Effect of sodium ascorbate on tumor induction in rats treated with morpholine and sodium nitrite, and with nitrosomorpholine

Cancer Let:tars, 2 (1976) 101--108

© Elsevier/North-Holland, Biomedical Press

EFFECT OF SODIUM ASCORBATE ON TUMOR INDUCTION IN R A T S

TREATED W~THMORPHOLINE AND SODIUMNITRITE,AND WITH NITROSOMORPHOLINE SIDNEY S. MIRVISH, ALAIN F. PELFRENE, HUMBERTO GARCIA and PHI"LIPPE SHUBIK The Eppley lnstitut¢ for Research in Cancer, University o f Nebraska Medical Center, Omaha, Nebrast~a 68105 (U.S.A.) (Received 16 June 19'76)

SUMMARY Groups of male MRC Wistar rats were treated for 2 years either with morpholine (10 g/kg food) together with sodium nitrite (3 g/1 drinking water) or with N-nitrosomorpholine (NM, 0.15 g/1 drinking water). In b o t h ,cases, a g~oup of r.~::tswas given sodium ascorbate (22.7 g/Itg food) in addition ~to these treatments. When ascorbate was present, the liver tumors induced by morpholine and nitrite showed a 1.7-fold longer induction period, a slightly lower incidence, mad an absence of metastases in the lungs, indicating t h a t ascorbate had inhibited the in vivo formation o f NM. Ascorbate did not ~fffect liver tumor ~nduction by the preformed NM. The group treated with morpholine~ nitrite, and ascorbate had a 54% incidence of forestomach tumors, including an 18% i: midence of squamous cell carcinomas, possibly because ascorbate promote~ NM action in this organ.

INTRODUCTION In 1972 we repc,rted that ascorbate blocked the in vitro formation of N-nitroso compounds from nitrite and amines or amides, presumably because ascorbate reduced the nitrite [12]. Accordingly, we suggested that ascorbate might be used to inhibit in vivo formation of carcinogenic N-nitroso compounds. Subsequently, ascorbate inhibition of the acute toxicity caused by amines administered with nitrite was reported from several laboratories (l~eviewed in [8] and [1!] ). In tumorigenesis experiments, ascorbate inhibited (a) lung adenoma induction in A-strain mice by nitrite and amines or amides [13,14], (b) tra.usplacentat induction of neurogenic tumors in rats and hamsters by ethylurea and nitrite [7,8], (c) induction of esophageal tumors in rats by N-methylbenzy!amine and nitrite [ 3], and (d) lung t u m o r induction in rats by aminopyrine and nitrite ~5]. We here report the effect of ascorbate on tumor induction in rats by morpholine and nitrite, and by N-nitrosomorpholine (NM).

102

MATERIALSAND METHODS Morpholineand NaNO2 [both American Chemical Society (ACS) grade] were obtained from Fisher Scientific Company (Pittsburgh, Pa.), and L-ascorbic acid was purchased from Sigma Chemical Company (S~. Louis, Me.). NM was synthesized from morphotine [4]. The powdered and pelleted +Jod was Wayne Lab-Blox (Allied Mills Inc., Chicago, Ill.). Male MRC-Wistar rats from the Eppley colony were u.~;d. Groups 1~4, each consisting of 40 rats, were kept 5/cage and treatP5 5 days/week :[or 2 years, from 8 weeks of age (Table 1). Pelleted feJd and tap water without additives were administered for life to controls (group 5), and ~o all.other groups on weekends and before treatment was started. Stock solution,s of each chemical in distilled water were prepared at 20 times the final concentration, adjusted to pH 5 with HC1 or NaOH (morphoIine and ascorbate only), and stored at 4°C for not more than 4 weeks. The ascorbate stock solution was prepared with 408 g ascorbic acid/1 solution, corresponding after NaOH neutralization to 454 g Na ascorbate/L Every 2--3 days, the NaNO2 and NM stock solutions were diluted with dis'~ilted water a~Jd given ad iibitum as drinking water in brown bottles. The morpholine and ascorbate stock solutions were blended with powdered food [14]. For selected cages, the consumption/ cage of water and (less accurately) food was measured over 2~t.h periods, at w~;ekly intervals, l%ats were maintained until they were moribund or died. At autopsy, organs were fixed in 10% bufferedformalin and sections stained with hematoxylin and eosin. RESULTS

These are summarized in Tables I and 2. The added ascorbate only slightly affected the food and water consumption (Table I). In group I, treated with morpholine and nitrite, 65% of the rats developed liw~r tumors (Table 2). in group 2, treated with morpholine, nitrite and ascerbate, the 49% incidence of rats with liver tumors was slightly, but not significantly, less than that in group I . The mean latent period for the liver tumors increased 1.7-fold from 54 weeks in group I to 93 weeks in group 2; this difference was significant (P < 0.O'01), as determined by comparing the means and standard deviations (Table 1). The 2 groups contained similar proportions c,f liver cell carcinomas and Kupffer cell sarcomas. Unlike group 1, group 2 had no rats with liver tumor metastases in the lungs. In addition, 21 rats (154%) in group 2 developed forestomach tumors (14 squamous cell papillomas and 7 squamous cell carcinomas). In group 3, treated with NM, 38% of the rats developed ~.ivel:tumors, with a mean laten~ period of 29 weeks. The incidence and latent period for liver tumors in group 4, treated wi~h NM and .':~scorbate, were similar to those in group 3. The hi~st;ologietypes of liver ~umor were also similar in-these 2 groups, both of which showed more Kupffer cell sarcomas than liver cell carcinomas.

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In these groups, the Kupffer cell sarcomas produced lung metastases more frequently than did the liver ceil carcinomas. Forestomach tumors occu.r~ed in 1 rat of group 3, and 4 rats of group 4, and included 1 squamous cell carcinoma in the latter group. In group 5 (untreated), 35%of the rats developedtumors, mostly of endocrine glands, which were typical for untreated MRC Wistar rats of our colony (some of these tumors also occurred in groups 1--4). Group 5 showed no liver tumors and 2 forestomach papillomas. DISCUSSION

The induction of liver tumors in rats by NM has been reported several times [4,6,17]. The liver tumor types were similar to those reported previously from this Institute for NM-treated rats [6] ; although in the earlier report the main tumor type, in addition to liver cell carcinoma, was described as hemangioendothelioma rather than Kupffer cell sarcoma. However, these two terms are among numerous synonyms describing the identical entity, i e., malignant vascular tumors developed into the live~'parenchyma [1,2,9]. The induction of liver tumors by morpholine and nitrite confirms similar experiments in other laboratories [ 15,17,19,21]. Unlike most of those studies, however, we administered the morpholine and nitrite in different vehicles (food and drinking water) to ensure that NM was not produced before ingestion. In the ascorbate experiments, the presence of morpholine and ascorbate in one vehicle (food) and nitrite in another (drinking water) was designed so that morpholine and ascorbate would compete with each ol~her for the nitrite. The results for groups 3 and 4 showed that ascorba'~e did not affect liver tumor induction by NM. This resembles the finding by Ivankovic et al. [7] that ascorbate did not affect transplacental carcinogenesis in rats by ethylnitrosourea, but differs from our observation [14] that ascorbate (in food) slightly increased lung adenoma induction in mice by NM and by mononitrosopiperazine (in drinking water). The results for groups 1 and 2 demonstrated that a,~corbate inhibited liver tumor induction by morpholine and nitrite, causing a significant increase in induction period and a slight reduction in tumor incidence. If we assume, as a first approximation, that liver tumor production was proportional to the NM dose (as for lung adenoma induction in n~ice by NM [14] ), the results indicate that in vivo NM formation ~vas reduced by about 50%. The effect was less than the 89% inhibition of lung adenoma induction in A-strain mice, where the same ascorbate level (2.3 g/kg food) was administered with morpho~ine (6.33 g/kg food) and NaNO.~ (!.0 g/1 water) [14]. The forestomach papillomas and carcinomas in group 2 (t~eated with morpholine, nitrite and ascorbate) could have been due simply to the action NM, since the longer survival of this group, compared to group 1., would allow more time for forestomach tumors to develop. However, in previous

107 s t u d i e s .~nvolving several dose levels, N M d i d n o t c a u s e t h e s e ~umors [ 4 , 6 , 1 7 ] . A l t e r n a t i v e l y , a s c o r b a t e c o u l d have p r o m o t e d t h e i n d u c t i o n o f f o r e s t o m a c h t u m o r s b y NM. This view is s u p p o r t e d b y t h e o c c u r r e n c e of 4 f o r e s t o m a c h t u m o r s , i n c l u d i n g 1 c a r c i n o m a , in g r o u p 4 ( t r e a t e d w i t h NM a n d a s c o r b a t e ) , d e s p i t e t h e s h o r t l i f e s p a n o f t h i s g r o u p . A t h i r d p o s s i b i l i t y is t h a t a s c o r b a t e r e a c t e d in t h e s t o m a c h w i t h n i t r i t e o r m o r p h o l i n e t o p r o d u c e a locally a c t i n g c a r c i n o g e n . A f o u r t h possibility, tha~ a s c o r b a t e a l o n e ~nduced t h e s e t u m o r s , appeaxs u n l i k e l y since p r e v i o u s c a r c i n o g e n i c i W t e s t s w i t h a s c o r b a t e gave n e g a t i v e results [14,16,201~. F u r t h e r f e e d i n g e x p e r i m e n t s are p l a n n e d t o determine why the forestomach tumors were induced. ACKNOWLEDGMENTS We t h a n k Dr. NeiI D r a k e f o r help in a n a l y z i n g t h e d a t a , a n d Miss F r a d a G o l d e n b e r g f o r c o m p e t e n t t e c h n i c a l assistance. T h e s t u d i e s w e r e s u p p o r t e d b y Public H e a l t h Service c o n t r a c t N O 1 CP 3 3 2 7 8 f r o m t h e Division o£ C a n c e r C a u s a t i o n a n d P r e v e n t i o n , tlae N a t i o n a l C a n c e r I n s t i t u t e , a n d b y g r a n t BC-39C f r o m t h e A m e r i c a n C a n c e r S o c i e t y . REFERENCES

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