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Apparent and real thresholds: a study on two mutagens
260 tions at the dilute and leaden loci of the mouse. If heterozygotes for dilute and leaden are irradiated in late foetal life then occasional abnormal pigment cells of the dilute or leaden phenotype can be clearly seen in hair follicles when whole mounts of epidermis are made shortly after birth. These must be the products of forward mutation, among nearly 106 normal follicular pigment cells per young mouse. The reverse mutation m e t h o d depends on the pinkeyed dilute mutation, removing pigmentation from the retinal melanocytes. By use of an unstable allele, pun, which has a high rate of reversion to wild-type, it has been shown that dark back-mutated melanocytes can be detected with ease in the cleared unpigmented retinal pigment epithelium, which is a single layer of about 10 s cells per eye. Attempts are being made to extend this method to epidermal pigment cells also.
187 Seiler, J.P., Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland)
Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens as a valid mammalian short-term testing procedure Friedman and Staub (Mutation Res., 37 (1976) 67--76) have described a mammalian short-term test system utilizing the inhibition of thymidine uptake into testicular DNA by mutagens and carcinogens. This system has been validated in our laboratory on over 200 different chemical compounds with or without mutagenic or carcinogenic activity. The system appears to work very efficiently with only a small percentage of false negatives or false positives. To the former belong especially the thiourea-type carcinogens, for which a nonmutational mode of action is considered. Special emphasis is placed on the good separation of carcinogenic from non-carcinogenic isomers, e.g. the pairs methionine vs. ethionine, 2-amino- vs. 4-amino-biphenyl etc. Also carcinogens inactive in the Ames-test, e.g. dieldrin and 1,2-dimethylhydrazine, can be detected in this system. As it is an in vivo mammalian system it is of more direct significance for man than any other short term test yet known.
188 Seiler, J.P., Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland)
Apparent and real thresholds: a study on two mutagens The two mutagenic compounds carbendazim (methyl benzimidazol-2-yl carbamate, MBC) and N-nitroso-ethylenethiourea (N-nitroso-ETU) were investigated with the micronucleus test in mice. For both compounds concentrations can be defined which do not produce any increase in the number of micronucleated erythrocytes. During these investigations blood-level determinations were performed for both compounds. The apparent threshold concentration for nitroso-ETU could thus be explained by the (extrapolated) zero blood concentration in mice receiving doses of around 20 mg/kg. For MBC, however, a
261 measurable blood concentration exists, at which no effects on micronuclei formation are seen. As the mode of action of MBC involves disturbances of the mitotic apparatus and no direct action on the chromosomes, the influence of this c o m p o u n d on the association of the spindle fiber protein, tubulin, was investigated. Inhibition of tubulin association was found to begin at exactly the same M B C concentration at which also the onset of micronuclei formation takes place. In c o n t r a s t to the apparent threshold in the case of nitroso-ETU which is due only to the pharmacokinetics of this substance, MBC exhibits a real threshold which is due to the minimum concentration needed to produce a recognizable degree of tubulin association inhibition.
189 Shirasu, Y., M. Moriya and K. Kato, Institute of Environmental Toxicology, Kodaira, T o k y o 187 (Japan) Mutagenicity screening of feed additives in microbial systems Mutagenicity testing was performed on 13 feed additives using rec-assay and reverse mutation tests with and without a metabolic activation system. The strains used were H17 (rec ÷) and M45 (rec-) of B. subtilis for rec-assay and 5 strains of S. typhimurium TA series (TA1535, TA1537, TA1538, TA98, and TA100) and E. coli WP2 hcr for reversion assay. Among the feed additives tested, Carbadox, Furazolidone, Panazon, and Zoalene were positive in both the assays. The former 3 were mutagenic for TA100, TA98, and WP2 hcr, while Zoalene was mutagenic for all the strains. These c o m p o u n d s did not require metabolic activation for their mutagenic activities. Furthermore, host-mediated assay and urinary assay were conducted on the 4 positive samples to investigate their mutagenicity in vivo. Since TA100 was most sensitive to the mutagenic activities of the 4 compounds, the strain was employed for the studies. Carbadox and Furazolidone were highly mutagenic in b o t h the assays. Although Panazon was strongly mutagenic in vitro like Furazolidone at low concentrations, host-mediated assay failed to reveal the mutagenicity of this c o m p o u n d at the same dose levels as Furazolidone. High doses were required for detection of the mutagenicity of Zoalene in the host-mediated assay. Both compounds were also much less active than the other two c o m p o u n d s in the urinary assay. Addition of ~-glucuronidase did not enhance the mutagenic activities of any collected urine. Abbreviations: Furazolidone, 3-(5onitrofurfurylideneamino)-2-oxazolidinone; Panazon, bis(5nitrofurfurylidene)acetone guanylhydrazone hydrochloride; Carbadox, methyl 3-(2-quinoxalinylmethylene) carbazate-N,N'-dioxide; Zoalene, 3,5-dinitro-o-toluamide.
190 Simmon a, V.F., L. Hedden a, D. Poole a and R. Tardiff b, a Stanford Research Institute, Menlo Park, Calif. and b Environmental Protection Agency, Cincinnati, Ohio (U.S.A.)
187 Seiler, J.P., Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland)
Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens as a valid mammalian short-term testing procedure Friedman and Staub (Mutation Res., 37 (1976) 67--76) have described a mammalian short-term test system utilizing the inhibition of thymidine uptake into testicular DNA by mutagens and carcinogens. This system has been validated in our laboratory on over 200 different chemical compounds with or without mutagenic or carcinogenic activity. The system appears to work very efficiently with only a small percentage of false negatives or false positives. To the former belong especially the thiourea-type carcinogens, for which a nonmutational mode of action is considered. Special emphasis is placed on the good separation of carcinogenic from non-carcinogenic isomers, e.g. the pairs methionine vs. ethionine, 2-amino- vs. 4-amino-biphenyl etc. Also carcinogens inactive in the Ames-test, e.g. dieldrin and 1,2-dimethylhydrazine, can be detected in this system. As it is an in vivo mammalian system it is of more direct significance for man than any other short term test yet known.
188 Seiler, J.P., Swiss Federal Research Station, CH-8820 Waedenswil (Switzerland)
Apparent and real thresholds: a study on two mutagens The two mutagenic compounds carbendazim (methyl benzimidazol-2-yl carbamate, MBC) and N-nitroso-ethylenethiourea (N-nitroso-ETU) were investigated with the micronucleus test in mice. For both compounds concentrations can be defined which do not produce any increase in the number of micronucleated erythrocytes. During these investigations blood-level determinations were performed for both compounds. The apparent threshold concentration for nitroso-ETU could thus be explained by the (extrapolated) zero blood concentration in mice receiving doses of around 20 mg/kg. For MBC, however, a
261 measurable blood concentration exists, at which no effects on micronuclei formation are seen. As the mode of action of MBC involves disturbances of the mitotic apparatus and no direct action on the chromosomes, the influence of this c o m p o u n d on the association of the spindle fiber protein, tubulin, was investigated. Inhibition of tubulin association was found to begin at exactly the same M B C concentration at which also the onset of micronuclei formation takes place. In c o n t r a s t to the apparent threshold in the case of nitroso-ETU which is due only to the pharmacokinetics of this substance, MBC exhibits a real threshold which is due to the minimum concentration needed to produce a recognizable degree of tubulin association inhibition.
189 Shirasu, Y., M. Moriya and K. Kato, Institute of Environmental Toxicology, Kodaira, T o k y o 187 (Japan) Mutagenicity screening of feed additives in microbial systems Mutagenicity testing was performed on 13 feed additives using rec-assay and reverse mutation tests with and without a metabolic activation system. The strains used were H17 (rec ÷) and M45 (rec-) of B. subtilis for rec-assay and 5 strains of S. typhimurium TA series (TA1535, TA1537, TA1538, TA98, and TA100) and E. coli WP2 hcr for reversion assay. Among the feed additives tested, Carbadox, Furazolidone, Panazon, and Zoalene were positive in both the assays. The former 3 were mutagenic for TA100, TA98, and WP2 hcr, while Zoalene was mutagenic for all the strains. These c o m p o u n d s did not require metabolic activation for their mutagenic activities. Furthermore, host-mediated assay and urinary assay were conducted on the 4 positive samples to investigate their mutagenicity in vivo. Since TA100 was most sensitive to the mutagenic activities of the 4 compounds, the strain was employed for the studies. Carbadox and Furazolidone were highly mutagenic in b o t h the assays. Although Panazon was strongly mutagenic in vitro like Furazolidone at low concentrations, host-mediated assay failed to reveal the mutagenicity of this c o m p o u n d at the same dose levels as Furazolidone. High doses were required for detection of the mutagenicity of Zoalene in the host-mediated assay. Both compounds were also much less active than the other two c o m p o u n d s in the urinary assay. Addition of ~-glucuronidase did not enhance the mutagenic activities of any collected urine. Abbreviations: Furazolidone, 3-(5onitrofurfurylideneamino)-2-oxazolidinone; Panazon, bis(5nitrofurfurylidene)acetone guanylhydrazone hydrochloride; Carbadox, methyl 3-(2-quinoxalinylmethylene) carbazate-N,N'-dioxide; Zoalene, 3,5-dinitro-o-toluamide.
190 Simmon a, V.F., L. Hedden a, D. Poole a and R. Tardiff b, a Stanford Research Institute, Menlo Park, Calif. and b Environmental Protection Agency, Cincinnati, Ohio (U.S.A.)