Z. Krebsforsch. 74, 179--184 (1970)

Carc inogenic i ty of N i t r o s o t h i o m o r p h o l i n e and 1 -Ni trosop iperaz ine in Rats*

H v M ~ T o GAneIA, LAlCRY KEEFEIr WILLIAM LIJINSKY CHAnL~S E. M. Wv,~Yo~**

Eppley Institute for Research in Cancer University of Nebraska College of Medicine

Omaha, Nebraska U.S.A.

Received January 7, 1970

Carcinogene Wirkung von 2Vitrosothiomorpholine und 1-Nitrosopiperazine bet Ratten

Zusammen/assung. Zwei cyclische ~itrosamine, nitroso-thiomorpholine und 1-Nitroso- piperazine, wurden Ratten in Langzeit-Versuchen im Trinkwasser appliziert (50 mg/l bzw. 200 rag/l). Nitroso-thiomorpholine fiihrte zu benignen und malignen Tumoren des Oesophagus und der Zunge. Nitroso-piperazine zeigte ebenfalls tumorigene Wirkung ffir eine groBe Zahl yon Organen und Geweben, allerdings keine Organ-Spezifit/~t. Nitroso-piperazine erwies sich als yon sehr viel geringerer akuter Toxicit~tt als Dinitroso-piperazin.

Summary. Two cyclic nitrosamines, nitrosothiomorpholine and 1-nitrosopiperazine, have been tested by long term feeding to rats in drinking water, at 50 mg/1 and 200 mg/L Nitro- sothiomorpholine produced tumors, both benign and malignant, of the esophagus andtongue. Nitrosopiperazine appeared to be definitely tumorigenic, although it showed no selectivity of site for tumor induction and induced tumors in a wide range of organs and tissues. Nitro- sopiperazine was very much less acutely toxic than dinitrosopiperazine.

The two cyclic N-n i t rosamines , n i t roso th iomorpho l ine (I) and 1-ni t rosopipera- zinc (II) , were considered for biological t es t ing because of the i r close s t ruc tu ra l s imi la r i ty to n i t rosomorphol ine ( I I I ) , which is a p o t e n t h e p a t o t o x i n and l iver carc inogen in ra t s (Banaseh and Mfiller; D r u e k r e y et al).

H S N 0

/ \ / \ / \ CH~ CH2 CH2 CH2 CI-I~ CH, J I I I ] r

CH2 Ctt2 CH2 CH2 CH2 CH~

I I I NO NO NO

I I I I I I

Materials and Methods Nitrosothiomorpholine. The amine to be nitrosated was prepared by the slow addition of

50 g of 3-thiomorpholinone (m. p. 89--92 ~ Aldrich Chemical Co.) as the dry powder to a magnetically stirring slurry of 20 g of powdered lithium alumininm hydride in a convenient

* This investigation was supported by Contract @ 43--68-959 from the National Cancer Institute, U.S.P.H.S.

** We wish to thank Miss P. Johns, Mrs. J. Stroud for skilled technical assistance. 13 z. Krebsforsch. Bd. 74

180 H. Garcia, L. Keefer, W. Lijinsky and C. E. M. Wenyon:

volume of anhydrous diethyl ether (Sommers and Horrom). The reaction mixture was refluxed for 4 hours, whereupon stirring and heating were interrupted until the next day. After refluxing for an additional 8 hours, the mixture was again allowed to stand overnight. The excess metal hydride was decomposed by the cautious addition, with vigorous stirring, of several hundred ml of ether saturated with water, followed by several drops of water. The resulting slurry was filtered, and the filter cake was washed thoroughly with ether. The filtrate was concentrated under reduced pressure, then distilled in vacuo. The yield was 27 g of thiomorpholine, b. p. 51--53 ~ at 9 mm.

To prepare the nitroso compound, 62 g of thiomorpholine was dissolved in an excess of aqueous sodium nitrite solution. The reaction mixture was cooled to --5 ~ in ice/salt, and 100 ml of concentrated hydrochloric acid was added slowly with stirring. After warming to room temperature, the mixture was saturated with sodium carbonate and extraetedseveral times wi~h chloroform. The combined extracts were dried over calcium chloride, filtered and concentrated using a rotary evaporator. The remaining yetlow oil was twice distilled in vacuo, giving 55 g, b. p. 87--90 ~ at 0.2 mm. The distillate eventually crystallized to a pale yellow solid, m. p. 50--51 ~

Analysis: C 36.36, H 6.16, N 20.92, S 24.02; talc. for C4HsN~OS -- 36.34O/o C, 6.10% H, 21.19% N, 24.27% S.

The mass spectrum and nuclear magnetic resonance spectrum of the product were con- sistent with the structure N-nitrosothiomorpholine.

1-Nitrosoplperazine was prepared according to Berg. Anhydrous piperazine (52 g) was dissolved in 200 ml of 4 N HC1 and cooled to --5 ~ in ice/salt. A solution of 44 g NaNO~ in 84 ml water was added drop by drop with stirring; the stirring was continued for 15 minutes after addition of the nitrite. The solution was adjusted to pH 6 with 50% NaOH and the dinitroso- piperazine removed by filtration. The filtrate was made strongly alkaline and extracted with 3 • 75 ml of chloroform. The chloroform was removed from the combined extracts using a rotary evaporator and the residual oil distilled in vacuo; the major portion distilling at 80--85 ~ (0.1 ram) was collected as 1-nitrosopiperazine. The mass spectrum was consistent with this structure and the I~VIR spectrum indicated high purity of the product, with virtual absence of dinitrosopiperazine, as well as piperazine and other impurities.

Animal Treatment. The animals were MRC rats, bred in our colony, both male and female, and were 9 to 10 weeks old at the beginning of the treatment.

The acute toxicity of both nitrosamines was determined by administration by garage to groups of 4 male rats. Nitrosopiperazine was dissolved in water and nitrosothiomorpholine was dissolved in olive oil; the doses given ranged from 50 to 3200 mg/kg body weight. The animals were observed for 48 hours following the treatment and the number of animals dying in each group was recorded.

For chronic administration the animals were divided into groups of 5 per cage and fed Rockland diet in pellets ad libitum. The nitrosamines were administered at two concentrations in drinking water, 50 mgfl and 200 mg/1. The treatment consisted of 100 ml of solution given to each cage (5 animals) each night, 5 nights per week; during daylight and at weekends the animals were given tap water. Th;s method of administration has been found very satisfactory for nitrosamines (Goodall, Lijinsky, and Tomatis). The nitrosamine treatments were continued for a measured time and the animals observed until death. Each animal was subjected to complete autopsy and all lesions observed were examined histologically.

Results

The acute toxicities were calculated according to Weft and the LDs0's were 800mg/kg for ni t rosothiomorphol ine and 22 60mg/ kg for 1-nitrosopiperazine. Necropsy showed with ni trosopiperazine b ra in hemorrhage, lung congestion with petechiae on the surface and alveolar edema, cloudy degenerat ion in l iver cellsl and cloudy degenerat ion of t ubu la r cells in the kidney, with vacuolar degenerat ion of the epithelia of collector tubules . Wi th ni t rosothiomorpholine, the pathology observed was b ra in hemorrhage and int raalveolar hemorrhage in the lungs.

Careinogenieity of :Nitrosothiomorpholine and I-Nitrosopiperazine in Rats 181

Table 1. Survival o/rats treated with nitrosothiomorpholine and 1-nitrosoplperazine

Compound Sex Con- Dura- Total Number of animals surviving at weeks cen- tion dose

0 20 40 60 70 80 90 100 110 120 130 tra- weeks mg tion rag/1

•itroso- ~ 50 38 thio- 9 50 38 morpholine ~ 200 38

200 38

Nitroso- ~ 50 60 piper- 9 50 60 azine ~ 200 60

200 60

190 11 11 10 8 7 5 3 0 190 10 9 9 9 9 8 8 5 0 760 10 10 7 4 3 0 760 17 17 6 3 2 0

300 10 10 10 10 10 9 8 8 8 7 300 10 10 10 10 l0 10 10 9 9 9

1200 10 10 9 6 4 3 2 0 1200 10 10 10 9 5 3 3 0

Table 2. Tumors in rats treated with nitrosothiomorpholine and 1-nitrosop@erazine

Compound Treat ment

No. of No. of Weeks of Type of tumor Animals TBA ~ treatment

at death

Nitrosothio- morpholine 50 rag/1 11 3

10 9

200mg/1 I0 3

17

69 77 88 88

86 87

91

56

58 61 62 68 69

46 52

55 55 61 62

71 72 73

Adenocarcinoma nasal cavity t~eticulum cell sarcoma Esthesoneuroepit.helioma Squamous cell papilloma of stomach

and reticulum cell sarcoma

Fibroadenoma of mammary gland Invasive squamous cell carcinoma of

tongue Adenoma of hypophisis

Carcinoma in situ of tracheal epithelium

Papillomata of esophagus Squamous cell carcinoma of esophagus Papillomata of esophagus Esthesoepithelioma Papilloma of esophagus

Squamous cell carcinoma of esophagus Squamous cell carcinoma of esophagus

and squamous cell carcinoma of tongue

Papilloma of esophagus Papilloma of esophagus Squamous cell carcinoma of tongue Squamous cell carcinoma of the nasal

cavity Mammary carcinoma Papilloma of esophagus Papilloma of esophagus

13"

182 H. Garcia, L. Keefer, W. Lijinskyand C.E.M. Wenyon:

Table 2. (Continued)

Compound Treat- ment

No. of No. of Weeks of Type of tumor Animals TBA �9 treatment

at death

1-Nitroso- piperazine

Untreated controls

50 rag/1 10 c7

10

200 rag/1 10

10 ~ 7

4 92 121

123 125

84 117 117 117 124 124 124 124

67 71

104 51 74 78 82 94 99

100

57 3 12 72 72 75 75 75 79 92

109 110 114 114 127

13 ~ 4 107 113 114 136

Hepatoma Reticulum cell sarcoma and he-

mangioma Subcutaneous hemangioma Carcinoma of adrenal Adenoma of pituitary gland Retieulum cell sarcoma Bronchial polyp Cystadenoma of thyroid Pituitary adenocarcinoma Astrocytoma Adenocarcinoma of endometrium Mammary fibroadenoma and leio-

mioma of the vagina

Papilloma of the trachea Squamous cell carcinoma of mouth Adenomatous polyp nasal cavity Leiomiosarcoma of uterus Mammary fibroadenoma Esthesoneuroepithelioma Hemangiosarcoma of uterus Squamous cell carcinoma nasal cavity Pleomorphic sarcoma sub. tissue Leiomioma of uterus and subcuta-

neous sclerosing hemangioma

Pituitary adenoma Pituitary adenoma Pituitary adenoma Pituitary adenoma Adrenal cortical adenoma Malignant meseothelioma Adenoearcinoma of lungs Interstitial cell tumor of testis Adenocarcinoma of prostate Pituitary adenoma Interstitial cell tumor of testis Reticulum cell sarcoma Fibroadenoma of breast Pituitary adenoma Fibroadenoma of breast Endometrial carcinoma

a TBA = Tumor bearing animals.

The survival of the rats t rea ted with the two ni t rosamines is given ill Table 1. Those given ni trosopiperazine appeared to survive as well as un t r ea t ed control rats. The to ta l dose received by the animals was: ni t rosothiomorphol ine 760 mg

Carcinogenicity of Nitrosothiomorpholine and 1-Nitrosopiperazine in Rats 183

and 190 mg in 38 weeks of t rea tment ; 1-nitrosopiperazine 1200 mg and 300 mg in 60 weeks of t reatment .

The incidence of tumors in the various groups is shown in Table 2, which also lists the tumors found in a large group of untreated control rats. The overall incidence of tumors was high with both compounds, although there seemed little predilection for one particular site, especially by nitrosopiperazine. To avoid an erroneous summation of the results by us, all of the tumors observed have been listed individually.

The earliest tumor elicited by nitrosothiomorpholine was found at 46 weeks in a female given the higher dose, and was a squamous cell carcinoma of the esophagus. The earliest tumor in animals t reated with 1-nitrosopiperazine was found at 51 weeks in a female given the higher dose, and was a leiomiosarcoma of the uterus.

Discussion

As with other groups of similar nitroso compounds, the tumorigenic effect of nitrosothiomorpholine and 1-nitrosopiperazine are quite different, and they also differ markedly in this regard from the structurally closely similar compounds nitrosomorpholine and 1,4-dinitrosopiperazine.

Whereas nitrosomorpholine is a very potent liver carcinogen in rats (perhaps the most potent), and also produces tumors of the nasal cavity (Druckrey et al; Thomas), nitrosothiomorpholine gives rise mainly to tumors of the esophagus and tongue, together with occasional nasal cavity tumors; obviously replacement of the oxygen a tom in nitrosomorpholine by a sulfur a tom reduces the carcinogenic potency of the molecule markedly.

Similarly, whereas dinitrosopiperazine produces tumors of the esophagus and more rarely of the liver and nose in rats (Druckrey et al. ; Thomas), 1-nitrosopipera- zinc gives rise to tumors a t a var iety of different sites, but seems to have no predilection for any one site. There is no doubt tha t both nitrosothiomorpholine and 1-nitrosopiperazine are carcinogenic, since the tumor incidence in animals t reated with either compound is much higher than tha t in untreated controls.

The two nitrosamines considered here are not only less carcinogenic than nitrosomorpholine and dinitrosopiperazine, but are also much loss toxic than the lat ter two, and do not cause death from liver damage as do the latter.

The explanation of these differences is obscure. I t might be suggested tha t the lower acute toxicity of nitrosothiomorpholine, and its lower carcinogenic potency, is due to its much lower solubility in water than nitrosomorpholine. However, 1- nitrosopiperazine is more soluble in water than is nitrosomorpholine, yet has very low toxicity. Dinitrosopiperazine is no more water soluble than nitrosothiomorpho- line and yet is a much more potent carcinogen than the latter, and also is more acutely toxic. The fact tha t 1-nitrosopiperazine is a base (perhaps leading to easy excretion) might have some bearing on its low toxicity and carcinogenicity, but there seems no easily understood pat tern to the differences in biological effect among the four nitroso compounds considered here. I t seems doubtful tha t studies of the distribution of the compounds in the body would, in themselves, provide an explanation of the differences in careinogenicity. Rather, it is probable tha t the explanation lies in a var iety of differences in metabolism, and tha t there is no simple answer, susceptible of easy investigation.

18~ Gareia et al. : Careinogenieity of Nitrosothiomorpholine and 1-Nitrosopiperazine in Rats

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Dr. William Lijinsky Eppley Institute for Research in Cancer University of Nebraska College of Medicine 42nd and Dewey Avenue, Omaha, Nebraska 68105, USA