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The mutagenic action of some nitrothiazoles and nitrothiophenes
117
between results in plate and in liquid tests. In order to keep the reaction mixtures as simple as possible, t w o chemicals which -- in partial confirmation of previous findings -- to n o t require microsome-mediated activation were selected, namely 4-nitro-o-phenylenediamine (4-NOPD) and 2,5-diaminoanisole (2,5DAA); the mutation system employed was detection of h/s* revertants (from his D30s2) in strain S. typhimurium TA 1538. The results can be summarized as follows: both c o m p o u n d s are mutagenic in the plate test, 4-NOPD being a b o u t ten times more active than 2,5-DAA. In liquid tests at 37 °C, using stationary and growing cells at incubation times up to 4 h, no increase in mutation frequency above the spontaneous level can be observed, even in the case of 2,5DAA, at concentrations which inactivate substantially the cell. With incubation times longer than 20 h, however, 4-NOPD treated bacterial suspensions show a more than 100-fold increase of mutation frequency without any inactiviation of colony forming ability; experiments with 2,5-DAA are in progress. The signification of these results for understanding of the mechanisms of hair dye mutagenicity as for routine mutagenicity testing procedures will be discussed.
30 C.E. Voogd, National Institute of Public Health, Bilthoven (Netherlands)
The mutagenic action of some nitrothiazoles and nitrothiophenes It is well-known, that some heterocyclic c o m p o u n d s with a nitro-group, e.g. nitrofurans and nitroimidazoles, possess a mutagenic action. As some nitrothiazoles were used for growth promoting purposes, we investigated the mutagenic properties of these c o m p o u n d s as they may be mutagenic. The fluctuation test was used to demonstrate an increase of the mutationfrequency. A KlebsieUa pneumoniae mutant requiring uracil and proline for growth was used as a testorganism. We found that 2-acetamido-5-nitrothiazole is a p o t e n t mutagenic compound. The mutagenic activity of N(5-nitrothiazole-2-yl) benzamide is somewhat less followed b y 2-bromo-5-nitrothiazole and 2-amino-5-nitrothiazole. Furthermore 2-amino-5-chlorothiazole and in a lesser degree 2-aminothiazole are also mutagenic. The mutagenic activity of 4-nitroisothiazole is equal to that of 2amino-5-nitrothiazole. Even w i t h o u t a nitrogen atom in the nucleus these compounds are mutagenic. So 2-nitrothiophene is more mutagenic than 3-nitrothiophene. With thiazole, isothiazole and thiophene no mutagenic action could be demonstrated. Although the presence of a nitro-group seems a prerequisite for a strong mutagenic action of these compounds, the relation between chemical structure and mutagenic activity of these substances remains unclear.
31 S. Igali and R.C. Von Borstel, "Frederic Joliot-Curie" National Research Institute of Radiobiology and Radiohygiene, Budapest (Hungary) and Dept. of Genetics, University of Alberta, E d m o n t o n (Canada)
118 Concentration-time comparison of thioxanthenones, indazoles and nitrofurans on Saccbaromyces cerevisiae We have reported that when given under conditions where it is not mutagenic (stationary phase at pH 5.9) indazole IA-5 kills yeast cells more efficiently when IA-5 is delivered as a function of concentration rather than one of time (Igali and yon Borstel, Mutation Res., 26, (1974) 452). This holds true for IA-4 as well (Meadows, 1973, M.S. Thesis, Univ. of Alberta). On the other hand, the thioxanthenone hycanthone, when given under conditions where it is mutagenic (stationary phase either pH 5.9 or pH 7.0), kills cells more efficiently when delivered as a function of time (Igali and von Borstel, ibid.). In general we have found that the above principle of concentration being more effective than time exposure holds true also for the thioxanthenone lucanthone and the indazole IA-3 when given under conditions where they are not mutagenic (stationary phase at pH 7.0). But the indazole IA-6 and the nitrofuran SQ 18,506 do not exhibit appreciable concentration-time differences when given under conditions where they are mutagenic (stationary ohase pH 7.0). This holds true for the nitrofuran AF-2 as well (Shahin and yon Borstel, unpublished data). The results are consistent with the notion that mutagens either do not show time-concentration differences or else are more sensitive when time is the criteria, the non mutagens are more sensitive when concentration is the criteria. Finally it is clear that when measuring mutagenicity and survival of S. cerevisiae with different compounds exposure must be measured as a function of both time and concentration until a theory is established for this phenomenon of concentration-time difference in killing efficiency.
32 B. Herbold and W. Buselmaier, Institut fiir Humangenetik und Anthropologie, Diisseldorf, and Institut ffir Anthropologie und Humangenetik, Heidelberg (Federal Republic of Germany) Comparative investigations with different bacterial strains Investigations with the liver microsomal system were made by the deep rough test set of Ames (Salmonella typhimurium TA 1535, TA 1536, TA 1537, TA 1538) and Salmonella typhimurium G. 46. Eleven substances with four different mechanisms had been tested (alkylating agents, antagonists, acridines and radicalating agents), including carcinogens, cytostatica, fungicides and other pharmacia. These were in detail: DMN, DEN, MNNG, cyclophosphamide, Captan, trypaflavine, azathioprine, 6-mercaptopurine, amethopterine, isoniazide and hydrazine. We found 10 of these substances as mutagens. Most of them caused only base-pair substitutions. Just amethopterine remained negative. It will be supposed that this system will assess all tested mechanisms and will be more sensitive than the host-mediated assay. This
work
was
sponsored b y the Deutsche Forschungsgemeinschaft.
between results in plate and in liquid tests. In order to keep the reaction mixtures as simple as possible, t w o chemicals which -- in partial confirmation of previous findings -- to n o t require microsome-mediated activation were selected, namely 4-nitro-o-phenylenediamine (4-NOPD) and 2,5-diaminoanisole (2,5DAA); the mutation system employed was detection of h/s* revertants (from his D30s2) in strain S. typhimurium TA 1538. The results can be summarized as follows: both c o m p o u n d s are mutagenic in the plate test, 4-NOPD being a b o u t ten times more active than 2,5-DAA. In liquid tests at 37 °C, using stationary and growing cells at incubation times up to 4 h, no increase in mutation frequency above the spontaneous level can be observed, even in the case of 2,5DAA, at concentrations which inactivate substantially the cell. With incubation times longer than 20 h, however, 4-NOPD treated bacterial suspensions show a more than 100-fold increase of mutation frequency without any inactiviation of colony forming ability; experiments with 2,5-DAA are in progress. The signification of these results for understanding of the mechanisms of hair dye mutagenicity as for routine mutagenicity testing procedures will be discussed.
30 C.E. Voogd, National Institute of Public Health, Bilthoven (Netherlands)
The mutagenic action of some nitrothiazoles and nitrothiophenes It is well-known, that some heterocyclic c o m p o u n d s with a nitro-group, e.g. nitrofurans and nitroimidazoles, possess a mutagenic action. As some nitrothiazoles were used for growth promoting purposes, we investigated the mutagenic properties of these c o m p o u n d s as they may be mutagenic. The fluctuation test was used to demonstrate an increase of the mutationfrequency. A KlebsieUa pneumoniae mutant requiring uracil and proline for growth was used as a testorganism. We found that 2-acetamido-5-nitrothiazole is a p o t e n t mutagenic compound. The mutagenic activity of N(5-nitrothiazole-2-yl) benzamide is somewhat less followed b y 2-bromo-5-nitrothiazole and 2-amino-5-nitrothiazole. Furthermore 2-amino-5-chlorothiazole and in a lesser degree 2-aminothiazole are also mutagenic. The mutagenic activity of 4-nitroisothiazole is equal to that of 2amino-5-nitrothiazole. Even w i t h o u t a nitrogen atom in the nucleus these compounds are mutagenic. So 2-nitrothiophene is more mutagenic than 3-nitrothiophene. With thiazole, isothiazole and thiophene no mutagenic action could be demonstrated. Although the presence of a nitro-group seems a prerequisite for a strong mutagenic action of these compounds, the relation between chemical structure and mutagenic activity of these substances remains unclear.
31 S. Igali and R.C. Von Borstel, "Frederic Joliot-Curie" National Research Institute of Radiobiology and Radiohygiene, Budapest (Hungary) and Dept. of Genetics, University of Alberta, E d m o n t o n (Canada)
118 Concentration-time comparison of thioxanthenones, indazoles and nitrofurans on Saccbaromyces cerevisiae We have reported that when given under conditions where it is not mutagenic (stationary phase at pH 5.9) indazole IA-5 kills yeast cells more efficiently when IA-5 is delivered as a function of concentration rather than one of time (Igali and yon Borstel, Mutation Res., 26, (1974) 452). This holds true for IA-4 as well (Meadows, 1973, M.S. Thesis, Univ. of Alberta). On the other hand, the thioxanthenone hycanthone, when given under conditions where it is mutagenic (stationary phase either pH 5.9 or pH 7.0), kills cells more efficiently when delivered as a function of time (Igali and von Borstel, ibid.). In general we have found that the above principle of concentration being more effective than time exposure holds true also for the thioxanthenone lucanthone and the indazole IA-3 when given under conditions where they are not mutagenic (stationary phase at pH 7.0). But the indazole IA-6 and the nitrofuran SQ 18,506 do not exhibit appreciable concentration-time differences when given under conditions where they are mutagenic (stationary ohase pH 7.0). This holds true for the nitrofuran AF-2 as well (Shahin and yon Borstel, unpublished data). The results are consistent with the notion that mutagens either do not show time-concentration differences or else are more sensitive when time is the criteria, the non mutagens are more sensitive when concentration is the criteria. Finally it is clear that when measuring mutagenicity and survival of S. cerevisiae with different compounds exposure must be measured as a function of both time and concentration until a theory is established for this phenomenon of concentration-time difference in killing efficiency.
32 B. Herbold and W. Buselmaier, Institut fiir Humangenetik und Anthropologie, Diisseldorf, and Institut ffir Anthropologie und Humangenetik, Heidelberg (Federal Republic of Germany) Comparative investigations with different bacterial strains Investigations with the liver microsomal system were made by the deep rough test set of Ames (Salmonella typhimurium TA 1535, TA 1536, TA 1537, TA 1538) and Salmonella typhimurium G. 46. Eleven substances with four different mechanisms had been tested (alkylating agents, antagonists, acridines and radicalating agents), including carcinogens, cytostatica, fungicides and other pharmacia. These were in detail: DMN, DEN, MNNG, cyclophosphamide, Captan, trypaflavine, azathioprine, 6-mercaptopurine, amethopterine, isoniazide and hydrazine. We found 10 of these substances as mutagens. Most of them caused only base-pair substitutions. Just amethopterine remained negative. It will be supposed that this system will assess all tested mechanisms and will be more sensitive than the host-mediated assay. This
work
was
sponsored b y the Deutsche Forschungsgemeinschaft.