Z. Krebsforsch.90, 79--86 (1977)

Zeitschrift fiJr Krebsforschung und Klinische Onkologie 9 Springer-Verlag1977

Transplacental Effects of Nitrosamines in Syrian Hamsters III. Dimethyl- and Dipropylnitrosamine* J. Althoff**, P. Pour, C. Grandjean, and S. Marsh Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 USA; Abteilungftir experimentellePathologie,MedizinischeHochschuleHannover, D-3000 Hannover, Karl-Weichert-Allee9, Federal Republic of Germany

Summary. The aliphatic nitrosamines dimethylnitrosamine (DMN), diethylnitrosamine (DEN), dipropylnitrosamine (DPN), and dibutylnitrosamine (DBN) reached fetal tissue in quantitatively measurable amounts after subcutaneous administration to pregnant Syrian hamsters. The compounds were present for at least 2 h in maternal blood, placenta, fetus, and amniotic fluid; D B N was still measurable after 6 h. Only a weak or borderline transplacental effect was seen when incidences and latencies of neoplasms in the respiratory and digestive tracts of the Fl-generation were compared with those of the Pgeneration after exposure to a single dose of D M N or DPN. However, some tumor types occurred at relatively high rates in the young, but were seen only occasionally in their mothers or in this hamster colony in general.

Transplacentare Effekte von Nitrosaminen in Syrischen Hamstern III. Dimethyl- und Dipropylnitrosamin Zusammenfassung. Die aliphatischen

Nitrosamine Dimethylnitrosamin (DMN), Di~ithylnitrosamin (DEN), Dipropylnitrosamin (DPN) und Dibutylnitrosamin (DBN) erreichen nach subcutaner Verabreichung an tr~ichtige, Syrische Hamster in quantitativ bestimmbaren Mengen das fetale Gewebe. Die Verbindungen waren im m~itterlichen Blut, in Placenta, Fetus und Fruchtwasser wenigstens 2 Std nachweisbar; D B N zeigte mei3bare Werte noch nach 6 Std. Der Vergleich der H~iufigkeit und Latenzzeit der Tumoren, die in den Luftwegen und im Verdauungstrakt der F1-Generation gefunden wurden, mit denen in der P-Generation nach Behandlung mit einer Einzeldosis von D M N oder D P N lieB nur eine schwache oder grenzwertige, transpla* Supported by U.S. Public Health Service Contract NO1 CP33278 from the National Cancer Institute, NIH ** The authors thank K.Multly and D. Ogrowsky for technical assistance, J. Villareal for graphic illustration and M. Susman for editorial help Offprint requests to: Prof. Dr. J. Althoff(address see above)

80

J. Althoffet al. centare W i r k u n g erkennen. Jedoch entwickelten sich bei d e n J u n g e n einige T u m o r a r t e n , die n u r gelegentlich bei d e n M i i t t e r n oder a n d e r e n H a m s t e r n dieser K o l o n i e gefunden wurden.

Introduction M o s t n i t r o s a m i n e s tested thus far have been f o u n d to be toxic a n d carcinogenic in one or several a n i m a l species a n d it is possible the same effects m a y be applicable in man. N i t r o s a m i n e s m a y be formed in vivo from n i t r o u s acid (nitrite) a n d c o r r e s p o n d i n g amines (Sander, 1968, 1971; Sen et al., 1969; Lijinsky et al., 1972; S h a n k a n d Newberne, 1973). Some of these n i t r o s o c o m p o u n d s have been described as being occasionally f o u n d in small a m o u n t s in the e n v i r o n m e n t , as well as in food ( P r e u s s m a n n , 1975). Such c o m p o u n d s include dimethyl- ( D M N ) , diethyl- (DEN), dipropyl- (DPN), a n d d i b u t y l n i t r o s a m i n e (DBN). Hence, it is of interest to d e t e r m i n e whether, following exposure of p r e g n a n t hamsters, these potential h u m a n carcinogens reach the fetal tissue. Experiments were carried out in Syrian hamsters, a n d the l o n g - t e r m t r a n s p l a c e n t a l effect of a single dose of D M N or D P N was d e t e r m i n e d in this species.

Material and Methods After mating, randomly bred, 12-week-old Syrian hamsters from the Eppley colony, were housed individually in plastic cages. The day of mating was considered the first day of pregnancy. Animals received a pelleted diet (Wayne, Allied Mills, Chicago, IlL) and water ad libitum. The hamsters and their young were checked twice daily and weighed at 4-week intervals. DMN and DEN were synthesized and purified by Dr. W. Lijinsky (Frederic Cancer Center, Maryland, USA). DPN and DBN were prepared by Dr. F.W. Kriiger (German Cancer Research Center, Heidelberg, FRG). Animals were treated subcutaneously (s.c.) with 12.5mg/kg b.w. DMN or 100 mg/kg b.w. DPN. For comparison, 100 mg/kg b.w. DEN and 100 mg/kg b.w. DBN were also investigated. After treatment, animals were sacrificed, and blood, amniotic fluid, placental and fetal tissues of 3 hamsters per interval were pooled (a total of 100 pregnant hamsters were used). Tissues were either processed immediately or stored at -90 ~ C until processing. Homogenates were prepared with an equal volume of distilled H20 [PCU-2 homogenizer, Brinkman Instruments] and centrifuged sequentially at 2000g and 10000g each for 10 min. Supernatants were shaken with an equal volume of diethyl ether, and mixtures separated by centrifugation at 2000 xg for 10 min. The ether layer was evaporated to a known volume using dry nitrogen. After analysis of the extracts by gas liquid chromatography (GLC), data were compared to those of the authentic compounds. A 2-m column of 15% diethyleneglycolsuccinate(DEGS) on chromasorb W. was used for GLC analyses. Column temperature and retention times of the authentic compounds were: DMN: 90~C, 43 min; DEN: 110~ C, 3.2 min; DPN: 130~C, 3.2 min; DBN: 150~C, 4 min. Authentic standards were run prior to and after analyses of the sample extracts. To determine the long-term transplacental effects of 12.5 mg/kg b.w. DMN or 100 mg/kg b.w. DPN, a singles.c. dose was given at day 8, 10, 12 or 14 of gestation to groups of 5--6 animals. (A dose of 25 mg/kg b.w. DMN was not tolerated by pregnant hamsters.) Newborn (DMN-treated, 226; DPNtreated, 208) were counted and examined for gross malformations within 24 h after delivery. The F 1generation (DMN: 90 females, 110 males; DPN: 93 females, 84 males) was separated from the Pgeneration after a 4-week lactation period, housed by sex in groups of 5, and kept for life. Animals were killed when moribund. After autopsy, organs were fixed in 10% buffered formalin and bones were decalcified with Decal (Omega Chemical Corp., Cold Spring on Hudson, N.Y.). Paraplastembedded tissues were sectioned and strained with hematoxylin and eosin. Step sections were prepared from nasal cavities,larynx, and trachea with stem bronchi attached. Calculations are based on the number of histologically examined hamsters (effective number of animals), since some animals were lost due to partial or complete cannibalism. As previously reported, 21 untreated females and their young served as controls (Parts 1 and 2).

Transplacental Effects of Nitrosamines in Syrian Hamsters. III

81

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Results In the present investigation all symmetric, aliphatic nitrosamines examined were found in the maternal blood after s.c. administration, and reached the placenta, fetus and amniotic fluid (Figs. 1--4). In the case of DMN, maximal concentrations occurred in all tissues about 1 h after injection, and only traces were found after 4 h. Maternal blood and placental levels were similar, and significantly higher than the amounts determined in amniotic fluid and fetus; the lowest levels were found in the latter. DEN reached a maximum in the maternal blood about 30 rain after administration, its concentration in amniotic fluid was maximal between 30 min and 1 h, and was higher than that in the maternal blood for the first 2 h. Maximal levels in the placenta occurred after 45 min and again exceeded those of maternal blood at the same time. The amount in the fetus was the lowest for all times examined and was fairly constant between 30 min and 2 h, after which time the level rapidly decreased. The compund essentially disappeared from these tissues after 4 h. DPN was examined in 3 separate experiments involving pooled material from 3 hamsters and showed 2 maximal levels for maternal blood and amniotic fluid, at 45 and 90 rain, respectively, after s.c. injection. The placenta and fetus had the highest DPN concentrations at 90 rain, and at this time similar high levels were seen in maternal blood, placenta, fetus, and amniotic fluid; DPN was not detected after 4h. DBN was detectable in very small amounts, especially in the placenta and fetus, and maintained in an almost constant level up to 6 h after treatment. No grossly visible malformations were noted in the Fl-generation. The litter size was 10.8+2 (DMN) and 9.9_ 1.8 (DPN) in the nitrosamine-treated groups (10.4+ 2.5 in controls). Postnatal mortality in the first 4 weeks was 12% (DMN) and 15% (DPN), compared to 2% in control. Average survival, age of tumorbearing animals and tumor incidence and multiplicity are given in Table 1. The young in the DPN groups outlived their mothers, and the age of tumor-bearing animals was higher in the F l-generation. A single dose of D M N primarily affected

83

Transplacental Effects of Nitrosamines in Syrian Hamsters. III Table 1. Transplacental effect of D M N and D P N Group

P-control Fa-control P-DMN F1 P-DPN F1

Effective no. of animals

20 203 19 194 20 159

Age in weeks at death

Age of TBA at death

(x--+s)

(x--+s)

T u m o r inciNo. of tumors dence in % and per TBA a (No. of TBA)

5 7 + 18 61 -+ 19 62-+13 65-+ 19 52-+ 13 70-+20

64-+ 18 76-+ 18 64-+15 75-+20 56-+ 15 80-+20

40 (8) 18 (37) 42 (8) 35 (67) 60 (12) 41 (65)

1.5 1.2 1.6 1.3 1.4 1.4

a Does not include multiple tumors in one organ TBA = Tumor-bearing animals P: parent generation; F a = 1 st generation D M N (1 • 12.5 mg/kg b.w. to s.c. to P-generation) D P N (1 • 100 mg/kg b.w. to s.c. to P-generation)

the digestive tract of the P-generation (Table 2). Hamsters treated once with DPN during pregnancy developed respiratory tract tumors. Both nitrosamines had a transplacental effect. Only a few neoplasms of the respiratory epithelium were observed in the young exposed to D M N in utero. A papillary polyp of the anterior portion (F~: day 8 of gestation) and an adenocarcinoma of the posterior portion (Fx: day 14 of gestation) were found in the nasal cavities, as well as a laryngeal papillary polyp (Fa: day i0 of gestation). DPN caused respiratory neoplasms in other segments. No nasal cavity tumors were observed. Papillary polyps were found in the larynx (P: 2 tumors; FI: 2 tumors, day 14 of gestation) and trachea (P: 55%, FI: 1 tumor, day 14 of gestation). One bronchiogenic adenocarcinoma was found (P) compared to one adenoma observed in controls. The respiratory tract tumor incidence in hamsters exposed transplacentally to DPN at day 14 of pregnancy corresponded to 7%. Other epithelial neoplasms of the lungs were metastases (P-DMN: adrenal cortical carcinoma; F-DPN: uterine adenocarcinoma). Digestive system tumors occurred in a relatively high incidence in DMN-treated hamsters and their offspring, and in the young of animals after DPN injection. A squamous cell carcinoma of the upper lip was found (F1-DPN). Squamous cell papillomas developed in the palate (F-DMN, F-DPN), pharynx (F-DPN), and forestomach (F-DMN: 3%; P-DPN: 1 x ; F-DPN: 8%; control 3 %). Also seen were adenomas of the bile ducts (P-DMN: 26 % ; F-DMN: 4%; FDPN: 2.5%) and pancreatic ducts (P-DMN: 1 x ; F-DMN: 3%; F-DPN: 4%; 83 __+20 weeks), and adenocarcinomas of bile ducts (control: 1 x ), pancreatic ducts (control: 2 x ) and colon (P-DMN: 1 x ; F-DMN: 5%; F-DPN: 4%; control: 2%). Discussion

The aliphatic nitrosamines examined in the present investigation reached the maternal blood after s.c. injection, passed the placenta into the fetus and were

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" Renal adenoma (F1-DMN); nephroblastoma (F1-DPN); urinary transitional cell papilloma (P DPN); Cowper's gland adenoma (F 1 DPN); seminoma (control); ovarian papillary cystadenoma (F1-DMN, FI-DPN), mesenchymal tumors (FI-DPN, controi); uterine leiomyofibromas (P-DMN, F1-DPN: 2 x, control 2%), adenocarcinomas (P-DMN, FI-DPN: 2 x ) b Parathyroid adenomas (P-DMN, F - D M N : 5%, P DPN, F t - D P N : 8%, control: 4%); thyroid adenomas (P DMN, F I - D M N : 4%, F1-DPN: 4%, control: 2%); pancreas insulomas (FI-DMN: 2%, F1-DPN : 2 x, control); adrenal cortical adenomas (P-DMN, F I-DMN: 12%, F 1-DPN: 10%, control: 7%) Hemangioendotheliomas of the lung (control: 2 x ), liver (P DMN, F I - D M N : 2%, FI-DPN: 2 x ), spleen (FI-DMN: 2%, Fx-DPN ) axillary subcutaneous tissue (control); hemangioendotheliosarcoma of axillary subcutaneous tissue (F~ DPN); malignant lymphomas (F1 DMN, F~-DPN: 2 x, control) a Malignant schwannoma of eyelid (F~ DPN), retromesenteric region (control); uterine granular cell tumors (F 1 DMN, F1-DPN); osteosarcoma of rib (F 1 DMN); chondrosarcoma of skull (F1 DPN ) TBA = Tumor-bearing animals P = Parent generation; F~ = lst-generation RES = Reticuloendothelial system

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Transplacental Effects of Nitrosamines in Syrian Hamsters. III

85

also present in the amniotic fluid. In addition to qualitative data obtained by thinlayer chromatography [see studies of DEN, DBN, nitrosohexamethyleneimine (N6MI): Mohr et al. (1965), Althoff et al. (1976)], specific quantitative values found by GLC showed that maximal concentration periods, duration and disappearance varied in the different tissues and may be characteristic for each compound. Since equal amounts were administered [DEN, DPN, DBN, and nitrosopiperidine (NP), see Part 2], differences might partially be explained by the varying chemical structures of these compounds, which may influence reabsorption in subcutaneous tissue, solubility in the serum, or metabolism and excretion rates. D M N and DBN levels in the fetus were less than 25% of those in the placenta (as seen also for NP and N6MI), whereas amounts of DEN in fetal tissues approached those of the placenta and maternal blood. The reasons for higher levels or earlier maxima of unchanged nitrosamines in amniotic fluid relative to those found in the fetus (DEN: also maternal blood) are not yet understood. The possibility of additional paraplacental routes, concentration mechanisms or contamination during amniocenthesis must be investigated. The relatively low levels of nitrosamines which pass the placenta might be partially related to their weak carcinogenic effect on the fetus. Following D M N exposure, the cholangioma incidence was considerably lower in the F~-generation than in the mothers, and lower than the rate found for non-pregnant adult hamsters receiving a single dose (Tomatis and Ceils, 1967). This tumor type, not induced by DPN in adult hamsters (Pour et al., 1973), also occurred in the young, but not at all in hamsters treated only once during gestation. Cholangiocarcinomas, which were reported after multiple D M N doses (Tomatis et al., 1964; Herrold, 1967; Kowalewsky and Todd, 1971 ; Haas et al., 1973), were not seen in animals treated once during pregnancy or in their offspring. The same was true for hepatocellular neoplasms, which were also reported after chronic D M N treatment (Tomatis et al., 1964; Tomatis and Ceils, 1967; Kowalewsky and Todd, 1971 ; Haas et al., 1973; StenNick et al., 1973). However, borderline transplacental effects were considered for some other tumors, including respiratory neoplasms in the nasal cavities, larynx and trachea, pancreas duct adenomas, colon adenocarcinomas, and vascular tumors in the liver and spleen. These tumors either did not occur or were rarely observed in treated mothers, except for DPN-exposed hamsters. Although their incidence was low in the young, they developed only occasionally in controls or in general in this breeding colony (Pour et al., 1976). The occurrence of some of these tumors in DMN- or DPN-treated adult hamsters (Tomatis et al., 1964; Herrold, 1967; Tomatis and Ceils, 1967; Haas et al., 1973; Stenb~ick et al., 1973; Pour et al., 1973) may support the view that they were associated with intrauterine exposure of progeny. Related to the wide spectrum of neoplasms induced by D M N or DPN in hamsters, however, the findings have indicated that the F1-generation is transplacentally less endangered by the carcinogenic effects than is the P-generation. Other investigations in rats and hamsters allowed similar conclusions (Mohr et al., 1966; Alexandrov, 1968; Mohr et al., 1975; Ivankovic, 1975; Althoff et al., 1976). The results may also imply that some types of tumors not seen in treated pregnant hamsters can develop during the lifespan of the young, even though the basic stimulus was triggered in utero during gestation.

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References Alexandrov, V. A.: Blast omogenic effect of dimethylnitrosamine on pregnant rats and their offspring. Nature (Lond.) 218, 280--281 (1968) Althoff, J., Pour, P., Grandjean, C., Eagen, M. : Transplacental effect of nitrosamine in Syrian hamsters. I. Dibutylnitrosamine and nitrosohexamethyleneimine. Z. Krebsforsch. 86, 69--75 (1976) Haas, H., Mohr, U., Kriiger, F. W. : Comparative studies with different doses of N-nitrosomorpholine, N-nitrosopiperidine, N-nitrosomethylurea, and dimethylnitrosamine in the Syrian golden hamster. J. Natl. Cancer Inst. 51, 1295--1301 (1973) Herrold, K. M. : Histogenesis of malignant liver tumors induced by dimethylnitrosamine. An experimental study in Syrian hamsters. J. Natl. Cancer Inst. 39, 1090--1111 (1967) Ivankovic, S.: Prgnatale Carcinogenese. In: Handbuch der allgemeinen Pathologie, 6. Band, Geschwiilste-Tumors 3, 7. Teil. Berlin: Springer 1975 Kowalewski, K., Todd, E. F.: Carcinoma of the gallbladder induced in hamsters by insertion of cholesterol pellets and feeding dimethylnitrosamine. Proc. Soc. Exp. Biol. Med. 136, 4 8 2 4 8 6 (1971) Lijinsky, W., Keefer, L., Conrad, E., Van de Bogart,R.: Nitrosation of tertiary amines and some biologic implications. J. Natl. Cancer Inst. 49, 1239--1249 (1972) Mohr, U., Althoff, J., Authaler,A.: Diaplacental effect of the carcinogen diethylnitrosamine in the golden hamster. Cancer Res. 26, 2349---2352 (1966) Mohr, U., Authaler,A., Althoff, J.: Der Nachweis von Diiithylnitrosamin in Goldhamsterembryonen nach Behandlung des Muttertieres. Naturwissenschaften 52, 188--189 (1965) Mohr, U., Reznik-Schiiller, H., Reznik, G., Hilfrich, J.: Transplacental effects of diethylnitrosamine in Syrian hamster as related to different days of administration during pregnancy. J. Natl. Cancer Inst. 55, 6 8 1 ~ 8 3 (1975) Pour, P., Kr tiger, F. W., Cardesa, A., Althoff, J., Mohr, U.: Carcinogenic effect of di-n-propylnitrosamine in Syrian golden hamsters. J. Natl. Cancer Inst., 51, 1019--1027 (1973) Pour, P, Kmoch, N., Greiser, E., Mohr, U., Althoff, J., Cardesa, A.: Spontaneous tumors and common diseases in two colonies of Syrian hamsters. I. Incidence and sites. J. Natl. Cancer Inst. 56, 931-935 (1976) Sander,J.: Kann Nitrit in der menschlichen Nahrung Ursache einer Krebsentstehung dutch Nitrosaminbildung sein? Arch. Hyg. (Bed.) 151, 22--28 (1967) Sander,J.: Untersuchungen fiber die Entstehung cancerogener Nitrosoverbindungen im Magen yon Versuchstieren und ihre Bedeutung for den Menschen. Arzneimittel-Forsch. (Drug Res.) 21, 1572-1580, 1703--1707, 2034--2039 (1971) Sen, N. P., Smith, D. C., Schwinghamer, L.- Formation of N-nitrosamines from secondary amines and nitrite in human and animal gastric juice. Food Cosmet. Toxicol. 7, 301--307 (1969) Shank,R.C., Newberne, P.M.: Nitrite-morpholine induced hepatomas. Food. Cosmet. Toxicol. 10, 887--888 (1972) Stenb~ick, F. G., Ferrero, A,, Montesano,R., Shubik, P.: Synergistic effect of ferric oxide on dimethylnitrosamine carcinogenesis in the Syrian golden hamster. Z. Krebsforsch. 79, 31--38 (1973) Tomatis, L., Cefis, F.: The effects of multiple and single administration of dimethylnitrosamine to hamsters. Tumori 53, 447~452 (1967) Tomatis, L., Magee, P. N., Shubik, P.: Induction of liver tumors in the Syrian golden hamsters by feeding dimethylnitrosamine. J. Natl. Cancer Inst. 33, 341--345 (1964)

Received September 20,1976; accepted June 15,1977