Z. Krebsforsch. 79, 141--144 (1973)

�9 by Springer-Verlag 1973

Kurze Originalmittei lung �9 Short Original Communicat ion

Studies of the Tumorigenic Effect in Feeding of Nitrosamino Acids and of low Doses of Amines and Nitrite to Rats *

Humber to Gareia and Wil l iam Lijinsky**, ***

Eppley Institute for Research in Cancer, University of Nebraska Medical Center 42nd and Dewey Avenue, Omaha, Nebraska 68105

Received November 20, 1972, accepted March 27, 1973

Summary. Three nitrosamino acids, nitrosoproline, nitrosohydroxyproline and nitroso- pipecolic acid were not tumorigenic by chronic administration at 0.015% in drinking water to MRC rats for 75 weeks. Similar administration for 75 weeks of mixtures of sodium nitrite (0.050/o) with each of five secondary amines (0.025~ pyrrolidine, piperidine, piperazine, morpholine and heptamethyleneimine was not tumorigenic ; neither was feeding of piperidine (0.1~ ~- sodium nitrite (0.2o/o). It is deduced that formation of nitrosamines in vivo from the free amines and nitrite in these conditions was insufficient to give rise to tumors.

Unte r suchungen zur Tumorgenese nach Ff i t te rung von Nit rosaminos~uren u n d niederen Dosen von Aminen mit Ni t r i t an R a t t e n

Zusammen/assung. Drei Nitrosaminos~uren, Nitrosoprolin, Nitrosohydroxyprolin und Nitrosopipecolins~ure erwiesen sich bei chronischer Gabe zu 0,015~ in Trinkwasser tiber 75 Wochen an MRC Ratten als nicht tumorigen. ,~hnliche Verabreichung yon Gemischen von Natrium nitrit (0,05O/o) jeweils mit einem yon ftinf sekund~ren Aminen (0,025O/o) und zwar Pyrrolidin, Piperidin, Piperazin, Morpholin und Heptamethylenimin erwies sich ebenfalls nicht als tumorigen, ebensowenig Fiitterung yon Piperidin (0,1~ und Natriumnitrit (0,2O/o). Es wird angenommen, dab unter den Versuchsbedingungen, die Bildung yon Nitrosaminen aus freiem Amin und Nitrit in vivo zur Erzeugung yon Tumoren nicht ausreichend war.

I t has been demonst ra ted tha t secondary amines fed to animals together with ni t r i te a t high doses form ni t rosamines which give rise to tumors (Sander and Bfirkle, 1969; Greenbla t t et al., 1971). We invest igated whether sufficient nitros- amine would be formed at considerable lower doses of amines and ni t r i te to give rise to tumors. I n addit ion, we tested three n i t rosamino acids which can be formed easily from the amino acids and ni t r i te in acid so]ution (Lij insky et al., 1970).

The t r ea tments were as described previously (Garcia and Lij insky, 1972), each cage of 5 MI~C rats, 8 - 1 0 weeks old, b~ing given 10i) ml of solution contain- ing n i t rosamino acid, amine or amine + nitr i te , as dr inking water each day five days per week. The t r ea tments lasted 75 weeks, after which the animals were kept un t i l death and subjected to complete pathological examinat ion.

* Supported by Contact # 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

1 Z. Krebsforsch. Bd. 79

142 H. Garcia and W. Lijinsky:

T h e s u r v i v a l of t h e e x p e r i m e n t a l r a t s a n d t h e d i s t r i b u t i o n o f t u m o r s in t h e m

is g i v e n i n T a b l e 1. S u r v i v a l i n all g r oups , i n c l u d i n g con t ro l s , was v e r y s imi l a r ,

s h o w i n g n o e v i d e n c e o f t o x i c i t y f r o m a n y o f t h e t r e a t m e n t s . T h e r e w a s n o signifi-

c a n t d i f f e rence b e t w e e n u n t r e a t e d or n i t r i t e t r e a t e d c o n t r o l s a n d t r e a t e d g r o u p s

i n p r o p o r t i o n of t u m o r b e a r i n g a n i m a l s n o r i n t h e t u m o r s c o m m o n l y f o u n d i n

c o n t r o l r a t s , w h i c h a re m a i n l y p i t u i t a r y a d e n o m a s a n d t u m o r s o f b r e a s t , u t e r u s

or t e s t i s . R u r e or a b s e n t were t h e t y p ~ s of t u m o r c o m m o n l y i n d u c e d b y n i t r o s a m i n e s

in r a t s .

Table 1. Tumors in Rats Treated with

Total No. of animals at week Treatment dose No. No.

(g) 0 20 40 60 80 100 TBA ~ TF ~

Nitrosoproline (0.015%) 1.1 153 15 14 14 14 12 4 l l 15 ~ 15 15 15 13 9 8 7

Nitrosohydroxyproline 1.1 15 3 15 15 15 12 9 5 10 (0.015%) 15 2 15 15 15 14 10 10 4

Nitrosopipecolic acid 1.1 153 15 15 12 11 6 10 5 (0.015%) 15 ~ 15 15 15 15 10 9 6

Morpholine (0.025%) + 1.8 153 15 15 14 14 l l 5 10 sodium nitr i te (0.05O/o) 3.7 15 ~ 15 15 14 14 10 13 2

Pyrrolidine (0.0250/0) -[- 1.8 153 15 14 14 14 10 4 11 sodium nitr i te (0.05%) 3.7 15 ~ 15 15 15 15 13 10 5

Piperazine (0.025%) 1.8 15 3 15 14 14 14 9 5 10 15 ~ 15 15 14 14 12 8 7

Piperazine (0.025O/o) + 1.8 153 15 15 15 15 11 8 7 sodium nitr i te (0.05O/o) 3.7 15 ~ 15 14 14 14 l l 13 2

t teptamethyleneimine 1.8 153 15 15 11 9 6 10 5 (0.025%)

15 ? 15 15 14 14 13 7 8

Heptamethyleneimine (0.025%) ~ 1.8 153 15 15 14 12 11 5 10 sodium nitr i te (0.050/O) 3.7 15 $ 15 15 15 13 l l 13 2

Piperidine (0.10/O) 3.7 153 15 15 14 14 11 4 11 15 ~ 15 15 15 14 13 7 8

Piperidine (0.025O/o) ~- 1.8 153 15 15 12 11 11 2 13 sodium nitr i te (0.05O/o) 3.7 15 ~ 15 15 14 13 10 9 6

Piperidine (0.1%) 3.7 15~ 15 15 15 15 12 7 8 sodium nitr i te (0.2O/o) 7.5 15 ~ 15 15 15 14 11 7 8

Sodium nitr i te (0.2O/o) 15.0 15 3 15 15 15 15 13 9 6 15 ~ 15 15 14 13 12 10 5

Untrea ted Control 15 3 15 14 14 14 11 5 10 15 $ 15 15 15 14 13 4 11

TBA ~ Tumor bearing animals, TF = Tumor free.

Studies of the Tumorigenic 143

The t h r e e n i t r o s a m i n o ac ids a p p e a r e d to be n o n c a r c i n o g e n i c in M R C ra t s w h e n

fed a t o t a l dose o f m o r e t h a n 1 g r a m per an ima l , showing t h a t t h e d e c a r b o x y l a t i o n

o f n i t r o s a m i n o ac ids to n i t r o s a m i n e s (L i j in sky a n d Eps t e in , 1970) d id no t t a k e

p lace to a n y s igni f icant e x t e n t in r a t s in vivo; a s imi la r n e g a t i v e r e su l t was obta i -

n e d w i t h n i t ro sop ro l ine in mice ( G r e e n b l a t t a n d L i j insky , 1972).

N i t r o s a m i n e s can be f o r m e d b y r eac t i on o f s e c o n d a r y a m i n e s w i t h n i t r i t e in

t h e s t o m a c h of e x p e r i m e n t a l an ima l s (Sander a n d B/ i rk le , 1969; Sen et al., 1969;

G r e e n b l a t t et al., 1972). Morpho l i ne a n d n i t r i t e fed to ra t s a t h igh doses f o r m e d

nitrosamino acids, amines and nitrite

animals with tumors of

Pitui- Breast Testis Uterus/ Adrenal Pan- Skin Other tary Vagina creas

0 0 2 0 0 0 1 6 1 0 2 0 0 0

1 1 1 0 1 0 1 7 2 0 3 0 0 0

1 1 4 0 1 0 1 5 2 0 0 0 0 0

4 0 1 0 0 0 1 12 3 0 1 1 0 1

1 0 1 0 1 1 1 9 1 0 0 0 0 0

1 0 3 0 0 2 0 6 1 0 1 0 0 0

3 0 4 0 0 4 0 12 1 0 0 0 0 0

3 0 1 0 1 0 1

3 2 0 3 1 1 0

0 0 2 0 0 0 2 7 3 0 3 0 0 0

1 0 2 0 0 1 1 4 1 0 1 0 0 0

2 0 0 0 0 0 0 8 0 0 3 0 0 0

2 0 3 0 0 1 2 4 1 0 0 0 0 0

1 0 2 0 1 1 3 6 2 0 1 0 0 1

0 0 3 0 0 1 0 3 i 0 1 0 0 0

1 salivary gland, 1 small intestine 1 salivary gland

1 thymus 1 soft tissue

2 nasal cavity 1 ovary, 1 myometrium, 1 soft

tissue

1 spleen 1 thyroid

1 soft tissue 1 brain

1 soft tissue

1 liver, 1 brain 2 soft tissue

2 large intestine, 2 brain, 2 thy- roid, 1 parathyroid

1 spleen, 1 soft tissue 1 stomach, 1 liver, 1 large intestine, 1 brain

1 brain 2 ovary, 1 thymus

1 liver

1 brain 1 salivary gland, 1 ovary

3 soft tissue 1 stomach, 1 thymus

1 soft tissue

11"

144 H. Garcia and W. Lijinsky: Studies of the Tumorigenic

sufficient n i t rosomorphol inc to give rise to l iver t umors (Sander and Bfirkle, 1969). However , in our tes ts of lower doses of morphol ine and ni t r i te , which are closer to those which man might experience, no l iver t umors were found. Nei ther d id the o ther four secondary amines t e s ted wi th n i t r i te form sufficient n i t rosamine to give rise to tumors . All of these amines are s t rong bases (pK~ 8 .4 -11 .2 ) and fo rmat ion of the n i t rosamine is p r o b a b l y not favored in d i lu te solution. Also we fed the animals solut ions of the free amines (pH 9 - 1 0 ) toge ther wi th n i t r i te , to p reven t fo rmat ion of n i t rosamine outside the animal . This might have reduced the ac id i ty of the r a t s tomach contents f rom the usual p H 3 - 5 (similar to t h a t in man ' s s tomach dur ing digest ion of a meal, Rhodes et al.), and could m a r k e d l y reduce the ex ten t of the n i t rosa t ion react ion. B y contras t , all five n i t rosamines were po t en t carcinogens, inducing tumors of specific organs in a lmos t 100% of these ra t s when fed a t 100 p .p .m. (Garcia and Li j insky, 1972).

The resul ts of these exper iments confirm t h a t fo rmat ion of n i t rosamines in vivo f rom amines and n i t r i t e can be very smal l a t low concent ra t ions (Mirvish, 1970). The y ie ld of n i t rosamine can, however, be more significant wi th some amines (even t e r t i a ry amines) t h a n with others (Li j insky et al., 1972). Also, while the ra t s in these exper imen t s were t r ea t ed for 11/2 years, ingest ion of ni t r i tes and ami- nes b y man can cont inue for a l i fet ime of 60 years or more.

References

Garcia, H., Lijinsky, W.: Tumorigenicity of five cyclic nitrosamines in MRC rats. Z. Krebs- forsch. 77, 257--261 (1972).

Greenblatt, M., Kommineni, V., Conrad,E, Wallcave, L., Lijinsky, W. : In vivo conversion of phenmetrazine into its N-nitroso derivate. Nature New Biol. 236, 25--26 (1972).

Greenblatt, M., Lijinsky, W.: Failure to induce tumors in Swiss mice after concurrent admi- nistration of amino acids and sodium nitrite. J. nat. Cancer Inst. 48, 1389--1392 (1972).

Greenblatt,M., Mirvish, S., So, B.T.: Nitrosamine studies: Induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. nat. Cancer Inst. 46, 1029--1034 (1971).

Lijinsky, W., Conrad, E., Van de Bogart, R. : Nitrcsamines formed by drug/nitrite interactions. Nature (Lond.) 289, 165--167 (1972).

Lijinsky, W., Epstein, S.S.: Nitrosamines as environmental carcinogens. Nature (Lond.) 225, 21--23 (1970).

Lijinsky, W., Kcefer, L., Loo, J. : The preparation and prol3erties of some nitrosamino acids. Tetrahedron 26, 5153--5153 (1970).

Mirvish, S. S. : Kinetics of dimethylamie nitrosation in relation to nitrosamine carcinogenesis. J. nat. Cancer Inst. 44, 633--639 (1970).

Rhodes, J., Barnardo, D.E., Phillips, S.F., Rovelstad, R.A., Hofmann, A. F. : Increased reflux of bile into the stomach in patients with gastric ulcer. Gastroenterology 57,241--252 (1969)

Sander, J., Biirkle, G. : Induktion maligner Tumoren bei Ratten durch gleichzeitigeVerftitte- rung yon Nitrit und sekundKren Aminen. Z. Krebsforsch. 73, 54--66 (1969).

Sen, N.P., Smith, D.C., Schwinghamer, L. : Formation of N-nitrosamines from secondary ami- nes and nitrite in human and animal gastric juice. Food Cosmet. Toxicol. 7, 301--307 (1969).

Humberto Garcia Eppley Institute for Research in Cancer University of Nebraska Medical Center 42nd and Dewey Avenue Omaha, Nebraska 68105 USA

William Lijinsky Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37830 USA