyroid adenoma;H, cheek pouch papibboma;I, urinary bladder papibboma;J, mesenterichemangioendothelioma; K, malignant melanoma; L, thyroid carcinoma; M, pharyngeal papibboma;N, adrenal adenoma; P, preputial carcinoma; 0,bipoma.

female. No tumors of the renal pelvis, ureter, and urethrawere found; however, hyperplasia of the urothelium wasseen in 5 females and 4 males (8%) of Group B. One of thesefemale hamsters (at 30 weeks) had a transitional cell papilloma of the urinary bladder in addition to a renal adenoma.All animals with urogenital tract tumors had simultaneouslyneoplasms of other organs.

Squamous cell papilbomas of the vagina occurred in bothGroup A (23%) and Group B (37%), as early as 12 weeks.These lesions, which were often multiple in origin, werelocated close to the orifice.

In males, a preputial squamous cell carcinoma (46 weeks)and Cowper's gland adenomas (Group A, 15%; Group B,10%) were found. However the latter developed also incontrols (25%).

Tumors of Other Sites. Keratmnizingsquamous cell papibbomasof the forestomach were found in a higher incidencein experimental animabs(Group A, 23%; Group B, 11%) thanin control (5%) hamsters, and earlier in treated hamsters (at24 weeks) than in nontreated animals (at 46 weeks) (Table8). In contrast to control hamsters, which had solitary papilbomas, most treated animals (3 females, 3 males) developedmultiple (2 to 4) neoplasms. Tumors originating in othertissues are listed in Table 8.

DISCUSSION

BAP was shown to be a potent carcinogen in Syriangolden hamsters after both a single s.c. injection and foblowing chronic treatment. Whereas the pancreas was themost affected organ after a single injection, followed by theliver, respiratory tract, and kidneys, repeated application ofthe compound resulted in a tumor incidence higher in thelungs than in the pancreas. However, most hamsters receiving weekly treatments died between 20 and 30 weeks, andanimals treated with a single injection survived for an add itional 10 to 20 weeks. This observation indicates that development of respiratory tract tumors is related less to the total

dose of BAP than to the frequency of treatment. In contrast,the development of kidney tumors seems to be time dependent, but not necessarily dose dependent, as seen inother studies (4, 5). Apparently, the same is true for thepancreatic neoplasms, with the exception that the tumorlatency in this organ seems to be shorter. However, thisassumption requires further evidence.

The susceptibility of the hamster respiratory tract, liver,and kidneys to various nitrosamines has been shown inmany studies. BAP and BHP (9, 10, 14) also induced a highincidence of pancreatic neoplasms. The latency, anatomical distribution, morphological patterns, and biological behavior of these lesions were similar after BAP or BHP treatment. The same appears to be valid for neoplasms of therespiratory tract, liver, and kidneys. Only the incidence andmultiplicity of nasal tumors were remarkably higher in hamsters treated with the highest dose of BHP than in thecorresponding group with BAP, although the average survival of BHP-treated hamsters corresponded to the treatment period with BAP (20 weeks). The morphology, latency,and distribution of neoplasms in the laryngotracheal tract,as well as in the lungs, liver, and kidneys, were similar afterBHP and BAP administration, despite different treatmentperiods. As with BHP, BAP treatment also resulted in induction of papillary polyps and adenocarcinomas in the gallbladder.

The effect of N-nitroso-bis(2-oxopropyl)amine differedfrom that of BAP and BHP, in that N-nitroso-bis(2-oxopropyl)ammneinduced relatively few neoplasms of the liver andkidneys, and no upper respiratory tract tumors, but causeda high incidence of pancreatic tumors at lower doses thandid BAP or BHP (7).

The similar biological effects of BHP and BAP in terms oftoxicity and tumor spectrum suggested that both cornpounds may be similarly metabolized. BHP was the onlymetabolite identified in 24-hr urine of hamsters given BAP,and it was excreted to about the same extent as after BHPadministration. BAP was rapidly deacetylated in VIVOto BHPvia the monoacetylated analog HPAP. Deacetylation could

2882 CANCER RESEARCH VOL. 36

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A Further Pancreatic Carcinogen

2. Kruger,F.W.MetabolismofNitrosaminesInVivo.II.OntheMethylationof Nucleic Acids by Aliphatic Di-n-alkylnitrosamines In Vivo, Caused by/3-Oxidation: The Increased Formation of 7-Methylguaninc after Application of /3-Hydroxypropyl-propylnitrosamine Compared to That after Application of Di-n-propylnitrosamine. z. Krebsforsch., 79: 90-97, 1973.

3. KrUger,F.W.,andBertram,B. Metabolismof NitrosaminesIn Vivo.Ill.On the Methylation of Nucleic Acids by Aliphatic Di-n-alkylnitrosaminesIn Vivo Resulting from /3-Oxidation: The Formation of 7-Methylguanineafter Application of 2-Oxopropyl-nitrosamine and Methyl-propyl-nitrosamine.Z.Krebsforsch.,80:189-196,1973.

4. Mohr, U., and Hilfrich, J. Brief Communication: Effect of a Single Doseof N-Diethylnitrosaminc on the Rat Kidney. J. NatI. Cancer Inst. , 49:1729-1731, 1972.

5. Pour, P., Althoff, J., Cardesa, A, KrUger, F. W., and Mohr, U. Effect ofBeta-Oxidized Nitrosamines on Syrian Golden Hamster. II. 2-Oxopropyln-propylnitrosamine. J. Natl. Cancer Inst., 52: 1869-1874, 1974.

6. Pour,P.,Althoff,J.,Gingell,A.,Kupper,A.,KrUger,F.W.,Schm8hl,D.,and Mohr, U. A Further Pancreatic Carcinogen in Syrian Golden Hamsters: N-Nitroso-bis(2-acetoxypropyl)amine. Cancer Letters, 1: 197-202,1976.

7. Pour, P., Althoff, J., Kruger, F. W., Schmà hl, D. , and Mohr, U. Inductionof Pancreatic Neoplasms by 2,2'-Dioxopropyl-N-propylnitrosamine. Cancer Letters, 1: 3—6.1975.

8. Pour,P.,KrUger,F.w., Althoff,J., Cardesa,A.,andMohr,U.EffectofBeta-Oxidized Nitrosamines on Syrian Golden Hamsters. I. 2-Hydroxypropyl-n-propylnitrosamine. J. NatI. Cancer Inst., 52: 1245-1249, 1974.

9. Pour, P., Kruger, F. W., Althoff, J. , Cardesa, A. , and Mohr, U. Cancer ofthe Pancreas Induced in the Syrian Golden Hamster. Am. J. Pathol. , 76:349-358, 1974.

10. Pour, P., KrUger, F. W., Althoff, J., Cardesa, A., and Mohr, U. Effect ofBeta-Oxidized Nitrosamines on Syrian Golden Hamsters. III. 2,2'-Dihydroxy-di-n-propylnitrosamine. J . NatI. Cancer Inst. , 54: 141-1 46, 1975.

11 . Pour, P. , KrUger, F. W., Cardesa, A., Althoff, J. , and Mohr, U. Carcinogenic Effect of Di-n-propylnitrosamine in Syrian Golden Hamsters. J.NatI. Cancer Inst. 51: 1019-1027, 1973.

12. Pour, P., KrUger, F. W., Cardesa, A. , Althoff, J., and Mohr, U. Tumorigenicity of Methyl-n-propylnitroeamine in Syrian Golden Hamsters. J.NatI. Cancer Inst. 52: 457-462, 1974.

13. Pour, P. . Mohr, U., Cardesa, A., Althoft, J. , and Kmoch, N. SpontaneousTumors and Common Diseases in Two Colonies of Syrian Golden Hamsters. Part Ill. Respiratory Tract and Digestive System Tumors. J. NatI.Cancer Inst. , in press.

14. Pour, P., Mohr, U., Cardcea, A., Althoff, J., and Kniger, F. W. PancreaticNeoplasms in Animal Model. Morphological, Biological and ComparativeStudies.Cancer,36:379-389,1975.

15. Weil, C. S. Tables for Convenient Calculation of Median Effective Dose(LD5O or ED5O)and Instructions in Their Use. Biometrics, 8: 249-263,1952.

also be performed in vitro by whole blood, but it remainsunknown whether plasma, liver, or other esterase activity ismostly responsible for BHP formation in vivo. It seems likelythat the carcinogenic effect of BAP in hamsters is mediatedto a large extent by its facile metabolism to BHP.

Although similar, the carcinogenic effects of BHP andBAP were not identical. BHP application led to developmentof pancreatic neoplasms in all treated hamsters, whereasonly 58% of BAP-treated animals exhibited this tumor. Thiscould be due to the comparatively short BAP treatmentperiod, or to incomplete metabolism to BHP. BAP treatmentalso induced multiple papillomas in the cheek pouch, forestomach, and vagina, as well as a squamous cell carcinomaof the preputium. The development of cheek pouch papillomas and the preputial carcinoma seems to be a specificeffect of BAP. These tumor types were not observed in ourcontrol hamster colony, nor have they been reported to beinduced with other nitroso compounds tested in hamstersthus far. Forestomach and vaginal papilbomas are within thetumor spectrum of our hamster colony (13); however, incontrast to spontaneous neoplasms, the lesions in BAPtreated hamsters were mostly multiple and occurred in aremarkably higher incidence and with a shorter latency.Therefore, it can be assumed that BAP itself has a localeffect in addition to its systemic action mediated probablythrough metabolism to BHP. This may be due to excretionof BAP, via either saliva, feces, or urine, and to self-contamination. Further studies in this regard are under way.

REFERENCES

1. KrUger. F. W. Metabolismus von Nitrosaminen In Vivo. I. Ubcr die /3-oxidation aliphatischer Di-n-alkylnitrosamine: Die Bildung von 7-Methylguanin neben 7-Propyl-bzw. 7-Butyl-guanin nach Applikation von Di-nPropyl-oder Di-n-butylnitrosamin. z. Krebsforsch., 76: 145-154, 1971.

Fig. 1. Tubular adenoma of pancreas composed of channels of various calibers lined by low cuboidal or flat cells. This type of adenoma, which seems tooriginate from ductules, showed frequent papillary infolding of the epithelial lining; some Iuminae contained mucus, cell debris, or, occasionally,erythrocytes. Male hamster, 46 weeks, Group B-2. H & E, x 250.

Fig. 2. Papillary adenoma, which derived from a large pancreatic duct in the head region, composed of tall columnar mucus cells with a few interspersedgoblet cells. Lymphocytic infiltration (lower left portion) was frequently observed around these types of neoplasms. Female, 30 weeks, Group B-3. H & E. x250.

Fig. 3. Intraductal papilloma arising from major duct in head region of pancreas. Abundant connective tissue stalks covered by single-layered regularepithelium distinguished this lesion from intraductal carcinoma (see Fig. 4). Female, 30 weeks, Group B-3. H & E, x 150.

Fig. 4. Intraductal carcinoma in body of pancreas. Formation of “glandsin the gland―and lack of supporting connective tissue in epithelial bridgescharacterize this malignant lesion. Male, 46 weeks, Group B-3. H & E, x 250.

Fig. 5. Adenocarcinoma in pancreas body composed of atypical glandular structures partially filled with mucus. Hyperplastic islet (upper left corner) andremnant of acinar cells (upper right corner) are seen. Female, 46 weeks, Group 8-2. H & E, x 100.

Fig. 6. Acinar cell carcinoma, found in both body and tail region of pancreas, shows well-differentiated (top) and poorly differentiated areas. Tumor cellscontained cytoplasmic cosinophilic granules in apical cell portion (inset). Some signet ring-like cells are intermingled (lower portion). This carcinoma hadinvaded the peritoneum and this photomicrograph was taken from an area remote from original tumor. This type of carcinoma was found in only 1 hamster.Female, 39 weeks, Group A-4. H & E, x 250.

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P. Pour et a!.

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1976;36:2877-2884. Cancer Res Parviz Pour, Jürgen Althoff, Ralph Gingell, et al. Carcinogen in Syrian Golden Hamsters

-Nitroso-bis(2-acetoxypropyl)amine as a Further PancreaticN

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