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Formation of mutagens in cooked foods. III. Isolation of a potent mutagen from beef
Cancer Letters, 9 (1980) 177-183 o Elsevier/North-Holland Scientific Publishers Ltd.
177
FORMATION OF MUTAGENS IN COOKED FOODS. III. ISOLATION OF A POTENT MUTAGEN FROM BEEF
NEIL E. SPINGARN*v’>‘, HIROSHI KASAI’, LORETTO L. VUOLO’, SUSUMU NISHIMURA*, ZIRO YAMAIZUIW, TAKASHI SUGIMURA’*4, TAIJIRO MATSUSHIMA4 and JOHN H. WFJSBURGER’ ‘Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595 (U.S.A.); zBiology Division and ‘Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan); and 4Department of Molecular Oncology, Institute of Medical Sciences, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo 108 (Japan} (Received 18 January 1980) (Revised version received 28 January 1980) (Accepted 28 January 1980)
SUMMARY
The major mutagenic component of fried beef has been isolated using a series of chromatographic steps. The pure compound has been analyzed by low and high resolution mass spectroscopy and nuclear magnetic resonance spectroscopy. The results indicate that the molecular weight of this extremely mutagenic compound is 198, with an elemental composition of C11H1,,N4.The compound is different from the known mutagenic pyrolysis pro*cts of amino acids or proteins.
INTRODUCTION
Mutagenic activity has been found in a variety of cooked foods by several laboratories [ 2,7,10,11]. Sugumura et al. [ 121 have hypothesized that pyrolysis of proteins or amino acids forms the highly mutagenic compounds responsible for this activity in cooked foods. Such mutagenic pyrolysis products have now been detected in broiled fish [14], but only in trace quantities. A recent report demonstrated that a major mutagenic component from broiled sardines is not identical to any of the known mutagenic amino acid or protein pyrolysis products [ 31.
Address all correspondence to: Neil E. Sp’ingarn, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595, U.S.A.
178
Alternatively, it has been proposed that the mutagenic activity results from non-enzymic browning reactions in food [9]. Browning reactions can occur at any temperature, but are accelerated by heating. This may explain why mutagenic activity can form from meat at temperatures as low as 100°C [ 111. Pyrolysis of amino acids and proteins does not occur until much higher temperatures are reached [ 4,6]. Due to the potential significance of potent mutagens in cooked foods [11,12], elucidation of the structure of the active principles followed by careful bioassay in several systems is considered of the utmost importance. We have isolated the principle mutagen formed from the cooking of beef and provide structural information. MATERIALS
Mutagen
AND METHODS
isolation
The mutagen was isolated from 2 sources, fried beef and commercial beef extract. The primary isolation employed Difco beef extract due to ease of handling. The findings were subsequently confirmed with 4.5 kg of fried beef using identical isolation procedures. Difco beef extract (2.25 kg) was heated in small batches for 1 h on a hot plate, extracted with 10 liters of 0.1 M HCl, and subsequently brought to pH 2 with HCl. After saturation with (NH4)#04 and filtration through glass wool, the aqueous phase was extracted with 6 liters of CHzClz on a continuous extractor shielded from ambient light. After evaporation of the organic layer to dryness on a rotary evaporator, the residue was designated the acidic fraction: The aqueous layer was then adjusted to pH 9.5-10 with NH,OH and reextracted with 9 liters of CH2C12.Drying the organic layer yielded the basic fraction. The basic fraction, which contained >95% of the mutagenic activity, was subjected to silica gel chromatography (1 kg of E. Merck silica gel 60, 70-230 mesh, 9.5 X 33 cm column) using 3 liters of each of 4 eluents: 100% ethyl acetate, 5% methanol in ethyl acetate, 30% methanol in ethyl acetate and 100% methanol. The most active fraction, always in the 30% methanol eluate, was then chromatographed on Sephadex LH-20 (Pharmacia, 25-100 pm, 4.7 X 40 cm column) using methanol as eluent. The principle active fractions (which eiuted as a single band) were combined, evaporated to dryness, and resubjected to acid/base partition, as described above employing minimal quantities of water and dichloromethane. This purified, highly mutagenic fraction was dissolved in a small volume of 15% methanol in water containing 0.4% formic acid and subjected to high performance liquid chromatography (HPLC). Conditions for the HPLC were as follows: Shimadzu LC-3A chromatograph, pBondapak/C18 columns (Waters, 3.9 X 300 mm, 2 in series), ambient temperature, 1 ml/min flow rate of 15% methanol in water containing 0.04% formic acid, and detection with a Shimadzu UVD-2 UV flow spectrophotometer.
179
Mass spectrometry Direct inlet low and high resolution mass spectra were obtained using a JEOL OlSG mass spectrometer. Nuclear magnetic resonance analysis The active fraction from the HPLC was lyphilized to dryness. After dissolution in CDC13 a Fourier-transform 270 MHz (FT-NMR) was obtained using a Briicker spectrometer. Mutagenesis assay Salmonella typhimurium TA98 [ 51 was kindly supplied by Dr. Bruce N. Ames, University of California, Berkeley, CA. Liver homogenate supernatant (S-9) was obtained from rats induced with polychlorinated biphenyls [ 11. The standard pour plate procedure [l,ll], the pour plate technique employing S-9 Mix containing NADPH and NADH instead of NADP [8], and the preincubation technique [ 131 were used to assay the mutagenic activity, each giving quantitatively similar results. RESULTS
AND DISCUSSION
Using a series of chromatographic procedures, the predominant active mutagenic principle from cooked beef has been purified. Table 1 shows the results from each step during purification. The mutagenic activity chromatographed primarily as a single band until the stage of HPLC, when activity was found in 2 distinct fractions (Fig. 1). Mutagenic activity followed UV absorption for the early peak, and HPLC peak shape and the UV spectrum suggested that this isolated material was pure. In addition, the specific activity of the mutagen thus isolated was found to be approx. 23,000 revertants/~g when assayed on strain TA98 with S-9 activation. This is greater activity than any mutagen yet assayed in the Sufmonella test system.
TABLE
1
ISOLATION
OF THE MUTAGEN Weight
Crude beef extract Acid/base partition Silica gel chromatography LH-20 chromatography Acid/base partition HPLC =Due to the inaccuracy bGiven for strain TA98
(mg)
2,250,ooo 790 160 57 2.4 O.la
Specific Activityb (revertantslpg) 0.02 15 45 70 1,500 23,000a
of a weight of such a low mass, these figures are approximate. with S-9 activation.
;i; --
ib..J 41
TIME
\
(MIN)
Fig. 1. Reversed-phase HPLC of purified basic fraction from LH-20 column for beef. Presented are absorbance at 254 nm and mutagenicity (revertants/plate) vs. time. Inset box shows that reinjection of the isolated material contains one peak with no shoulders. 0.7% of each fraction was applied to the plate. Conditions are described in the text.
Purification of the other mutagenic principle(s) from cooked beef is still underway. Table 2 gives the direct inlet mass spectrum of the isolated mutagen, showing a molecular ion at m’/e = 198. A high resolution mass spectrum was also obtained. Table 2 also gives the elemental composition of several peaks in the spectrum. The molecular weight of the mutagen was measured to be 198.0927. This is very close to that measured for Glu-P-l*, of molecular formula C1HION4 (m’/e = 198.0921). Although the molecular formulas of the 2 compounds are the same, the mutagen from beef demonstrated distinctly different behavior on TLC and HPLC. In addition, the UV spectrum of the beef mutagen (hmm = 213, 264 nm) is unlike that of Glu-P-1. Fourier-transform nuclear magnetic resonance analysis of the active principle (Table 3) indicated that both a-heterocyclic and phenylic aromatic protons are present. A low field singlet signifies the probable presence of a methyl group which, based on its chemical shift, is attached to one ofthe nitrogens. Additional evidence for this is the mass spectral peak at m’/e = *Glu-P-1 is a mutagen formed by pyrolysis of glutamic acid. It has been identified as 2-amino-6-methyldipyrrido(l,2-a : 3’,2’-d)imidazole [15].
181 TABLE 2 MASS SPECTRUM OF THE MUTAGEN FROM BEEF m+/ea
Relative abundance
199 198.0927 197 183.0693 170.0739 156 129 119 111 105 99 97 91 85 83 71 69 57
12 82 34 24 20 26 22 27 21 32 28 33 26 31 46 100 51 82
Ion formula’
C, ,%N, C,,H,N, C,,H,N,
aFractional masses and ion formulas are based on high resolution measurements.
185 (M-CH3). Based on these data, a general structure can be suggested. The data fit well with a structure containing a methyl group and an amino group attached to a 3-ring skeleton (two B-membered and one 5-membered rings fused together) containing 10 carbons and 3 nitrogens. However, a large number of structural isomers can be proposed from the available data. It is interesting to note that a relatively large proportion of the mutagenic activity present in heated beef extract or fried beef consisted of a single component. This finding was unexpected due to the complex composition of beef. In contrast, mutagenic activity from broiled fish is due to many TABLE 3 FOURIER-TRANSFORM 270 MHz NUCLEAR MAGNETIC RESONANCE SPECTRAL DATA OF THE MUTAGEN FROM BEEF IN CDCl, Chemical shift (ppm)
Peak data
Assignment
3.70 5.52 7.46 7.57 7.88 8.70 8.87
singlet, 3 H singlet (broad), 2 H doubled doublet, 1 H doublet, 1 H doublet, 1 H doublet, 1 H doublet, 1 H
N-CH, -NH,
Aromatic protons
182
active compounds (3, and unpublished observations). Based on behavior on TLC and HPLC, the mutagen from beef was found not to be identical to any of the known mutagens from amino acid or protein pyrolysates. Isolation of amounts of the mutagen sufficient for such investigations as X-ray crystallographic analysis is underway. The ubiquity of this material is also being studied. Once the structure is assigned, the compound will be synthesized and tested for carcinogenicity in a variety of test systems in order to permit determination of potential risk of such cooked foods to humans. ACKNOWLEDGEMENTS
This work was supported in part by the US National Cancer Institute’s US-Japan Cooperative Cancer Program; the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan; USPHS Grant CA-24217 and fellowship CA-06357; and the Pricess Takamatsu Cancer Research Fund. We thank Dr. K. Wakabayashi for assistance with mutagenicity assays and Clare T. Garvie-Gould for assistance with sample preparation. REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, F. (1975) Methods for detecting carcinogens and mutagens with SaZmoneZZa/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-361. 2 Commoner, B., Vithayathil, A.J., Dolara, P., Nair, S., Madyastha, P. and Cuca, G.C. (1978) Formation of mutagens in beef and beef extract during cooking. Science, 201,913-916. 3 Kasai, H., Nishimura, S., Nagao, M., Takahashi, Y. and Sugimura, T. (1979) Fraction ation of a mutagenic principle from broiled fish by high-pressure liquid chromatography. Cancer Letters, 7, 343-348. 4 Matsumoto, T., Yoshida, D., Mizusaki, S. and Okamoto, H. (1977) Mutagenic activity of amino acid pyrolysates in Salmonella typhimurium TA98. Mutat. Res., 48, 279286. 5 McCann, J., Spingarn, N., Kobori, J. and Ames, B. (1975) Detection of carcinogens as mutagens: improved tester strains incorporating an R-factor plasmid. Proc. Natl. Acad. Sci. USA, 72, 979-983. 6 Nagao, M., Honda, M., Seino, Y., Yahagi, T., Kawachi, T. and Sugumura, T. (1977) Mutagenicity of protein pyrolysates. Cancer Letters, 2, 335-340. 7 Nagao, M., Honda, M., Seino, Y., Yahagi, T. and Sugimura, T. (1977) Mutagenicities of smoke condensates and the charred surfaces of fish and meat. Cancer Letters, 2, 221-226. 8 Nagao, M., Yahagi, T., Seino, Y., Sugimura, T. and Ito, N. (1977) Mutagenicities of quinoline and its derivatives. Mutat. Res. 42, 335-342. 9 Spingarn, N.E. and Garvie, C.T. (1979) Formation of mutagens in sugar-ammonia model systems. J. Agric. Food Chem., 27, 1319-1321. 10 Spingarn, N.E., Slocum, L.A. and Weisburger, J.H. (1980) Formation of mutagens in cooked foods. II. Starchy foods. Cancer Letters, 9 (1980) 7-l 2. 11 Spingarn, N.E. and Weisburger, J.H. (1979) Formation of mutagens in cooked foods. I. Beef. Cancer Letters, 7, 259-264.
183 12 Sugimura, T., Nagao, M., Kawachi, T., Honda, M., Yahagi, T., Seino, Y., Sato, S., Matsukura, N., Matsushima, T., Shirai, A., Sawamura, M. and Matsumoto, H. (1977) Mutagen-carcinogens in food with special reference to highly mutagenic pyrolytic products in broiled foods. In: Origins of Human Cancer, 1561-1577. Editors: H.H. Hiatt, J.D. Watson and J.A. Winsten, Cold Spring Harbor, NY. 13 Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T., and Okamoto, T. (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48, 121-129. 14 Yamaizumi, Z., Shiomi, T., Kasai, H., Nishimura, S., Takahashi, Y., Nagao, M. and Sugimura, T. (1980) Detection of potent mutagens, Trp-P-l and Trp-P-2, in broiled fish. Cancer Letters, 9 (1980) 75-83. 15 Yamamoto, T., Tsuji, K., Kosuge, T., Ikamoto, T., Shudo, K., Takeda, K., Iitaka, Y., Yamaguchi, K., Seino, Y., Yahagi, T., Nagao, M. and Sugimura, T. (1978) Isolation and structure determination of mutagenic substances in Lglutamic acid pyrolysate. Proc. Jpn. Acad., 54B, 248-250.
177
FORMATION OF MUTAGENS IN COOKED FOODS. III. ISOLATION OF A POTENT MUTAGEN FROM BEEF
NEIL E. SPINGARN*v’>‘, HIROSHI KASAI’, LORETTO L. VUOLO’, SUSUMU NISHIMURA*, ZIRO YAMAIZUIW, TAKASHI SUGIMURA’*4, TAIJIRO MATSUSHIMA4 and JOHN H. WFJSBURGER’ ‘Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595 (U.S.A.); zBiology Division and ‘Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104 (Japan); and 4Department of Molecular Oncology, Institute of Medical Sciences, University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo 108 (Japan} (Received 18 January 1980) (Revised version received 28 January 1980) (Accepted 28 January 1980)
SUMMARY
The major mutagenic component of fried beef has been isolated using a series of chromatographic steps. The pure compound has been analyzed by low and high resolution mass spectroscopy and nuclear magnetic resonance spectroscopy. The results indicate that the molecular weight of this extremely mutagenic compound is 198, with an elemental composition of C11H1,,N4.The compound is different from the known mutagenic pyrolysis pro*cts of amino acids or proteins.
INTRODUCTION
Mutagenic activity has been found in a variety of cooked foods by several laboratories [ 2,7,10,11]. Sugumura et al. [ 121 have hypothesized that pyrolysis of proteins or amino acids forms the highly mutagenic compounds responsible for this activity in cooked foods. Such mutagenic pyrolysis products have now been detected in broiled fish [14], but only in trace quantities. A recent report demonstrated that a major mutagenic component from broiled sardines is not identical to any of the known mutagenic amino acid or protein pyrolysis products [ 31.
Address all correspondence to: Neil E. Sp’ingarn, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595, U.S.A.
178
Alternatively, it has been proposed that the mutagenic activity results from non-enzymic browning reactions in food [9]. Browning reactions can occur at any temperature, but are accelerated by heating. This may explain why mutagenic activity can form from meat at temperatures as low as 100°C [ 111. Pyrolysis of amino acids and proteins does not occur until much higher temperatures are reached [ 4,6]. Due to the potential significance of potent mutagens in cooked foods [11,12], elucidation of the structure of the active principles followed by careful bioassay in several systems is considered of the utmost importance. We have isolated the principle mutagen formed from the cooking of beef and provide structural information. MATERIALS
Mutagen
AND METHODS
isolation
The mutagen was isolated from 2 sources, fried beef and commercial beef extract. The primary isolation employed Difco beef extract due to ease of handling. The findings were subsequently confirmed with 4.5 kg of fried beef using identical isolation procedures. Difco beef extract (2.25 kg) was heated in small batches for 1 h on a hot plate, extracted with 10 liters of 0.1 M HCl, and subsequently brought to pH 2 with HCl. After saturation with (NH4)#04 and filtration through glass wool, the aqueous phase was extracted with 6 liters of CHzClz on a continuous extractor shielded from ambient light. After evaporation of the organic layer to dryness on a rotary evaporator, the residue was designated the acidic fraction: The aqueous layer was then adjusted to pH 9.5-10 with NH,OH and reextracted with 9 liters of CH2C12.Drying the organic layer yielded the basic fraction. The basic fraction, which contained >95% of the mutagenic activity, was subjected to silica gel chromatography (1 kg of E. Merck silica gel 60, 70-230 mesh, 9.5 X 33 cm column) using 3 liters of each of 4 eluents: 100% ethyl acetate, 5% methanol in ethyl acetate, 30% methanol in ethyl acetate and 100% methanol. The most active fraction, always in the 30% methanol eluate, was then chromatographed on Sephadex LH-20 (Pharmacia, 25-100 pm, 4.7 X 40 cm column) using methanol as eluent. The principle active fractions (which eiuted as a single band) were combined, evaporated to dryness, and resubjected to acid/base partition, as described above employing minimal quantities of water and dichloromethane. This purified, highly mutagenic fraction was dissolved in a small volume of 15% methanol in water containing 0.4% formic acid and subjected to high performance liquid chromatography (HPLC). Conditions for the HPLC were as follows: Shimadzu LC-3A chromatograph, pBondapak/C18 columns (Waters, 3.9 X 300 mm, 2 in series), ambient temperature, 1 ml/min flow rate of 15% methanol in water containing 0.04% formic acid, and detection with a Shimadzu UVD-2 UV flow spectrophotometer.
179
Mass spectrometry Direct inlet low and high resolution mass spectra were obtained using a JEOL OlSG mass spectrometer. Nuclear magnetic resonance analysis The active fraction from the HPLC was lyphilized to dryness. After dissolution in CDC13 a Fourier-transform 270 MHz (FT-NMR) was obtained using a Briicker spectrometer. Mutagenesis assay Salmonella typhimurium TA98 [ 51 was kindly supplied by Dr. Bruce N. Ames, University of California, Berkeley, CA. Liver homogenate supernatant (S-9) was obtained from rats induced with polychlorinated biphenyls [ 11. The standard pour plate procedure [l,ll], the pour plate technique employing S-9 Mix containing NADPH and NADH instead of NADP [8], and the preincubation technique [ 131 were used to assay the mutagenic activity, each giving quantitatively similar results. RESULTS
AND DISCUSSION
Using a series of chromatographic procedures, the predominant active mutagenic principle from cooked beef has been purified. Table 1 shows the results from each step during purification. The mutagenic activity chromatographed primarily as a single band until the stage of HPLC, when activity was found in 2 distinct fractions (Fig. 1). Mutagenic activity followed UV absorption for the early peak, and HPLC peak shape and the UV spectrum suggested that this isolated material was pure. In addition, the specific activity of the mutagen thus isolated was found to be approx. 23,000 revertants/~g when assayed on strain TA98 with S-9 activation. This is greater activity than any mutagen yet assayed in the Sufmonella test system.
TABLE
1
ISOLATION
OF THE MUTAGEN Weight
Crude beef extract Acid/base partition Silica gel chromatography LH-20 chromatography Acid/base partition HPLC =Due to the inaccuracy bGiven for strain TA98
(mg)
2,250,ooo 790 160 57 2.4 O.la
Specific Activityb (revertantslpg) 0.02 15 45 70 1,500 23,000a
of a weight of such a low mass, these figures are approximate. with S-9 activation.
;i; --
ib..J 41
TIME
\
(MIN)
Fig. 1. Reversed-phase HPLC of purified basic fraction from LH-20 column for beef. Presented are absorbance at 254 nm and mutagenicity (revertants/plate) vs. time. Inset box shows that reinjection of the isolated material contains one peak with no shoulders. 0.7% of each fraction was applied to the plate. Conditions are described in the text.
Purification of the other mutagenic principle(s) from cooked beef is still underway. Table 2 gives the direct inlet mass spectrum of the isolated mutagen, showing a molecular ion at m’/e = 198. A high resolution mass spectrum was also obtained. Table 2 also gives the elemental composition of several peaks in the spectrum. The molecular weight of the mutagen was measured to be 198.0927. This is very close to that measured for Glu-P-l*, of molecular formula C1HION4 (m’/e = 198.0921). Although the molecular formulas of the 2 compounds are the same, the mutagen from beef demonstrated distinctly different behavior on TLC and HPLC. In addition, the UV spectrum of the beef mutagen (hmm = 213, 264 nm) is unlike that of Glu-P-1. Fourier-transform nuclear magnetic resonance analysis of the active principle (Table 3) indicated that both a-heterocyclic and phenylic aromatic protons are present. A low field singlet signifies the probable presence of a methyl group which, based on its chemical shift, is attached to one ofthe nitrogens. Additional evidence for this is the mass spectral peak at m’/e = *Glu-P-1 is a mutagen formed by pyrolysis of glutamic acid. It has been identified as 2-amino-6-methyldipyrrido(l,2-a : 3’,2’-d)imidazole [15].
181 TABLE 2 MASS SPECTRUM OF THE MUTAGEN FROM BEEF m+/ea
Relative abundance
199 198.0927 197 183.0693 170.0739 156 129 119 111 105 99 97 91 85 83 71 69 57
12 82 34 24 20 26 22 27 21 32 28 33 26 31 46 100 51 82
Ion formula’
C, ,%N, C,,H,N, C,,H,N,
aFractional masses and ion formulas are based on high resolution measurements.
185 (M-CH3). Based on these data, a general structure can be suggested. The data fit well with a structure containing a methyl group and an amino group attached to a 3-ring skeleton (two B-membered and one 5-membered rings fused together) containing 10 carbons and 3 nitrogens. However, a large number of structural isomers can be proposed from the available data. It is interesting to note that a relatively large proportion of the mutagenic activity present in heated beef extract or fried beef consisted of a single component. This finding was unexpected due to the complex composition of beef. In contrast, mutagenic activity from broiled fish is due to many TABLE 3 FOURIER-TRANSFORM 270 MHz NUCLEAR MAGNETIC RESONANCE SPECTRAL DATA OF THE MUTAGEN FROM BEEF IN CDCl, Chemical shift (ppm)
Peak data
Assignment
3.70 5.52 7.46 7.57 7.88 8.70 8.87
singlet, 3 H singlet (broad), 2 H doubled doublet, 1 H doublet, 1 H doublet, 1 H doublet, 1 H doublet, 1 H
N-CH, -NH,
Aromatic protons
182
active compounds (3, and unpublished observations). Based on behavior on TLC and HPLC, the mutagen from beef was found not to be identical to any of the known mutagens from amino acid or protein pyrolysates. Isolation of amounts of the mutagen sufficient for such investigations as X-ray crystallographic analysis is underway. The ubiquity of this material is also being studied. Once the structure is assigned, the compound will be synthesized and tested for carcinogenicity in a variety of test systems in order to permit determination of potential risk of such cooked foods to humans. ACKNOWLEDGEMENTS
This work was supported in part by the US National Cancer Institute’s US-Japan Cooperative Cancer Program; the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan; USPHS Grant CA-24217 and fellowship CA-06357; and the Pricess Takamatsu Cancer Research Fund. We thank Dr. K. Wakabayashi for assistance with mutagenicity assays and Clare T. Garvie-Gould for assistance with sample preparation. REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, F. (1975) Methods for detecting carcinogens and mutagens with SaZmoneZZa/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-361. 2 Commoner, B., Vithayathil, A.J., Dolara, P., Nair, S., Madyastha, P. and Cuca, G.C. (1978) Formation of mutagens in beef and beef extract during cooking. Science, 201,913-916. 3 Kasai, H., Nishimura, S., Nagao, M., Takahashi, Y. and Sugimura, T. (1979) Fraction ation of a mutagenic principle from broiled fish by high-pressure liquid chromatography. Cancer Letters, 7, 343-348. 4 Matsumoto, T., Yoshida, D., Mizusaki, S. and Okamoto, H. (1977) Mutagenic activity of amino acid pyrolysates in Salmonella typhimurium TA98. Mutat. Res., 48, 279286. 5 McCann, J., Spingarn, N., Kobori, J. and Ames, B. (1975) Detection of carcinogens as mutagens: improved tester strains incorporating an R-factor plasmid. Proc. Natl. Acad. Sci. USA, 72, 979-983. 6 Nagao, M., Honda, M., Seino, Y., Yahagi, T., Kawachi, T. and Sugumura, T. (1977) Mutagenicity of protein pyrolysates. Cancer Letters, 2, 335-340. 7 Nagao, M., Honda, M., Seino, Y., Yahagi, T. and Sugimura, T. (1977) Mutagenicities of smoke condensates and the charred surfaces of fish and meat. Cancer Letters, 2, 221-226. 8 Nagao, M., Yahagi, T., Seino, Y., Sugimura, T. and Ito, N. (1977) Mutagenicities of quinoline and its derivatives. Mutat. Res. 42, 335-342. 9 Spingarn, N.E. and Garvie, C.T. (1979) Formation of mutagens in sugar-ammonia model systems. J. Agric. Food Chem., 27, 1319-1321. 10 Spingarn, N.E., Slocum, L.A. and Weisburger, J.H. (1980) Formation of mutagens in cooked foods. II. Starchy foods. Cancer Letters, 9 (1980) 7-l 2. 11 Spingarn, N.E. and Weisburger, J.H. (1979) Formation of mutagens in cooked foods. I. Beef. Cancer Letters, 7, 259-264.
183 12 Sugimura, T., Nagao, M., Kawachi, T., Honda, M., Yahagi, T., Seino, Y., Sato, S., Matsukura, N., Matsushima, T., Shirai, A., Sawamura, M. and Matsumoto, H. (1977) Mutagen-carcinogens in food with special reference to highly mutagenic pyrolytic products in broiled foods. In: Origins of Human Cancer, 1561-1577. Editors: H.H. Hiatt, J.D. Watson and J.A. Winsten, Cold Spring Harbor, NY. 13 Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T., and Okamoto, T. (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48, 121-129. 14 Yamaizumi, Z., Shiomi, T., Kasai, H., Nishimura, S., Takahashi, Y., Nagao, M. and Sugimura, T. (1980) Detection of potent mutagens, Trp-P-l and Trp-P-2, in broiled fish. Cancer Letters, 9 (1980) 75-83. 15 Yamamoto, T., Tsuji, K., Kosuge, T., Ikamoto, T., Shudo, K., Takeda, K., Iitaka, Y., Yamaguchi, K., Seino, Y., Yahagi, T., Nagao, M. and Sugimura, T. (1978) Isolation and structure determination of mutagenic substances in Lglutamic acid pyrolysate. Proc. Jpn. Acad., 54B, 248-250.