Z. Krebsforsch. 90, 119--126 (1977)

Zeitschrift~r Kreb~n~hung unclKlinische Onkologie 9 Springer-Verlag 1977

Transplacental Effect of Nitrosamines in Syrian Hamsters IV. Metabolites of Dipropyl-and Dibutylnitrosamine* J. Althoff, C. Grandjean, and P. Pour Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105, USA

Summary. The present investigations showed that assumed and established metabolites of dipropylnitrosamine and dibutylnitrosamine reach the Syrian hamster fetus after subcutaneous (s.c.) treatment of their mothers (at day 14 of gestation). The compounds [2-hydroxypropylpropylnitrosamine, HPPN; 2oxopropylpropylnitrosamine, OPPN; methylpropylnitrosamine, MPN; Nnitrosobis(2-hydroxypropyl)amine, BHP; and 4-hydroxybutylbutylnitrosamine, HBBN] were still present in the examined tissue (maternal blood, placenta, fetus, amniotic fluid) 4--6 h after s.c. injection. The overall incidence of transplacentally induced tumors was lower in the F 1- than in the P-generation and comparatively longer latencies were also observed in the F 1generation. However, in some groups low incidences were found of tumors which did not occur in the mothers (i.e., nasal cavities: BHP, HBBN; trachea: HBBN; lungs: HPPN, BHP, HBBN; liver: OPPN, MPN, BHP, HBBN). Compared to exposure at early gestation, the transplacental carcinogenic effect increased at day 14 of gestation. Neoplasms originating in other organs were not associated with a transplacental effect of the examined nitrosamines. Transplacentare Wirkungen von Nitrosaminen bei Hamstern. IV. Metaboliten yon Dipropyl- und Dibutylnitrosamin Zusammenfassung. Die Untersuchungen haben gezeigt, dab angenommene sowie gefundene Metaboliten des Dipropyl- und Dibutylnitrosamins die Feten Syrischer Hamster via Placenta nach subcutaner (s.c.) Behandlung der Muttertiere (14. Tag der Tragzeit) erreichen. Die Verbindungen (2-hydroxypropylpropylnitrosamin, HPPN; 2-oxopropylpropylnitrosamin, OPPN; methylpropylnitrosamin, MPN; N-nitrosobis(2-hydroxypropyl)amin, BHP; 4hydroxybutylbutylnitrosamin, HBBN) waren noch 4 6 Std nach subcutaner Injektion in den untersuchten Geweben (miJtterliches Blut, Placenta, Fetus, Fruchtwasser) nachweisbar. Die allgemeine Tumorh~iufigkeit transplacentar * Supported by U.S. Public Health Service Contract NO1 CP33278 from the Division of Cancer Cause and Prevention, National Cancer Institute

120

J. Althoffet al.

induzierter Tumoren war geringer in der F 1- als in der P-Generation, w~ihrend vergleichsweise die Latenzzeiten verliingert waren. Dariiber hinaus fanden sich jedoch Geschwiilste in geringer H/iufigkeit bei einigen Gruppen, die bei Muttertieren nicht gefunden wurden (NasenhiShle: BHP, HBBN; Trachea: HBBN; Lunge: HPPN, BHP, HBBN; Leber: OPPN, MPN, BHP, HBBN). Fiir den Laryngo-Tracheal-Trakt erh6hte sich die transplacentare carcinogene Wirkung nach Behandlung am 14. Tag der Tragzeit in Vergleich zur Exposition an einem friiheren Zeitpunkt der Gestation. Tumoren anderer Organe wurden nicht auf eine transplacentare Wirkung der untersuchten Nitrosamine bezogen.

Introduction

Previous examinations have shown that some nitrosamines reach fetal tissues unaltered and in measurable amounts after subcutaneous (s.c.) treatment of Syrian hamsters during gestation (Mohr et al., 1965; Althoff et al., 1976, 1977). However, the tumor incidence in target organs was found to be lower in offspring (F 1-generation) than in mothers (P-generation). These observations were tentatively explained by a lack of fetal enzyme systems which may activate the nitrosamines, and by the comparatively low concentrations which actually reached the fetus. Since some nitrosamine metabolites were linked to the carcinogenicity of the parent compound and may be considered more proximate carcinogens, transplacental studies were initiated in Syrian hamsters using S-oxidized dipropynitrosamine (DPN) derivatives. For comparative purposes, an ~-hydroxylated metabolite of dibutylnitrosamine (DBN) was also examined. Moreover, we also attempted to demonstrate the presence of these metabolites in maternal blood, placenta, fetus, and amniotic fluid. Material and Methods Randomly bred, 12-week-old Syrian hamsters from the Eppley colony were housed individually in plastic cages after mating. They received a pelleted diet (Wayne, Allied Mills, Chicago, I113 and water ad libitum. The day of mating was considered the first day of pregnancy. P-generation hamsters were treated s.c. once, by a single injection of the assumed or proven metabolites of selected aliphatic nitrosamines at day 8, 10, 12 or 14 of gestation. Groups of 15 animals received 100 mg/kg b.w. 2hydroxypropylpropylnitrosamine (HPPN), 2-oxopropylpropylnitrosamine (OPPN), methylpropylnitrosamine (MPN), N-nitrosobis(2-hydroxypropyl)nitrosamine (BHP) or 4-hydroxybutylbutylnitrosamine (HBBN). These compounds were synthesized and purified by Dr. F.W.Krtiger, Institute for Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg. Within 24 h after delivery, offspring were counted and examined for malformations; after 4 weeks, they were separated from their mothers and housed by sex in groups of 5. P and F~ hamsters were checked twice daily, weighed at 4-week intervals, and maintained for life. Animals were killed when moribund. After complete autopsy, organs were fixed in 10% buffered formalin, and bones were decalcified with Decal (Omega Chemical Corporation, Cold Spring on Hudson, New York). Paraplast-embedded tissues were sectioned and stained with hematoxylin and eosin. Graded sections were obtained from nasal cavities, larynx, trachea with attached stem bronchi and urinary bladder (HBBN-treated hamsters only). Calculations were based on the number of histologically examined animals (effective no. of animals). Data concerning the controls were reported previously (see Parts 1--3). To examine the maximal concentration periods of the compounds in maternal blood and fetal tissues, 102 hamsters were treated s.c. with 100 mg/kg b.w. of each compound on day 14 of gestation.

Transplacental Effectof Nitrosamines in Syrian Hamsters. IV

121

The animals were sacrificed in groups of 3 at 0.25 h, and at 0.5, 0,75, 1.0, 1.5,2, 4, and 6 h after injection. Maternal blood, placenta, fetus, and amniotic fluid of 3 animals were pooled, homogenized in H20, and extracted (3 • ) with diethylether. Tissue homogenates from BHP-treated animals were extracted with ethylacetate. Recovery of administered nitrosamines from the homogenate varied (HPPN: 70%; DPPN: 40%; MPN: 80%; BHP: 45%; HBBN: 80%). The extracts were analyzed by gas-liquid chromatography (GLC) on a 2-m glass column of SE-30 (3.4%) on Chromasorb W-HP. MPN extracts were examined by GLC on a 2-m column of DEGS (15%) on Chromasorb W-HP. The flow rate was 20 ml/min, and retention times of all samples were compared to those of authentic standards under identical conditions. The temperatures used and retention times for each compound were: HPPN: 100~C, 6 min; OPPN: 100~C, 2.67 min; MPN: 140~C, 4.67 min; BHP: 130~C, 3.25 min; HBBN: 130~C, 5.5 min.

Results All the nitrosamines were found unchanged in maternal blood and fetal tissue 15 rain after s.c. injection. However, as shown in Figure 1, the time of maximal concentration for the compounds and the tissue affected varied. The highest peak for all tissue samples was at 30 min after O P P N treatment. Also at 30 rain, maximal levels were recorded in maternal blood for both H P P N and MPN, in the placenta and fetus for H P P N , and in the fetus for MPN. At 45 min, a high peak resulted in maternal blood and placenta for B H P and in the placenta for HBBN. At 60 min, H B B N showed the highest level in maternal blood, placenta and amniotic fluid, whihe maximal M P N and B H P concentrations were found in amniotic fluid. H P P N reached its highest concentration in amniotic fluid at 90 min, and MPN, in the placenta at 180 min. M P N revealed higher levels in the placenta fetus and amniotic fluid than in maternal blood after 2 h and was still present in measurable amounts in all samples at 4 and 6 h, as were B H P and H P P N . Considering the recovery rates of the different nitrosamines, lesser amounts (approximately 0.1--0.5) occurred in the placenta, compared to maternal blood concentrations. In the fetus, the nitrosamine level approximated that of maternal blood after H P P N treatment, whereas values were approximately 50% in the other groups. In addition to the unchanged compounds, several as yet unknown compounds were detected, including 3 comparatively more volatile compounds in the H P P N group, 2 less volatile compounds in the O P P N group and 1 less volatile c o m p o u n d in the HBBN-treated animals. N o additional peaks were observed after M P N or B H P administration in any of the examined tissues. These unknown compounds were not detected in control urine. N o grossly visible malformations were found after transplacental exposure of hamsters to a single dose of oxidized nitrosamines. The litter size between groups varied only slightly (A: 10.4 + 2.5; B: 9.6 + 1.8; C: 10.7 + 2.3; D: 10.3 + 2.5; E: 9.4 + 1.2; F: 10.6 + 2.6). However, postnatal mortality (at 4 weeks) in the treated groups increased, compared to controls (A:2%, B:7%, C : 6 % , D : 8 % , E: 13%, F:10%). D a t a are given in Table 1 regarding animals which survived the lactation period. Offspring outlived their mothers and the overall t u m o r incidence was commonly higher in P than F 1 hamsters, whereas the age of tumor-bearing animals was lower in mothers than in their offspring. N o marked differences relative to t u m o r multiplicity and average survival of tumor-bearing animals were noted between the groups and subgroups.

122

70

J. Althoff et al.

~glg

I

/~ ,o

o

MATERNAL BLOOD

50--

501 gig

FETUS

~ _l~172 6 ~O.om

~-' / ~~~"

IO~'-

L

~

,

I ~ "-A--,'~...____HBBN

I

,

0 I~l,

I

I

9

OPPN I

I

3

h

00PPN I,

,

i

,

Iii, I

I

t $

I h

50 -,ug/g

~-~

-

.~f ~,.~

;.~c~,,,

,oi[

_o ~00PPoN

I

,

,

AMNIOTIC FLUID

~lI

,

~ H - ~ p B N BN I~F I

I

/

.

'xt'x\~_A

/

[~

I

I

3

h

BHP

[]

OPPN

t

,

I

,

Ill, I

I

I 3

I h

Fig. 1. Time of maximal concentration of nitrosamines m maternal blood, fetus, placenta, and amniotic fluid

Neoplasms of the Respiratory Tract Tumors of respiratory epithelium occurred in all treated groups. Their incidence was low for the nasal cavities (A: 0%; B: P, 5%; F1, 1%; C: 0%; D: P, 13%; F 1 , 1 % ; E : F 1, 1%;F:F1, 1%). Histologically, nasal cavity tumors were papillary polyps, papillomas, and adenocarcinomas; their appearance in the young was unrelated to the day of in

Transplacental Effect of Nitrosamines in Syrian Hamsters. IV

123

Table 1. Transplacental effects of oxidized aliphatic nitrosamines in Syrian hamsters Group Genera- Effective Age at tion and no. of death treatment animals (wks_+SD)

Tumor No. of Organ distribution of tumors with incidence in % incidence tumors and age of TBA at death (wks+SD) % (No. of per TBA TBA) Respiratory a Digestive Endocrine b Other ~

A

P-control 20 F1 203

64+18 76_+18

40 (8) 18(37)

1.5 1.2

B

P-HPPN F1

19 173

52-+16 80+22

53(10) 36(62)

1.3 1.5

47(49-+17) 6(76-+20)

C

P-OPPN F1

20 198

55+13 79-+21

40 (8) 24(47)

D

P-MPN F1

16 173

59_+t0 77_+22

E

P-BHP F1

17 166

F

P-HBBN F,

19 180

5(53) 0--

10(77~85) 6(84+22)

25(69+ 7) 10(53,55) 12(75_+21) 3 (75+ 13)

5(54) 12(91_+22)

11(62,73) 20(86_+23)

5(73) 8(85+27)

1.3 1.2

15(57_+ 3) 15(54+ 2) 15(59_+21) 4(82_+22) 6(85_+22) 12(78_+20)

5(42) 6(77_+25)

31 (5) 32(55t

1.8 1.6

25(58_+11) 4(76_+44)

6(65) 12(82_+16)

57_+13 82_+24

59(10) 37(61)

1.4 1.5

12(48,49) 2(86_+18)

29(70_+ 9) 24(59_+14) 24(59_+14) 11(88_+24) 20(85_+25) 5(85_+241

57_+18 82_+24

32 (6) 37(67)

1.7 1.2

0-3(81_+30)

0 16(84_+22)

16(64_+18) 21(52_+16) 8(88_+25) 24(81_+18)

t3 (54, 62) 12(72_+20)

10(75,75) 7(85_+171

The incidence of respiratory neoplasms was comparatively higher in some groups of Fl-hamsters exposed at day 14of gestation(Groups:B, 22% ;C, 15% ;D, 2% ;E, 4% ; F , 3%) b Parathyroid adenomas (A: 20% p, 2% FI; B: 8% FI; C: 4% FI; D: 6% FI; E: 1 x p; F: 2 x p, 7% FI); thyroid adenomas (A: 2 x P ; B: 4% F1; C: 2% FI; D: 4% Fl; E: 2% FI; F: 2 x P , 5% F 0 ; thyroid adenocarcinomas(E:2xF1;F:2xF0; pancreas insulomas (A: I • B: l x P , 2% F 1 ; C : I • 3% Ft; D: 2 • F 1; E: 5% F 1; F: 3% F,); pancreas islet cell carcinomas (C: 2 x F 1; E: 1 • F l); adrenal cortical adenomas (A: 7% F1; B: l x P , 10% F l, C: l x P , 5% F t ; D: 8% FI; E: I x P , 9% Ft; F: l x P , 11% F0;adrenal cortical carcinomas (E: 1 x F , ; F: 2 • F 0 ; adrenal pheochromocytomas (A: 1 • FI) Harderian gland adenomas (B: 2 • F1; E: 2% F1; F: 2 x FI); cutaneous malignant melanomas (F: 2 x F1); trichoepitheliomas(E: 1 x F0;Schwannomas--subcutaneous(D: 1 • F1; E: 1 x F0;mesentery(A: 1 • P; B: 1 x FI; D: 2 • FI); hemangioendothelioma--subcutaneous (A: 1 x F0;lung (A: 2 • F1); spleen (B: 2 x F 1; C: 1 • P, 2% F, ; D: 2% FI; E: l • F: l x F l ) ; mesentery (C: l x F l ) ; malignant lymphomas (A: I • B: 2% FI; C: 1 • D: 1 x P, 2% FI; F: 2 x FI); osteosarcomas-costal (B: 1 x F , ; D: 2 x F0; pelvic (E: 1 x F 0 ; renal carcinomas (C: 1 x F1 ; D: 1 • F l); urinary bladder papillomas (B: 1 • P, 1 x F 1; D: 1 • F,); ovarian granulosa cell tumors (A: 1 x F 1; D: 1 x F 1; E: 2 x P, 2 • F 1); uterine adenocarcinomas (D: 3% F 1; F: 1 x P, I x F 1); uterine leiomyofibromas (A: 1 x P, 2 x FI; D: 2 x F1; E: 1 x P, 1 x F 0 ; uterine granular cell tumors (D: 1 x F1); vaginal squamous cell carcinomas (E: 1 x F1); mammary adenocarcinomas (B: 1 x F l; E: 1 • P); Cowper gland adenomas (B:2xFI;C:2• 0

utero exposure. The frequency of neoplasms in the laryngotracheal tract was significantly higher in the P-generations of Groups B--E (42%, 15%, 12%, respectively) than in their young (15%, 4%, 1%, 0%, respectively); the incidence was similarly high in offspring of Groups C (16%) and F (3%) and in their mothers after exposure at day 14 of gestation. The larynx was free of neoplastic growth in Groups C, D, and F (parent hamsters) and E (F 1 animals). Tracheal tumors were also not found in Group E or in progeny of Group F. Neoplasms of stem bronchi were seen only in parent hamsters of Groups B, D, and E. Most of these tumors were papillary polyps; only 1 laryngeal papilloma (B: F1), 2 epidermoid carcinomas (B: F t, C: Ft) and 1 tracheal adenocarcinoma (D: FI) were found. Most neoplasms in this respiratory segment of the young occurred after exposure at day 14 of pregnancy; only 6 animals deleloped tumors following treatment at days 10

124

J. Althoffet al.

or 12. Lung neoplasms were not seen in animals of Group C or in the P-generation of Groups B, E, and F. The tumor incidence was higher in mothers (P, 13%, F1, 2%) of Group D only; however, in offspring of the other groups the incidence was comparatively low (B: 5%, E : 4 % ; F: 1%). No dependence was observed on the day of exposure during pregnancy. All lung tumors were bronchiogenic adenomas (also found in 1 control) and adenocarcinomas.

Digestive Tract Neoplasms Papillomas occurred in the pharynx (C: P, 5 % and D : F 1, 2%) and in the forestomach of all groups. In the latter organ, incidences varied between 2 and 5%, but the tumors were relatively frequent in Group E (P, 12%) and did not occur in mothers of Group F. One of the forestomach tumors was a squamous cell carcinoma (D: F 0. Different types of neoplasms developed in the liver. Most tumors were cholangiomas, which ranged in incidence from 0% (A: Fl) to 12% (E: P). In Groups B (5%) and D (4%), these neoplasms were found only in the young. One cholangiocarcinoma was seen in a control (A: P); papillary polyps of the gall bladder occurred in some animals (B:P, 5%; F 1, 2%; F: F1, 1%). However, hepatocellular neoplasms (1 adenoma, C: F1) and 2 carcinomas with metastases into the lung, were seen only in offspring of treated groups, as were 1 Kuppfer cell sarcoma (F) and hemangioendotheliomas (C--F: 1% each). The pancreas of all groups showed ductal adenomas; 1 adenocarcinoma occurred (E: P), and the incidence ranged between 1% and 4% in the Fl-generation and between 0% (Am D) and 5%--6% (E and F) in mothers. Except for Group C, colon adenocarcinomas were found in all groups. Their frequency was between 1 and 4% in the offspring; one of these neoplasms occurred in a control (A: P). Discussion There is human and experimental evidence that drugs and chemicals can endanger the Fl-generation following exposure in utero (Tomatis, 1973; Tomatis and Mohr, 1973; Ivankovic, 1975). In terms of mechanisms, dose dependencies and relation to the gestation period little is known. Transplacental investigations in animals revealed that nitrosamines commonly affect the young to a lesser extent than their mothers (Mohr et al., 1965; Ivankovic, 1975; Althoff et al., 1976, 1977) and that the carcinogenic effect is more evident after intrauterine exposure at the end of pregnancy than directly after placentation (Mohr et al., 1975). Since it was demonstrated that several aliphatic and cyclic nitrosamines pass the placenta unaltered into the fetus and amniotic fluid at measurable amounts (Mohr et al., 1965; Althoff et al., 1976, 1977), the relatively weak carcinogenicity for the young was tentatively attributed to comparatively low concentrations in fetal tissue, insufficiently competent enzyme systems for formation of the ultimate carcinogens, or highly active repair mechanisms. These possible interpretations may be also valid for the overall transplacental effect of some fl-oxidized DPN intermediates and the DBN metabolite, because compounds were present in the fetus after treatment of mothers, and the total tumor incidence did not increase in offspring nor did the latency of induced neoplasms decrease. Some tumors occurred in other organs of the young, and may be considered as resulting from a borderline

Transplacental Effect of Nitrosamines in Syrian Hamsters. IV

125

transplacental effect. They were found after chronic administration of these compounds to adult hamsters and were seen, in some instances, in the treated Pgeneration. Such neoplasms include those originating in the pharynx (papillomas), liver (cholangiomas, hepatocellular tumors, hemangioendotheliomas), pancreas (duct adenomas), kidneys (adenocarcinomas), and urinary bladder (transitional papillomas). Urinary tract neoplasms were not found in P- or Fl-hamsters treated with HBBN or DBN, although the substances are bladder carcinogens (Druckrey et al., 1964; Ivankovic and Bticheler, 1968; Wood et al., 1970; Althoff et al., 1971, 1976). Tumors of other sites were not related to a transplacental effect, since they represented single observations, occurring in controls at similar incidences, or develop occasionally in this breeding colony (Pour et al., 1976). Comparative studies with equitoxic doses of DPN fl-oxidized metabolites, as well as of the DBN w-hydroxylated intermediates, did not show unequivocally a correlation between the carcinogenicity of metabolites and their parent compounds (Druckrey et al., 1964, 1967; Althoff et al., 1971, 1973, 1975; Pour et al., 1973--1975; Kriiger et al., 1974). This could be anticipated for metabolites considered to be more proximate carcinogens (Tomatis, 1973). In the present transplacental study, equal doses (100 mg/kg b.w.) led to a respiratory tumor incidence in the different groups (0--47%) which did not reach those observed after DPN treatment (60%). Also the carcinogenic response varied considerably when doses were related to the LDs0 (HPPN: 0.07; OPPN: 0.08; MPN: 0.2; BHP: 0.02; HBBN: 0.03); i.e., a high equitoxic dose (MPN) was less effective than a low equitoxic dose (HPPN). However, the young of some groups exposed in utero at day 14 of gestation showed respiratory tumor incidences which were higher (HPPN, OPPN) than those in the young after DPN exposure (7%). The data suggest that fetal respiratory tissue is more sensitive for the effect of these oxidized metabolites than for the parent compounds. We thank Nancy Hays for technical assistance and Mardelle Susman for editorial aid.

References Althoff, J., Grandjean, C., Marsh, S., Pour, P., Takahashi, M.: Transplacental effects of nitrosamines in Syrian hamsters. II. Nitrosopiperidine. Z. Krebsforsch. (in press) Althoff, J., Kl:iiger, F.W.: Carcinogenicity of 4-hydroxybutylbutylnitrosamine in Syrian hamsters. Cancer Letters I, 15--19 (1975) Althoff, J., Kriiger, F.W., Mohr, U., Schmiihl, D.: Dibutylnitrosamine carcinogenesis in Syrian golden and Chinese hamsters. Proc. Soc. Exp. Biol. Med. 136, 168--173 (1971) Althoff, J., Pour, P., Grandjean, C., Marsh, S.: Transplacental effects of nitrosamines in Syrian hamsters. III, Dimethyl- and dipropylnitrosamine. Z. Krebsforsch. (in press) Althoff, J., Pour, P., Grandjean, C., Eagen, M.: Transplacental effects of nitrosamines in Syrian hamsters. I. Dibutylnitrosamine and nitrosohexamethyleneimine. Z. Krebsforsch. 86, 69--75 (1976) Druckrey, H., Preussmann, R., Ivankovic, S., Schmidt, C. H., Mennel, H.D., Stahl, K. W.: Selektive Erzeugung yon Blasenkrebs an Ratten durch Dibutyl- und N-Butyl-N-butanol(4)-nitrosamin. Z. Krebsforsch. 66, 280--290 (1964) Druckrey, H., Preussmann, R., Ivankovic, S., Schm~ihl,D.: Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitrosoverbindungen an BD-Ratten. Z. Krebsforsch. 69, 103--201 (1967) Ivankovic, S. : Pr~inatale Carcinogenese. Handbuch der allgemeinen Pathologie, 6. Band, GeschwiilsteTumore 3, 7. Teil. Berlin: Springer 1976 I vankovic, S., Biicheler, J.: Leber- und Blasencarcinome beim Meerschweinchen nach Di-n-butylnitrosamin. Z. Krebsforsch. 71, 183--185 (1968)

126

J. Althoff et al.

KriJger, F.W., Pour, P., Althoff, J.: Induction of pancreas tumors by di-iso-propanolnitrosamine. Naturwissenschaften 61, 328 (1974) Mohr, U., Althoff, J., Authaler, A.: Diaplacental effect of the carcinogen diethylnitrosamine in the golden hamster. Cancer Res. 26, 2349--2352 (1966) M ohr, U., Authaler, A., Althoff, J. : Der Nachweis von Di~ithylnitrosamin in Goldhamsterembryonen nach Behandlung des Muttertieres. Naturwissenschaften 52, 188--189 (1965) Mohr, U., Reznik-SchiJller, H., Reznik, G., Hilfrich, J.: Transplacental effects of diethylnitrosamine in Syrian hamsters as related to different days of administration during pregnancy. J. Natl. Cancer Inst. 55, 6 8 1 ~ 8 5 (1975) Pour, P, Althoff, J., Cardesa, A., Kriiger, F.W., Mohr, U.: Effect of fl-oxidized nitrosamines on Syrian golden hamsters. II. 2-Oxopropyl-n-propylnitrosamine.J. Natl. Cancer Inst. 52, 1869--1874 (1974) 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) Pour, P., Kri~ger,F.W., Althoff, J., Cardesa, A., Mohr, U.: Effect of fl-oxidized nitrosamines in Syrian golden hamsters. I. 2-Hydroxypropyl-n-propylnitrosamine. J. Natl. Cancer Inst. 52, 1245--1249 (1974) Pour, P., Kriiger, 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, Kriiger, F.W., Cardesa, A., Althoff, J., Mohr, U.: Tumorigenicity of methyl-n-propylnitrosamine in Syrian golden hamsters. J. Natl. Cancer Inst. 52, 457~462 (1974) Tomatis, L. : Transplacental carcinogenesis. Oncology, Vol 1, p.5. London: Butterworth 1973 Wood, M., Flaks, A., Clayson, D. B.: The carcinogenic activity of dibutylnitrosamine in IFXC57 mice. Europ. J. Cancer 6, 433---440 (1970) Received April 13, 1977; accepted June 10, 1977