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Involvement of free radicals in the formation of heterocyclic amines and prevention by antioxidants
Cancer Letters 143 (1999) 123±126
Involvement of free radicals in the formation of heterocyclic amines and prevention by antioxidants Kiyomi Kikugawa* School of Pharmacy, Tokyo University of Pharmacy and Life Science (Formerly Tokyo College of Pharmacy), 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received 3 November 1998; received in revised form 21 January 1999; accepted 25 January 1999
Abstract It was found that free radical intermediates, pyrazine cation radial and carbon-centered radicals, generated in the Maillard reaction of sugars/amino acids, were involved in the production of mutagenic and carcinogenic imidazoquinoquizaline heterocyclic amines. Prevention of the generation of these radical intermediates by phenolic antioxidants effectively inhibited the generation of the heterocyclic amines. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Heterocyclic amines; Antioxidants
1. Introduction Mutagenic and carcinogenic heterocyclic amines are produced in cooked or processed meats and ®shmeats. The formation of the mutagenic imidazoquinoline-type and imidazoquinoxaline-type heterocyclic amines in most cooked or processed meats has been elucidated to be due to the Maillard reaction of sugars/ amino acids/creatin(in)e. It has been assumed that the initial stage is the formation of dihydropyrazines (or pyridines) and aldehydes by the Maillard reaction of sugars/amino acids, and the ®nal stage is the condensation of these components with creatinine. A hypothesis for the mechanisms of mutagen formation in model systems involving the free radical Maillard intermediates, pyrazine (or pyridine) cation radicals, has appeared [1]. In the present investigation, we examined, by using the heated model system and bonito meat in which imidazoquinoxaline-type * Tel.: 181-426-76-4503; fax 181-426-76-4508. E-mail address: [email protected] (K. Kikugawa)
heterocyclic amines were generated, whether the free radical Maillard intermediates are involved in mutagen formation, and whether phenolic antioxidants inhibit the mutagen formation by scavenging the radical intermediates. 2. Prevention of the formation of the imidazoquinoxaline-type mutagens by phenolic antioxidants It has been reported that various imidazoquinoxaline-type heterocyclic amine mutagens, MeIQx, 7,8DiMeIQx, 4,8-DiMeIQx and IQx, are produced by heating a mixture of glucose/glycine/creatinine in diethylene glycol±water, and they are mutagenic to Salmonella typhimurium TA98 strain with metabolic activation. Effects of phenolic antioxidants, butylated hydroxyanisole (BHA), propyl gallate (PG), sesamol, esculetin and epigallocatechin gallate (EGCG) on the generation of the imidazoquino-xaline-type mutagens were examined.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00140-8
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K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 1. Inhibitory effect of BHA (A), PG (B), sesamol (C), esculetin (D) and EGCG (E) on mutagen formation in the heated mixture of glucose/ glycine/creatinine.
A solution of glucose (0.2 M)/glycine (0.4 M)/creatinine (0.4 M) in diethylene glycol±water (8:2, vol./ vol.) was heated at 1208C for 2 h in the absence and presence of each of the phenolic antioxidants at concentrations between 1±100 mM. After heating, the mutagenicity of the reaction mixtures was tested on S. typhimurium TA98 strain with metabolic activation. It is likely that all the phenolic antioxidants inhibited the generation of the mutagenicity in a dose-dependent manner (Fig. 1). Most phenolics at the concentration of 100 mM appeared to reduce the generation of mutagenicity to lower than 50% of the original mutagenicity. The mutagens generated in the mixtures of glucose (0.02 M)/glycine (0.04 M)/creatinine (0.04 M) in diethylene glycol±water at 1208C for 2 h in the absence and presence of BHA or EGCG were puri®ed Table 1 Effect of pretreatment of bonito meat with EGCG and green tea extract on the mutagen formation during heating-and-drying at 100 or 1108C for 24 h Pretreatment
His 1 revertants/g weight % Mutagenicity
Water 118 0.5% EGCG 55 Water 1582 5% Green tea extract 450
100 47 100 28
using the established blue rayon adsorption method [2] before the assay. It was found that these antioxidants effectively prevented the mutagen formation. Thus, BHA at 20 and 100 mM reduced the mutagenicity to about 60 and 20%, respectively, and EGCG at 20 and 100 mM reduced the mutagenicity to about 40 and 2%, respectively. 3. Prevention of the formation of the imidazoquinoxaline-type mutagens in heated-anddried bonito meat MeIQx and 4,8-DiMeIQx, were generated in smoked-and-dried bonito (Katsuobushi in Japanese) [3]. Preventive effect of EGCG and green tea extract on the mutagen formation in bonito meat during experimental heating-and-drying was examined. Thus, pieces of raw bonito meat were boiled in 0.5% EGCG solution or 5% green tea extract for 40 min so that the antioxidant permeated into the meat. The boiled pieces of bonito meat were then heated-anddried at above 1008C for 24 or 48 h. The mutagenicity of the heated-and-dried bonito meat was tested on S. typhimurium TA98 strain with metabolic activation after blue rayon extraction. The number of His 1 revertant colonies was dramatically reduced on pretreatment with EGCG or green tea extract (Table 1).
K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 2. ESR spectra of the heated mixtrure of glucose/glycine in diethylene glycol±water in the absence and presence of antioxidants.
In the processing of smoked-and-dried bonito in the factory, green tea extract was found effective in preventing mutagen formation. 4. Electron spin resonance studies of the heated model systems Characteristic multi-line ESR signals due to 1,4di(carboxymethyl)pyrazine cation radical [4] appeared after 5 min heating of a mixture of glucose (0.2 M)/glycine (0.4 M) in diethylene glycol±water (8:2, vol./vol.), which disappeared during the subsequent 5 min heating and the stable broad signal appeared after heating for more than 20 min. However, addition of creatinine at 0.4 M to the mixture caused the reduction of the intensity of the signals. The effect of the antioxidants on the generation of the pyrazine cation radical was investigated by heating the mixture of glucose/glycine in diethylene glycol±water (Fig. 2) in the presence of BHA, sesamol and EGCG (each 0.1 M). It was found that the intensities of the ESR signals due to the pyrazine cation radical decreased in the presence of these antioxidants. These antioxidants may prevent the formation of the cation radical or destroy the cation radical. 5. Electron spin resonance spin trapping studies of the heated model systems When a solution of glucose (0.2 M)/glycine (0.4 M)
125
in diethylene glycol±water was heated at 1208C for 5 min and was added to a solution of DMPO (Fig. 3) or PBN, the six-line signals due to the spin adducts of carbon-centered radical(s) were observed. The effect of the antioxidants (0.1 M) and creatinine (0.4 M) on the formation of the spin adducts of DMPO and PBN in the heating of glucose (0.2 M)/glycine (0.4 M) in diethylene glycol±water was investigated. The intensities of the signals of the spin adducts were reduced by BHA, sesamol and EGCG (each 0.1 M). Addition of creatinine (0.4 M) did not affect the spin adduct formation, indicating that the carbon-centered radical(s) were generating in the presence of creatinine. The intensities of the spin adduct signals in the presence of creatinine were effectively reduced by BHA, sesamol and EGCG (each 0.1 M). 6. Discussion The present results offer three important suggestions for the formation of the imidazoquinoxalinetype heterocyclic amines. One is the mechanistic consideration for the formation of these mutagens. The present investigation gave evidence for the participation of the unstable free radical Maillard intermediates, the pyrazine cation radical and certain carbon-centered radical(s) (Fig. 4), in the mutagen formation. The present results gave an important indication of the effectiveness of the phenolic antioxidants in minimizing the development of the imidazoquinoxalinetype heterocyclic amines in cooking and processing of meat and ®shmeat. All the phenolic antioxidants examined here reduced the mutagen development in
Fig. 3. ESR spectra of the spin adducts of DMPO from the heated mixture of glucose/glycine in diethylene glycol±water in the absence and presence of antioxidants.
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K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 4. Involvement of pyratine cation radical and carbon-centered radical in the imidazoquinoxaline-type heterocyclic amine mutagens.
the heated model systems composed of glucose/ glycine/creatinine and in the heated-and-dried bonito meat. Although relatively high concentrations of these antioxidants were required for the effective prevention of mutagen formation in the model system, green tea extract containing EGCG at concentrations practically applicable effectively reduced the mutagen formation during the heating-and-drying of bonito meat. The third important point of the present results suggests that prevention of the formation of the unstable free radical Maillard intermediates by other means can reduce the formation of heterocyclic amine mutagens in cooking and processing of meat and ®shmeat. It has been shown that a high water content reduces the mutagen formation [5]. In the present study, it was found that a high amount of water in the model system composed of glucose/glycine prevented the formation of the pyrazine cation radical and the carbon-centered radical(s). Hence, water
might be effective in prevention of mutagen formation through the free radical mechanisms. References [1] A.M. Pearson, C. Chen, J.I. Gray, S.D. Aust, Mechanism(s) involved in meat mutagen formation and inhibition (hypothesis paper), Free Rad. Biol. Med. 13 (1992) 161±167. [2] H. Hayatsu, Cellulose bearing covalently linked copperphthalocyanine trisulphonate as an adsorbent selective for polycyclic aompounds and its use in studies of environmental mutagen and carcinogens, J. Chromatogr. 597 (1992) 37±56. [3] K. Kikugawa, T. Kato, H. Hayatsu, The presence of 2-amino3,8-imethylimidazo[4,5]quinoxaline in smoked dry bonito (Katsuobushi), Jpn. J. Cancer Res. 77 (1986) 99±102. [4] M. Namiki, T. Hayashi, Development of novel free radicals during the amino-carbonyl reaction of sugars with amino acids, J. Agric. Food Chem. 23 (1975) 487±491. [5] K. Kikugawa, T. Kato, Effect of water content on the generation of mutagenicity in heated ®sh meats, Eisei Kagaku 33 (1987) 62±65.
Involvement of free radicals in the formation of heterocyclic amines and prevention by antioxidants Kiyomi Kikugawa* School of Pharmacy, Tokyo University of Pharmacy and Life Science (Formerly Tokyo College of Pharmacy), 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received 3 November 1998; received in revised form 21 January 1999; accepted 25 January 1999
Abstract It was found that free radical intermediates, pyrazine cation radial and carbon-centered radicals, generated in the Maillard reaction of sugars/amino acids, were involved in the production of mutagenic and carcinogenic imidazoquinoquizaline heterocyclic amines. Prevention of the generation of these radical intermediates by phenolic antioxidants effectively inhibited the generation of the heterocyclic amines. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Heterocyclic amines; Antioxidants
1. Introduction Mutagenic and carcinogenic heterocyclic amines are produced in cooked or processed meats and ®shmeats. The formation of the mutagenic imidazoquinoline-type and imidazoquinoxaline-type heterocyclic amines in most cooked or processed meats has been elucidated to be due to the Maillard reaction of sugars/ amino acids/creatin(in)e. It has been assumed that the initial stage is the formation of dihydropyrazines (or pyridines) and aldehydes by the Maillard reaction of sugars/amino acids, and the ®nal stage is the condensation of these components with creatinine. A hypothesis for the mechanisms of mutagen formation in model systems involving the free radical Maillard intermediates, pyrazine (or pyridine) cation radicals, has appeared [1]. In the present investigation, we examined, by using the heated model system and bonito meat in which imidazoquinoxaline-type * Tel.: 181-426-76-4503; fax 181-426-76-4508. E-mail address: [email protected] (K. Kikugawa)
heterocyclic amines were generated, whether the free radical Maillard intermediates are involved in mutagen formation, and whether phenolic antioxidants inhibit the mutagen formation by scavenging the radical intermediates. 2. Prevention of the formation of the imidazoquinoxaline-type mutagens by phenolic antioxidants It has been reported that various imidazoquinoxaline-type heterocyclic amine mutagens, MeIQx, 7,8DiMeIQx, 4,8-DiMeIQx and IQx, are produced by heating a mixture of glucose/glycine/creatinine in diethylene glycol±water, and they are mutagenic to Salmonella typhimurium TA98 strain with metabolic activation. Effects of phenolic antioxidants, butylated hydroxyanisole (BHA), propyl gallate (PG), sesamol, esculetin and epigallocatechin gallate (EGCG) on the generation of the imidazoquino-xaline-type mutagens were examined.
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00140-8
124
K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 1. Inhibitory effect of BHA (A), PG (B), sesamol (C), esculetin (D) and EGCG (E) on mutagen formation in the heated mixture of glucose/ glycine/creatinine.
A solution of glucose (0.2 M)/glycine (0.4 M)/creatinine (0.4 M) in diethylene glycol±water (8:2, vol./ vol.) was heated at 1208C for 2 h in the absence and presence of each of the phenolic antioxidants at concentrations between 1±100 mM. After heating, the mutagenicity of the reaction mixtures was tested on S. typhimurium TA98 strain with metabolic activation. It is likely that all the phenolic antioxidants inhibited the generation of the mutagenicity in a dose-dependent manner (Fig. 1). Most phenolics at the concentration of 100 mM appeared to reduce the generation of mutagenicity to lower than 50% of the original mutagenicity. The mutagens generated in the mixtures of glucose (0.02 M)/glycine (0.04 M)/creatinine (0.04 M) in diethylene glycol±water at 1208C for 2 h in the absence and presence of BHA or EGCG were puri®ed Table 1 Effect of pretreatment of bonito meat with EGCG and green tea extract on the mutagen formation during heating-and-drying at 100 or 1108C for 24 h Pretreatment
His 1 revertants/g weight % Mutagenicity
Water 118 0.5% EGCG 55 Water 1582 5% Green tea extract 450
100 47 100 28
using the established blue rayon adsorption method [2] before the assay. It was found that these antioxidants effectively prevented the mutagen formation. Thus, BHA at 20 and 100 mM reduced the mutagenicity to about 60 and 20%, respectively, and EGCG at 20 and 100 mM reduced the mutagenicity to about 40 and 2%, respectively. 3. Prevention of the formation of the imidazoquinoxaline-type mutagens in heated-anddried bonito meat MeIQx and 4,8-DiMeIQx, were generated in smoked-and-dried bonito (Katsuobushi in Japanese) [3]. Preventive effect of EGCG and green tea extract on the mutagen formation in bonito meat during experimental heating-and-drying was examined. Thus, pieces of raw bonito meat were boiled in 0.5% EGCG solution or 5% green tea extract for 40 min so that the antioxidant permeated into the meat. The boiled pieces of bonito meat were then heated-anddried at above 1008C for 24 or 48 h. The mutagenicity of the heated-and-dried bonito meat was tested on S. typhimurium TA98 strain with metabolic activation after blue rayon extraction. The number of His 1 revertant colonies was dramatically reduced on pretreatment with EGCG or green tea extract (Table 1).
K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 2. ESR spectra of the heated mixtrure of glucose/glycine in diethylene glycol±water in the absence and presence of antioxidants.
In the processing of smoked-and-dried bonito in the factory, green tea extract was found effective in preventing mutagen formation. 4. Electron spin resonance studies of the heated model systems Characteristic multi-line ESR signals due to 1,4di(carboxymethyl)pyrazine cation radical [4] appeared after 5 min heating of a mixture of glucose (0.2 M)/glycine (0.4 M) in diethylene glycol±water (8:2, vol./vol.), which disappeared during the subsequent 5 min heating and the stable broad signal appeared after heating for more than 20 min. However, addition of creatinine at 0.4 M to the mixture caused the reduction of the intensity of the signals. The effect of the antioxidants on the generation of the pyrazine cation radical was investigated by heating the mixture of glucose/glycine in diethylene glycol±water (Fig. 2) in the presence of BHA, sesamol and EGCG (each 0.1 M). It was found that the intensities of the ESR signals due to the pyrazine cation radical decreased in the presence of these antioxidants. These antioxidants may prevent the formation of the cation radical or destroy the cation radical. 5. Electron spin resonance spin trapping studies of the heated model systems When a solution of glucose (0.2 M)/glycine (0.4 M)
125
in diethylene glycol±water was heated at 1208C for 5 min and was added to a solution of DMPO (Fig. 3) or PBN, the six-line signals due to the spin adducts of carbon-centered radical(s) were observed. The effect of the antioxidants (0.1 M) and creatinine (0.4 M) on the formation of the spin adducts of DMPO and PBN in the heating of glucose (0.2 M)/glycine (0.4 M) in diethylene glycol±water was investigated. The intensities of the signals of the spin adducts were reduced by BHA, sesamol and EGCG (each 0.1 M). Addition of creatinine (0.4 M) did not affect the spin adduct formation, indicating that the carbon-centered radical(s) were generating in the presence of creatinine. The intensities of the spin adduct signals in the presence of creatinine were effectively reduced by BHA, sesamol and EGCG (each 0.1 M). 6. Discussion The present results offer three important suggestions for the formation of the imidazoquinoxalinetype heterocyclic amines. One is the mechanistic consideration for the formation of these mutagens. The present investigation gave evidence for the participation of the unstable free radical Maillard intermediates, the pyrazine cation radical and certain carbon-centered radical(s) (Fig. 4), in the mutagen formation. The present results gave an important indication of the effectiveness of the phenolic antioxidants in minimizing the development of the imidazoquinoxalinetype heterocyclic amines in cooking and processing of meat and ®shmeat. All the phenolic antioxidants examined here reduced the mutagen development in
Fig. 3. ESR spectra of the spin adducts of DMPO from the heated mixture of glucose/glycine in diethylene glycol±water in the absence and presence of antioxidants.
126
K. Kikugawa / Cancer Letters 143 (1999) 123±126
Fig. 4. Involvement of pyratine cation radical and carbon-centered radical in the imidazoquinoxaline-type heterocyclic amine mutagens.
the heated model systems composed of glucose/ glycine/creatinine and in the heated-and-dried bonito meat. Although relatively high concentrations of these antioxidants were required for the effective prevention of mutagen formation in the model system, green tea extract containing EGCG at concentrations practically applicable effectively reduced the mutagen formation during the heating-and-drying of bonito meat. The third important point of the present results suggests that prevention of the formation of the unstable free radical Maillard intermediates by other means can reduce the formation of heterocyclic amine mutagens in cooking and processing of meat and ®shmeat. It has been shown that a high water content reduces the mutagen formation [5]. In the present study, it was found that a high amount of water in the model system composed of glucose/glycine prevented the formation of the pyrazine cation radical and the carbon-centered radical(s). Hence, water
might be effective in prevention of mutagen formation through the free radical mechanisms. References [1] A.M. Pearson, C. Chen, J.I. Gray, S.D. Aust, Mechanism(s) involved in meat mutagen formation and inhibition (hypothesis paper), Free Rad. Biol. Med. 13 (1992) 161±167. [2] H. Hayatsu, Cellulose bearing covalently linked copperphthalocyanine trisulphonate as an adsorbent selective for polycyclic aompounds and its use in studies of environmental mutagen and carcinogens, J. Chromatogr. 597 (1992) 37±56. [3] K. Kikugawa, T. Kato, H. Hayatsu, The presence of 2-amino3,8-imethylimidazo[4,5]quinoxaline in smoked dry bonito (Katsuobushi), Jpn. J. Cancer Res. 77 (1986) 99±102. [4] M. Namiki, T. Hayashi, Development of novel free radicals during the amino-carbonyl reaction of sugars with amino acids, J. Agric. Food Chem. 23 (1975) 487±491. [5] K. Kikugawa, T. Kato, Effect of water content on the generation of mutagenicity in heated ®sh meats, Eisei Kagaku 33 (1987) 62±65.