- Email: [email protected]
Mutagenic activity of dopamine after nitrosation
wll
Mutation Research Letters
ELSEVIER
Mutation Research 347 (1995) 17-19
Mutagenic activity of dopamine after nitrosation S u n C h a n g h a o *, C h e n B i n g q i n g , W a n g S u y a n , L i u Z i c h e n g Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 199 Dong Da Zhi Street, Nan Gang District, Harbin, People's Republic of China Received 28 October 1994; revised 19 January 1995; accepted 28 February 1995
Abstract
Dopamine hydrochloride is reported to be a new mutagen precursor in this study. After treatment with nitrite under acidic conditions, dopamine hydrochloride showed direct-acting mutagenicity on Salmonella typhimurium TA100, TA98 and Escherichia coli WP2uvra. The addition of $9 mix did not affect the mutagenicity of nitrosated dopamine significantlyin these three strains. Meanwhile, a comparison of the mutagenicity of nitrosated dopamine with nitrosated tyramine was carried out. Keywords: Dopamine; Tyramine; Nitrosation; Mutagenicity 1. Introduction
Since dopamine and tyramine are very closely related structurally, differing only by a single ring hydroxyl group, and the mutagenicity of nitrosated tyramine has been known for many years (Orchiai and Wakabayashi, 1984), it is very important to know if nitrosated dopamine has mutagenic activity. Thus, in this paper, the mutagenicity of dopamine was examined a n d compared with that of tyramine, after nitrite treatment.
Factory (Shanghai, China). Tyramine hydrochloride was from Sigma Chemical Company. Bacterial strains S. typhimurium TA100 and TA98 were generously provided by Professor B. Ames. E. coli
WP2uvrA was generously provided by Professor Tomita and Professor Nakamura. Liver microsomal fractions ($9) were obtained from the livers of rats treated with pentachlorinated biphenyls in our laboratory. Nitrite treatment
2. Materials and methods Materials
Sodium nitrite and ammonium sulfamate were from Beijing Chemical Plant. Dopamine hydrochloride was from Tian Feng Pharmaceutical * Corresponding author.
Various concentrations (including 2, 3, 5, 10 mg/ml) of dopamine hydrochloride (aqueous) (3 ml) were adjusted to pH 1.5 with 6 N hydrochloric acid. To each concentration, 0.5 ml of 0.28 M NaNO2 was added (final concentration 40 mM), and the pH was again adjusted to 1.5. The mixtures were incubated for 1 h in a shaking waterbath (37°C) in the dark. The solution of dopamine hydrochloride (7.5 mg/ml) without nitrite treat-
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 5 - 7 9 9 2 ( 9 5 ) 0 0 0 1 3 - 5
S. Changhao et al. / Mutation Research 347 (1995) 17-19
18
Table 1 The mutagenicity of dopamine after nitrosation Precursor
Dose (~g/plate)
Number of revertants a
E. coliWP2uvrA
TA98
TA100 - $9
+ $9
- $9
+ $9
- $9
+ $9
Dopamine treated with nitrite
150 225 375 750
195 245 360 571
201 248 362 568
44 61 92 181
50 61 99 178
32 37 58 96
33 37 52 95
Dopamine untreated with nitrite
750
123
131
11
20
18
21
116
127
14
22
16
22
Distilled water treated with nitrite
a Data are the mean of two independent assays. Spontaneous revertants: TAI00: 135; TA98: 30; E. coli WP2uvrA: 19.
ment and distilled water with nitrite treatment were also incubated as controls under the same conditions. Then, the nitrosation reaction was stopped by adding 0.5 ml of 0.32 M ammonium sulfamate (final concentration 40 mM). 0.1 ml of the reaction mixtures was plated for the mutagenicity assay no longer than 3 h after the end of incubation. In the meantime, the various concentrations of tyramine hydrochloride were also assayed for mutagenicity, according to the above method.
Mutation test The mutation assay with S. typhimurium TA100 and TA98 with and without $9 mix was done with the preincubation method (Maron and Ames, 1983). The assay with E. coli WP2uvrA was performed by Araki's method (Araki and Muramatsu, 1984). Two independent assays were carried out with four doses using three plates of each dose. The criteria for positive mutagenicity were defined as a dose-dependent increase in the number of revertants induced by dopamine or tyramine after treatment with nitrite, and a number at least twice as high as that seen in controls. 3. Results
The revertant frequencies with dopamine after nitrosation are shown in Table 1. Dopamine
treated with nitrite showed a definite dose-dependent mutagenicity in S. typhimurium TA100 and TA98 and E. coli WP2uvrA without $9 mix, and the addition of $9 mix did not affect the mutagenicity significantly in all strains. It was also found that TA100 was more sensitive to nitrosated dopamine than TA98 and E. coli WP2uvrA. Similar results were observed with nitrosated tyramine (data not shown). Dopamine and tyramine (both at the dose of 225/~g/plate) treated with nitrite induced 245 and 928 revertants of S. typhimurium TA100 without $9 mix, respectively, indicating that the mutagenicity of nitrosated dopamine was weaker than that of nitrosated tyramine.
4. Discussion
Up to now, no reports on mutagenicity of nitrosated dopamine have been found. The results of this study suggest that nitrosated dopamine is mutagenic. The finding that nitrosated dopamine was less mutagenic than nitrosated tyramine could result either from a difference in mutagenic potencies of the nitrosation products or from a difference in the efficiency of nitrosation of the two amines. As a medicine, dopamine hydrochloride is widely used in clinical practice to treat various
S. Changhao et al./Mutation Research 347 (1995) 17-19 s h o c k s . T h e r e f o r e , it is n e c e s s a r y t o f u r t h e r s t u d y the health risk of human exposure to the mutagen precursor dopamine.
References Araki, A., M. Muramatsu and T. Matsushima (1984) Comparison of mutagenicities of N-nitrosamines on Salmonella
19
typhimurium TA100 and Escherichia coli WP2uvrA/ PKM101 using rat and hamster liver $9, Gann, 75, 8-16. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, 173-215. Ochiai, M., K. Wakabayashi, M. Magao and T. Sugimura (1984) Tyramine is a major mutagen precursor in soy sauce, being convertible to a mutagen by nitrite, Gann, 75, 1-3. Communicated by S.M. Galloway
Mutation Research Letters
ELSEVIER
Mutation Research 347 (1995) 17-19
Mutagenic activity of dopamine after nitrosation S u n C h a n g h a o *, C h e n B i n g q i n g , W a n g S u y a n , L i u Z i c h e n g Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 199 Dong Da Zhi Street, Nan Gang District, Harbin, People's Republic of China Received 28 October 1994; revised 19 January 1995; accepted 28 February 1995
Abstract
Dopamine hydrochloride is reported to be a new mutagen precursor in this study. After treatment with nitrite under acidic conditions, dopamine hydrochloride showed direct-acting mutagenicity on Salmonella typhimurium TA100, TA98 and Escherichia coli WP2uvra. The addition of $9 mix did not affect the mutagenicity of nitrosated dopamine significantlyin these three strains. Meanwhile, a comparison of the mutagenicity of nitrosated dopamine with nitrosated tyramine was carried out. Keywords: Dopamine; Tyramine; Nitrosation; Mutagenicity 1. Introduction
Since dopamine and tyramine are very closely related structurally, differing only by a single ring hydroxyl group, and the mutagenicity of nitrosated tyramine has been known for many years (Orchiai and Wakabayashi, 1984), it is very important to know if nitrosated dopamine has mutagenic activity. Thus, in this paper, the mutagenicity of dopamine was examined a n d compared with that of tyramine, after nitrite treatment.
Factory (Shanghai, China). Tyramine hydrochloride was from Sigma Chemical Company. Bacterial strains S. typhimurium TA100 and TA98 were generously provided by Professor B. Ames. E. coli
WP2uvrA was generously provided by Professor Tomita and Professor Nakamura. Liver microsomal fractions ($9) were obtained from the livers of rats treated with pentachlorinated biphenyls in our laboratory. Nitrite treatment
2. Materials and methods Materials
Sodium nitrite and ammonium sulfamate were from Beijing Chemical Plant. Dopamine hydrochloride was from Tian Feng Pharmaceutical * Corresponding author.
Various concentrations (including 2, 3, 5, 10 mg/ml) of dopamine hydrochloride (aqueous) (3 ml) were adjusted to pH 1.5 with 6 N hydrochloric acid. To each concentration, 0.5 ml of 0.28 M NaNO2 was added (final concentration 40 mM), and the pH was again adjusted to 1.5. The mixtures were incubated for 1 h in a shaking waterbath (37°C) in the dark. The solution of dopamine hydrochloride (7.5 mg/ml) without nitrite treat-
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 1 6 5 - 7 9 9 2 ( 9 5 ) 0 0 0 1 3 - 5
S. Changhao et al. / Mutation Research 347 (1995) 17-19
18
Table 1 The mutagenicity of dopamine after nitrosation Precursor
Dose (~g/plate)
Number of revertants a
E. coliWP2uvrA
TA98
TA100 - $9
+ $9
- $9
+ $9
- $9
+ $9
Dopamine treated with nitrite
150 225 375 750
195 245 360 571
201 248 362 568
44 61 92 181
50 61 99 178
32 37 58 96
33 37 52 95
Dopamine untreated with nitrite
750
123
131
11
20
18
21
116
127
14
22
16
22
Distilled water treated with nitrite
a Data are the mean of two independent assays. Spontaneous revertants: TAI00: 135; TA98: 30; E. coli WP2uvrA: 19.
ment and distilled water with nitrite treatment were also incubated as controls under the same conditions. Then, the nitrosation reaction was stopped by adding 0.5 ml of 0.32 M ammonium sulfamate (final concentration 40 mM). 0.1 ml of the reaction mixtures was plated for the mutagenicity assay no longer than 3 h after the end of incubation. In the meantime, the various concentrations of tyramine hydrochloride were also assayed for mutagenicity, according to the above method.
Mutation test The mutation assay with S. typhimurium TA100 and TA98 with and without $9 mix was done with the preincubation method (Maron and Ames, 1983). The assay with E. coli WP2uvrA was performed by Araki's method (Araki and Muramatsu, 1984). Two independent assays were carried out with four doses using three plates of each dose. The criteria for positive mutagenicity were defined as a dose-dependent increase in the number of revertants induced by dopamine or tyramine after treatment with nitrite, and a number at least twice as high as that seen in controls. 3. Results
The revertant frequencies with dopamine after nitrosation are shown in Table 1. Dopamine
treated with nitrite showed a definite dose-dependent mutagenicity in S. typhimurium TA100 and TA98 and E. coli WP2uvrA without $9 mix, and the addition of $9 mix did not affect the mutagenicity significantly in all strains. It was also found that TA100 was more sensitive to nitrosated dopamine than TA98 and E. coli WP2uvrA. Similar results were observed with nitrosated tyramine (data not shown). Dopamine and tyramine (both at the dose of 225/~g/plate) treated with nitrite induced 245 and 928 revertants of S. typhimurium TA100 without $9 mix, respectively, indicating that the mutagenicity of nitrosated dopamine was weaker than that of nitrosated tyramine.
4. Discussion
Up to now, no reports on mutagenicity of nitrosated dopamine have been found. The results of this study suggest that nitrosated dopamine is mutagenic. The finding that nitrosated dopamine was less mutagenic than nitrosated tyramine could result either from a difference in mutagenic potencies of the nitrosation products or from a difference in the efficiency of nitrosation of the two amines. As a medicine, dopamine hydrochloride is widely used in clinical practice to treat various
S. Changhao et al./Mutation Research 347 (1995) 17-19 s h o c k s . T h e r e f o r e , it is n e c e s s a r y t o f u r t h e r s t u d y the health risk of human exposure to the mutagen precursor dopamine.
References Araki, A., M. Muramatsu and T. Matsushima (1984) Comparison of mutagenicities of N-nitrosamines on Salmonella
19
typhimurium TA100 and Escherichia coli WP2uvrA/ PKM101 using rat and hamster liver $9, Gann, 75, 8-16. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, 173-215. Ochiai, M., K. Wakabayashi, M. Magao and T. Sugimura (1984) Tyramine is a major mutagen precursor in soy sauce, being convertible to a mutagen by nitrite, Gann, 75, 1-3. Communicated by S.M. Galloway