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Which standard tester set must be used for optimal routine screening in the Ames test?
350
methylbenzidine and benzo[a]pyrene-pyrene) in order to assess the sensitivity and specificity of the method. CHO-K1 were grown in 6-well plates. 24 h after, cells were exposed at 6 concentrations of the chemical to study, with and without metabolic activation during 4 h, 24, 48 and 72 h after, cells were fixed with methanol and stained with May-Grianwald-Giemsa. The percentage of micronucleated cells was scored for a total of 1000 cells. The test was positive with dimethyl- and diethyl-nitrosamine and with 2-AAF which suggests good sensitivity. Benzo[a]pyrene and benzidine were positive, tetramethylbenzidine and pyrene were negative which suggests good specificity. All positive chemicals gave dose and time effects. These results suggest that this test can be used for screening of chemicals and good predictivity of clastogenicity.
92 Maier, P., and B. Weibel, Institute of Toxicology, Swiss Federal Institute of Technology and University of Ztirich, Schwerzenbach (Switzerland) The mutagenic activity of aristoloehic acid in mammalian cells in vitro is modified by the oxygen tension in the culture medium
AA I and AA II, the two main components of aristolochic acid, were tested for their induction of 6-thioguanine-resistant mutants in vivo in the granuloma pouch assay and in vitro in the corresponding freshly isolated fibroblast-like cells. Oxygen tension determined in vivo in the subcutaneous tissue of rats was between 30 and 40 mm Hg. In vitro cells were exposed to the test compounds under this tissue pO 2 (5% O2) or under standard culture conditions (19% 02). In vitro AA I (10 # g / m l ) expressed a 6-fold higher mutagenic activity than AA II at equimolar dose. At tissue pO 2, AA I induced a 1.4-fold higher mutation frequency as compared to standard pO 2. AA II (20/~g/ml) behaved reciprocally. At low pO 2 the mutation frequency decreased 0.6-fold to that obtained under standard (hyperoxic) conditions. In addition oxygen tension dur-
ing the expression period influenced the mutation frequencies. Results from in vivo studies suggest that exposure under low pO2 in vitro corresponds more closely to the in vivo situation.
93 Pampfer, S., and C. Streffer, Institut far Medizinische Strahlenbiologie, Universitatsklinik u ~ Essen, Essen (F.R.G.) Micronucleus formation in 2-cell embryos after in vitro X-irradiation of capacitated spermatozoa
Capacitated mouse spermatozoa were exposed in vitro to various doses of X-rays and added 1 h later to a medium containing superovulated oocytes. The percentage of fertilized oocytes was determined after 24 h of incubation and 2-cell embryos were investigated for micronucleus formation. The results showed that: (a) the fertilization rate was drastically diminished after sperm exposure to 470 cGy whereas smaller doses had no significant effect on the percentage of fertilized oocytes, (b) the mean number of micronuclei per 2-cell embryo increased with dose and the dose-response relationship for micronucleus formation fitted well to a linear-quadratic equatiom and (c) the number of embryos containing at least one micronucleus increased with a linear function of the irradiation dose. Based on these observations, it was concluded that: (a) the fertilization capacity is reduced only if a relatively high dose of X-rays is applied, (b) chromosome aberrations which lead to micronucleus formation in embryos conceived with irradiated spermatozoa are caused mainly by independent one-hit events, and (c) the prenatal mortality observed by other authors after in vivo exposure of spermatozoa is directly correlated with the early appearance of micronuclei in embryos since the proportion of prenatally lost embryos and the proportion of embryos containing micronuclei are both linearly dependent on the irradiation dose.
94 Bonneau, D., and A. Cordier, Rh6ne-Poulenc
351 Sant6, Centre de Recherches de Vitry, D6partement de Toxicologie, 13, quai Jules Guesde, 94400 Vitry sur Seine (France) Which standard tester set must be used for optimal routine screening in the Ames test?
The aim of this study was to select the best strain set to choose among the 7 Ames strains (TA1535, TA100, TA1537, TA97, TA1538, TA98, TA102) commonly used in routine screening. The Ames tests were carried out with 79 compounds (44 reference compounds described as positive in the literature and 35 compounds found positive in our in-house screening) in the same experimental conditions. It must be noted that all tested compounds were positive without metabolic activation. The Ames recommendation to use TA100, TA97, TA98 and TA102 was justified by the number of compounds found positive with these strains taken into account each strain separately. But this standard tester set (strains taken into account all together) did not show the best detection power with the reference compounds as well as with our in-house compounds. The most sensitive tester set to detect mutagenicity of the reference compounds was TA1535, TA1537, TA1538 and TA102 (98%) when the standard set only detected 84%. The most sensitive sets to detect 100% of our in-house compounds were the 3 following: TA100, TA1537, TA1538 - - TA100, TA97, TA1538 - - TA100, TA1538, TA98 when any sets (possible combination of 3 strains proposed by Ames) did not detect 100% of these positive compounds. In conclusion, for the purpose of routine screening, the standard tester set proposed by Ames did not appear to be the more sensitive in our experimental conditions.
95 Vanparys, Ph. 1, p.j. Lewi 2 and R. M a r s b o o m l , 1 Department of Toxicology, and 2 Department of Information Science, Janssen Pharmaceutica, Turnhoutseweg 30, 2340 Beerse (Belgium) Visualization of mutagenic potency by Spectramap
Spectral map analysis (SMA) has been developed for the mapping of biological activity pro-
files of drugs (Lewi, 1976, 1986). Basically, spectral mapping is a graphic technique for the study of interactions of drugs with tests. But, SMA can also be used to visualize results obtained in the Salmonella reversion assay. Chemicals are, irrespectively of their potencies, mapped according to their specificity for the S. typhimurium strains. Conversely, strains are irrespectively of their sensitivities, located on the same map according to their specificities for the chemicals. Chemicals are represented by means of circles, the areas of which are made proportional to their average potency. Strains are represented by means of squares whose areas vary proportionally to their average sensitivity. For the purpose of illustration, SMA is applied to the mutagenic activity of nitrofuran and nitrothiazole derivatives on S. typhimurium strains TA97, TA98, TA1538, TA100 and TA1535. References
Lewi, P.J., Arzn.-Forsch. (Drug Res.), 26 (1976) 1295-1300. Lewi, P.J., Eur. J. Med. Chem. (1986) in press.
96 Grinfeld, S., and P. Jacquet, Mammalian Genetics Laboratory, Department of Biology, C E N / S C K , B-2400 Mol (Belgium) Existence of a particular radiation-induced G 2 arrest in the mouse egg of the B A L B / c strain
Pregnant superovulated female mice of the B A L B / c strain were irradiated with 1 Gy of X-rays at hourly intervals during the first cellular cycle of the eggs. Embryos were flushed out of the oviducts on the following day, and classified into uncleaved or two-cell embryos. Two-cell embryos were cultured for 4 days and examined at the end of this period for their development into blastocysts. Two types of effects were found, depending on the time of X-irradiation. When irradiation was delivered between 2 and 10 h after superovulation, cultured 2-cell embryos developed normally up to the morula stage, where a high mortality occurred. On the other hand, when irradiation was given between 5 and 12 h after superovulation (i.e. at the onset of the first D N A synthesis), a high propor-
methylbenzidine and benzo[a]pyrene-pyrene) in order to assess the sensitivity and specificity of the method. CHO-K1 were grown in 6-well plates. 24 h after, cells were exposed at 6 concentrations of the chemical to study, with and without metabolic activation during 4 h, 24, 48 and 72 h after, cells were fixed with methanol and stained with May-Grianwald-Giemsa. The percentage of micronucleated cells was scored for a total of 1000 cells. The test was positive with dimethyl- and diethyl-nitrosamine and with 2-AAF which suggests good sensitivity. Benzo[a]pyrene and benzidine were positive, tetramethylbenzidine and pyrene were negative which suggests good specificity. All positive chemicals gave dose and time effects. These results suggest that this test can be used for screening of chemicals and good predictivity of clastogenicity.
92 Maier, P., and B. Weibel, Institute of Toxicology, Swiss Federal Institute of Technology and University of Ztirich, Schwerzenbach (Switzerland) The mutagenic activity of aristoloehic acid in mammalian cells in vitro is modified by the oxygen tension in the culture medium
AA I and AA II, the two main components of aristolochic acid, were tested for their induction of 6-thioguanine-resistant mutants in vivo in the granuloma pouch assay and in vitro in the corresponding freshly isolated fibroblast-like cells. Oxygen tension determined in vivo in the subcutaneous tissue of rats was between 30 and 40 mm Hg. In vitro cells were exposed to the test compounds under this tissue pO 2 (5% O2) or under standard culture conditions (19% 02). In vitro AA I (10 # g / m l ) expressed a 6-fold higher mutagenic activity than AA II at equimolar dose. At tissue pO 2, AA I induced a 1.4-fold higher mutation frequency as compared to standard pO 2. AA II (20/~g/ml) behaved reciprocally. At low pO 2 the mutation frequency decreased 0.6-fold to that obtained under standard (hyperoxic) conditions. In addition oxygen tension dur-
ing the expression period influenced the mutation frequencies. Results from in vivo studies suggest that exposure under low pO2 in vitro corresponds more closely to the in vivo situation.
93 Pampfer, S., and C. Streffer, Institut far Medizinische Strahlenbiologie, Universitatsklinik u ~ Essen, Essen (F.R.G.) Micronucleus formation in 2-cell embryos after in vitro X-irradiation of capacitated spermatozoa
Capacitated mouse spermatozoa were exposed in vitro to various doses of X-rays and added 1 h later to a medium containing superovulated oocytes. The percentage of fertilized oocytes was determined after 24 h of incubation and 2-cell embryos were investigated for micronucleus formation. The results showed that: (a) the fertilization rate was drastically diminished after sperm exposure to 470 cGy whereas smaller doses had no significant effect on the percentage of fertilized oocytes, (b) the mean number of micronuclei per 2-cell embryo increased with dose and the dose-response relationship for micronucleus formation fitted well to a linear-quadratic equatiom and (c) the number of embryos containing at least one micronucleus increased with a linear function of the irradiation dose. Based on these observations, it was concluded that: (a) the fertilization capacity is reduced only if a relatively high dose of X-rays is applied, (b) chromosome aberrations which lead to micronucleus formation in embryos conceived with irradiated spermatozoa are caused mainly by independent one-hit events, and (c) the prenatal mortality observed by other authors after in vivo exposure of spermatozoa is directly correlated with the early appearance of micronuclei in embryos since the proportion of prenatally lost embryos and the proportion of embryos containing micronuclei are both linearly dependent on the irradiation dose.
94 Bonneau, D., and A. Cordier, Rh6ne-Poulenc
351 Sant6, Centre de Recherches de Vitry, D6partement de Toxicologie, 13, quai Jules Guesde, 94400 Vitry sur Seine (France) Which standard tester set must be used for optimal routine screening in the Ames test?
The aim of this study was to select the best strain set to choose among the 7 Ames strains (TA1535, TA100, TA1537, TA97, TA1538, TA98, TA102) commonly used in routine screening. The Ames tests were carried out with 79 compounds (44 reference compounds described as positive in the literature and 35 compounds found positive in our in-house screening) in the same experimental conditions. It must be noted that all tested compounds were positive without metabolic activation. The Ames recommendation to use TA100, TA97, TA98 and TA102 was justified by the number of compounds found positive with these strains taken into account each strain separately. But this standard tester set (strains taken into account all together) did not show the best detection power with the reference compounds as well as with our in-house compounds. The most sensitive tester set to detect mutagenicity of the reference compounds was TA1535, TA1537, TA1538 and TA102 (98%) when the standard set only detected 84%. The most sensitive sets to detect 100% of our in-house compounds were the 3 following: TA100, TA1537, TA1538 - - TA100, TA97, TA1538 - - TA100, TA1538, TA98 when any sets (possible combination of 3 strains proposed by Ames) did not detect 100% of these positive compounds. In conclusion, for the purpose of routine screening, the standard tester set proposed by Ames did not appear to be the more sensitive in our experimental conditions.
95 Vanparys, Ph. 1, p.j. Lewi 2 and R. M a r s b o o m l , 1 Department of Toxicology, and 2 Department of Information Science, Janssen Pharmaceutica, Turnhoutseweg 30, 2340 Beerse (Belgium) Visualization of mutagenic potency by Spectramap
Spectral map analysis (SMA) has been developed for the mapping of biological activity pro-
files of drugs (Lewi, 1976, 1986). Basically, spectral mapping is a graphic technique for the study of interactions of drugs with tests. But, SMA can also be used to visualize results obtained in the Salmonella reversion assay. Chemicals are, irrespectively of their potencies, mapped according to their specificity for the S. typhimurium strains. Conversely, strains are irrespectively of their sensitivities, located on the same map according to their specificities for the chemicals. Chemicals are represented by means of circles, the areas of which are made proportional to their average potency. Strains are represented by means of squares whose areas vary proportionally to their average sensitivity. For the purpose of illustration, SMA is applied to the mutagenic activity of nitrofuran and nitrothiazole derivatives on S. typhimurium strains TA97, TA98, TA1538, TA100 and TA1535. References
Lewi, P.J., Arzn.-Forsch. (Drug Res.), 26 (1976) 1295-1300. Lewi, P.J., Eur. J. Med. Chem. (1986) in press.
96 Grinfeld, S., and P. Jacquet, Mammalian Genetics Laboratory, Department of Biology, C E N / S C K , B-2400 Mol (Belgium) Existence of a particular radiation-induced G 2 arrest in the mouse egg of the B A L B / c strain
Pregnant superovulated female mice of the B A L B / c strain were irradiated with 1 Gy of X-rays at hourly intervals during the first cellular cycle of the eggs. Embryos were flushed out of the oviducts on the following day, and classified into uncleaved or two-cell embryos. Two-cell embryos were cultured for 4 days and examined at the end of this period for their development into blastocysts. Two types of effects were found, depending on the time of X-irradiation. When irradiation was delivered between 2 and 10 h after superovulation, cultured 2-cell embryos developed normally up to the morula stage, where a high mortality occurred. On the other hand, when irradiation was given between 5 and 12 h after superovulation (i.e. at the onset of the first D N A synthesis), a high propor-