Die Nahrung 34 (1990) 7,661-664

Institute of Nutrition and Food Hygiene, Havana, Cuba (Direktor: Dra. MILEA COBAS)

Mutagenicity of N-nitrosomorpholine biosynthesized from morpholine in the presence of nitrate and its inhibition by ascorbic acid

A. PfftEZ', S. I. FERNANDEZ~, M. 0. GARCIA-ROCHP, A. DE LAS CAGIGAS', A. CASTILLO', 0. FONSECAj and M. HERR ERA^

N-nitrosomorpholine biosynthesis and inhibition in mice treated with nitrate, morpholine and ascorbic acid, was studied by means of the mutagenic activity in urinesamples. The preincubation method was used and Salmo- nella ryphimurium TA 1535 was the test strain. The results suggest that N-nitrosomorpholine biosynthesis was carriedout when theanimalswerefedhighdosesofnitrate, howeverascorbicacid wasaninhibitor ofthis process.

Introduction

Dieteticfactorshavebeenestimatedtobethecause of35 %ofallcancercases[ 1l.Thisafirma- tion is supported because through the foodstuff the humans are exposed to a great number of genotoxic and/or carcinogenic compounds or their precursors such as natural constituents, food additives or cooking process-derived products [2].

N-nitroso compounds are one of the mutagenic and carcinogenic agents identified in the diet; their endogenous formation from precursors has been established [3, 41. In vivo N- nitrosation from nitrite as precursor has been demonstrated by many workers, however in Cuba the daily intake of nitrate is more systematic and of higher level [5], which also can participate in the synthesis of N-nitroso compounds [6,7]. Therefore it is important to know whether the daily intake of nitrate combined with nitrosable amines can yield such a level of nitrosamine which induce measurable mutagenic damages. This aspect is the aim the present study.

Materials and methods

This study is integrated to a long-term test for carcinogenicity. 12 groups ofOF 1 mice(6 animals for eachgroup) were treated during 6 months. The different doses ofthe test substances (morpholine, nitrate and ascorbic acid) were administered daily in the water (Table 1). Taking into consideration the security range for the extrapolation from animals to humans, the three doses of nitrate, 333,666 and 2000 mg/kg of body weight are the daily intake estimated for the Cuban population, a level near the acceptable daily intake and a severe dose, respectively.

The dose of morpholine was found to be nitrosated in a previous study [7]. Both levels of ascorbic acid have shown to inhibit the endogenous synthesis of nitrosamine [S].

__

' Institute of Nutrition and Food Hygiene, Havana; ' University of Havana, Havana, Cuba.

National Center for Scientific Research, Havana;

662 Die Nahrung 34 (1990) 7

Table 1 Daily doses of the test compounds [mg/kg of body weight]

Groups NaNO, Morpholine Ascorbic acid

I* I1 I11 IV V VI VII VIII IX X XI XI1

- 333 666

2000

333 666

2000 333 333

2000 2000

- - 250 250 250 250 250 250 250 250

- I5 30 15 30

* control group

Mutagenicity assay

The urine samples from each group were collected during 24 h and stored at -20 "C until the mutagenicity test. Before the assay they were passed through a millipore filter (0.45 pm).

An overnight culture (108 cells/ml) of Salmonella typhimuriurn TA 1535 was used as the test strain. The liquid preincubation method was carried out: 0.1 ml of inoculum, 100, 200 or 400 pl of urine sample, 500 pl of j - glucuronidase and 0.5 ml of the mix or 0.5 ml of buffer phosphate pH = 7.4 were mixed during 20 min at 37 "C, then 2 ml of soft agar were added and poured onto minimum media petri dishes, which were incubated at 37 "C during 48 h. The S, fraction was from aroclor 1254 induced Sprague Dawley male rats, weighing about 200 g, the liver was the donor tissue.

Results and discussion

Table 2 shows the mean of revertants from two plates. Results of groups I, 11,111, IV and V show that nitrate and morpholine did not induce mutagenic activity "per se", nevertheless the combined administration of both substances did it (group VIII). This is supposed to be an effect of N-nitrdsomorpholine biosynthesis due to the reduction. of nitrate to nitrite and the following transformation of this compound into the stomach to nitrous acid, which in turn, forms nitrous anhydride that could react with morpholine to yield N-nitosomorpholine [9].

These results were not seen with the lower doses of nitrate (group VI and VII) and can be explained because of the different transformations that this compound should undergo to be able to react with morpholine.

These results suggest that the daily intake of nitrate estimated for the Cuban population does not represent a genotoxic risk, even when it is combined with morpholine, which is a secondary and weakly basic amine, hence easily nitrosated [lo].

When the higher level of nitrate plus morpholine were administered together with both doses of ascorbic acid (groups XI and XII) the mutagenicity of group VIII disappeared, showing once again the antimutagenic activity of vitamin C, which is known to act reducing nitrous anhydride to nitric oxide, thus decreasing the concentration of N20, available for nitrosation of amines [ 1 11.

I%REZ/FERN~JDEZ et al. : Mutagenicity of N-nitrosomorpholine 663

Table 2 Mutagenicity of the urine samples*

Samples [PI] Groups

I I1 111 IV V VI

- + - + - + - + - + - + 100 1.6 1.4 1.2 1.2 1.2 1.2 0.8 1.4 1.9 1.2 1.0 1.5

200 1.1 1.9 1.1 1.7 1.1 1.1 1.8 1.9 1.9 1.1 1.5 1.7

400 1.8 1.8 1.0 1.7 0.9 1.3 1.8 1.9 1.8 1.0

Samples [pl] Groups

VII VIII IX X XI XI1

- + - + - + - + - + - + 100 1.8 1.4 6.0 8.3 1.0 1.4 1.0 1.0 1.0 1.7 0.7 1.0

200 1.8 1.9 7.1 11.7 1.4 1.6 0.5 1.2 0.7 0.8 0.6 0.7

400 1.9 1.9 7.4 10.4 1.3 0.9 1.0 1.1 0.5 1.0 0.5 0.8

- 0.5 ml of buffer phosphate + 0.5 ml of the S, mix.

induced revertants/spontaneous revertants

The lower dose of ascorbic acid represents 900 mg for a person weighing 60 kg. Although it is much higher than the nutritional recommendations it is lower than the doses used by other workers, giving relevance to this finding.

Zusammenfassung

A. Hmz, S. I. FERNANDEZ, M. 0. G A R ~ A Roc&, A. DE LAS CAGIGAS, A. CASTILLO, G. FONSECA und M. HERRERA: Mutagenitiit von aus Morpholin in Gegenwart von Nitrat biosynthetisiertem N-Nitrosomorpholin und dessen Inhibierung durch Ascorbinsaure

Die Biosynthese von N-Nitrosomorpholin und dessen Inhibierung bei Mausen, denen Nitrat, Morpholin und Ascorbinsirure verabfolgt worden war, wurde anhand der mutagenen Aktivitat von Urinprobenuntersucht. Es wurde die Prainkubationsmethode angewendet ; als Teststamm diente Salmonella typhimurium TA 1535. Die Ergebnisse zeigen, daD eine Biosynthese von N-Nitrosomorpholin bei Verabfolgung hoher Nitratdosen erfolgt ; Ascorbinsaure wirkt als Inhibitor dieses Prozesses.

664 Die Nahrung 34 (1990) 7

References

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[9] REVELLE, R., in: Basic and applied mutagenesis. Ed. by AMIR MUHAMMED and R. C. VON BORSTEL. p, I I . 99 (1987).

Basic Life Science, Vol. 34. Plenum Press, New York, London 1985. [lo] MIRVISH, S. S., J. Toxicol. Environm. Health 2, 1267 (1977). [ 1 11 FIALA, E. S., B. S. REDDY and H. W. JOHN, Ann. Rev. Nutr. 5,295 (1985).

A. PEREZ ~ERDUELES, Institute of Nutrition and Food Hygiene, Infanta 1158, La Habana 3, Cuba

Received November 13, 1989