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Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol
Mutation Research 415 Ž1998. 219–226
Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol Minoru Higashimoto a , Hirokuni Yamato b, Takemi Kinouchi c , Yoshinari Ohnishi a
c,)
Faculty of Pharmaceutical Sciences, Tokushima Bunri UniÕersity, Tokushima 770-8514, Japan b Faculty of Domestic Sciences, Tokushima Bunri UniÕersity, Tokushima 770-8514, Japan c School of Medicine, The UniÕersity of Tokushima, Tokushima 770-8503, Japan Received 6 January 1998; revised 26 May 1998; accepted 26 May 1998
Abstract It has been shown that the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid ŽMTCCA., a mutagen precursor in soy sauce treated with nitrite, was strongly increased when it was treated with nitrite in the presence of alcohols. We found that the mutagenicity of MTCCA treated with 50 mM nitrite at pH 3, 378C for 60 min in the presence of 7.5% ethanol was reduced by the addition of citrus fruits sudachi Ž Citrus sudachi ., lemon Ž C. limon. and yuzu Ž C. junos ., to the reaction mixture. The mutagenicity-reducing activity per weight of flavedos Žouter colored portions of peel. of the citrus fruits was considerably higher than that of the juices. The juices of the other citrus fruits commercially available in Japan also had mutagenicity-reducing activity against the nitrite-treated MTCCA. Among the many components of citrus fruits, dietary fibers lignin and pectin showed strong antimutagenic activity in the reaction mixture, suggesting that the mixed fractions of these components including lignin, pectin, D-limonene, naringin, hesperidin, ascorbic acid and citric acid reduce the mutagenicity of MTCCA in the reaction mixture containing nitrite and ethanol. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antimutagenicity; Citrus fruits; Soy sauce; Nitrosation; Ethanol
1. Introduction N-nitroso compounds which are easily produced under the acidic condition in the stomach may induce stomach cancer w1,2x. Wakabayashi et al. w3x ) Corresponding author. Tel.: q81-886-33-7069; Fax: q81886-33-7069; E-mail: [email protected]
reported that Japanese soy sauce had the highest mutagenicity among various food stuffs on treatment with nitrite and suggested a relationship of food consumption by the Japanese and their high gastric cancer mortality. Tyramine is a major mutagen precursor in soy sauce on treatment with nitrite and 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid ŽMTCCA. is a minor mutagen precursor in the
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M. Higashimoto et al.r Mutation Research 415 (1998) 219–226
soy sauce w4,5x. Fujita et al. w6x reported that 3-diazotyramine, a mutagen produced by nitrite treatment of tyramine, induces squamous cell carcinomas in the oral cavity of rats, although it has not been demonstrated that soy sauce induces stomach cancers w7–9x. The chemical structure of a mutagen produced from MTCCA and nitrite and carcinogenicity of the mutagen have not been clarified yet. However, when MTCCA is treated with nitrite in the presence of ethanol, mutagenicity of the product is very high w10x, while the mutagenicity of tyramine that has been treated with nitrite in the presence of ethanol is very low w11x. It has been reported that citrus fruits reduce the mutagenicity of known mutagens w12x and the risk of stomach cancer w13–15x. Japanese use some acid citrus fruits for foods cooked with soy sauce. We attempted to reduce the mutagenicity of nitrite-treated MTCCA in the presence of ethanol by adding three kinds of acid citrus fruits. The effects of six other citrus fruits on the mutagenicity were also studied. In addition, we studied the antimutagenicity of the components of the citrus fruits to elucidate the mechanisms of the mutagenicity suppression.
2. Materials and methods 2.1. Citrus fruits, soy sauce and chemicals Sudachi Ž Citrus sudachi Shirai., yuzu Ž C. junos Tanaka., unshu Ž C. unshiu Marc.., hassaku Ž C. hassaku Tanaka. and iyokan Ž C. iyo Tanaka., produced in Japan, lemon Ž C. limon Burm.., grapefruit Ž C. paradisi Macf.. and navel orange Ž C. sinensis Osbeck., produced in the USA, and lime Ž C. aurantifolia Swingle., produced in Mexico, were purchased at local markets in Japan. Sudachi, lemon and yuzu were fractionated into juice and flavedo Žthe outer colored portion of peel.. For the other citrus fruits, only the juices were used. The citrus samples were prepared aseptically by using sterile glassware and flame sterilization. Well-washed citrus fruits were cut in half and hand-squeezed to obtain their juice samples. The flavedo portions were grated. One gram of each grated sample was ground with water
in a mortar with a pestle and was adjusted to a volume of 10 ml. A typical brand of soy sauce commercially available in Japan was used. MTCCA ŽCAS No. 5470-37-1. was purchased from Sigma ŽSt. Louis, MO, USA.. Lignin Ždealkaline, CAS No. 9005-53-2., pectin Žcitrus, CAS No. 9000-69-5., Dlimonene ŽCAS No. 5989-27-5., naringin ŽCAS No. 10236-47-2., hesperidin ŽCAS No. 520-26-3. and tyramine ŽCAS No. 51-67-2. were obtained from Tokyo Kasei Kogyo ŽTokyo, Japan.. Lignin was also obtained from Kanto Chemical ŽTokyo.. Ascorbic acid ŽCAS No. 50-81-7. was from Wako. Citric acid ŽCAS No. 5949-29-1. was from Kanto. The other chemicals were of reagent grade. 2.2. Nitrite treatment Chemicals were dissolved or suspended in sterilized water. Pectin was used as a suspension in dimethyl sulfoxide. Soy sauce was used after filtration through a sterilized membrane filter Žpore size, 0.45 mm.. Aqueous solutions of 1 ml containing 0.06–0.6 mg of MTCCA Žor 0.3–1.2 mg of tyramine or 0.05 ml of soy sauce., 0–0.15 ml of ethanol and 0–0.1 ml of citrus juice sample or citrus flavedo sample were mixed in brown tubes with 1 ml of 0.1 M sodium nitrite and adjusted to pH 3.0 with 6 N HCl with monitoring by a pH meter equipped with a small electrode. The reaction mixtures were incubated at 378C for 60 min in the dark. The nitrosation was stopped by addition of 1 ml of 0.1 M ammonium sulfamate to decompose the residual nitrite. The effect of each citrus sample was examined at three or six doses. All the nitrite treatments were performed in triplicate. The nitrite-treated sample was immediately used for the mutation assay. 2.3. Mutation test The mutation test with Salmonella typhimurium strain TA100 was conducted in duplicate by the preincubation procedure of Maron and Ames w16x in the absence of S9 mix. A mixture containing 0.1 ml of a nitrite-treated sample, 0.5 ml of buffer solution ŽpH 7.4. and 0.1 ml of an overnight culture of strain TA100 was preincubated at 378C for 20 min, mixed
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with 2 ml of soft agar and plated on agar plates. The Hisq revertant colonies were counted after incubation at 378C for 48 h. The mutation was assayed with brown tubes under a yellow lamp in the dark. The numbers of spontaneous revertants Ž106 " 14. were subtracted in all the data on mutagenicity. A positive control, 2-Ž2-furyl.-3-Ž5-nitro-2-furyl.-acrylamide ŽAF-2, 10 ng. induced 418 " 72 Hisq revertants from strain TA100 in the absence of S9 mix.
3. Results 3.1. Effects of citrus juice and flaÕedo on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol The mutagenicity of MTCCA treated with nitrite in the presence of 7.5% ethanol was dose-dependently reduced by the addition of 2.5–100 ml of juice samples of the acid citruses sudachi, lemon and yuzu per 2 ml of the reaction mixture as shown in Fig. 1. It was reduced to 29.5, 35.7 and 31.0% by the addition of 100 ml of sudachi, lemon and yuzu juice, respectively. It was strongly reduced by the addition of 2.5–100 ml Žequivalent to 0.25–10 mg of original flavedo. of flavedo samples of the acid citruses as shown in Fig. 2. It was reduced to 4.6, 18.2 and
Fig. 1. Effects of citrus juices on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1210"43 Hisq revertants from strain TA100 without S9 mix.
Fig. 2. Effects of citrus flavedos on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1210"43 Hisq revertants from strain TA100 without S9 mix.
18.4% by the addition of 10 mg of sudachi, lemon and yuzu flavedo, respectively. The juices of the other citrus fruits commercially available in Japan had similar mutagenicity-reducing activity as shown in Fig. 3. The mutagenicity of nitrite-treated MTCCA was reduced to 26.7, 27.6, 28.9, 29.3, 32.5 and 39.1% by the addition of 100 ml of juice sample of
Fig. 3. Effects of six citrus juices on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1078"87 Hisq revertants from strain TA100 without S9 mix.
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Table 1 Antimutagenicity of the components of citrus fruits against the mutagenicity of MTCCA treated with nitrite in the presence of ethanol Compound Experiment I Control Lignin
Experiment II Control Pectin
D-Limonene
Naringin Hesperidin Ascorbic acid Citric acid
Dose Žmgr2 ml.
Hisq revertantsa
Relative mutagenicity b Ž%.
– 1 0.5 0.25 0.1 0.05 0.025
1167 " 135 96 " 32 149 " 41 175 " 32 350 " 51 537 " 119 735 " 149
100 8 13 15 30 46 63
– 3 2 1 1 0.1 1 0.1 1 0.1 1 0.1 5 1
1116 " 91 498 " 67 679 " 81 856 " 93 536 " 95 770 " 77 502 " 92 825 " 94 540 " 51 816 " 94 531 " 47 793 " 77 789 " 81 1042 " 75
100 45 61 77 48 69 45 74 48 73 48 71 71 93
a
Hisq revertants from strain TA100 without S9 mix were means" S.D. per 2 mg of MTCCA. 100% is mutagenicity of the nitrite-treated MTCCA in the presence of ethanol. Number of spontaneous revertants Ž112 " 12. were subtracted.
b
navel orange, iyokan, lime, hassaku, unshu and grapefruit, respectively. No mutagenicity was detected in citrus juice samples and citrus flavedo samples after nitrite treatment at the same volumes
as those described above Ždata not shown.. No bactericidal activity was observed in nitrite-treated citrus samples after preincubation in the Ames mutation assay Ždata not shown..
Table 2 Antimutagenicity of citrus components added during and after the nitrosation of MTCCA in the presence of ethanol Compound
Dose Žmgr2 ml.
Control Lignin Pectin D-Limonene Naringin Hesperidin Ascorbic acid Citric acid
– 1 3 1 1 1 1 5
a
Hisq revertants when adding a compounda During nitrosation
After nitrosation
1128 " 127 82 " 33 420 " 48 605 " 54 593 " 65 615 " 73 646 " 69 870 " 98
– 117 " 20 1112 " 126 635 " 62 1135 " 98 1135 " 122 1178 " 143 1160 " 130
Hisq revertants from strain TA100 without S9 mix were means" S.D. per 2 mg of MTCCA. Number of spontaneous revertants Ž113 " 17. was subtracted.
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Table 3 Effects of sudachi and lemon on the mutagenicity of MTCCA, tyramine and soy sauce treated with nitrite in the absence and presence of ethanol Substrate
Citrus sample
Dose a
Žy. Ethanol
Žq. Ethanol
Hisq revertantsb Relative mutagenicity c Ž%. Hisq revertantsb Relative mutagenicity c Ž%. MTCCA
– Sudachi juice
1008 " 91 329 " 15 291 " 19 241 " 51 134 " 16 124 " 26 76 " 18
100 32.6 28.9 23.9 13.3 12.3 7.5
– Sudachi juice
769 " 49 797 " 63 899 " 48 929 " 76 719 " 97 755 " 91 826 " 33
100 103.6 116.9 120.8 93.5 98.2 107.4
40 " 15 47 " 19 84 " 31 125 " 29 51 " 7 69 " 18 89 " 17
5.2 6.1 10.9 16.3 6.6 9.0 11.6
– Sudachi juice
760 " 45 708 " 43 679 " 11 655 " 26 682 " 23 623 " 64 591 " 52
100 93.1 89.4 86.2 89.7 82.0 77.8
386 " 18 358 " 31 318 " 27 279 " 11 294 " 17 233 " 28 150 " 10
50.8 47.1 41.9 36.7 38.7 30.6 19.7
1008 " 91 374 " 21 280 " 40 249 " 21 225 " 26 186 " 18 116 " 25
100 37.1 27.8 24.7 22.3 18.5 11.5
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
Tyramine
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
Soy sauce
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
MTCCA
– Lemon juice
Lemon flavedo
Tyramine
– Lemon juice
Lemon flavedo
Soy sauce
– Lemon juice
Lemon flavedo
a
25 ml 50 100 2.5 mg 5 10
876 " 54 25 ml 959 " 82 50 1092 " 70 100 1244 " 81 2.5 mg 851 " 99 5 910 " 52 10 1027 " 81 25 ml 50 100 2.5 mg 5 10
694 " 41 680 " 19 654 " 78 581 " 31 630 " 80 600 " 68 521 " 48
12 100 " 430 5570 " 640 4500 " 470 3570 " 220 3290 " 270 1580 " 340 560 " 160
12 100 " 740 6450 " 420 5020 " 700 4310 " 420 4400 " 530 3550 " 330 2200 " 500
1200 553 446 354 326 157 55.2
1200 640 498 428 437 352 218
100 109.5 124.7 142.0 97.1 103.9 117.2
42 " 8 106 " 11 123 " 23 128 " 36 98 " 26 119 " 29 137 " 23
4.8 12.1 14.0 14.6 11.2 13.6 15.6
100 98.0 94.3 83.7 90.8 86.4 75.0
391 " 13 324 " 25 316 " 20 287 " 24 291 " 23 248 " 16 149 " 13
56.3 46.7 45.6 41.3 42.0 35.8 21.5
Dose is ml of juice or mg of flavedo in 2 ml of the nitrosation reaction mixture. Hisq revertants from strain TA100 without S9 mix were means" S.D. per 20 mg of MTCCA, 20 mg of tyramine of 3.3 ml of soy sauce. Numbers of spontaneous revertants Ž106 " 12. were subtracted. c 100% is mutagenicity of nitrite-treated substrate in the absence of ethanol. b
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3.2. Effects of components of citrus fruits on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol These effects were investigated and the results are shown in Table 1. The doses used were decided from the reported values in juices, flavedos or peels of citrus fruits w17–27x. The mutagenicity of nitritetreated MTCCA in the presence of ethanol was dose-dependently reduced to 63.2–8.2% by addition of 0.025–1 mg of lignin. The lignins obtained from two makers gave approximately the same results. The mutagenicity was reduced to 45–48% by 3 mg of pectin, or 1 mg of D-limonene, naringin, hesperidin or ascorbic acid. It was weakly reduced by 2 mg of pectin, 0.1 mg of D-limonene, naringin, hesperidin or ascorbic acid, or 5 mg of citric acid. Within these components, the lignin and D-limonene were observed to have similar antimutagenicity when they were added both during and after the nitrosation of MTCCA, however, the other components had the antimutagenicity only when they were simultaneously nitrosated with MTCCA in the presence of ethanol as shown in Table 2. The other components, 1 mg of cellulose, sugars Žsucrose, glucose and fructose. and organic acids Žmalic acid and succinic acid. and 0.1 mg of synephrine and amino acids Žasparagine, glutamine, asparaginic acid, glutaminic acid, proline, valine and serine., had little mutagenicity-reducing activity when they were added during nitrosation of MTCCA in the presence of ethanol Ždata not shown.. 3.3. Effects of sudachi and lemon on the mutagenicity of MTCCA, tyramine or soy sauce treated with nitrite in the absence and presence of ethanol The sudachi samples strongly reduced the mutagenicity of nitrite-treated MTCCA in both the absence and the presence of ethanol as shown in Table 3. On the other hand, the mutagenicity of nitritetreated tyramine in the presence of ethanol was slightly increased by the addition of sudachi. Sudachi considerably reduced the mutagenicity of nitritetreated soy sauce in the presence of ethanol. The lemon samples were also found to have mutagenicity-reducing activity in a manner similar to that of the sudachi samples ŽTable 3..
4. Discussion Soy sauce may be one of the most important Japanese food stuffs concerning the relation between nitrite-treated food and stomach cancer as stated in Section 1. Among the mutagen precursors in soy sauce, MTCCA is a most potent one when it is treated with nitrite in the presence of alcohols w11x. The mutagenicity of nitrite-treated MTCCA was considerably reduced by the citrus fruits added to the reaction mixture although that of nitrite-treated tyramine, another important mutagen precursor in soy sauce, was a little increased ŽTable 3.. It must be due to the contribution of MTCCA in soy sauce that sudachi and lemon reduced considerably the mutagenicity of soy sauce treated with nitrite and ethanol. The mutagenicity-reducing activity per weight of flavedos of the citrus fruits was considerably higher than that of their juices because the flavedos contain a greater amount of active components except citric acid than the juices w17–27x. Several components in the citrus fruits were found to have mutagenicity-reducing activity against nitrite-treated MTCCA as shown in Table 1. Among the components, lignin had the strongest inhibitory effect. Grapefruit and oranges contain 0.03–0.24 g of lignin in 100 g of the peeled portion and they contain 0.12–1.2 g of lignin in 100 g of the peel w17,19,23–26x. Pectin, which is a major component of the citrus dietary fibers, also had considerable mutagenicity-reducing activity. Yuzu, unshu, grapefruit and orange contain 0.03– 0.23 g of pectin in 100 g of the peeled portion, and sudachi, lemon, yuzu, unshu, grapefruit and orange contain 3.0–4.6 g of pectin in 100 g of the peel w17,19,26,27x. If all of the citrus fruits contain as much of the dietary fibers lignin and pectin as these six do, they must play a major role in the antimutagenicity of citrus fruits. However, a method for analyzing lignin is not well established and the commercial lignin is known to have a different structure and chemical properties from lignified plant cell walls. Experimental data suggest that the pectin reduces production of the mutagen from MTCCA treated with nitrite and ethanol and that the lignin reduces development of the mutagen produced ŽTable 2.. The flavonoids naringin and hesperidin were antimutagenic, as previously reported w28,29x, but were
M. Higashimoto et al.r Mutation Research 415 (1998) 219–226
not very abundant in the citrus fruits w17x. Although citric acid is one of the most abundant organic acids in citrus juices Ž1–6%., its antimutagenicity against the nitrite-treated MTCCA was limited. The antimutagenicity of the citrus fruits may be the result of an additive or synergistic action as described by Bala and Grover w12x, by not only the known components but also other factorŽs.. The reason why D-limonene, an essential oil contained in the citrus flavedo, has antimutagenicity not only before but also after the nitrite treatment of MTCCA, must be investigated further. Some citrus fruits such as sudachi, lemon and yuzu, so-called acid citruses, are eaten with soy sauce in the Japanese menu as refreshing flavors. The acid taste results from the organic acids such as citric acid and ascorbic acid present in the juice and the sweet smell results mainly from the essential oils present in the flavedo. The experimental data suggest that 9.5 ml of juice and 1.9 g of flavedo obtained from a normal size sudachi Ž35 g. have a possibility of reducing by 48% and 80%, respectively, the mutagenicity of MTCCA and nitrite with ethanol in 1 l, equivalent to our stomach volume after a meal, of an acidic medium ŽFigs. 1 and 2.. The dietary fibers lignin and pectin can account for 16–108% of the antimutagenicity of the juice and 13–69% of that of the flavedo ŽTable 1., if the sudachi contains as much fiber as the citruses described above and the antimutagenicity is the same as that of the commercial fibers. The citrus fruits may bring us an additional healthful benefit if they prevent the development of mutagens in our stomach in a manner similar to that described above. References w1x S.S. Mirvish, Formation of N-nitroso compounds: chemistry, kinetics, and in vivo occurrence, Toxicol. Appl. Pharmacol. 31 Ž1975. 325–351. w2x P.E. Hartman, Nitrates and nitrites: Ingestion, pharmacodynamics, and toxicology, in: F.J. de Serres, A. Hollaender ŽEds.., Chemical Mutagens, Vol. 7, Plenum, New York, 1982, pp. 211–294. w3x K. Wakabayashi, M. Ochiai, H. Saito, M. Tsuda, Y. Suwa, M. Nagao, T. Sugimura, Presence of 1-methyl-1, 2, 3, 4-tetrahydro-b-carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound, in soy sauce, Proc. Natl. Acad. Sci. U.S.A. 80 Ž1983. 2912–2916.
225
w4x M. Ochiai, K. Wakabayashi, M. Nagao, T. Sugimura, Tyramine is a major mutagen precursor in soy sauce, being convertible to a mutagen by nitrite, Gann 75 Ž1984. 1–3. w5x M. Higashimoto, K. Matano, Y. Ohnishi, Augmenting effect of a nonmutagenic fraction in soy sauce on mutagenicity of 3-diazotyramine produced in the nitrite-treated sauce, Jpn. J. Cancer Res. ŽGann. 79 Ž1988. 1284–1292. w6x Y. Fujita, K. Wakabayashi, S. Takayama, M. Nagao, T. Sugimura, Induction of oral cavity cancer by 3-diazotyramine, a nitrosated product of tyramine present in foods, Carcinogenesis 8 Ž1987. 527–529. w7x W.C. MacDonald, J.W. Dueck, Long-term effect of shoyu ŽJapanese soy sauce. on the gastric mucosa of the rat, J. Natl. Cancer Inst. 56 Ž1976. 1143–1145. w8x I. Hirono, M. Funahashi, C. Kaneko, H. Ogino, M. Ito, A. Yoshida, Gastric lesions in rats fed salted food materials commonly eaten by Japanese, Nutr. Cancer 14 Ž1990. 127– 132. w9x I. Kato, S. Tominaga, K. Matsumoto, A prospective study of stomach cancer among a rural Japanese population: a 6-year survey, Jpn. J. Cancer Res. 83 Ž1992. 568–575. w10x M. Higashimoto, T. Yamamoto, T. Kinouchi, H. Matsumoto, Y. Ohnishi, Mutagenicity of 1-methyl-1,2,3,4,-tetrahydro-bcarboline-3-carboxylic acid treated with nitrite in the presence of alcohols, Mutat. Res. 367 Ž1995. 43–49. w11x M. Higashimoto, T. Yamamoto, T. Kinouchi, Y. Handa, H. Matsumoto, Y. Ohnishi, Mutagenicity of soy sauce treated with nitrite in the presence of ethanol or alcoholic beverages, Mutat. Res. 345 Ž1995. 155–166. w12x S. Bala, I.S. Grover, Antimutagenicity of some citrus fruits in Salmonella typhimurium, Mutat. Res. 222 Ž1989. 141–148. w13x H.A. Risch, M. Jain, N.W. Choi, J.G. Fodor, C.J. Pfeiffer, G.R. Howe, L.W. Harrison, K.J.P. Craib, A.B. Miller, Dietary factors and the incidence of cancer of the stomach, Am. J. Epidemiol. 122 Ž1985. 947–959. w14x E. Buiatti, D. Palli, A. Decarli, D. Amadori, C. Avellini, S. Bianchi, R. Biserni, F. Cipriani, P. Cocco, A. Giacosa, E. Marubini, R. Puntoni, C. Vindigni, J. Fraumeni Jr., W. Blot, A case-control study of gastric cancer and diet in Italy, Int. J. Cancer 44 Ž1989. 611–616. w15x H. Boeing, B.R. Frentzel, M. Berger, V. Berndt, W. Gores, M. Korner, R. Lohmeier, A. Menarcher, H.F. Mannl, M. Meinhardt, R. Muller, H. Ostermeier, F. Paol, K. Schwemmle, ¨ K.H. Wagner, J. Wahrendorf, Case-control study on stomach cancer in Germany, Int. J. Cancer 47 Ž1991. 858–864. w16x D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 Ž1983. 173–215. w17x S. Ito ŽEd.., Science of Fruits, Asakura Pub., Tokyo, 1991 Žin Japanese.. w18x K. Kimura, T. Taninaka, Acid Citrus: Acid Citrus in Shikoku Island, Harada Pub., Tokushima, 1988 Žin Japanese.. w19x Nippon Kajyu Kyokai ŽEd.., Kajyu-kajitsu Inryo Jiten, Asakura Pub., Tokyo, 1978 Žin Japanese.. w20x Resources Council, Science and Technology Agency, Japan ŽEd.., Standard Tables of Food Composition in Japan, 4th edn., Printing Bureau, Ministry of Finance, Tokyo, 1982. w21x Resources Council, Science and Technology Agency, Japan
226
w22x
w23x w24x
w25x
M. Higashimoto et al.r Mutation Research 415 (1998) 219–226 ŽEd.., Standard Tables of Food Composition in Japan, Dietary fiber, Printing Bureau, Ministry of Finance, Tokyo, 1992. S.V. Ting, R.L. Rouseff, Citrus Fruits and Their Products: Analysis and Technology, Marcel Dekker, New YorkrBasel, 1986. D.A.T. Southgate, P.J. Van Soest, Fiber analysis tables, Am. J. Clin. Nutr. 31 Ž1978. S281–S284. E.D. Lund, J.M. Smoot, Dietary fiber content of some tropical fruits and vegetables, J. Agric. Food Chem. 30 Ž1982. 1123–1127. E.D. Lund, J.M. Smoot, N.T. Hall, Dietary fiber content of eleven tropical fruits and vegetables, J. Agric. Food Chem. 31 Ž1983. 1013–1016.
w26x R.J. Braddock, T.R. Graumlich, Composition of fiber from citrus peel, membranes, juice vesicles and seeds, Lebensm. Wiss. Technol. 14 Ž1981. 229–231. w27x A. Kawabata, S. Sawayama, K. Uryu, A study on the contents of peptic substances in fruits, vegetable fruits and nuts, Jpn. J. Nutr. 32 Ž1974. 9–18, Žin Japanese with English abstract.. w28x A.R. Francis, T.K. Shetty, R.K. Bhattacharya, Modulating effect of plant flavonoids on the mutagenicity of N-methylX N -nitro-N-nitrosoguanidine, Carcinogenesis 10 Ž1989. 1953–1955. w29x M. Calomme, L. Pieters, A. Vlietinck, D.V. Berghe, Inhibition of bacterial mutagenesis by Citrus flavonoids, Planta Med. 62 Ž1996. 222–226.
Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol Minoru Higashimoto a , Hirokuni Yamato b, Takemi Kinouchi c , Yoshinari Ohnishi a
c,)
Faculty of Pharmaceutical Sciences, Tokushima Bunri UniÕersity, Tokushima 770-8514, Japan b Faculty of Domestic Sciences, Tokushima Bunri UniÕersity, Tokushima 770-8514, Japan c School of Medicine, The UniÕersity of Tokushima, Tokushima 770-8503, Japan Received 6 January 1998; revised 26 May 1998; accepted 26 May 1998
Abstract It has been shown that the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid ŽMTCCA., a mutagen precursor in soy sauce treated with nitrite, was strongly increased when it was treated with nitrite in the presence of alcohols. We found that the mutagenicity of MTCCA treated with 50 mM nitrite at pH 3, 378C for 60 min in the presence of 7.5% ethanol was reduced by the addition of citrus fruits sudachi Ž Citrus sudachi ., lemon Ž C. limon. and yuzu Ž C. junos ., to the reaction mixture. The mutagenicity-reducing activity per weight of flavedos Žouter colored portions of peel. of the citrus fruits was considerably higher than that of the juices. The juices of the other citrus fruits commercially available in Japan also had mutagenicity-reducing activity against the nitrite-treated MTCCA. Among the many components of citrus fruits, dietary fibers lignin and pectin showed strong antimutagenic activity in the reaction mixture, suggesting that the mixed fractions of these components including lignin, pectin, D-limonene, naringin, hesperidin, ascorbic acid and citric acid reduce the mutagenicity of MTCCA in the reaction mixture containing nitrite and ethanol. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antimutagenicity; Citrus fruits; Soy sauce; Nitrosation; Ethanol
1. Introduction N-nitroso compounds which are easily produced under the acidic condition in the stomach may induce stomach cancer w1,2x. Wakabayashi et al. w3x ) Corresponding author. Tel.: q81-886-33-7069; Fax: q81886-33-7069; E-mail: [email protected]
reported that Japanese soy sauce had the highest mutagenicity among various food stuffs on treatment with nitrite and suggested a relationship of food consumption by the Japanese and their high gastric cancer mortality. Tyramine is a major mutagen precursor in soy sauce on treatment with nitrite and 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid ŽMTCCA. is a minor mutagen precursor in the
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soy sauce w4,5x. Fujita et al. w6x reported that 3-diazotyramine, a mutagen produced by nitrite treatment of tyramine, induces squamous cell carcinomas in the oral cavity of rats, although it has not been demonstrated that soy sauce induces stomach cancers w7–9x. The chemical structure of a mutagen produced from MTCCA and nitrite and carcinogenicity of the mutagen have not been clarified yet. However, when MTCCA is treated with nitrite in the presence of ethanol, mutagenicity of the product is very high w10x, while the mutagenicity of tyramine that has been treated with nitrite in the presence of ethanol is very low w11x. It has been reported that citrus fruits reduce the mutagenicity of known mutagens w12x and the risk of stomach cancer w13–15x. Japanese use some acid citrus fruits for foods cooked with soy sauce. We attempted to reduce the mutagenicity of nitrite-treated MTCCA in the presence of ethanol by adding three kinds of acid citrus fruits. The effects of six other citrus fruits on the mutagenicity were also studied. In addition, we studied the antimutagenicity of the components of the citrus fruits to elucidate the mechanisms of the mutagenicity suppression.
2. Materials and methods 2.1. Citrus fruits, soy sauce and chemicals Sudachi Ž Citrus sudachi Shirai., yuzu Ž C. junos Tanaka., unshu Ž C. unshiu Marc.., hassaku Ž C. hassaku Tanaka. and iyokan Ž C. iyo Tanaka., produced in Japan, lemon Ž C. limon Burm.., grapefruit Ž C. paradisi Macf.. and navel orange Ž C. sinensis Osbeck., produced in the USA, and lime Ž C. aurantifolia Swingle., produced in Mexico, were purchased at local markets in Japan. Sudachi, lemon and yuzu were fractionated into juice and flavedo Žthe outer colored portion of peel.. For the other citrus fruits, only the juices were used. The citrus samples were prepared aseptically by using sterile glassware and flame sterilization. Well-washed citrus fruits were cut in half and hand-squeezed to obtain their juice samples. The flavedo portions were grated. One gram of each grated sample was ground with water
in a mortar with a pestle and was adjusted to a volume of 10 ml. A typical brand of soy sauce commercially available in Japan was used. MTCCA ŽCAS No. 5470-37-1. was purchased from Sigma ŽSt. Louis, MO, USA.. Lignin Ždealkaline, CAS No. 9005-53-2., pectin Žcitrus, CAS No. 9000-69-5., Dlimonene ŽCAS No. 5989-27-5., naringin ŽCAS No. 10236-47-2., hesperidin ŽCAS No. 520-26-3. and tyramine ŽCAS No. 51-67-2. were obtained from Tokyo Kasei Kogyo ŽTokyo, Japan.. Lignin was also obtained from Kanto Chemical ŽTokyo.. Ascorbic acid ŽCAS No. 50-81-7. was from Wako. Citric acid ŽCAS No. 5949-29-1. was from Kanto. The other chemicals were of reagent grade. 2.2. Nitrite treatment Chemicals were dissolved or suspended in sterilized water. Pectin was used as a suspension in dimethyl sulfoxide. Soy sauce was used after filtration through a sterilized membrane filter Žpore size, 0.45 mm.. Aqueous solutions of 1 ml containing 0.06–0.6 mg of MTCCA Žor 0.3–1.2 mg of tyramine or 0.05 ml of soy sauce., 0–0.15 ml of ethanol and 0–0.1 ml of citrus juice sample or citrus flavedo sample were mixed in brown tubes with 1 ml of 0.1 M sodium nitrite and adjusted to pH 3.0 with 6 N HCl with monitoring by a pH meter equipped with a small electrode. The reaction mixtures were incubated at 378C for 60 min in the dark. The nitrosation was stopped by addition of 1 ml of 0.1 M ammonium sulfamate to decompose the residual nitrite. The effect of each citrus sample was examined at three or six doses. All the nitrite treatments were performed in triplicate. The nitrite-treated sample was immediately used for the mutation assay. 2.3. Mutation test The mutation test with Salmonella typhimurium strain TA100 was conducted in duplicate by the preincubation procedure of Maron and Ames w16x in the absence of S9 mix. A mixture containing 0.1 ml of a nitrite-treated sample, 0.5 ml of buffer solution ŽpH 7.4. and 0.1 ml of an overnight culture of strain TA100 was preincubated at 378C for 20 min, mixed
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with 2 ml of soft agar and plated on agar plates. The Hisq revertant colonies were counted after incubation at 378C for 48 h. The mutation was assayed with brown tubes under a yellow lamp in the dark. The numbers of spontaneous revertants Ž106 " 14. were subtracted in all the data on mutagenicity. A positive control, 2-Ž2-furyl.-3-Ž5-nitro-2-furyl.-acrylamide ŽAF-2, 10 ng. induced 418 " 72 Hisq revertants from strain TA100 in the absence of S9 mix.
3. Results 3.1. Effects of citrus juice and flaÕedo on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol The mutagenicity of MTCCA treated with nitrite in the presence of 7.5% ethanol was dose-dependently reduced by the addition of 2.5–100 ml of juice samples of the acid citruses sudachi, lemon and yuzu per 2 ml of the reaction mixture as shown in Fig. 1. It was reduced to 29.5, 35.7 and 31.0% by the addition of 100 ml of sudachi, lemon and yuzu juice, respectively. It was strongly reduced by the addition of 2.5–100 ml Žequivalent to 0.25–10 mg of original flavedo. of flavedo samples of the acid citruses as shown in Fig. 2. It was reduced to 4.6, 18.2 and
Fig. 1. Effects of citrus juices on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1210"43 Hisq revertants from strain TA100 without S9 mix.
Fig. 2. Effects of citrus flavedos on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1210"43 Hisq revertants from strain TA100 without S9 mix.
18.4% by the addition of 10 mg of sudachi, lemon and yuzu flavedo, respectively. The juices of the other citrus fruits commercially available in Japan had similar mutagenicity-reducing activity as shown in Fig. 3. The mutagenicity of nitrite-treated MTCCA was reduced to 26.7, 27.6, 28.9, 29.3, 32.5 and 39.1% by the addition of 100 ml of juice sample of
Fig. 3. Effects of six citrus juices on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol. 100% was 1078"87 Hisq revertants from strain TA100 without S9 mix.
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Table 1 Antimutagenicity of the components of citrus fruits against the mutagenicity of MTCCA treated with nitrite in the presence of ethanol Compound Experiment I Control Lignin
Experiment II Control Pectin
D-Limonene
Naringin Hesperidin Ascorbic acid Citric acid
Dose Žmgr2 ml.
Hisq revertantsa
Relative mutagenicity b Ž%.
– 1 0.5 0.25 0.1 0.05 0.025
1167 " 135 96 " 32 149 " 41 175 " 32 350 " 51 537 " 119 735 " 149
100 8 13 15 30 46 63
– 3 2 1 1 0.1 1 0.1 1 0.1 1 0.1 5 1
1116 " 91 498 " 67 679 " 81 856 " 93 536 " 95 770 " 77 502 " 92 825 " 94 540 " 51 816 " 94 531 " 47 793 " 77 789 " 81 1042 " 75
100 45 61 77 48 69 45 74 48 73 48 71 71 93
a
Hisq revertants from strain TA100 without S9 mix were means" S.D. per 2 mg of MTCCA. 100% is mutagenicity of the nitrite-treated MTCCA in the presence of ethanol. Number of spontaneous revertants Ž112 " 12. were subtracted.
b
navel orange, iyokan, lime, hassaku, unshu and grapefruit, respectively. No mutagenicity was detected in citrus juice samples and citrus flavedo samples after nitrite treatment at the same volumes
as those described above Ždata not shown.. No bactericidal activity was observed in nitrite-treated citrus samples after preincubation in the Ames mutation assay Ždata not shown..
Table 2 Antimutagenicity of citrus components added during and after the nitrosation of MTCCA in the presence of ethanol Compound
Dose Žmgr2 ml.
Control Lignin Pectin D-Limonene Naringin Hesperidin Ascorbic acid Citric acid
– 1 3 1 1 1 1 5
a
Hisq revertants when adding a compounda During nitrosation
After nitrosation
1128 " 127 82 " 33 420 " 48 605 " 54 593 " 65 615 " 73 646 " 69 870 " 98
– 117 " 20 1112 " 126 635 " 62 1135 " 98 1135 " 122 1178 " 143 1160 " 130
Hisq revertants from strain TA100 without S9 mix were means" S.D. per 2 mg of MTCCA. Number of spontaneous revertants Ž113 " 17. was subtracted.
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Table 3 Effects of sudachi and lemon on the mutagenicity of MTCCA, tyramine and soy sauce treated with nitrite in the absence and presence of ethanol Substrate
Citrus sample
Dose a
Žy. Ethanol
Žq. Ethanol
Hisq revertantsb Relative mutagenicity c Ž%. Hisq revertantsb Relative mutagenicity c Ž%. MTCCA
– Sudachi juice
1008 " 91 329 " 15 291 " 19 241 " 51 134 " 16 124 " 26 76 " 18
100 32.6 28.9 23.9 13.3 12.3 7.5
– Sudachi juice
769 " 49 797 " 63 899 " 48 929 " 76 719 " 97 755 " 91 826 " 33
100 103.6 116.9 120.8 93.5 98.2 107.4
40 " 15 47 " 19 84 " 31 125 " 29 51 " 7 69 " 18 89 " 17
5.2 6.1 10.9 16.3 6.6 9.0 11.6
– Sudachi juice
760 " 45 708 " 43 679 " 11 655 " 26 682 " 23 623 " 64 591 " 52
100 93.1 89.4 86.2 89.7 82.0 77.8
386 " 18 358 " 31 318 " 27 279 " 11 294 " 17 233 " 28 150 " 10
50.8 47.1 41.9 36.7 38.7 30.6 19.7
1008 " 91 374 " 21 280 " 40 249 " 21 225 " 26 186 " 18 116 " 25
100 37.1 27.8 24.7 22.3 18.5 11.5
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
Tyramine
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
Soy sauce
25 ml 50 100 Sudachi flavedo 2.5 mg 5 10
MTCCA
– Lemon juice
Lemon flavedo
Tyramine
– Lemon juice
Lemon flavedo
Soy sauce
– Lemon juice
Lemon flavedo
a
25 ml 50 100 2.5 mg 5 10
876 " 54 25 ml 959 " 82 50 1092 " 70 100 1244 " 81 2.5 mg 851 " 99 5 910 " 52 10 1027 " 81 25 ml 50 100 2.5 mg 5 10
694 " 41 680 " 19 654 " 78 581 " 31 630 " 80 600 " 68 521 " 48
12 100 " 430 5570 " 640 4500 " 470 3570 " 220 3290 " 270 1580 " 340 560 " 160
12 100 " 740 6450 " 420 5020 " 700 4310 " 420 4400 " 530 3550 " 330 2200 " 500
1200 553 446 354 326 157 55.2
1200 640 498 428 437 352 218
100 109.5 124.7 142.0 97.1 103.9 117.2
42 " 8 106 " 11 123 " 23 128 " 36 98 " 26 119 " 29 137 " 23
4.8 12.1 14.0 14.6 11.2 13.6 15.6
100 98.0 94.3 83.7 90.8 86.4 75.0
391 " 13 324 " 25 316 " 20 287 " 24 291 " 23 248 " 16 149 " 13
56.3 46.7 45.6 41.3 42.0 35.8 21.5
Dose is ml of juice or mg of flavedo in 2 ml of the nitrosation reaction mixture. Hisq revertants from strain TA100 without S9 mix were means" S.D. per 20 mg of MTCCA, 20 mg of tyramine of 3.3 ml of soy sauce. Numbers of spontaneous revertants Ž106 " 12. were subtracted. c 100% is mutagenicity of nitrite-treated substrate in the absence of ethanol. b
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3.2. Effects of components of citrus fruits on the mutagenicity of MTCCA treated with nitrite in the presence of ethanol These effects were investigated and the results are shown in Table 1. The doses used were decided from the reported values in juices, flavedos or peels of citrus fruits w17–27x. The mutagenicity of nitritetreated MTCCA in the presence of ethanol was dose-dependently reduced to 63.2–8.2% by addition of 0.025–1 mg of lignin. The lignins obtained from two makers gave approximately the same results. The mutagenicity was reduced to 45–48% by 3 mg of pectin, or 1 mg of D-limonene, naringin, hesperidin or ascorbic acid. It was weakly reduced by 2 mg of pectin, 0.1 mg of D-limonene, naringin, hesperidin or ascorbic acid, or 5 mg of citric acid. Within these components, the lignin and D-limonene were observed to have similar antimutagenicity when they were added both during and after the nitrosation of MTCCA, however, the other components had the antimutagenicity only when they were simultaneously nitrosated with MTCCA in the presence of ethanol as shown in Table 2. The other components, 1 mg of cellulose, sugars Žsucrose, glucose and fructose. and organic acids Žmalic acid and succinic acid. and 0.1 mg of synephrine and amino acids Žasparagine, glutamine, asparaginic acid, glutaminic acid, proline, valine and serine., had little mutagenicity-reducing activity when they were added during nitrosation of MTCCA in the presence of ethanol Ždata not shown.. 3.3. Effects of sudachi and lemon on the mutagenicity of MTCCA, tyramine or soy sauce treated with nitrite in the absence and presence of ethanol The sudachi samples strongly reduced the mutagenicity of nitrite-treated MTCCA in both the absence and the presence of ethanol as shown in Table 3. On the other hand, the mutagenicity of nitritetreated tyramine in the presence of ethanol was slightly increased by the addition of sudachi. Sudachi considerably reduced the mutagenicity of nitritetreated soy sauce in the presence of ethanol. The lemon samples were also found to have mutagenicity-reducing activity in a manner similar to that of the sudachi samples ŽTable 3..
4. Discussion Soy sauce may be one of the most important Japanese food stuffs concerning the relation between nitrite-treated food and stomach cancer as stated in Section 1. Among the mutagen precursors in soy sauce, MTCCA is a most potent one when it is treated with nitrite in the presence of alcohols w11x. The mutagenicity of nitrite-treated MTCCA was considerably reduced by the citrus fruits added to the reaction mixture although that of nitrite-treated tyramine, another important mutagen precursor in soy sauce, was a little increased ŽTable 3.. It must be due to the contribution of MTCCA in soy sauce that sudachi and lemon reduced considerably the mutagenicity of soy sauce treated with nitrite and ethanol. The mutagenicity-reducing activity per weight of flavedos of the citrus fruits was considerably higher than that of their juices because the flavedos contain a greater amount of active components except citric acid than the juices w17–27x. Several components in the citrus fruits were found to have mutagenicity-reducing activity against nitrite-treated MTCCA as shown in Table 1. Among the components, lignin had the strongest inhibitory effect. Grapefruit and oranges contain 0.03–0.24 g of lignin in 100 g of the peeled portion and they contain 0.12–1.2 g of lignin in 100 g of the peel w17,19,23–26x. Pectin, which is a major component of the citrus dietary fibers, also had considerable mutagenicity-reducing activity. Yuzu, unshu, grapefruit and orange contain 0.03– 0.23 g of pectin in 100 g of the peeled portion, and sudachi, lemon, yuzu, unshu, grapefruit and orange contain 3.0–4.6 g of pectin in 100 g of the peel w17,19,26,27x. If all of the citrus fruits contain as much of the dietary fibers lignin and pectin as these six do, they must play a major role in the antimutagenicity of citrus fruits. However, a method for analyzing lignin is not well established and the commercial lignin is known to have a different structure and chemical properties from lignified plant cell walls. Experimental data suggest that the pectin reduces production of the mutagen from MTCCA treated with nitrite and ethanol and that the lignin reduces development of the mutagen produced ŽTable 2.. The flavonoids naringin and hesperidin were antimutagenic, as previously reported w28,29x, but were
M. Higashimoto et al.r Mutation Research 415 (1998) 219–226
not very abundant in the citrus fruits w17x. Although citric acid is one of the most abundant organic acids in citrus juices Ž1–6%., its antimutagenicity against the nitrite-treated MTCCA was limited. The antimutagenicity of the citrus fruits may be the result of an additive or synergistic action as described by Bala and Grover w12x, by not only the known components but also other factorŽs.. The reason why D-limonene, an essential oil contained in the citrus flavedo, has antimutagenicity not only before but also after the nitrite treatment of MTCCA, must be investigated further. Some citrus fruits such as sudachi, lemon and yuzu, so-called acid citruses, are eaten with soy sauce in the Japanese menu as refreshing flavors. The acid taste results from the organic acids such as citric acid and ascorbic acid present in the juice and the sweet smell results mainly from the essential oils present in the flavedo. The experimental data suggest that 9.5 ml of juice and 1.9 g of flavedo obtained from a normal size sudachi Ž35 g. have a possibility of reducing by 48% and 80%, respectively, the mutagenicity of MTCCA and nitrite with ethanol in 1 l, equivalent to our stomach volume after a meal, of an acidic medium ŽFigs. 1 and 2.. The dietary fibers lignin and pectin can account for 16–108% of the antimutagenicity of the juice and 13–69% of that of the flavedo ŽTable 1., if the sudachi contains as much fiber as the citruses described above and the antimutagenicity is the same as that of the commercial fibers. The citrus fruits may bring us an additional healthful benefit if they prevent the development of mutagens in our stomach in a manner similar to that described above. References w1x S.S. Mirvish, Formation of N-nitroso compounds: chemistry, kinetics, and in vivo occurrence, Toxicol. Appl. Pharmacol. 31 Ž1975. 325–351. w2x P.E. Hartman, Nitrates and nitrites: Ingestion, pharmacodynamics, and toxicology, in: F.J. de Serres, A. Hollaender ŽEds.., Chemical Mutagens, Vol. 7, Plenum, New York, 1982, pp. 211–294. w3x K. Wakabayashi, M. Ochiai, H. Saito, M. Tsuda, Y. Suwa, M. Nagao, T. Sugimura, Presence of 1-methyl-1, 2, 3, 4-tetrahydro-b-carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound, in soy sauce, Proc. Natl. Acad. Sci. U.S.A. 80 Ž1983. 2912–2916.
225
w4x M. Ochiai, K. Wakabayashi, M. Nagao, T. Sugimura, Tyramine is a major mutagen precursor in soy sauce, being convertible to a mutagen by nitrite, Gann 75 Ž1984. 1–3. w5x M. Higashimoto, K. Matano, Y. Ohnishi, Augmenting effect of a nonmutagenic fraction in soy sauce on mutagenicity of 3-diazotyramine produced in the nitrite-treated sauce, Jpn. J. Cancer Res. ŽGann. 79 Ž1988. 1284–1292. w6x Y. Fujita, K. Wakabayashi, S. Takayama, M. Nagao, T. Sugimura, Induction of oral cavity cancer by 3-diazotyramine, a nitrosated product of tyramine present in foods, Carcinogenesis 8 Ž1987. 527–529. w7x W.C. MacDonald, J.W. Dueck, Long-term effect of shoyu ŽJapanese soy sauce. on the gastric mucosa of the rat, J. Natl. Cancer Inst. 56 Ž1976. 1143–1145. w8x I. Hirono, M. Funahashi, C. Kaneko, H. Ogino, M. Ito, A. Yoshida, Gastric lesions in rats fed salted food materials commonly eaten by Japanese, Nutr. Cancer 14 Ž1990. 127– 132. w9x I. Kato, S. Tominaga, K. Matsumoto, A prospective study of stomach cancer among a rural Japanese population: a 6-year survey, Jpn. J. Cancer Res. 83 Ž1992. 568–575. w10x M. Higashimoto, T. Yamamoto, T. Kinouchi, H. Matsumoto, Y. Ohnishi, Mutagenicity of 1-methyl-1,2,3,4,-tetrahydro-bcarboline-3-carboxylic acid treated with nitrite in the presence of alcohols, Mutat. Res. 367 Ž1995. 43–49. w11x M. Higashimoto, T. Yamamoto, T. Kinouchi, Y. Handa, H. Matsumoto, Y. Ohnishi, Mutagenicity of soy sauce treated with nitrite in the presence of ethanol or alcoholic beverages, Mutat. Res. 345 Ž1995. 155–166. w12x S. Bala, I.S. Grover, Antimutagenicity of some citrus fruits in Salmonella typhimurium, Mutat. Res. 222 Ž1989. 141–148. w13x H.A. Risch, M. Jain, N.W. Choi, J.G. Fodor, C.J. Pfeiffer, G.R. Howe, L.W. Harrison, K.J.P. Craib, A.B. Miller, Dietary factors and the incidence of cancer of the stomach, Am. J. Epidemiol. 122 Ž1985. 947–959. w14x E. Buiatti, D. Palli, A. Decarli, D. Amadori, C. Avellini, S. Bianchi, R. Biserni, F. Cipriani, P. Cocco, A. Giacosa, E. Marubini, R. Puntoni, C. Vindigni, J. Fraumeni Jr., W. Blot, A case-control study of gastric cancer and diet in Italy, Int. J. Cancer 44 Ž1989. 611–616. w15x H. Boeing, B.R. Frentzel, M. Berger, V. Berndt, W. Gores, M. Korner, R. Lohmeier, A. Menarcher, H.F. Mannl, M. Meinhardt, R. Muller, H. Ostermeier, F. Paol, K. Schwemmle, ¨ K.H. Wagner, J. Wahrendorf, Case-control study on stomach cancer in Germany, Int. J. Cancer 47 Ž1991. 858–864. w16x D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 Ž1983. 173–215. w17x S. Ito ŽEd.., Science of Fruits, Asakura Pub., Tokyo, 1991 Žin Japanese.. w18x K. Kimura, T. Taninaka, Acid Citrus: Acid Citrus in Shikoku Island, Harada Pub., Tokushima, 1988 Žin Japanese.. w19x Nippon Kajyu Kyokai ŽEd.., Kajyu-kajitsu Inryo Jiten, Asakura Pub., Tokyo, 1978 Žin Japanese.. w20x Resources Council, Science and Technology Agency, Japan ŽEd.., Standard Tables of Food Composition in Japan, 4th edn., Printing Bureau, Ministry of Finance, Tokyo, 1982. w21x Resources Council, Science and Technology Agency, Japan
226
w22x
w23x w24x
w25x
M. Higashimoto et al.r Mutation Research 415 (1998) 219–226 ŽEd.., Standard Tables of Food Composition in Japan, Dietary fiber, Printing Bureau, Ministry of Finance, Tokyo, 1992. S.V. Ting, R.L. Rouseff, Citrus Fruits and Their Products: Analysis and Technology, Marcel Dekker, New YorkrBasel, 1986. D.A.T. Southgate, P.J. Van Soest, Fiber analysis tables, Am. J. Clin. Nutr. 31 Ž1978. S281–S284. E.D. Lund, J.M. Smoot, Dietary fiber content of some tropical fruits and vegetables, J. Agric. Food Chem. 30 Ž1982. 1123–1127. E.D. Lund, J.M. Smoot, N.T. Hall, Dietary fiber content of eleven tropical fruits and vegetables, J. Agric. Food Chem. 31 Ž1983. 1013–1016.
w26x R.J. Braddock, T.R. Graumlich, Composition of fiber from citrus peel, membranes, juice vesicles and seeds, Lebensm. Wiss. Technol. 14 Ž1981. 229–231. w27x A. Kawabata, S. Sawayama, K. Uryu, A study on the contents of peptic substances in fruits, vegetable fruits and nuts, Jpn. J. Nutr. 32 Ž1974. 9–18, Žin Japanese with English abstract.. w28x A.R. Francis, T.K. Shetty, R.K. Bhattacharya, Modulating effect of plant flavonoids on the mutagenicity of N-methylX N -nitro-N-nitrosoguanidine, Carcinogenesis 10 Ž1989. 1953–1955. w29x M. Calomme, L. Pieters, A. Vlietinck, D.V. Berghe, Inhibition of bacterial mutagenesis by Citrus flavonoids, Planta Med. 62 Ž1996. 222–226.