Z. Krebsforsch. 77, 257--261 (1972) �9 by Springer-Verlag 1972

Tumorigenicity of Five Cyclic Nitrosamines in MRC Rats*

HUMBEI~TO GARCIA and WILLTAM LIJINSKY** ***

Eppley Institute for Research in Cancer University of Nebraska Medical Center

Received February 21, 1972, accepted March 3, 1972

Summary. Five cyclic nitrosamines have been fed to groups of 30 MRC rats at a concentra- tion of 100 rag/liter in drinking water. Nitroso-3-pyrroline produced only tumors of the liver, and was the weakest carcinogen of the five. Nitrosopiperidine induced tumors of liver, eso- phagus and respiratory tract. Nitrosomorpholine induced tumors of liver and the nasal cavity. Nitrosoheptamethyleneimine induced squamous tumors of lung, and tumors of the esophagus and trachea and was the most potent carcinogen of the five. Dinitrosopiperazine induced tumors of the nasal cavity. The reasons for the pronounced organ specificities of these nitros- amines are discussed.

Tumorerzeugende Wirkung von ffinf cyclischen Ni t rosaminen bei MRC-Ra t t en

Zusammen/assung. Gruppen von je 30 MRC-Ratten wurden in einer Konzentration von 100 mg/1 im Trinkwasser Nitrosamine gegeben. Es entstanden nach Nitroso-3-pyrrolin, dem schw~chsten Cancerogen, nur Lebertumoren, nach Nitrosopiperidin Tumoren yon Leber, Sl3eiscrShre und Respirationstrakt, nach Nitrosomorpholin Tumoren von Leber und Nasen- hShle, nach Nitrosoheptamethylenimin, dcm st/irksten Carcinogen, Plattenepitheltumoren der Lunge und Tumoren yon SpeiserShre und Trachea, nach Dinitrosopiperazine Tumoren der NasenhShle. Die Griinde fiir die ausgcsprochene Organspezifit/it dieser Nitrosoamine werden bcsproehcn.

I n conjunct ion with our studies of the possible in vivo format ion of ni t rosamines from ni t r i te and several cyclic imines, five corresponding cyclic ni t rosamines were fed to MRC rats. The five ni t rosamines, N-nitrosopiperidine, N-nitrosomorpholine, N-nitroso-3-pyrroline, N-ni t rosoheptamethylene imine and N,N'-dini t rosopipera- zinc, were fed concurrent ly a t the same dose level in the dr inking water. All bu t ni t rosopyrrol ine have been previously tested in rats, bu t only ni t rosoheptamethyl- eneimine has been studied in rats of the MRC strain. As was expected, there were sharp organ specificities in the d is t r ibut ion of tumors induced by these nitrosamines.

Materials and Methods

Chemicals: Nitrosopiperidine, nitrosomorpholine, dinitrosopiperazine and nitrosohepta- mcthyleneimine were prepared by reaction of the corresponding cyclic imines with sodium nitrite in strongly acid solution, as has been previously described (Druckrey et al., 1967;

* Supported by Contract No. 43-68-959 from the National Cancer Institute, U. S. Public Health Service.

** Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37830.

*** We wish to thank Mrs. J. Stroud for valuable technical assistance.

258 H. Garcia and W. Lijinsky:

Lijinsky et al., 19611). The compounds we used were purified by fractional distillation in vacuo, or by crystallization (for dinitrosopiperazine), and the purity of each was established as better than 99O/o by mass spectrometry and NMI~ spectrometry.

N-Nitroso-3-pyrroline: 3-Pyrroline, 22 g, was dissolved in 25 ml water, 30 g of ice were added and 60 ml concentrated HC1. The solution was cooled in ice and 45 g of sodium nitrite was added slowly. After 2 hrs the solution was extracted with 3 • 80 ml of methylene chloride. The extract was washed with a small volume of water, dried with anhydrous sodium sulfate and the methylene chloride removed by evaporation in a stream of nitrogen. The residual light brown oil was distilled under reduced pressure and the center cut was collected. The pale yellow oil solidified on standing and was crystallized from ether, giving 13 g of almost colorless crystals, m.p. 32.5--33 ~

The mass spectrum and nuclear magnet ic resonance spectrum of the product were consistent with the s tructure of N-nitroso-3-pyrroline, and no significant impuri t ies were detected.

A n i m a l treatments. The test animals were 8 - -10 week old male and female MRC rats of the colony ma in ta ined at the Eppley Ins t i tu te . They were housed in plastic cages, five per cage, and were fed Rockland diet in pellets ad libitum. To each cage of five animals was given 100 ml of n i t rosamine solution in water each night, 5 nights per week. Dur ing the day and at weekends the animals received tap water. All, or almost all, of the 100 ml of n i t rosamine solution was consumed overnight. I n this way, as in previous ni t rosamine tests (Goodall et al.), the dose of ni t rosamine could be quantified. The concentra t ion of all five nitros- amines was s tandardized at 100 mg per liter, or 0 . 7 - 1 millimolar, each ra t re- ceiving approximate ly 2 mg of n i t rosamine per day, 5 days a week. The t r e a t me n t lasted for at least 50 weeks, except with n i t rosoheptamethyleneimine, in which group all of the animals were dead at the 31st week of t rea tment . I n all of the other groups some animals died with tumors before the end of the t rea tment .

Animals were allowed to die na tu ra l ly or were killed when mor ibund and were completely autopsied. All tumors and gross lesions were examined histologically.

Results and Discussions

The survival of rats t rea ted with the five ni t rosamines is given in Table 1, and the d is t r ibut ion of tumors of various organs as a result of the t r ea tmen t s is listed in Table 2.

A large var ie ty of tumors was seen, all of which have been described in previous l i terature. Tumors of the trachea, pha rynx and larnx were squamous cell papillomas. Tumors of the lungs were well differentiated kerat iniz ing squamous cell carcinomas. Tumors of the liver were classified as liver cell carcinoma, with a large vascular component , or hemangioendothel iomas of the liver. The major i ty of the nasal cavi ty tumors were esthesoneuroepitheliomas, with a few adenocarci- nomas and squamous cell carcinomas.

A major i ty of the animals t rea ted with nitrosopiperidine, ni t rosomorpholine and n i t rosoheptamethyleneimine had tumors of more t h a n one organ, whereas this was rare in the animals t rea ted with dini trosopiperazine and nitroso-3- pyrroline. This indicated tha t the act ion of these ni t rosamines in inducing tumors

1 There was an error in the description of the preparation of nitrosoheptamethyleneimine (and of nitrosooctamethyleneimine) previously reported (Lijinsky et a/., 1969). The volume of hydrochloric acid used should read 100 ml in each case not 30 ml and 40 ml, as stated.

Tumorigenicity of Five Cyclic Nitrosamines in MRC Rats 259

Table 1. Survival of ra ts t reated with nitrosopiperidine, nitrosomorpholine, nitrosopyrroline, dinitrosopiperazine and nitrosoheptamethyleneimine

Compound Sex Concen- Dura- Total Number of animals at week t ra t ion tion Dose (rag/C) (weeks) (mg) 0 10 20 30 40 50 60 70 80 90 100 110

Nitroso- ~ 100 50 500 15 15 15 15 14 7 0 piperidine ~ 100 50 500 15 14 I4 13 9 1 0

Nitroso- 3 100 50 500 15 15 15 15 11 1 0 morpholine ~ 100 50 500 15 15 15 13 2 0

Nitroso-3- 3 100 60 600 15 15 15 15 15 13 12 12 11 l0 Pyrroline ~ 100 60 600 15 15 15 15 15 14 10 8 4 2

Nitroso- 3 100 50 500 15 15 15 15 13 l0 2 1 0 piperazine ~ 100 50 500 15 15 15 15 14 6 0

Nitrosohepta- ~ 100 30 300 15 15 14 6 0 methyleneimine ~) 100 30 300 15 15 14 2 0

7 0 1 0

Table 2. Tumors in rats t reated with nitrosopiperidiue, nitrosomorpholine, nitrosopyrroline, dinitrosopiperazine and nitrosoheptamethyleneimine

Corn- Sex No. TBA Number of animals with tumors of pound Ani-

mals Tra- Pha- La- Lung Liver Exo- Nasal Other autop- chea rynx rynx pha- Cavity sied gus

Nitroso- ~ 15 15 0 5 1 0 2 l0 0 Tongue 2 piperidine ~ 14 13 2 4 2 0 8 12 2 Tongue 1,

Stomach 1

Nitroso- ~ 15 15 0 0 1 0 13 2 9 Stomach 1 morpho- ~ 14 13 0 0 0 0 13 1 5 Mammary line gland i

Nitroso- ~ 15 10 0 0 0 0 2 0 2 Tongue 1, 3-pyrro- Skin 2, Sali- line vary gland 1,

Pi tu i tary gland l, Pan- creas 1, Testis 4

$ 15 11 0 0 0 0 8 0 0 Pi tu i tary gland 4, Re- troperitoneal liposarcoma 1, Skin 1

Di ~ 15 13 0 2 0 0 1 1 12 nitroso- ~ 14 13 0 0 0 0 1 0 13 Large piperazine intestine 1

Nitroso- ~ 15 14 8 0 0 8 0 10 0 Tongue 1, hepta- Brain 1 methyl- g? 15 14 4 0 0 9 0 12 1 Tongue 1, Thy- eneimine mus 1, Small

intestine 1

TBA: Tumor Bearing Animals.

260 H. Garcia and W. Lijinsky:

in one site did not preclude their having a similar tumorigenic action in another organ; tha t is, there was no indication of the nitrosamine being "used up" in inducing tumors.

Of the five nitrosamines, nitrosopyrroline was the weakest carcinogen. Although the total dose of this compound was higher tha t tha t of the other four, the animals given nitrosopyrroline showed the longest survival and the lowest incidence of tumors. Apart from liver tumors and tumors of the nasal cavity, there was no indication that the tumors in this group were a consequence of the treatment, since the other scattered tumors were those that would be expected in old animals of this strain. The pat tern of tumors induced by nitrosopyrroline (liver), and the relatively low effectiveness of this compound, were similar to the results obtained with the saturated analog nitrosopyrrolidine (Greenblatt and Lijinsky). The presence of the double bond in nitrosopyrroline did not, therefore, appear to have an influence on the carcinogenicity of the 5-membered cyclic nitrosamine.

On the other hand, nitrosoheptamethyleneimine appeared to be the most potent carcinogen among the five nitrosamines. The animals given this compound received the smallest total dose and all were dead at the 31st week ot t reatment , all but two having tumors of the lung or esophagus, or both. The lung tumors induced by this compound have been described previously (Li]insky et al.).

Nitrosopiperidine, nitrosomorpholine and dinitrosopiperazine appeared to be about equal carcinogenic potency, giving rise to tumors in almost 100~ of the animals treated with them. They did, however, differ markedly in their target organ specificity. ~itrosomorpholine produced liver carcinomas in every animal surviving the treatment, and several of these also had tumors of the nasal cavity. Nitrosomorpho]ine has been reported many times to induce liver tumors (Banasch und Mfiller) and tumors of the nasal cavity (Druckrey et al., Thomas). Nitroso- piperidine also gave rise to liver tumors in a number of the rats, but mostly females (as was the case with nitroso-3-pyrroline, but was obviously a less potent liver carcinogen than nitrosomorpholine. On the other hand, almost all of the ani- mals treated with nitrosopiperidine had tumors of the esophagus, which were produced only rarely by nitrosomorpholine. In this pat tern of tumor induction nitrosopiperidine resembled its higher homolog, nitrosohexamcthyleneimine (Goodall et al.), and differed from the next higher homolog, nitrosoheptamcthyl- eneimine, but was quite different from its lower homolog, nitrosopyrrolidine; nitrosopyrrolidine, like nitrosopyrroline, produced only liver tumors in these rats (Greenblatt and Lijinsky).

Dinitrosopiperazine gave rise to tumors of the nasal cavity in almost all of the animals given it, males as well as females, and to almost no other tumors. Only two animals developed liver tumors, in contrast with the reports of Druck- rey that dinitrosopiperazine was a liver carcinogen (Druckrey et al.).

These profound differences in tumor response of the same animals to very closely similar t reatment with five related nitrosamines are difficult to interpret. Differences in organ distribution because of variations in solubility are unlikely, since both nitrosomorpholine and nitrosopyrroline are very soluble in water, nitrosopiperidine and dinitrosopiperazine are moderately soluble in water and nitrosoheptamethylencimine is hardly soluble.

Tumorigenicity of Five Cyclic Nitrosamines in MRC Rats 261

Dinitrosopiperazine and nitrosomorpholine both gave rise to tumors of the nasal cavity; nevertheless, there must be profound differences in behavior between these two compounds, since nitrosomorpholine is a very potent liver carcinogen, while dinitrosopipcrazine could hardly be called a liver carcinogen on the basis of these results.

Nitrosopiperidine differs form nitrosoheptamethylcneimine only by two carbon atoms in the ring, yet the latter produces lung tumors, but no liver tumors, and both induce a high incidence of esophageal tumors. In addition, is is very hard to explain why nitrosoheptamcthyleneiminc is so potent a carcinogen that all of the animals receiving it died with tumors after 30 weeks of treatment. There is no apparent difference in chemical activity between these two compounds that could explain the differences in tumor response.

As for other hypotheses of the mechanism of action of nitrosamines, we have been unable to detect alkylation of nucleic acids in target organs by nitroso- morpholine, nitrosopiperidine or dinitrosopiperazinc (Lijinsky et al., unpublished data); although there was some very small degree of interaction of the labeled nitrosamines with nucleic acids, this was no greater in target than in nontarget organs. The activation of a latent virus seems unlikely, since there is no logical reason why nitrosoheptamethyleneimine should activate such a virus in the lung, while nitrosopiperidine or the other nitrosamines should not. Similarly, why should nitrosomorpholine activate a virus in the liver, and dinitrosopiperazine not ? I t is highly unlikely that liver cells would not receive similar exposure to all of these nitrosamines. We are exploring other modes of chemical interaction of these nitrosamines with cell macromolecules, together with distribution studies in an attempt to explain the profound differences in tumorigenicity.

References

Banasch, P., Mi~ller, H.-A.: Lichtmikroskopische Untersuchungen i~ber die Wirkung yon N-Nitrosomorpholin auf die Leber yon Ratte und Maus. Arzneim.-Forseh. 14, 805 (1964).

Druckrey, H., Preussmann, R., Ivankovic, S., Sehm~hl, D. : Organotrope carcinogene Wir- kungen bci 65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten. Z. Krebsforsch. 69, 103 (1967).

Garcia, H., Keefer, L., Lijinsky, W., Wenyon, C. E. M. : Carcinogenicity of nitrosothiomorpho- line and 1-nitrosopiperazine in rats. Z. Krebsforsch. 74, 179 (1970).

Goodall, C.M., Lijinsky, W., Tomatis, L.: Tumorigenicity of N-nitrosohexamethyleneimine. Cancer Res. 28, 1217 (1968).

Greenblatt, M., Lijinsky, W. : Neoplasms of liver induced by nitrosopyrrolidine in MRC rats. J. nat. Cancer Institute (in press).

Lijinsky, W., Tomatis, L., Wenyon, C.E.M.: Lung tumors in rats treated with N-nitroso- heptamethyleneimine and N-nitrosooctamethyleneimine. Proc. Soc. exp. Biol. (N.Y.) 130, 945 (1969).

Thomas, C. : Zur Morphologie der Nasenh5hlentumoren bei der Ratte. Z. Krebsforsch. 67, 1 (1965).

Dr. Humberto Garcia University of Nebraska Medical Center 42nd & Dewey Ave Omaha, Nebraska 68105/USA