THE MUTAGENICITY
OF SOY BEAN SAUCE
J. Y. LIN, H.-l. WANG and Y.-C. YEH Institute
Abstract-Soy
of Biochemistry,
bean sauce is commonly
College Taipei,
of .Medicine,
(Received
19 July
Taiwan,
National
Taiwan University.
R.O.C. 1978)
used as a food material in Taiwan where the incidence of
hepatoma and gastric cancer is high. We have therefore investigated the mutagenicity of soy bean saucein vitro. When treated with nitrite at the 2000ppm level soy bean sauce produced a mutagenic substance as demonstrated using the Salmonella/mammalian microsome mutagenicity test. All 21 different brands of soy bean sauce showed similar results. Most mutagenic material was formed when the nitrite level was 2000 nom and the nH was 3; ascorbic acid prevented the formation of mutagenic products in nitrite-treated soy bean sauce.
INTRODUCTION
Carcinomas of the liver and stomach are the most common malignant tumours among Chinese people living in Taiwan, and cancer has been the second commonest cause of death since 1,964 (Lin, Chang dc Chen, 1977; Yeh, 1966). In a search for the aetiological factors contributing to these malignant tumours, the short-term carcinogenicity testing method developed by Ames (Ames, McCann & Yamasaki, 1975) was used to examine the extracts of food materials particularly favoured by Chinese people in the preparation of food. The samples were treated with or without sodium nitrite at pH 3 and five strains TA98, TAlOO, TAlS35, TA 1537 and TA1538 of histidine-requiring auxotrophs of Salmonella typhimurium kindly supplied by B. N. Ames were used for these experiments. EXPERIMENTAL
Materials. N-Methyl-N’-nitro-N-nitrosoguanidine and 2-aminofluorene were obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Picrolonic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). NADP and glucose 6-phosphate were obtained from Calbiochem (San Diego, CA, USA). The food materials were purchased from the local stores in Taipei City. Extraction of samples. About 600ml of. each soy bean sauce sample were first concentrated under reducing pressure with a flush evaporator to 2OOmL Except for the dose-response experiment and ascorbic-acid treated samples, 2000ppm NaN02 was added to 3OOml aliquots of soy bean sauce. Nothing was added to the control samples. The reaction was carried out for 1 hr at 25°C and pH 3, and it was terminated by the addition of 2000ppm ammonium sulphamate. The reaction mixtures were cooled to 4°C and extracted twice with 200ml ether. The combined ether extracts were reduced under a stream of nitrogen to QSml, and then diluted with S-Sml of dimethylsulphoxide (DMSO) for assaying. Mutagenicity assay.The mutagenicity assaywas carried out using S. typhimurium according to the 329
method of Ames et al. (1975). S-9 fraction, 9000 g liver supernatant, ‘from Aroclor-treated male albino rats (SOOmg/kg),was used for metabolic activation of the mutagens possibly present in the samples (Ames et al. 1975). The samples were incubated at 37°C for 48 hr. and then the results were recorded. N-MethylN’-nitro-N-nitrosoguanidine (2 pg/plate), and picroionic acid (250 pg/plate) were used as positive controls with TAlCQ 1535 and with TA98, 1537 and 1538 respectively. Neither of these compounds requires metabolic activation, 2-aminofluorene (250 pg/plate) was used as ,a positive control with the S-9 fraction for TAlS38. Each assay was performed twice quite independently, and the data presented are the average of two experiments. In order to determine the optimal pH for producing the highest numbers of revertant colonies, the samples were treated with 2ooOppm sodium nitrite at pH 1,2,3 and 4. To determine the optimum nitrite level, nitrite levels of 1000. 2000, 3fD0, 4000,’ 5000 and 10,000 ppm were added to the 3O@ml samples of soy bean sauce. The quantity of the final DMSO extract of the nitrite-treated soy bean sauce was also varied to determine whether there was a dose-response. The effect of adding 13,000 ppm ascorbic acid was also determined using the reaction mixture containing SOOOppmof sodium nitrite at pH 3.
RESULTS
For the indicator organisms, TAlS3S and 100, in the presence of g-9 fraction, the optimum pH was found to be 3 (Fig. 1). A positive dose-re!ponse. relationship was found between the concentration of sodium nitrite and the number of revertant colonies for the indicator organisms S. typhimurium TAlS35 and 100 in the presence of S-9 fraction (Figs 2a and b). Nitrite alone does not cause an increase in the number of revertant colonies at concentrations of up to 500 pg/plate. The increase in the number of revertant colonies was proportional to the amounts of DMSO extraqt applied, using tester strains TAlOO and, 1535 (Figs 3a and b). Control samples of soy bean sauce without
300-
IOO-
-
I
lncubatlon
2
3
pH
4
(4
/
n
005
01
Volume’ of som*,ml
/ plate
5
/
l
l
ppm
10000
Volume
of sample
0.03
of S-9 fraction,
, ml I plate
response of S. typhimurium code name OP) treated with
of NoNO,,
5000
WO25
Concn
3Ow
Fig. 2. The mutagenic soy bean sauce (sample
1000
to
O-l
strains various
5clo
Km
1500
3mo
5ooo
lmoo
O-I
r
0.3
Concn
0.5
, ; g / plate
to
10000
2
of S. typhimurium strains to various amounts of (MNNG).
of MNNG
I
Concn of Na NO,, ppm 1535(b) in the presence of S-9 fraction, of nitrite.
1000
Fig. 4. The mutagenic response TAIOO (-u-) and 1535 (-¤-) N-methyl-N’-nitro-N-nitrosoguanidine
TA 100(a) and concentrations
12=Y /
900
0 ‘C P 8 t 600 s t
f ,”
(a)
strains TAlOO(a) and 1535(b) in the presence name OP) pretreated with 2GOO ppm nitrite.
(
strains of S-9 treated
02
Fig. 3. The mutagenic response of S. ryphimurium various amounts of soy bean sauce (sample code
o-025
z@py,
600 -
nook
Fig. I. The mutagenic response of S. typhimurium, TAIOO (-o--) and 1535 (-L) in the presence fraction, to soy bean sauce (sample code name OP) with 2OOOppm nitrite, at various pHs.
% 0 z
i
i ?I a? 200
g
I-k
0) z = 4QO\
500-
The mutagenicity of soy bean sauce
nitrite treatment had no effect on strains TAlOO and 1535. Incubation with ascorbic acid, prevented the formation of the mutagenic materials as tested by using strains S. typhimurium TAIOO and 1535. The results of dose-response experiments with N-methyl-N’-nitro-N-nitrosoguanidine using TAI 535 and 100 are shown in Fig. 4. Picrolonic acid (25Occgl plate) produced an average of 1700. 430 and 1265 revertant colonies per plate with TA98, I537 and 1538 respectively. 2-Aminofluorene (10 pg/plate) produced 234 revertant colonies with TA 1538 in the presence of s-9.
331
the reversion of the tester strains, such as S. typhimurium TAl538. These mutagens could originate from the materials used in the preparation of soy bean sauce or from metabolic products prqduced by fungi or bacteria during fermentation. These two possibilities and the nature of nitrosable compounds are currently being investigated. The animal experiments to examine whether the mutagens found in nitrosated soy bean sauce are also carcinogens are being carried out. Acknowledgement-This work was supported in part by a grant from the National Council of Science, Republic of China.
DISCUSSION
All 21 different brand names of soy bean sauce, brought from the local stores of Taipei City, contain an unknown substance which reacts to form a compound of the alkylnitrosoureido type by nitrosation in vitro. The mutagenic activity of the resulting compound was revealed using strains of S. typhimurium (TA1535 and 100) that are susceptible to mutation through base-pair substitution. Although less revertants were obtained if the S-9 fraction was not added during the analysis, there was very little difference in the number of revertants produced by the extracts of soy bean sauce in the presence or absence of S-9 fraction after the mutagen(s) were purified by thinlayer chromatography. Soy bean sauce is a very popular flavour for use in Chinese cooking. The 16 million people on this island annually consume I4 to 15 million dozen bottles (each bottle containing 600 ml) of soy bean sauce. Chinese people in Taiwan are currently using nitrite in the food industry for several purposes, including protection against botulism in meat and fish products (Chen & Lin. 1977; Lin, 1978). Nitrosamines and nitrosamides were first reported to be toxic and carcinogenic by Barnes & Magee (1954), and these compounds rpay play a part in the occurrence of cancer in man since it was shown that nitrosamines can be formed from their precursors in animal organs (Sander, 1971). and that feeding of amine and nitrite together produced tumours in animals (Druckrey, 1975; Greenblatt, Mirvish & So, 1971; Lijinsky & Taylor, 1977; Magee & Barnes, 1954; Sugimura & Kawachi, 1973). Nitrosable products have been detected in fish (Marquardt, Rufino & Weisburger, 1977; Neurath. Diinger, Pein, Ambrosius & Schreiber, 1977), which was suspected of being an aetiological factor in the high incidence of gastric cancer in Japan. Comparing the mutagenic effects of nitrite-treated raw fish with soy bean sauce, @8ml of soy bean sauce is equivalent to I g of raw fish in its power to induce the reversion of S. typhimurium TAl535 and 100 based on the data presented by Marquardt, Rufino & Weisburger in 1977. It was also found that some of the nitrite-treated sauce samples also contained mutagen(s) that caused
REFERENCES
Ames, B. N.. McCann, J. & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonrlla/mammalian-microsome mutagenicity test. Mutation
31, 347.
Res.
Barnes, J. M. & Magee, P. N. (1954). Some toxic properties of dimethylnitrosamine. Br. J. ind. Med. 11. 167. Chen, L. C. & Lin, C. Y. (1977). Studies on adapted additive and substitute of nitrite in cured meat products (in Chinese). National Science Council Monthly (R.O.C.) 5. 107. Druckrey, H. (1975). Chemical carcinogenesis of N-nitroso derivatives Gann Monogr. 17, 107. Greenblatt, M.. Mirvish. S. & So, B. T. (1971). Nitrosamine studies: Induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. natn. Cancer Inst. 46, 1029. Lijinsky. W. & Taylor, H. W. (1977). Feeding tests in rats on mixtures of nitrite with secondary and tertiary amines of environmental importance. Fd Cosmet. Toxicol. 15, 269. Lin, J. K. (1978). Studies on the levels and toxicities of nitrite and nitrosamine in Chinese Food (in Chinese). National
Science
Council
Monthly
(R.O.C.)
6. 155.
tin, T. M., Chang L. C. & Chen. K. P. (1977). A statistic analysis on mortality of malignant neoplasms in Taiwan. J. Formosan
Med.
Ass. 76, 656.
McCann, J.. Choi. E., Yamasaki, E. & Ames, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/ mammalian microsome test: assay of 300 chemicals. Proc. natn.
Acad.
Sci. U.S.A.
72. 5135.
Magee, P. N. & Barnes. J. M. (1967). Carcinogenic nitroso compounds. Adu. Cancer Res. 10, 163. . Marquardt. H., Rufino, F. & Weisburger, J. H. (1977). On the aetiology of gastric cancer: mutagenicity of food extracts after incubation with nitrite. Fd Cosmet. Toxicol. 15, 97. Neurath. G. B., Diinger, M., Pein. F. G., Ambrosius, D. & Schreiber. 0. (1977). Primary and secondary amines in the human environment. Fd Cosmet. Toxicol. 15.275. Sander. J. (1971). Untersuchungen iiber die Entstehung cancerogener Nitrospverbindungen im Magen von Versuchstieren und ihre Bedeutung ftir den Menschen. l-3 Mitteilung. Ar:neimitteCForsch. 21, 1572. t707 & 2034. Sugimura. T. & Kawaachi, T. (1973). Experimental Stomach Cancer. In Methods in Cancer Research. Edited by H. Bush. p. 245. Academic Press. Inc., New York. Yeh. S. (1966). Some geographic aspects of most common diseases in Taiwan. Part 11. Infection and. cancer. Inr. Path. 7, 81.
OF SOY BEAN SAUCE
J. Y. LIN, H.-l. WANG and Y.-C. YEH Institute
Abstract-Soy
of Biochemistry,
bean sauce is commonly
College Taipei,
of .Medicine,
(Received
19 July
Taiwan,
National
Taiwan University.
R.O.C. 1978)
used as a food material in Taiwan where the incidence of
hepatoma and gastric cancer is high. We have therefore investigated the mutagenicity of soy bean saucein vitro. When treated with nitrite at the 2000ppm level soy bean sauce produced a mutagenic substance as demonstrated using the Salmonella/mammalian microsome mutagenicity test. All 21 different brands of soy bean sauce showed similar results. Most mutagenic material was formed when the nitrite level was 2000 nom and the nH was 3; ascorbic acid prevented the formation of mutagenic products in nitrite-treated soy bean sauce.
INTRODUCTION
Carcinomas of the liver and stomach are the most common malignant tumours among Chinese people living in Taiwan, and cancer has been the second commonest cause of death since 1,964 (Lin, Chang dc Chen, 1977; Yeh, 1966). In a search for the aetiological factors contributing to these malignant tumours, the short-term carcinogenicity testing method developed by Ames (Ames, McCann & Yamasaki, 1975) was used to examine the extracts of food materials particularly favoured by Chinese people in the preparation of food. The samples were treated with or without sodium nitrite at pH 3 and five strains TA98, TAlOO, TAlS35, TA 1537 and TA1538 of histidine-requiring auxotrophs of Salmonella typhimurium kindly supplied by B. N. Ames were used for these experiments. EXPERIMENTAL
Materials. N-Methyl-N’-nitro-N-nitrosoguanidine and 2-aminofluorene were obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Picrolonic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). NADP and glucose 6-phosphate were obtained from Calbiochem (San Diego, CA, USA). The food materials were purchased from the local stores in Taipei City. Extraction of samples. About 600ml of. each soy bean sauce sample were first concentrated under reducing pressure with a flush evaporator to 2OOmL Except for the dose-response experiment and ascorbic-acid treated samples, 2000ppm NaN02 was added to 3OOml aliquots of soy bean sauce. Nothing was added to the control samples. The reaction was carried out for 1 hr at 25°C and pH 3, and it was terminated by the addition of 2000ppm ammonium sulphamate. The reaction mixtures were cooled to 4°C and extracted twice with 200ml ether. The combined ether extracts were reduced under a stream of nitrogen to QSml, and then diluted with S-Sml of dimethylsulphoxide (DMSO) for assaying. Mutagenicity assay.The mutagenicity assaywas carried out using S. typhimurium according to the 329
method of Ames et al. (1975). S-9 fraction, 9000 g liver supernatant, ‘from Aroclor-treated male albino rats (SOOmg/kg),was used for metabolic activation of the mutagens possibly present in the samples (Ames et al. 1975). The samples were incubated at 37°C for 48 hr. and then the results were recorded. N-MethylN’-nitro-N-nitrosoguanidine (2 pg/plate), and picroionic acid (250 pg/plate) were used as positive controls with TAlCQ 1535 and with TA98, 1537 and 1538 respectively. Neither of these compounds requires metabolic activation, 2-aminofluorene (250 pg/plate) was used as ,a positive control with the S-9 fraction for TAlS38. Each assay was performed twice quite independently, and the data presented are the average of two experiments. In order to determine the optimal pH for producing the highest numbers of revertant colonies, the samples were treated with 2ooOppm sodium nitrite at pH 1,2,3 and 4. To determine the optimum nitrite level, nitrite levels of 1000. 2000, 3fD0, 4000,’ 5000 and 10,000 ppm were added to the 3O@ml samples of soy bean sauce. The quantity of the final DMSO extract of the nitrite-treated soy bean sauce was also varied to determine whether there was a dose-response. The effect of adding 13,000 ppm ascorbic acid was also determined using the reaction mixture containing SOOOppmof sodium nitrite at pH 3.
RESULTS
For the indicator organisms, TAlS3S and 100, in the presence of g-9 fraction, the optimum pH was found to be 3 (Fig. 1). A positive dose-re!ponse. relationship was found between the concentration of sodium nitrite and the number of revertant colonies for the indicator organisms S. typhimurium TAlS35 and 100 in the presence of S-9 fraction (Figs 2a and b). Nitrite alone does not cause an increase in the number of revertant colonies at concentrations of up to 500 pg/plate. The increase in the number of revertant colonies was proportional to the amounts of DMSO extraqt applied, using tester strains TAlOO and, 1535 (Figs 3a and b). Control samples of soy bean sauce without
300-
IOO-
-
I
lncubatlon
2
3
pH
4
(4
/
n
005
01
Volume’ of som*,ml
/ plate
5
/
l
l
ppm
10000
Volume
of sample
0.03
of S-9 fraction,
, ml I plate
response of S. typhimurium code name OP) treated with
of NoNO,,
5000
WO25
Concn
3Ow
Fig. 2. The mutagenic soy bean sauce (sample
1000
to
O-l
strains various
5clo
Km
1500
3mo
5ooo
lmoo
O-I
r
0.3
Concn
0.5
, ; g / plate
to
10000
2
of S. typhimurium strains to various amounts of (MNNG).
of MNNG
I
Concn of Na NO,, ppm 1535(b) in the presence of S-9 fraction, of nitrite.
1000
Fig. 4. The mutagenic response TAIOO (-u-) and 1535 (-¤-) N-methyl-N’-nitro-N-nitrosoguanidine
TA 100(a) and concentrations
12=Y /
900
0 ‘C P 8 t 600 s t
f ,”
(a)
strains TAlOO(a) and 1535(b) in the presence name OP) pretreated with 2GOO ppm nitrite.
(
strains of S-9 treated
02
Fig. 3. The mutagenic response of S. ryphimurium various amounts of soy bean sauce (sample code
o-025
z@py,
600 -
nook
Fig. I. The mutagenic response of S. typhimurium, TAIOO (-o--) and 1535 (-L) in the presence fraction, to soy bean sauce (sample code name OP) with 2OOOppm nitrite, at various pHs.
% 0 z
i
i ?I a? 200
g
I-k
0) z = 4QO\
500-
The mutagenicity of soy bean sauce
nitrite treatment had no effect on strains TAlOO and 1535. Incubation with ascorbic acid, prevented the formation of the mutagenic materials as tested by using strains S. typhimurium TAIOO and 1535. The results of dose-response experiments with N-methyl-N’-nitro-N-nitrosoguanidine using TAI 535 and 100 are shown in Fig. 4. Picrolonic acid (25Occgl plate) produced an average of 1700. 430 and 1265 revertant colonies per plate with TA98, I537 and 1538 respectively. 2-Aminofluorene (10 pg/plate) produced 234 revertant colonies with TA 1538 in the presence of s-9.
331
the reversion of the tester strains, such as S. typhimurium TAl538. These mutagens could originate from the materials used in the preparation of soy bean sauce or from metabolic products prqduced by fungi or bacteria during fermentation. These two possibilities and the nature of nitrosable compounds are currently being investigated. The animal experiments to examine whether the mutagens found in nitrosated soy bean sauce are also carcinogens are being carried out. Acknowledgement-This work was supported in part by a grant from the National Council of Science, Republic of China.
DISCUSSION
All 21 different brand names of soy bean sauce, brought from the local stores of Taipei City, contain an unknown substance which reacts to form a compound of the alkylnitrosoureido type by nitrosation in vitro. The mutagenic activity of the resulting compound was revealed using strains of S. typhimurium (TA1535 and 100) that are susceptible to mutation through base-pair substitution. Although less revertants were obtained if the S-9 fraction was not added during the analysis, there was very little difference in the number of revertants produced by the extracts of soy bean sauce in the presence or absence of S-9 fraction after the mutagen(s) were purified by thinlayer chromatography. Soy bean sauce is a very popular flavour for use in Chinese cooking. The 16 million people on this island annually consume I4 to 15 million dozen bottles (each bottle containing 600 ml) of soy bean sauce. Chinese people in Taiwan are currently using nitrite in the food industry for several purposes, including protection against botulism in meat and fish products (Chen & Lin. 1977; Lin, 1978). Nitrosamines and nitrosamides were first reported to be toxic and carcinogenic by Barnes & Magee (1954), and these compounds rpay play a part in the occurrence of cancer in man since it was shown that nitrosamines can be formed from their precursors in animal organs (Sander, 1971). and that feeding of amine and nitrite together produced tumours in animals (Druckrey, 1975; Greenblatt, Mirvish & So, 1971; Lijinsky & Taylor, 1977; Magee & Barnes, 1954; Sugimura & Kawachi, 1973). Nitrosable products have been detected in fish (Marquardt, Rufino & Weisburger, 1977; Neurath. Diinger, Pein, Ambrosius & Schreiber, 1977), which was suspected of being an aetiological factor in the high incidence of gastric cancer in Japan. Comparing the mutagenic effects of nitrite-treated raw fish with soy bean sauce, @8ml of soy bean sauce is equivalent to I g of raw fish in its power to induce the reversion of S. typhimurium TAl535 and 100 based on the data presented by Marquardt, Rufino & Weisburger in 1977. It was also found that some of the nitrite-treated sauce samples also contained mutagen(s) that caused
REFERENCES
Ames, B. N.. McCann, J. & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonrlla/mammalian-microsome mutagenicity test. Mutation
31, 347.
Res.
Barnes, J. M. & Magee, P. N. (1954). Some toxic properties of dimethylnitrosamine. Br. J. ind. Med. 11. 167. Chen, L. C. & Lin, C. Y. (1977). Studies on adapted additive and substitute of nitrite in cured meat products (in Chinese). National Science Council Monthly (R.O.C.) 5. 107. Druckrey, H. (1975). Chemical carcinogenesis of N-nitroso derivatives Gann Monogr. 17, 107. Greenblatt, M.. Mirvish. S. & So, B. T. (1971). Nitrosamine studies: Induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. natn. Cancer Inst. 46, 1029. Lijinsky. W. & Taylor, H. W. (1977). Feeding tests in rats on mixtures of nitrite with secondary and tertiary amines of environmental importance. Fd Cosmet. Toxicol. 15, 269. Lin, J. K. (1978). Studies on the levels and toxicities of nitrite and nitrosamine in Chinese Food (in Chinese). National
Science
Council
Monthly
(R.O.C.)
6. 155.
tin, T. M., Chang L. C. & Chen. K. P. (1977). A statistic analysis on mortality of malignant neoplasms in Taiwan. J. Formosan
Med.
Ass. 76, 656.
McCann, J.. Choi. E., Yamasaki, E. & Ames, B. N. (1975). Detection of carcinogens as mutagens in the Salmonella/ mammalian microsome test: assay of 300 chemicals. Proc. natn.
Acad.
Sci. U.S.A.
72. 5135.
Magee, P. N. & Barnes. J. M. (1967). Carcinogenic nitroso compounds. Adu. Cancer Res. 10, 163. . Marquardt. H., Rufino, F. & Weisburger, J. H. (1977). On the aetiology of gastric cancer: mutagenicity of food extracts after incubation with nitrite. Fd Cosmet. Toxicol. 15, 97. Neurath. G. B., Diinger, M., Pein. F. G., Ambrosius, D. & Schreiber. 0. (1977). Primary and secondary amines in the human environment. Fd Cosmet. Toxicol. 15.275. Sander. J. (1971). Untersuchungen iiber die Entstehung cancerogener Nitrospverbindungen im Magen von Versuchstieren und ihre Bedeutung ftir den Menschen. l-3 Mitteilung. Ar:neimitteCForsch. 21, 1572. t707 & 2034. Sugimura. T. & Kawaachi, T. (1973). Experimental Stomach Cancer. In Methods in Cancer Research. Edited by H. Bush. p. 245. Academic Press. Inc., New York. Yeh. S. (1966). Some geographic aspects of most common diseases in Taiwan. Part 11. Infection and. cancer. Inr. Path. 7, 81.
