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Aristolochic acid is a direct mutagen in Salmonella typhimurium
Mutation Research, 105 (1982) 201-204 Elsevier Biomedical Press
201
Aristolochic acid is a direct m u t a g e n in Salmonella
typhimurium Giinter Robisch a, Oskar Schimmer a and Waltraud G6ggelmann b Institut ffir Botanik und Pharmazeutische Biologie der Universit#t Erlangen-Nfirnberg, 8520 Erlangen~ and Abteilung far Toxikologie, GSF Miinchen, 8042 Neuherberg ~ (Federal Republic of Germany) (Accepted 24 June 1982)
Extracts from plants used to be a major source of medicins. Although little information on the potential risk to health is available such extracts are still widely used in human therapy. Aristolochic acid (Fig. 1) is a natural plant product found in certain species of Aristolochiaceae. Extracts from the leaves and roots of Aristolochia clematitis L. have been recommended for the therapy of arthritis, rheumatism and festering wounds. Moreover, several species of Aristolochiaceae are active against bacteria and fungi. Further investigations [1, 2] have indicated that aristolochic acid increases phagocytosis in leukocytes of rabbits, guinea pigs and rats and influences the defence mechanism against Herpes simplex infections of rabbit eyes [3]. Toxicological studies have shown that aristolochic acid has a carcinogenic effect on rats [4]. A dose of 10 mg/kg/day over 3 months led to papillomatosis of the stomach with malign neoplasms. Within a period of 4 months after treatment multiple carcinomas were found in the stomach and in other tissues such as liver, kidney, bladder, lung and skin. In 1981 as a result of these data, the German Federal Health Office withdrew the licence of products containing aristolochic acid. We have used the method of Ames et al. [5] to determine the mutagenicity of aftstolochic acid (EGA-Chemie, D-7924 Steinheim). The experiments were performed with the Salmonella strains TA1535, TA100, TA1537, TA1538 and TA98 (about
~___~OCH3 Fig. 1. Aristolochic acid (8-methoxy-6-nitrophenanthro[3,4-d]l,3-dioxole-5-carboxylic acid).
Correspondence should be addressed to: Dr. W. Gfggelmann, Institut fiir Toxikologie und Biochemie, Abteilung fiir Toxikologie, Gesellschaft ffir Strahlen- und Umweltforschung Miinchen, D-8042 Neuherherg (Federal Republic of Germany).
0165-7992/82/0000-0000/$02.75
© Elsevier Biomedical Press
202
TA 1537
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Fig. 2. N u m b e r o f revertant colonies o f the indicated strains as a function of the dose of aristolochic acid dissolved in DMSO. Tests were performed without and with 1.25 mg protein per plate of Clophen AS0-activated rat-liver $9 and about 1 x l0 s bacteria per plate. Each point gives the m e a n value of 3 plates of 1 representative Expt. out o f 3. TA1537 showed without $9 mix 11 ± 3 spontaneous revertants and 182 ± 15 induced revertants (50/~g 9-aminoacridine per plate) and with $9 mix 15 ± 2 spontaneous revertants and 113 ± 16 induced revertants (2.5 #g 2-aminoanthracene per plate). TA100 showed without $9 mix 144 ± 6 spontaneous revertants and 431 ± 27 induced revertants (1/tg sodium azide per plate) and with $9 mix 152 _+ 10 spontaneous revertants and 593 ± 10 induced revertants (1/zg 2-aminoanthracene per plate).
1 × l0 s bacteria per plate) without and with the addition of liver $9 from male male Wistar rats pretreated with a single oral dose of Clophen A50 (500 mg/kg in olive oil) 5 days before sacrifice. The $9 mix contained per mh 0.1 ml $9 (25 mg protein
203
per ml), 7 mM MgSO4, 4 mM NADP +, 20 mM D,L-isocitrate in 70 mM phosphate buffer (pH 7.4). As shown in Fig. 2 aristolochic acid was found to be a direct mutagen in strains TA1537 and TAI00. 200/~g aristolochic acid per plate increased the number of induced revertants in TA1537 by a factor of l0 relative to the control value. TA100 showed less mutagenicity. The presence of $9 mix had little effect on the observed number of induced revertants. Aristolochic acid showed no mutagenicity in TA1535, TA1538 and TA98 with and without $9 mix (Table 1). Morimoto et al. [6] have reported that Saishin, a commercial crude drug used in Japan, is mutagenic in both the Bacillus subtilis rec-assay and the Salmonella strain TA100 without metabolic activation. Saishin is derived from Asiasarium sieboldt F. Mackawa, a species of the family of Aristolochiaceae and may contain aristolochic acid which could account for the mutagenic effect. Although it is not possible to extrapolate from the concentrations of aristolochic acid in the bacterial test system to those applied in human therapy, the direct mutagenic action in Salmonella typhimurium points to the hazard of products containing aristolochic acid. Furthermore, these results imply that in vitro mutagenicity testing of plant products should also be included in a screening program to evaluate the toxicity of these chemicals.
TABLE 1 RESULTS OF M U T A G E N I C I T Y TESTING OF A R I S T O L O C H I C ACID IN SALMONELLA STRAINS a TA1535, TA1538 AND TA98 Aristolochic acid b (~g/plate)
Revertants per plate TA1535 - $9
0 10 50 100 160 200 Sodium azide (0.5/~g/plate)
17+ 17+_ 18_+ 22+ 21± 19+_
TA1538
+ $9
2 3 2 2 1 4
17± 15± 17+ 22+_ 18+_ 17_+
- $9
1 2 3 3 5 3
2 4 1 3 2 2
18+ 12+ 16+ 19-+ 21± 22+_
- $9
3 2 2 4 5 5
47± 49+ 54+_ 50+ 54± 56+-
639 _+35
5 4 9 9 4 7
58_+ 64_+ 64± 66_+ 66± 74±
4 4 5 6 4 2
133 _ 17
430 + 22
364 +_20
Data given the mean values of 3 plates _+ SD of 1 representative Expt. out of 3. b Added in 5 0 - 10 ttl DMSO to a total volume of 2.6 ml per assay.
a
+ $9
272+ 13
4-Nitro-o-phenylenediamine (5 #g/plate) 2-Aminoanthracene (1 ttg/plate)
17± 15± 16± 13± 11_+ 15___
TA98
+ $9
554 _ 21
204
References 1 M6se, J.R., and G. Lukas, Versuche iiber die Wirkungsweise yon Aristolochia clematitis L., Drug Res., 11 ( 1 9 6 1 ) 33 - 36. 2 M~se, J.R., Versuche fiber die Wirksamkeit yon Aristolochiasiiure, Planta Med., 11 (1963) 7 2 - 9 1 . 3 M6se, J.R., D. Stfinzner, M. Zirm, Ch. Egger-Biissing and F. Schmalzl, Experimentelle Beeinflussung der Herpes-simplex-Infektion des Kaninchen-Auges durch Aristolochiasiiure, Drug Res., 30 (1980) 1571 - 1573. 4 BGA, Zum Zulassungswiderruf Aristolochias~iure-haltiger Fertigarzneimittel, Pharm. Z., 126 (1981) 1373 - 1374. 5 Ames, B.N., J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microscome mutagenicity test, Mutation Res., 31 (1975) 3 4 7 - 364. 6 Morimoto, I., F. Watanabe, T. Osawa, T. Okitsu and T. Kada, Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay, Mutation Res., 97 (1982) 81 - 102.
201
Aristolochic acid is a direct m u t a g e n in Salmonella
typhimurium Giinter Robisch a, Oskar Schimmer a and Waltraud G6ggelmann b Institut ffir Botanik und Pharmazeutische Biologie der Universit#t Erlangen-Nfirnberg, 8520 Erlangen~ and Abteilung far Toxikologie, GSF Miinchen, 8042 Neuherberg ~ (Federal Republic of Germany) (Accepted 24 June 1982)
Extracts from plants used to be a major source of medicins. Although little information on the potential risk to health is available such extracts are still widely used in human therapy. Aristolochic acid (Fig. 1) is a natural plant product found in certain species of Aristolochiaceae. Extracts from the leaves and roots of Aristolochia clematitis L. have been recommended for the therapy of arthritis, rheumatism and festering wounds. Moreover, several species of Aristolochiaceae are active against bacteria and fungi. Further investigations [1, 2] have indicated that aristolochic acid increases phagocytosis in leukocytes of rabbits, guinea pigs and rats and influences the defence mechanism against Herpes simplex infections of rabbit eyes [3]. Toxicological studies have shown that aristolochic acid has a carcinogenic effect on rats [4]. A dose of 10 mg/kg/day over 3 months led to papillomatosis of the stomach with malign neoplasms. Within a period of 4 months after treatment multiple carcinomas were found in the stomach and in other tissues such as liver, kidney, bladder, lung and skin. In 1981 as a result of these data, the German Federal Health Office withdrew the licence of products containing aristolochic acid. We have used the method of Ames et al. [5] to determine the mutagenicity of aftstolochic acid (EGA-Chemie, D-7924 Steinheim). The experiments were performed with the Salmonella strains TA1535, TA100, TA1537, TA1538 and TA98 (about
~___~OCH3 Fig. 1. Aristolochic acid (8-methoxy-6-nitrophenanthro[3,4-d]l,3-dioxole-5-carboxylic acid).
Correspondence should be addressed to: Dr. W. Gfggelmann, Institut fiir Toxikologie und Biochemie, Abteilung fiir Toxikologie, Gesellschaft ffir Strahlen- und Umweltforschung Miinchen, D-8042 Neuherherg (Federal Republic of Germany).
0165-7992/82/0000-0000/$02.75
© Elsevier Biomedical Press
202
TA 1537
•
120.
,//~o
*S9
- S9
o
800 o
¢: 0 > i.
1
lb
sb
~0
1~,0
26o.,.,g©tot,
5b
~o
1~0
26o~g~p~te
TA 100 /.80-
320-
~6
Fig. 2. N u m b e r o f revertant colonies o f the indicated strains as a function of the dose of aristolochic acid dissolved in DMSO. Tests were performed without and with 1.25 mg protein per plate of Clophen AS0-activated rat-liver $9 and about 1 x l0 s bacteria per plate. Each point gives the m e a n value of 3 plates of 1 representative Expt. out o f 3. TA1537 showed without $9 mix 11 ± 3 spontaneous revertants and 182 ± 15 induced revertants (50/~g 9-aminoacridine per plate) and with $9 mix 15 ± 2 spontaneous revertants and 113 ± 16 induced revertants (2.5 #g 2-aminoanthracene per plate). TA100 showed without $9 mix 144 ± 6 spontaneous revertants and 431 ± 27 induced revertants (1/tg sodium azide per plate) and with $9 mix 152 _+ 10 spontaneous revertants and 593 ± 10 induced revertants (1/zg 2-aminoanthracene per plate).
1 × l0 s bacteria per plate) without and with the addition of liver $9 from male male Wistar rats pretreated with a single oral dose of Clophen A50 (500 mg/kg in olive oil) 5 days before sacrifice. The $9 mix contained per mh 0.1 ml $9 (25 mg protein
203
per ml), 7 mM MgSO4, 4 mM NADP +, 20 mM D,L-isocitrate in 70 mM phosphate buffer (pH 7.4). As shown in Fig. 2 aristolochic acid was found to be a direct mutagen in strains TA1537 and TAI00. 200/~g aristolochic acid per plate increased the number of induced revertants in TA1537 by a factor of l0 relative to the control value. TA100 showed less mutagenicity. The presence of $9 mix had little effect on the observed number of induced revertants. Aristolochic acid showed no mutagenicity in TA1535, TA1538 and TA98 with and without $9 mix (Table 1). Morimoto et al. [6] have reported that Saishin, a commercial crude drug used in Japan, is mutagenic in both the Bacillus subtilis rec-assay and the Salmonella strain TA100 without metabolic activation. Saishin is derived from Asiasarium sieboldt F. Mackawa, a species of the family of Aristolochiaceae and may contain aristolochic acid which could account for the mutagenic effect. Although it is not possible to extrapolate from the concentrations of aristolochic acid in the bacterial test system to those applied in human therapy, the direct mutagenic action in Salmonella typhimurium points to the hazard of products containing aristolochic acid. Furthermore, these results imply that in vitro mutagenicity testing of plant products should also be included in a screening program to evaluate the toxicity of these chemicals.
TABLE 1 RESULTS OF M U T A G E N I C I T Y TESTING OF A R I S T O L O C H I C ACID IN SALMONELLA STRAINS a TA1535, TA1538 AND TA98 Aristolochic acid b (~g/plate)
Revertants per plate TA1535 - $9
0 10 50 100 160 200 Sodium azide (0.5/~g/plate)
17+ 17+_ 18_+ 22+ 21± 19+_
TA1538
+ $9
2 3 2 2 1 4
17± 15± 17+ 22+_ 18+_ 17_+
- $9
1 2 3 3 5 3
2 4 1 3 2 2
18+ 12+ 16+ 19-+ 21± 22+_
- $9
3 2 2 4 5 5
47± 49+ 54+_ 50+ 54± 56+-
639 _+35
5 4 9 9 4 7
58_+ 64_+ 64± 66_+ 66± 74±
4 4 5 6 4 2
133 _ 17
430 + 22
364 +_20
Data given the mean values of 3 plates _+ SD of 1 representative Expt. out of 3. b Added in 5 0 - 10 ttl DMSO to a total volume of 2.6 ml per assay.
a
+ $9
272+ 13
4-Nitro-o-phenylenediamine (5 #g/plate) 2-Aminoanthracene (1 ttg/plate)
17± 15± 16± 13± 11_+ 15___
TA98
+ $9
554 _ 21
204
References 1 M6se, J.R., and G. Lukas, Versuche iiber die Wirkungsweise yon Aristolochia clematitis L., Drug Res., 11 ( 1 9 6 1 ) 33 - 36. 2 M~se, J.R., Versuche fiber die Wirksamkeit yon Aristolochiasiiure, Planta Med., 11 (1963) 7 2 - 9 1 . 3 M6se, J.R., D. Stfinzner, M. Zirm, Ch. Egger-Biissing and F. Schmalzl, Experimentelle Beeinflussung der Herpes-simplex-Infektion des Kaninchen-Auges durch Aristolochiasiiure, Drug Res., 30 (1980) 1571 - 1573. 4 BGA, Zum Zulassungswiderruf Aristolochias~iure-haltiger Fertigarzneimittel, Pharm. Z., 126 (1981) 1373 - 1374. 5 Ames, B.N., J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microscome mutagenicity test, Mutation Res., 31 (1975) 3 4 7 - 364. 6 Morimoto, I., F. Watanabe, T. Osawa, T. Okitsu and T. Kada, Mutagenicity screening of crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay, Mutation Res., 97 (1982) 81 - 102.