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Synthesis of pure dinitropyrenes
363 cant change in the 1H-NMR spectra of quercetin treated with the different-pH media was seen. These results suggest that pre-exposure at various pH values may play a role in the mutagenicity and/or the carcinogenicity of this type of compound.
7 Hachiya, N., M. Sato and Y. Takizawa, Akita University School of Medicine, Akita (Japan) Detection of DNA damage in mutagen-treated mammalian tissues by alkaline elution assay DNA strand breaks were determined by alkaline elution assay in mammalian tissues after administration of acrylonitrile or ethylene dibromide. It has been reported that acrylonitrile and ethylene dibromide induce no chromosome aberrations, micronuclei or dominant lethals, in spite of their apparent mutagenicity and carcinogenicity. The liver or brain was excised from mice or rats 2 h after intraperitoneal injection of the agents. Alkaline elution was carried out with the nuclei isolated from these tissues according to the method of Kohn. Eluted DNA was determined microfluorometrically as described by Bradley et al. DNA single strand breaks were induced in the liver of female rats by acrylonitrile, but not detected in the brain, one of the target tissues of acrylonitrile tumorigenesis. The alkali-labile site was formed in liver of mice after ethylene dibromide treatment. The alkaline elution assay is useful to evaluate the certain types of DNA damage induced in vivo.
8 Haresaku, M., M. Muramatsu, T. Matsushima, T. Takeuchi 1 and H. Umezawa 1, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo and 1 Institute of Microbial Chemistry, Tokyo (Japan) Mutagenicities of bleomyein Salmonella typhimurium TA 102
derivatives
on
Mutagenicities of 14 bleomycin derivatives were
tested on Salmonella typhimurium TA102 by the preincubation method (30 o C, 30 min), without the metabolic activation system. 12 derivatives having different structures of terminal amine showed specific mutagenicities of 1.6-20 × 10 s His ÷/mg. Bleomycin A 2, B2, B4 and A 5 (all metal-free compounds) showed specific mutagenicities of 2.5, 2.7, 5.8 and 10 × 105 His÷/mg, respectively. Bleomycin A 2 (Cu 2+ compound) showed 7.7 × 105 His ÷/mg and Cu 2÷ ion enhanced its mutagenicity and cytotoxicity. Bleomycin derivatives having chemically modified terminal amine structures had the strongest mutagenicities (14-20 x 10 5 His +/mg). Bleomycin acid having an OH group instead of a terminal amine group showed weaker mutagenicity of 4.6 × 10 4 His+/mg. The desamido derivative showed the weakest mutagenicity (7.9 × 103 His÷/mg) and removal of the amido group decreased its mutagenicity to one-fortieth of that of the parent compound.
9 Hashimoto, Y., and K. Shudo, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo (Japan) Synthesis of pure dinitropyrenes Pure isomers of dinitropyrene (1,3-, 1,6- and 1,8-dinitropyrenes) without contamination of other isomers were synthesized from the corresponding diaminopyrenes by two methods, A and B. Pyrene was nitrated with HNO 3 in CH3COOH to give a mixture of 1,3-, 1,6- and 1,8-dinitropyrenes which was reduced with NaSH in aqueous EtOH to yield a mixture of 1,3-, 1,6- and 1,8-diaminopyrenes. Each isomer of diaminopyrene was purified with silica gel column chromatography. Method A: A purified isomer of diaminopyrene was oxidized in a mixture of CH3CN, H202 and Na2WO4 to give the pure corresponding isomer of dinitropyrene. Method B: A purified isomer of diaminopyrene was diazotized with NaNO 2 and then treated with excess NaNO 2 and CuSO4 in water to give the pure isomer of corresponding dinitropyrene. The purity of each isomer of the dinitropyrenes was
364 estimated to be not less than 99.95% from high performance liquid chromatography.
10 Hayashi, M., T. Sofuni and M. Ishidate Jr., National Institute of Hygienic Sciences, Tokyo (Japan) Mechanism of micronuclei formation in mouse bone marrow
The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with mitomycin C, 6-mercaptopurine and 1-fl-D-arabinofuranosylcytosine were compared and subjected to the simulation study. Two parameters, the time between the final mitosis of the erythroid series and nucleus expulsion, and the duration of the polychromatic erythrocyte stage in the bone marrow were almost identical for the 3 chemicals. However, the coefficient of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) differed depending on the type of chromosomal aberrations induced by these chemicals. The DNA content of micronuclei was also compared to the length of acentric fragments induced by 1-fl-D-arabinofuranosylcytosine and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by chemicals are essential events for the induction of micronuclei in the polychromatic erythrocytes of mouse bone marrow.
11 Hayatsu, H., Y. Ohara, A. Murano 1 and K. Nakai 1, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, and 1 Osaka Research Laboratory, Sumitomo Chemical Co., Osaka (Japan) Characterization of a mutagen that was present in blue chalks as impurity
Previously, w e r e p o r t e d that the methanolic ex-
tract of blue chalks showed mutagenicity in
Salmonella typhimurium TA98 in the presence of $9, and that the mutagenic substance was an impurity in the blue pigment used for manufacturing the chalks (Hayatsu, H. et al. (1983) Mutation Res., 124, 1-7). As a result of this finding, the blue chalks presently in the market are no longer mutagenic. We have now purified this mutagen starting from 5 kg of the crude blue pigment (copper phthalocyanine). The material obtained (1.9 mg) was analyzed by mass spectroscopy, 1H-NMR, and 13C-NMR, and the structure was deduced to be 1,2,3-triazolo[1,5-c]quinazolin-l,5-diamine. The mutagenic activity of this compound, as measured on S. typhimurium TA98 ( + $9), was 2800 His + revertant colonies//~g.
12 Hisamatsu, Y., T. Nishimura, T. Shiozaki and H. Matsushita, Institute of Public Health, Tokyo (Japan) Mutagen formation by the photochemical reaction of pyrene with nitrogen dioxide
The photochemical reaction of pyrene with nitrogen dioxide (NO 2) has been studied by irradiation with a high-pressure mercury lamp in nitrogen atmosphere and in oxygen atmosphere. Pyrene dissolved in acetonitrile-ether (1 : 1) was deposited on a quartz fiber filter. Mutagenic activity of the product was measured in Salmonella typhimurium TA98 with and without $9 mix. In the absence of NO 2, the irradiation product in the nitrogen atmosphere was not mutagenic, but was mutagenic in the oxygen atmosphere. The product in the presence of 10 ppm of NO: in the oxygen atmosphere was far more mutagenic than in the nitrogen atmosphere. The mutagenicity of the products became greater in proportion to the NO 2 concentration and the irradiation time, but decreased when the irradiation time was longer than about 150 min. Formation of 1-nitropyrene was confirmed by HPLC.
7 Hachiya, N., M. Sato and Y. Takizawa, Akita University School of Medicine, Akita (Japan) Detection of DNA damage in mutagen-treated mammalian tissues by alkaline elution assay DNA strand breaks were determined by alkaline elution assay in mammalian tissues after administration of acrylonitrile or ethylene dibromide. It has been reported that acrylonitrile and ethylene dibromide induce no chromosome aberrations, micronuclei or dominant lethals, in spite of their apparent mutagenicity and carcinogenicity. The liver or brain was excised from mice or rats 2 h after intraperitoneal injection of the agents. Alkaline elution was carried out with the nuclei isolated from these tissues according to the method of Kohn. Eluted DNA was determined microfluorometrically as described by Bradley et al. DNA single strand breaks were induced in the liver of female rats by acrylonitrile, but not detected in the brain, one of the target tissues of acrylonitrile tumorigenesis. The alkali-labile site was formed in liver of mice after ethylene dibromide treatment. The alkaline elution assay is useful to evaluate the certain types of DNA damage induced in vivo.
8 Haresaku, M., M. Muramatsu, T. Matsushima, T. Takeuchi 1 and H. Umezawa 1, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Tokyo and 1 Institute of Microbial Chemistry, Tokyo (Japan) Mutagenicities of bleomyein Salmonella typhimurium TA 102
derivatives
on
Mutagenicities of 14 bleomycin derivatives were
tested on Salmonella typhimurium TA102 by the preincubation method (30 o C, 30 min), without the metabolic activation system. 12 derivatives having different structures of terminal amine showed specific mutagenicities of 1.6-20 × 10 s His ÷/mg. Bleomycin A 2, B2, B4 and A 5 (all metal-free compounds) showed specific mutagenicities of 2.5, 2.7, 5.8 and 10 × 105 His÷/mg, respectively. Bleomycin A 2 (Cu 2+ compound) showed 7.7 × 105 His ÷/mg and Cu 2÷ ion enhanced its mutagenicity and cytotoxicity. Bleomycin derivatives having chemically modified terminal amine structures had the strongest mutagenicities (14-20 x 10 5 His +/mg). Bleomycin acid having an OH group instead of a terminal amine group showed weaker mutagenicity of 4.6 × 10 4 His+/mg. The desamido derivative showed the weakest mutagenicity (7.9 × 103 His÷/mg) and removal of the amido group decreased its mutagenicity to one-fortieth of that of the parent compound.
9 Hashimoto, Y., and K. Shudo, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo (Japan) Synthesis of pure dinitropyrenes Pure isomers of dinitropyrene (1,3-, 1,6- and 1,8-dinitropyrenes) without contamination of other isomers were synthesized from the corresponding diaminopyrenes by two methods, A and B. Pyrene was nitrated with HNO 3 in CH3COOH to give a mixture of 1,3-, 1,6- and 1,8-dinitropyrenes which was reduced with NaSH in aqueous EtOH to yield a mixture of 1,3-, 1,6- and 1,8-diaminopyrenes. Each isomer of diaminopyrene was purified with silica gel column chromatography. Method A: A purified isomer of diaminopyrene was oxidized in a mixture of CH3CN, H202 and Na2WO4 to give the pure corresponding isomer of dinitropyrene. Method B: A purified isomer of diaminopyrene was diazotized with NaNO 2 and then treated with excess NaNO 2 and CuSO4 in water to give the pure isomer of corresponding dinitropyrene. The purity of each isomer of the dinitropyrenes was
364 estimated to be not less than 99.95% from high performance liquid chromatography.
10 Hayashi, M., T. Sofuni and M. Ishidate Jr., National Institute of Hygienic Sciences, Tokyo (Japan) Mechanism of micronuclei formation in mouse bone marrow
The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with mitomycin C, 6-mercaptopurine and 1-fl-D-arabinofuranosylcytosine were compared and subjected to the simulation study. Two parameters, the time between the final mitosis of the erythroid series and nucleus expulsion, and the duration of the polychromatic erythrocyte stage in the bone marrow were almost identical for the 3 chemicals. However, the coefficient of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) differed depending on the type of chromosomal aberrations induced by these chemicals. The DNA content of micronuclei was also compared to the length of acentric fragments induced by 1-fl-D-arabinofuranosylcytosine and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by chemicals are essential events for the induction of micronuclei in the polychromatic erythrocytes of mouse bone marrow.
11 Hayatsu, H., Y. Ohara, A. Murano 1 and K. Nakai 1, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, and 1 Osaka Research Laboratory, Sumitomo Chemical Co., Osaka (Japan) Characterization of a mutagen that was present in blue chalks as impurity
Previously, w e r e p o r t e d that the methanolic ex-
tract of blue chalks showed mutagenicity in
Salmonella typhimurium TA98 in the presence of $9, and that the mutagenic substance was an impurity in the blue pigment used for manufacturing the chalks (Hayatsu, H. et al. (1983) Mutation Res., 124, 1-7). As a result of this finding, the blue chalks presently in the market are no longer mutagenic. We have now purified this mutagen starting from 5 kg of the crude blue pigment (copper phthalocyanine). The material obtained (1.9 mg) was analyzed by mass spectroscopy, 1H-NMR, and 13C-NMR, and the structure was deduced to be 1,2,3-triazolo[1,5-c]quinazolin-l,5-diamine. The mutagenic activity of this compound, as measured on S. typhimurium TA98 ( + $9), was 2800 His + revertant colonies//~g.
12 Hisamatsu, Y., T. Nishimura, T. Shiozaki and H. Matsushita, Institute of Public Health, Tokyo (Japan) Mutagen formation by the photochemical reaction of pyrene with nitrogen dioxide
The photochemical reaction of pyrene with nitrogen dioxide (NO 2) has been studied by irradiation with a high-pressure mercury lamp in nitrogen atmosphere and in oxygen atmosphere. Pyrene dissolved in acetonitrile-ether (1 : 1) was deposited on a quartz fiber filter. Mutagenic activity of the product was measured in Salmonella typhimurium TA98 with and without $9 mix. In the absence of NO 2, the irradiation product in the nitrogen atmosphere was not mutagenic, but was mutagenic in the oxygen atmosphere. The product in the presence of 10 ppm of NO: in the oxygen atmosphere was far more mutagenic than in the nitrogen atmosphere. The mutagenicity of the products became greater in proportion to the NO 2 concentration and the irradiation time, but decreased when the irradiation time was longer than about 150 min. Formation of 1-nitropyrene was confirmed by HPLC.