Genotoxicity of nitrosated ranitidine

Mutation Research, 227 (1989) 13-16

13

Elsevier MUTLET 0242

Genotoxicity of nitrosated ranitidine J a s n a F r a n e k i r , Stefica S k u p n j a k I a n d Z d e n k a

Matija~evi6

Faculty of Food Technology and Biotechnology, 41000 Zagreb (Yugoslavia) and 1FoodIndustry 'Podravka', 43300 Koprivnica (Yugoslavia)

(Accepted25 April 1989)

Keywords: Ranitidine;Nitrite;Nitrosationin vitro; (Salmonella typhimurium; Saccharomycescerevisiae)

Summary The genotoxicity of ranitidine, widely used in the therapy of peptic ulcers, and of nitrosated ranitidine was examined in test systems with the bacteria Salmonella typhimurium for gene mutations, and with the yeast Saccharomyces cerevisiae D7 for reverse mutations and gene conversion. Under the experimental conditions applied, ranitidine was negative in both systems, while the product obtained by nitrosation in vitro was mutagenic for Salmonella strains TA100 and TA98 with and without metabolic activation. The largest increase of his + revertants, 3 times greater than the control, was obtained in strain TA 100 in the absence of $9 fraction. Nitrosated ranitidine was also recombinogenic for the yeast S. cerevisiae.

Many drugs and chemicals containing secondary amino groups have been shown to react with nitrite in acidic solution to give mutagenic and/or carcinogenic N-nitroso compounds. Ohta et al. (1988) reported that acetaminophen, a widely used antipyretic analgesic, forms a diazoquinone-type mutagen when treated with nitrite. Ranitidine is a histamine H2-receptor antagonist used for the treatment of peptic ulcer (Langman et al., 1980). In acidic conditions, ranitidine reacts with nitrite to give nitrosated derivatives. Combination of ranitidine with nitrite produces a variety of genotoxic effects on mammalian cells in Correspondence: Dr. J. Franekir, Facultyof Food Technology and Biotechnology,41000 Zagreb, Pierottijeva6 (Yugoslavia).

culture (Maura et al., 1983; Martelli et al., 1983), and mice treated in vivo (Brambilla et al., 1983). The genotoxicity of the nitrosation product of another histamine H2-receptor antagonist, cimetidine, was first demonstrated by De Flora and Picciotto in 1980. Using the nitrosation assay procedure (NAP), recommended by the WHO (IARC, 1980), the ability of ranitidine to react with nitrite and to form derivatives mutagenic and recombinogenic for bacteria and yeast was examined.

Materials and methods Chemicals

Ranitidine HC1 (CAS No. 66357-35-5), kindly

0165-7992/89/$ 03.50 © 1989ElsevierSciencePublishers B.V. (BiomedicalDivision)

14

supplied by 'Podravka' Food Factory (Koprivnica, Yugoslavia), was dissolved in distilled water. Sodium nitrite, obtained from 'Kemika' (Zagreb, Yugoslavia), was dissolved in distilled water.

Nitrosation conditions The solution of ranitidine was mixed with a molar excess of nitrate, the pH value was adjusted (3.3-3.7) by the addition of HCI, and the mixture was incubated at 37°C in a water-bath for 1 h. After incubation, the solution was neutralized by the addition of NaHCO3.

Mutagenicity assay in his- Salmonella typhimurium The mutagenicity of ranitidine in the Salmonella/mammalian microsome test was examined according to the standard plate-incorporation test procedure with the tester strains T A I 0 0 (hisG46, rfa, AuvrB, pKM101) and TA98 (hisD3052, rfa, AuvrB, pKMI01) (Maron and Ames, 1983). In assays to evaluate the mutagenicity o f nitrite-drug mixtures, solutions o f nitrite and drug were mixed, adjusted to pH 3.3-3.7 and incubated for 1 h at 37°C. After incubation each mixture was neutralized with phosphate-buffered saline (PBS), pH 7.4, prior to adding to the top agar. For metabolic activation $9 mix containing 10°70 liver $9 fraction from male Aroclor-treated rats (protein concentration: 37.8 mg/ml) was added to the top agar as well as to the bacterial cells and the compound tested. All the results are expressed as mean +_ standard deviation of triplicate plates. Aliquots (50/zl) were used for the genotoxicity assay~

Detection of the genetic effects on Saccharomyces cerevisiae The test was performed with the diploid yeast strain S. cerevisiae D7 (Zimmermann, 1975) with the genotype: ade2-40/ade2-119; trps-12/trps-27; ilVl-92/ilvl-92. Yeast cells from the mid-log phase were harvested and exposed to various concentrations of ranitidine, nitrite or their mixture, prepared beforehand, in phosphate buffer, pH = 7.4, for 2 h at 30°C. The cells were washed by cen-

trifugation, and adequate dilutions were spread on complete medium, on tryptophan-free medium and on isoleucine-free medium. Colonies counted on these 3 media allow us to calculate celll survival and the frequency of spontaneous or chemically induced gene convertants and mutants. Results and discussion In the concentrations examined, ranitidine was not toxic or mutagenic for the his- Salmonella typhimurium tester strains, either with or without the $9 mix. A mixture of ranitidine and nitrite, with an acidic pH, resulted in the formation of a mutagenic derivative(s) active in both S. typhimurium strains. The frequency of his ÷ revertants obtained by sodium nitrite combined with 2 doses of ranitidine is presented in Table 1. Several doses of ranitidine (in mixtures with nitrite) were tested but we present only therapeutic dose as well as the dose which showed significant mutagenicity (Tables 1 and 2). A slight increase in the number of revertants compared to the control was observed with the higher dose of nitrite alone. Moreover, when compared to nitrite combined with ranitidine the assay of ranitidine-nitrite mixtures (higher doses of nitrite and ranitidine) yielded an increased mutagenicity in both bacterial strains. Nitrite-ranitidine reversion in strains TA100 and TA98 containing G ' C base-pairs at the critical site for reversion is the result of base-substitution mutation (strain TA100) or frameshift mutation (strain TA98). Chemical studies have demonstrated that nitrosation products of a similar compound, cimetidine, generate a spectrum of DNA lesions similar to that of N-methyl-N'-nitro-N-nitrosoguanidine (Foster et al., 1980; Jensen and Hagee, 1981). The nitrosation product(s) of ranitidine was also found to be active in Salmonella strain TA102 which has an A" T base-pair at the site of reversion (De Flora et al., 1986). Nitroso derivatives of ranitidine elicited a significant dose-dependent increase in the DNA-repair assay by measuring unscheduled DNA synthesis (UDS) in rat hepatocyte primary cultures (Bichet et al., 1986).

15 TABLE 1 M U T A G E N I C I T Y OF R A N I T I D I N E A N D NITROSATED R A N I T I D I N E 1N Salmonella typhimurium Test substance

Concentration (mmol/l)

Number of his ÷ revertants Strain TA100 - $9

Distilled water Ranitidine Ranitidine Sodium nitrite Sodium nitrite Ranitidine + Sodium nitrite Ranitidine + Sodium nitrite

109 121 126 135 161

0.1 3 0.4 13 0.1 0.4 3 13

Strain TA98 + $9

-+ + _ + _+

14 5 6 13 9

142 154 158 161 174

-+ -+ _+ + _+

3 5 8 11 4

- $9

+ $9

17 20 24 23 23

28___5 31-+2 35-+5 31_+9 38___4

_+ 2 + 1 _+ 6 + 3 _+ 3

169 _+ 14

218 _

9

26 _+ 4

40+5

302 _+ 8

357 _

7

36 _+ 3

58±7

Results are expressed as mean + standard deviation.

To obtain more detailed information about the genetic consequences of nitroso derivatives, we also tested ranitidine and nitrite-ranitidine mixtures in the yeast Saccharomyces cerevisiae D7. The results of a typical experiment are presented in Table 2. Ranitidine or nitrite did not increase the frequency of revertants in S. cerevisiae, but the gene conversion frequencies were slightly higher than in the control. However, the gene conversion and mutation frequencies obtained by the nitrosation product(s) of ranitidine were increased 5 times over the control. The results obtained suggest that in addition to being mutagenic, the nitrosation product(s) of ranitidine also has recombinogenic potency.

Although no chemical characterization of nitrosation product(s) was attempted, the genotoxic effects observed deserve attention. In comparison with doses used for humans, it should be noted that the nitrite concentrations used in this study were higher than those present in gastric juice. Consequently, because nitrosation of amines depends on the square of t he concentration of nitrite in patients treated with this drug, the yield of nitroso derivatives should be minimal or even absent. Although the excellent therapeutic activity of ranitidine predicts the worldwide use of this histamine H2-receptor antagonist, every effort should be made to make a better benefit/risk evaluation possible.

TABLE 2 G E N O T O X I C I T Y OF R A N I T I D I N E A N D NITROSATED R A N I T I D I N E IN Saccharomyces cerevisiae D7 Test substance

Distilled water Ranitidine Ranitidine Sodium nitrite Sodium nitrite Ranitidine + Sodium nitrite Ranitidine + Sodium nitrite

Concentration (mmol/l)

0.1 3 0.4 13 0.1 0.4 3 13

Survival (o7o)

TRP convertants per 105 survivors

ILV revertants per 106 survivors

100 93 95 101 86

1.07 1.52 1.65 1.79 1.86

0.45 0.54 0.49 0.41 0.49

85

2.0

0.60

71

5.36

2.25

16

Acknowledgements T h i s w o r k w a s p a r t i a l l y s u p p o r t e d b y t h e Selfmanaged

Community

of

Interest

for

Scientific

Research of SR Croatia (No. 1.04.11.01.16)

and

the Federal Committee for Science and Technology of Yugoslavia (No. P-22).

References Bichet, N., D. Gouy, L. Manara, G. Mazue and P. Vie (1'986) Non-genotoxicity of CM 57755 a new histamine-H2 receptor antagonist, to hepatocytes in primary culture with and without nitrite-enriched human gastric juice, Summary of the XXVI EEMS Meeting, Brussels, 25-30 August. BrambiUa, G., M. Cavanna, P. Faggin, A. Maura, A. Pino, R. Ricci and L. Robbiano (1983) Genotoxic effects in rodents given high oral doses of ranitidine and sodium nitrite, Carcinogenesis, 4, 1281-1285. De Flora, S., and A. Picciotto (1980) Mutagenicity of cimetidine in nitrite-enriched human gastric juice, Carcinogenesis, 1, 925-930. De Flora, S., A. Camoirano, C. Basso, M. Astengo, P. Zanacchi and C. Bennicelli (1986) Bacterial genotoxicity of nitrosated famotidine, Mutagenesis, 1, 125-130. Foster, A.B., M. Jarman, D. Manson and H.Z. Schutten (1980) Structure and reactivity of nitrosoeimetidine, Cancer Lett., 9, 47-52.

IARC (1980) Monographs on the Evolution of the Carcinogenic Risk of Chemicals to Humans, Vol. 24, IARC, Lyon, pp. 297-314. Jensen, D.E., and P.N. Magee (1981) Methylation of DNA by nitrosocimetidine in vitro, Cancer Res., 41,230-236. Langman, M.J.S., D.A. Henry, G.C. Bell, W.R. Burnham and A. Ogilvy (1980) Cimetidine and ranitidine in duodenal ulcer, Br. Med. J., 281,473-474. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Martelli, A., E. Fugassa, A. Voci and G. Brambilla (1983) Unscheduled DNA synthesis induced by nitrosated ranitidine in primary cultures of rat hepatocytes, Mutation Res., 122, 373-376. Maura, A., A. Pino, I. Robbiano, E. Cajelli, R. Finollo, M. Cavanna and G. Brambilla (1983) DNA damage induced by nitrosated ranitidine in cultured mammalian cells, Toxicol. Lett., 18, 97-102. Ohto, T., H. Oribe, T. Kamayama, Y. Goto and S. Takitani (1988) Formation of a diazoquinone-type mutagen from acetaminophen treated with nitrite under acidic conditions, Mutation Res., 209, 95-98. Zimmermann, F.K. (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutations, Mutation Res., 28, 381-388. Communicated by M. Ala~evid