Mutagenicity of 24-hour duplicate of Japanese diet

ELSEVIER

Mutation Research 370 (1996)

Genetic Toxicology 203-208

Mutagenicity of 24-hour duplicate of Japanese diet Hiroshi Mano a, Norio Shibuya b, Hiroto Nakadaira a, Tatsuo Ohta ", T~iji Ishizu a, Osamu Yamazaki a, Shuko Takagi a, Kazuo Endoh a, Masaharu Yamamoto a. *, Tomio Hirohata c • Department of Hygiene and Preventive Medicine, Niigata University School of Medicine, Niigata, Japan b Department of Biology. Niigata College of Pharmacy, Niiguta, Japan C Department of Public Health. School of Medicine. Kyushu University. Fukuoka, Japan

Received

14 March 1996; revised 14 March 1996; accepted 23 May 1996

Abstract

In order to elucidate the genotoxicological characteristics of the Japanese diet, the mutagenicity of 24-h duplicate of the diet samples were investigated. The mutagenicity of blue rayon extract was examined in the Ames Salmonella/microsome assay. Thirty-two (91.4%) of 35 samples revealed mutagenicity toward Salmonella typhimurium TA98 in the presence of 89 mix. The mutagenic activities showed significant correlations with the consumption rates of broiled fish (r = 0.517. p = 0,()02 J) and broiled meat (r = 0.494, P = 0.(036). In other test conditions, 6 (17.1 %), 5 (14.3%) and 8 (22.9%) samples were mutagenic to Salmonella typhimurium TA98 without S9 mix, TAIOO with 89 mix and TAloo without 89 mix, respectively. Findings in the present study suggest that high consumption of broiled fish and broiled meat are important as the source of mutagens/carcinogens in the Japanese diet. In the present study, however, biological inference of these findings could not be made in relation to the occurrence of cancers. especially of the gastric cancer. which is the most prevalent form of cancer in Japan. Keywords:

Duplicate diet: Gastric cancer; Ames Salmonella/microsome test; Mutagenicity; Blue rayon

1. Introduction Although the mortality rate for gastric cancer has decreased in advanced countries, it is still one of the most important cause of cancer deaths in Japan [1-4]. In Japan. however. there are significant variations in mortality rates for gastric cancer [1]. It has been noted that the mortality rates are generally high in the north-eastern and low in the south-western

• Corresponding author. Tel.: + 81-25-223-6161, Ext. 2330: Fax: + 81-25-223-7971: E-mail: [email protected] 0165-1218/96/$15.00 Copyright © 1996 PII 50165-1218(96)00077-8

part of Japan. The Niigata prefecture where we live is one of the prefectures with high mortality rates in Japan. According to Nakadaira et a1. [5], high mortalities in other cancers of the alimentary tract such as those of the esophagus and rectum are also evident in Niigata. With regard to the determinants of the frequencies of these cancers in Japan, especially of stomach cancer. a large number of epidemiological studies has been conducted and demonstrated an association between particular dietary factors and cancer risks. For example. salted foods, smoked fish products and smoked meat products increase gastric cancer [6,7].

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H. Mono et al. / Mutation Research 370 (1996) 203-208

Fruit and fresh vegetables decrease the risk for gastric cancer [8]. Thus. dietary factors are related not only to the induction of gastric cancer but also its prevention. A large variety of mutagens and carcinogens. such as N-nitrosamines. mutagenic heterocyclic amines and polycyclic aromatic hydrocarbons, were detected in food [9-16]. Some chemicals in food have tumor-promoting effects [9]. Furthermore. particular habits of food preparation and cooking are reported as the causes of mutagen-formation [15.17.18]. According to Layton et aI. [16]. consumption of meat and fish products in the U.S. diet contributed for about 80% to the total cancer risk. Inadequate preservation of food also increases the risk for gastric cancer [3.19]. In the present study, we examined the mutagenicity of the 24-h duplicate of Japanese diet by using the Ames Salmonella/microsome test in order to clarify the genotoxicological characteristics of the Japanese diet as a whole. which is consumed especially in the high-risk area for gastric cancer.

2. Materials and methods 2. J. Collection of the 24-h duplicate diet samples Diet samples were collected from 35 adult volunteers in Niigata prefecture. Japan. Of the 35 volunteers, 17 were males (mean age 57.3. range 47-68) and 18 females (mean age 55.7. range 43-66). Each volunteer was selected from the native inhabitants with traditional Japanese-style food habits. Therefore the diet samples were thought to be characteristic of food of the high risk area. Duplicate portions of the 24-h diet. consisting of at least 3 meals a day. were sampled on given days in winter, 1991 and 1992. Samples were carried to our laboratory using sterilized polyvinyl packages at - 18°C. After measuring the weight of every each menu. samples were stored at - 20°C until analysis. 2.2. Sample preparation

All colIected foods. except for rice. were put into a mixer. Since rice was too adhesive when stirred in a mixer with the other foods. it was treated sepa-

Table 1 Mutagenicity of blue rayon extracts of 24-h duplicate of Japanese diet a Diet No. Revertant colonies/plate

rxroo

TA98

+ 59 I 2 3 4

5 6

7 8 9

10

11 12 13 14 15 16 17 18 19 20 21

22

23 24

25 26 27

28 29 30 31 32 33 34 35

98

74

121 153 76

108 133 68

131 75 92 80

mix - 59 mix 23 16 17

71 21 63 17

21 20 19

72

25

24 16 18 25 22 23 18

128

20

104

16

163 110

164 94

89

124 107

73 160 92 122 72

109 30

Age Sex

+ 59

mix - 59 mix

133 106 124 131 137 144 354 122 115 134 157 143 401 128

149 123 127

120 373 153

373 113

321 161

104 116 138 291 129

134 122 107

155 120

125

134

139

117 142

22

147

21 17

131

408 125 152

47 24 24 21 23 16 54 19

88 28 79

69

115

25 17

130

21

153

Consumer

b

122

140 166

129 124 153 304 131 377 126 119 151

128 147

364 106

153 212

355 162 118

137 102

413 146 129

61 60 47 53 50 57 53 52 50 60 54 52 57 59 67 68 43 03 62 61 55 58 61 61 51 63 54 54 58 58 52 59 57 54 47

Male Male Male Male Male Ferllalt: Femalt: Female Ft:malt: Female Male Male Female Female Male Male Female Female Female Female Male Ferllale Male Female Malt: Male Fenmle Female Male Malt: Female Male Male Female Female

a The Ames tests were performed using Sa/monel/a tl'phifllllril 11l1 TA98 and TAloo with or without metabolic activation. b The numbers of spontaneous revertants (solvent control) wt:re 16i2 (meani5D) for TA98 with 59 mix. ISi3 for TA98 without 59 mix. 90i 10 for TAloo with 59 mix and 95 ± 7 I"or TA100 without 89 mix. After subtracting the number 01" SPOnta_ neous revertants. each value in the table shows the mean 01" number of revertants in duplicated plates when I g diet equivalent per single plate was assayed. The numbers of revertants 01" the positive samples were shown in bold.

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H. Mano et al. / Mutation Research 370 ( 1996) 203-208

rately from the mixed food samples. Freeze-dried rice and food mixture were extracted with methyl alcohol/light petroleum (I : 1, v /v). Extraction was performed in a soxhlet apparatus. Residues were evaporated and dissolved in dimethyl sulfoxide (DMSO). These specimens were referred as 'crude samples'.

lent per single plate were assayed. When the mutagenic activity greater than twice the spontaneous level and a dose-response relationship in mutagenicity were observed, the samples were evaluated to be mutagenic. No bactericidal toxicity was observed in any conditions up to 1 g diet equivalent per single plate.

2.3. Blue rayon treatment

2.5. Food consumption rates

Crude samples were diluted about 100-fold with water. Two hundred mg of blue rayon (Funakoshi, Tokyo, Japan) was added to the solution. The mixture was stirred at room temperature for 30 min. After removal of the blue rayon, 200 mg of fresh blue rayon was added, and the treatment was repeated. Four hundred mg of recovered blue rayon was washed with water. The test compounds were eluted with 400 ml of methyl alcohol-concentrated ammonium hydroxide (50: 1, v Iv) by stirred the mixture for 30 min. This step was repeated. The eluates were evaporated and the residues were dissolved with DMSO.

The collected foods were divided into the following nine groups, namely; broiled fish, boiled fish, broiled meat, fried meat, fresh vegetables, boiled vegetables, salty foods, bean paste soup and fruit, The percentage of each group in total diet weight was calculated; this percentage was called 'food consumption rate'.

2.4. Mutagenicity assays The mutagenicities were examined with the Salmonella tester strains T A98 and TAl 00 with or without metabolic activation [20]. The Ames tests were performed according to the preincubation method [21] and the S9 mix containing 4% of S9 from liver microsomes of Sprague-Dawley rats treated with Aroclor 1254 was used. To each plate, 0.5 ml of S9 mix was added. Duplicate plates were used to test the mutagenicity at 3 different doses, thus, 250 mg, 500 mg and 1 g duplicate diet equiva-

2.6. Statistical analysis Student's t-test and Pearson's correlation coefficients were used to observe the statistical significance of differences in the mutagenic activities and the relationships between mutagenicity and food consumption rates, respectively.

3. Results Of 35 crude samples (without blue rayon treatment), only one (Case No. 11 in Table l) showed mutagenicity to Salmonella typhimurium T A98 with S9 mix and TAloo without 89 mix. In other samples, no mutagenicity was detected. In order to collect mutagens, the crude samples

Table 2 Average numbers of revenant colonies of mutagenically positive diet with blue rayon treatment Revenant colonies/plate Diet

Diet for males Diet for females

rxioo

TA98

+ S9 mix 105 ± 3()(n =

16) 113 ± 28(n = 16)

a

b

-S9 mix 66 55

± 8(n = 4) ± I)(n = 2)

+S9 mix

-S9mix

375 ± 3(n = 2) 353 ± 12)(n = 3)

371 ± S6(n - 4) 313±7()(n-4)

Mutagenicities were examined in the Ames test using Salmonella typhimurium TA98 and TAloo with or without metabolic activation. The numbers of spontaneous revenants (solvent control) were 16 ± 2 (mean ± SO) for TA98 with S9 mix. 15 ± 3 for TA98 without S9 mix. 90 ± 10 for TAloo with S9 mix and 95 ± 7 for without S9 mix. After subtracting the number of spontaneous revertants, each value in the table shows the mean ± SD when I g diet equivalent per single plate was assayed. a

b

H. Manoet al./ Mutation Research 370 (/9961 203-208

206

were treated with blue rayon. The results are shown in Table 1. Thirty-two (91.4%) of 35 samples were mutagenic to Salmonella typhimurium TA98 with metabolic activation. On other test conditions, 6 07.1%),504.3%) and 8 (22.9%) samples out of 35 samples were mutagenic to Salmonella typhimurium TA98 with S9 mix, TAIOO with S9 mix and TAIOO without S9 mix, respectively. When I g duplicate diet equivalent per single plate was assayed, toward Salmonella typhimurium TA98 with S9 mix, positive samples gave 4.3-1O.3-fold as many revertant colonies as the background level (Table 2). On the other hand, 2.2-4.8-fold increases of revertant colonies over the background level were observed on other test conditions with the same doses assayed. The greatest level of the mutagenicity was detected toward Salmonella typhimurium TA98 with S9 mix. No significant differences between men's and women's diets were observed in terms of frequencies of the mutagenically positive samples and average numbers of revertant colonies (Table 2), Table 3 shows an association between the average number of revertant colonies observed in the positive samples and food consumption rates. The differences in food consumption rates between men's and women's diets were not significant (data not shown). On the test condition using Salmonella t.vphimllrill/ll TA98 with S9 mix, consumption rates of broiled fish (r = 0.517, p = 0.0021) and broiled meat (r = 0.494 P = 0.0036) showed significant positive association~ with the number of revertant colonies of the positive samples. On other test conditions, however, no Significant correlation was found.

.5

4. Discussion

N

r-1!3{1.lI"l",,'Or-lI"l'O

;;:;°'1'~~8~~= 0 10000000

Of 35 crude samples examined without blue rayon treatment, only one (2.9%) was found to be mutagenic in the tester strain of TA9S with 59 mix. This is in contrast with the high positive rate of mutgenu-, ity (91.4%) in samples with blue rayon treatment. The most plausible explanation is that the mutagenic substances become diluted in crude samples by mixing all components of foods. On the other hand, the use of blue rayon in the extraction process for mutagens increases the concentration of conjugated aromatic substances in crude samples [22].

H. Mano et al. / Mutation Research 370 (1996) 203-208

With regard to an attempt for analyzing mutagenicity of Japanese diet, Kamiyama [6] conducted a survey in Akita prefecture, where the mortality rate for gastric cancer is also high as Niigata. Although the method of analysis and diagnostic criteria are different from ours, they presented an interesting finding that consumption of broiled fish and broiled meat was responsible for increasing a risk of mutagenicity. The present finding is well consistent with that of Kamiyama [6]. In the present investigation, however, identification of mutagenic chemicals was not undertaken. Previously, it has been reported that a large number of mutagenic heterocyclic amines (e.g., IQ, MeIQ, Trp-P-I, Glu-P-l etc.) were isolated from broiled fish and meat, and adsorbed by blue rayon with high recovery rates [22]. In addition, these chemicals have been known to show mutagenicity toward Salmonella typhimurium TA98 with S9 mix [23,24]. Based on this circumstantial evidence, there is no special reason to exclude the role of heterocyclic amines on mutagenesis in the Ames assay. In addition, a few test samples demonstrated mutagenic activity in other test conditions. This finding suggests that some other mutagenic chemicals exist in diet samples treated with blue rayon. Up to the present, however, nothing has been known as to whether or not unidentified but actually existed chemicals are responsible for the occurrence of gastric cancer in Japan. Further research is necessary to investigate the characteristics of unidentified mutagens/carcinogens in relation to the occurrence of gastric cancer.

Acknowledgements This work was supported in part by Yujin Memorial Grant, Niigata University School of Medicine, and Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan.

References [I] Kono, S. and T. Hirohata (1994) A review on the epidemiology of stomach cancer. J. Epidemiol., 4, I-II. [2] Devesa, S.S., WJ. Blot, BJ. Stone. B.A. Miller, R.E. Tarone

207

and J.F. Fraumeri, Jr. (1995) Recent cancer trends in. the United States. J. Natl. Cancer Inst., 87, 175-182. [3) Fuchs, C.S. and RJ. Mayer (1995) Gastric carcinoma. N. Engl. J. Med., 333, 32-41. [4) Thomas, R.M. and L.H. Sobin (1995) Gastrointestinal cancer. Cancer, 75, 154-170. [5) Nakadaira, H.. H. Mano, M. Tsunoda, K. Endoh and M. Yamamoto (I 990) Trends of mortality from major malignant neoplasms in Japan. Niigata. Igakkai Zasshi 104,960-968. [6) Kamiyama, S. (1981) Studies on regional difference of gastric cancer mortality and the dietary mutagenicity, Akita J. Med., 7, 227-236. [7) Honjo, S., S. Kono, T. Hirohata and S. Tokunaga (1992) Geographic correlation between stomach cancer mortality and food consumption in Japan. J. Epidemiol. 2, 41-49. [8) Kato, I.. S. Tominaga, Y. Ito, S. Kobayashi, Y. Yoshii, A. Matsuura. A. Kameya, T. Kano and A. Ikari (1992) A prospective study of atrophic gastritis and stomach cancer risk. Jpn. 1. Cancer Res., 83, 1137-1142, [9) Sugimura, T. (]982) Mutagens, carcinogens, and tumor promoters in our daily food. Cancer, 49, 1970-1984. [10) Ames, B.N. (1983) Dietary carcinogens and anticarcinogens. Science. 221, 1256-1264. [II) Willett. W.C. and B. MacMahon (1984) Diet and cancer - an overview (First of two parts). N. EngI. J. Med., 310, 633-638. [12) Willett, W.C. and B. MacMahon (1984) Diet and cancer - an overview (Second of two parts). N. EngI. J. Med. 310, 697-703. [13) Zhang. X-M., K. Wakabayashi, Z-C. Liu, T, Sugimura, and M. Nagao (1988) Mutagenic and carcinogenic amines in Chinese cooked foods. Mutation Res., 201, 181-188. [14] Rogers, A.E., S.H. Zeisel and J. Groopman (1993) Diet and carcinogenesis. Carcinogenesis, 14, 2015-2217. [15) Sugimura, T., M. Nagao and K. Wakabayashi (1994) Heterocyclic amines in cooked foods: Candidates for causation of common cancers. J. Natl. Cancer Inst., 86, 2-4. [16) Layton, OW., K.T. Bogen, M.G. Knize, F.T. Hatch, V,M, Johnson and J.S. Felton (1995) Cancer risk of heterocyclic amines in cooked foods: An analysis and implications for research. Carcinogenesis, 16, 39-52. [17] Sugimura, T. (1985) Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutation Res.. ISO, 33-41. [18] Felton, J.S. and M.G. Knize (1990) New mutagens from cooked food, in: M.W. Pariza, H-U. Aeschbacher, J.S. Felton and S. Sato (Eds.), Mutagens and Carcinogens in the Diet. Wiley-Liss, New York, pp. 19-38. [19) Shi, G.Z. (1992) Fungi N-nitrosamines and mutagens in the extract of the sour com-soybean cakes from high incidence area of stomach cancer. Chinese J. Prev. Moo. 26, 13-15. [20) Ames, B.N., J. McCann and E, Yamasaki (t975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res., 31, 347-364. [21) Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura, and M. Okada (t977) Mutagenicities of N-nitrosamines on Salmonella. Mutation Res., 48, 121-130.

208

H. Mano et al./ Mutation Research 370 (1996) 203-208

(22) Hayatsu, H.• S. Arimoto and K. Wakabayashi (1990) Methods for detection of mutagens in food. in: Hayatsu, H. (Eds.), Mutagens in Food: Detection and Prevention. CRC Press, Boca Raton. FL. pp. 101-112. (23) Hayatsu, H.• T. Hayatsu, Y. Wataya and H.F. Mower (l98S)

Fecal mutagenicity arising from ingestion of fried ground beef in the human. Mutation Res.• 143,207-211. (24) Kikugawa, K.. T. Kato and H. Hayatsu (1985) Mutagenicity of smoked, dried bonito products. Mutation Res.. 158. 35-44.