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Metabolic activation of hydroxamic acid to the ultimate mutagen
263
separately from that by the alkylating chemical, mitomycin C (MMC)when human lymphocytes were exposed to these two agents in combination. Whole-blood cultures of human lymphocytes in G O phase were exposed to ~,-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA) and sister-chromatid exchanges (SCEs) in short term cultures. Different culture times were used, i.e., 56 h for CA and 72 h for SCE. The frequency of chromosome-type aberrations (dicentric and ring) increased with increasing doses of ~,-rays. The dose-response relationships were the same with or without concomitant treatment with MMC. Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and T-rays. There was no mutual effect between MMC and ',/-rays concerning these two endpoints. 35 Ishihara, Y., S. Igarashi, K. Nakajima and S. Sakamoto, Shiraoka Research Station of Biological Science, Nissan Chemical Industries, Ltd., 1470, Shiraoka, Minami-saitama, Saitama 349-02 (Japan) An investiption of mutagenlelty of resorcinol using Drosophila eytogenetle assays Resorcinol (RES) is clastogenic in human peripheral lymphocytes (HPL) and CHL cells, but never in CHO cells. To investigate the cause of the difference, we used the Drosophila wing spot test (WST) developed by Wtirgler and his coworkers and the DNA-repair test (RecT) developed by Fujikawa. In these test, RES was not mutagenic at a concentration of 10 mg/ml. While HPL, CHL cells and CHO cells were treated with RES for 24 h at concentrations of 250 and 500 ~ g / m l , Drosophila was treated with each treatment medium. In WST the frequencies of wing spots were significantly increased by the treatment medium of HPL and CHL cells at the concentration of 500 ~g/ml, but never by that of CHO cells. In RecT, the sex ratio (male/female) was decreased by the medium of HPL and CHL cells at the concentration of 500 ~ g / m l but not
significantly by any medium. These results reflected those in chromosomal aberration assays. Therefore we suggest RES might be metabolized in HPL and CHL cells and these metabolites be mutagenic. On one hand the possibility remains that the mutation might be induced by aa active oxygen species followed by cell death, based on the fact that chromosomal aberrations appeared in CHL cells only at a severely cytotoxic concentration. 36 Ishii, Y., Y. Samejima, F. Saji and T. Nomura, Faculty of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565 (Japan) Differential sensitivity of mouse and human sperm to surfaetants The fertility of mouse sperm was estimated by scoring swollen heads of sperm which penetrated mouse eggs. 0.01% and 0.02% LAS (linear alkylbenzene sulfonate) reduced the fertility to 80% and 0% of that of untreated sperm, respectively. Natural soap did not show any effect up to 0.05%. These findings are similar to the effect of these surfactants on the development of fertilized mouse eggs. The fertility of human sperm can be estimated by means of Syrian hamster eggs. 0.01% and 0.02% LAS reduced the fertility of human sperm to 70% and 0%, respectively, it is noteworthy that natural soap also reduced the fertility of human sperm and no sperm fertility was observed when 0.06% soap was used. Thus human sperm was more sensitive to natural soap than mouse sperm, which might show that the fertilized human egg could be sensitive to natural soap. 37 lsobe, M., I. Ueki and E. Takabatake, Department of Toxicology., Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotogecho, Hirakata, Osaka 573-0! (Japan) Metabolic activation of hydroxamic acid to the ultimate mutagen Hydroxamic acid (R-CO-NH-OH) shows mutagenic activity toward Salmonella typhimurium TA
264 strains without $9 mix. It has been reported that isocyanate generated by the O-acetylation of hydroxamic acid and the following Lossen rearrangement in the bacterial cells may be an ultimate mutagen. In this study, we examined whether the proposed mechanism is involved in the bacterial mutagenesis. No significant relationship between the decomposition rate to isocyanate and the mutagenicity was found in benzohydroxamic acid acyl derivatives or in para-substituted benzohydroxamic acid O-acetates. 2-Naphthohydroxamic acid (NHA) showed strong mutagenic activity toward S. typhimurium TA97, TA98, TA98NR, TA98 1,8-DNP6, and YG1024. Though NHA O-acetate (NHAOAc) induced 3-5 times more His + revertants than NHA in the plate incorporation test, it was found that NHA is more mutagenic than NHAOAc in a preincubation experiment. The bacterial metabolic disappearance of NHA and NHAOAc in the preincubation mixture (2 /zg each) was 6% and 18%, respectively. Pentachlorophenol and 2,4-dinitrophenol dose-dependently inhibited the mutagenesis by NHA, but not by NHAOAc. Hydro~lamine reduced the mutagenic activity of both NHA and NHAOAc. These results indicate that the ultimate mutagenie species of hydroxamic acid is an acyl derivative(s) other than O-acetate or isocyanatc. 38 Itoh, S., C. Hattori, Y. Matsuura and H. Shimada, Developmental Research Laboratories, Daiichi Pharmaceutical Co. Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134 (Japan)
Micronucleus induction of metal compounds Many heavy metal compounds are known to have mutagenic effects on the chromosomes of cultured mammalian cells and carcinogenicity has also been reported in some metals such as chromium, selenium, nickel, cobalt, arsenic, beryllium and lead. We studied the in vivo clastogenicity in the mouse micronucleus test of various metal compounds: chromium (K2CrO4, CrCla), manganese (MnCI 2, MnSO4), selenium (H, SeO a, Na2SeO4) and nickel (NiCl2, NiSO4).
Metal compounds were injected intraperitoneally once or twice to ddY male mice, then the polychromatic erythrocytes (PCEs) from the bone marrow were examined for micronucleus induction. Significant increases of micronucleated PCEs (MNPCEs) were observed only in the group of animals treated with K2CrO4 and HeSeO 3. In these compounds, the incidence of MNPCEs seemed to be reduced by double dosing. In addition, the incidences of MNPCEs were significantly decreased by pretreatment with Bi(NO3)3, a potent inducer of metaUothionein. These results suggest that the mechanism of micronucleus induction of the metal compounds involves a participation of metallothionein in the bone marrow.
39 lwakura, K. ~, H. Tamura ~, A. Matsumoto ~, Y. Yamashita ~, S. Ajimi e, S. Ogura 2, K. Kakimoto a, T. Matsumoto 2 and M. Hayashi 3, ~ Toxicological Laboratories, Nippon Shinyaku Co., Ltd., Kyoto 601, e Chemicals Inspection & Testing Institute, Japan, Chemical Biotesting Center, Hita 877, Oita and 3 Division of Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, Tokyo 158 (Japan)
The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-13-D-arabinofuranosylcytosine(ara-C) Previous studies with ara-C have indicated that the maximum frequencies of micronucleated reticulocytes (MNRETs) in peripheral blood lagged about 24 h behind those of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow. To more closely estimate the transition time from MNPCEs to MNRETs, we made precise time course experiments with 12-h intervals. The frequencies of MNPCEs peaked about 24 h and those of MNRETs about 36 h after i.p. treatment. It seems likely, therefore, that the transition time is closer to 12 h than 24 h. The concentrations of ara-C in plasma peaked at 0.5 h after i.p. treatment with 25 and 50 mg/kg and the chemical disappeared within 3 h. These results
separately from that by the alkylating chemical, mitomycin C (MMC)when human lymphocytes were exposed to these two agents in combination. Whole-blood cultures of human lymphocytes in G O phase were exposed to ~,-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA) and sister-chromatid exchanges (SCEs) in short term cultures. Different culture times were used, i.e., 56 h for CA and 72 h for SCE. The frequency of chromosome-type aberrations (dicentric and ring) increased with increasing doses of ~,-rays. The dose-response relationships were the same with or without concomitant treatment with MMC. Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and T-rays. There was no mutual effect between MMC and ',/-rays concerning these two endpoints. 35 Ishihara, Y., S. Igarashi, K. Nakajima and S. Sakamoto, Shiraoka Research Station of Biological Science, Nissan Chemical Industries, Ltd., 1470, Shiraoka, Minami-saitama, Saitama 349-02 (Japan) An investiption of mutagenlelty of resorcinol using Drosophila eytogenetle assays Resorcinol (RES) is clastogenic in human peripheral lymphocytes (HPL) and CHL cells, but never in CHO cells. To investigate the cause of the difference, we used the Drosophila wing spot test (WST) developed by Wtirgler and his coworkers and the DNA-repair test (RecT) developed by Fujikawa. In these test, RES was not mutagenic at a concentration of 10 mg/ml. While HPL, CHL cells and CHO cells were treated with RES for 24 h at concentrations of 250 and 500 ~ g / m l , Drosophila was treated with each treatment medium. In WST the frequencies of wing spots were significantly increased by the treatment medium of HPL and CHL cells at the concentration of 500 ~g/ml, but never by that of CHO cells. In RecT, the sex ratio (male/female) was decreased by the medium of HPL and CHL cells at the concentration of 500 ~ g / m l but not
significantly by any medium. These results reflected those in chromosomal aberration assays. Therefore we suggest RES might be metabolized in HPL and CHL cells and these metabolites be mutagenic. On one hand the possibility remains that the mutation might be induced by aa active oxygen species followed by cell death, based on the fact that chromosomal aberrations appeared in CHL cells only at a severely cytotoxic concentration. 36 Ishii, Y., Y. Samejima, F. Saji and T. Nomura, Faculty of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565 (Japan) Differential sensitivity of mouse and human sperm to surfaetants The fertility of mouse sperm was estimated by scoring swollen heads of sperm which penetrated mouse eggs. 0.01% and 0.02% LAS (linear alkylbenzene sulfonate) reduced the fertility to 80% and 0% of that of untreated sperm, respectively. Natural soap did not show any effect up to 0.05%. These findings are similar to the effect of these surfactants on the development of fertilized mouse eggs. The fertility of human sperm can be estimated by means of Syrian hamster eggs. 0.01% and 0.02% LAS reduced the fertility of human sperm to 70% and 0%, respectively, it is noteworthy that natural soap also reduced the fertility of human sperm and no sperm fertility was observed when 0.06% soap was used. Thus human sperm was more sensitive to natural soap than mouse sperm, which might show that the fertilized human egg could be sensitive to natural soap. 37 lsobe, M., I. Ueki and E. Takabatake, Department of Toxicology., Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotogecho, Hirakata, Osaka 573-0! (Japan) Metabolic activation of hydroxamic acid to the ultimate mutagen Hydroxamic acid (R-CO-NH-OH) shows mutagenic activity toward Salmonella typhimurium TA
264 strains without $9 mix. It has been reported that isocyanate generated by the O-acetylation of hydroxamic acid and the following Lossen rearrangement in the bacterial cells may be an ultimate mutagen. In this study, we examined whether the proposed mechanism is involved in the bacterial mutagenesis. No significant relationship between the decomposition rate to isocyanate and the mutagenicity was found in benzohydroxamic acid acyl derivatives or in para-substituted benzohydroxamic acid O-acetates. 2-Naphthohydroxamic acid (NHA) showed strong mutagenic activity toward S. typhimurium TA97, TA98, TA98NR, TA98 1,8-DNP6, and YG1024. Though NHA O-acetate (NHAOAc) induced 3-5 times more His + revertants than NHA in the plate incorporation test, it was found that NHA is more mutagenic than NHAOAc in a preincubation experiment. The bacterial metabolic disappearance of NHA and NHAOAc in the preincubation mixture (2 /zg each) was 6% and 18%, respectively. Pentachlorophenol and 2,4-dinitrophenol dose-dependently inhibited the mutagenesis by NHA, but not by NHAOAc. Hydro~lamine reduced the mutagenic activity of both NHA and NHAOAc. These results indicate that the ultimate mutagenie species of hydroxamic acid is an acyl derivative(s) other than O-acetate or isocyanatc. 38 Itoh, S., C. Hattori, Y. Matsuura and H. Shimada, Developmental Research Laboratories, Daiichi Pharmaceutical Co. Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134 (Japan)
Micronucleus induction of metal compounds Many heavy metal compounds are known to have mutagenic effects on the chromosomes of cultured mammalian cells and carcinogenicity has also been reported in some metals such as chromium, selenium, nickel, cobalt, arsenic, beryllium and lead. We studied the in vivo clastogenicity in the mouse micronucleus test of various metal compounds: chromium (K2CrO4, CrCla), manganese (MnCI 2, MnSO4), selenium (H, SeO a, Na2SeO4) and nickel (NiCl2, NiSO4).
Metal compounds were injected intraperitoneally once or twice to ddY male mice, then the polychromatic erythrocytes (PCEs) from the bone marrow were examined for micronucleus induction. Significant increases of micronucleated PCEs (MNPCEs) were observed only in the group of animals treated with K2CrO4 and HeSeO 3. In these compounds, the incidence of MNPCEs seemed to be reduced by double dosing. In addition, the incidences of MNPCEs were significantly decreased by pretreatment with Bi(NO3)3, a potent inducer of metaUothionein. These results suggest that the mechanism of micronucleus induction of the metal compounds involves a participation of metallothionein in the bone marrow.
39 lwakura, K. ~, H. Tamura ~, A. Matsumoto ~, Y. Yamashita ~, S. Ajimi e, S. Ogura 2, K. Kakimoto a, T. Matsumoto 2 and M. Hayashi 3, ~ Toxicological Laboratories, Nippon Shinyaku Co., Ltd., Kyoto 601, e Chemicals Inspection & Testing Institute, Japan, Chemical Biotesting Center, Hita 877, Oita and 3 Division of Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, Tokyo 158 (Japan)
The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-13-D-arabinofuranosylcytosine(ara-C) Previous studies with ara-C have indicated that the maximum frequencies of micronucleated reticulocytes (MNRETs) in peripheral blood lagged about 24 h behind those of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow. To more closely estimate the transition time from MNPCEs to MNRETs, we made precise time course experiments with 12-h intervals. The frequencies of MNPCEs peaked about 24 h and those of MNRETs about 36 h after i.p. treatment. It seems likely, therefore, that the transition time is closer to 12 h than 24 h. The concentrations of ara-C in plasma peaked at 0.5 h after i.p. treatment with 25 and 50 mg/kg and the chemical disappeared within 3 h. These results