Search PubMed⌕ Search

Biomedical subjects

T Sofuni

Publications and source records attributed to T Sofuni.

At least 73 records · Page 4Linked to original sources
PubMed1997

Agreement of mutational characteristics of heterocyclic amines in lacI of the Big Blue mouse with those in tumor related genes in rodents.

The mutational spectra of carcinogenic heterocyclic amines (HCAs), 2-amino-3,4-dimethylimidazo[4,5-b]quinoline (MeIQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alphaC) were studied in the colon of Big Blue mice. In 90, 115 and 105 lacI mutants from mice fed 300 p.p.m. MeIQ, 400 p.p.m. PhIP and 800 p.p.m. A alphaC, respectively, 92, 115 and 105 mutations were identified. G:C-->T:A transversions predominated with these HCAs. Mutational hot spots for base-substitution mutations caused by MeIQ, PhIP and A alphaC were in distinct sequence contexts; at 5'-GC-3', in runs of guanine and in 5'-CGT-3', respectively. Further, 30 of 115 (26%) PhIP-induced mutations were G:C base pair deletions, and eight of these deletions were in 5'-GGGA-3'. The mutational characteristics of MeIQ in the lacI gene coincided well with those in the Ha-ras gene of MeIQ-induced mouse forestomach tumors and rat Zymbal gland tumors. The characteristic single-base deletion induced by PhIP in the lacI gene also coincided well with those in the Apc gene of PhIP-induced rat colon tumors. These results suggest that the mutational characteristics of each chemical are conserved across different genes in different species.

Animals↗
PubMed1997

Aneuploidy induction by benzo[a]pyrene and polyploidy induction by 7,12-dimethylbenz[a]anthracene in Chinese hamster cell lines V79-MZ and V79.

We found that different ploidy effects were induced in four Chinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1) treated through two cell cycles with polycyclic aromatic hydrocarbons in the absence of a metabolic activation system. 5-Bromodeoxyuridine was used to investigate cell cycle delay and sister chromatid exchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP) induced aneuploidy at 2.5-10 micrograms/ml in V79-MZ cells. 7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at 3.125-6.25 and 6.25-1.25 micrograms/ml in V79-MZ and V79 cells respectively. Higher concentrations caused cell cycle delay and, therefore, did not affect ploidy. BP and DMBA did not induce a significant increase in SCE frequency at the above doses. 3-Methylcholanthrene tested up to its solubility limit (10 micrograms/ml) did not induce numerical aberrations in any cell line. The clastogen mitomycin C, tested up to 0.01 microgram/ml, did not produce numerical aberrations but did significantly increase SCE frequency in all cell lines. The spindle poison colchicine, tested up to 0.1 microgram/ml, induced ploidy changes in the four cell lines that showed different sensitivities. Four cell lines showed no arylhydrocarbon hydroxylase activity, and V79-MZ, but not the other cells lines, showed high glutathione S-transferase activity. Aneuploidy induction by BP and polyploidy induction by DMBA in the absence of S9 mix in vitro have not been described before, and the finding might be due to the effect on tubulin. Due to their specificity and high sensitivity, the V79-MZ and V79 cell lines might be good systems for detecting aneuploidogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells.

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.

Alleles↗
PubMed1997

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

Animals↗
PubMed1997

X-ray- and ultraviolet-radiation-induced mutations in Muta mouse.

To evaluate the usefulness of the transgenic Muta mouse for investigating radiation-induced mutations in vivo, we have examined the effects of whole-body X irradiation and compared them to the effects of ultraviolet light. The spontaneous mutation frequencies in young adults were about 7 x 10(-5) in the spleen, liver and skin. The mutation frequencies 1 week after a lethal dose of X radiation (8 Gy) were 3.2, 2.6 and 2.7 times the spontaneous levels in the spleen, liver and skin, respectively. When the skin was irradiated with 10 kJ m-2 of UVB, the mutation frequency increased about 6 times. The mutation frequencies induced by an acute dose of 4 Gy or by a fractionated dose of 11.7 Gy (0.15 Gy x 78 times, 3 times/week) in the spleen and liver were less than 2-fold the spontaneous levels at 16 weeks after irradiation. A comparison of X and UV radiation was also conducted with cultured cells derived from the mouse embryo. UVC of 5 J m-2 raised the mutation frequency to 15 times that of unirradiated cells, while 10 Gy X rays raised it 2.6 times. The findings indicate that the Muta mouse is less sensitive to X-ray-induced mutation than UV-induced mutation.

Animals↗
PubMed1996

Chromosome painting analysis of spontaneous and methyl methanesulfonate-induced trifluorothymidine-resistant L5178Y cell colonies.

Spontaneous and methyl methanesulfonate-induced trifluorothymidine-resistant mutants in mouse lymphoma L5178Y cells were analyzed using fluorescence in situ hybridization with mouse probes specific for chromosome 11, on which the tk gene is located, and chromosome 3, as the control. 76.5% (13/17) of small-colony mutants (thought to be the result of chromosomal mutation) and 28.6% (4/14) of large-colony mutants (thought to be the result of gene mutation) showed rearranged chromosome 11. Of the mutants with abnormal chromosome 11 painting pattern, 5 small- and 2 large-colony mutants carried clonal aberrations, while the remaining 8 small- and 2 large-colony mutants showed mosaic aberrations. Most abnormalities in the small-colony mutants involved the distal region of one painted chromosome 11, where the tk+ gene maps. An increase, rather than a decrease, in chromosome 11 material was found in a majority of abnormally painted mutants. On the contrary, no rearrangements involving chromosome 3 were found in any small- and large-colony mutants analyzed except one large-colony mutant, which showed chromosome rearrangements involving both chromosome 11 and 3. The present study confirms that the majority of small-colony mutants in L5178Y cells have chromosome 11 rearrangements that can be detected by chromosome painting and that the majority of the chromosomal abnormalities in TFT-resistant mutants involved complex rearrangements.

Animals↗
PubMed1996

Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

Animals↗
PubMed1996

Construction of a new system for separate expression of mutagenesis proteins: the abilities to promote UV mutagenesis and interchangeability of MucA', MucB, SamA' and SamB proteins in Salmonella typhimurium.

The two distinct mucAB and samAB operons originally isolated from the plasmids of Salmonella typhimurium encode proteins engaged in induced mutagenesis. They represent two extreme cases among the so far characterized members of the enterobacterial umuDC family in respect to both the strength and the specificity of their effect. It is suggested that the MucA and SamA proteins are post-translationally processed to MucA' and SamA', respectively, which lack the N-terminal 25 amino acids and are the active species in mutagenesis. For the purpose of characterizing the individual activities of these proteins, we developed a new system for their SOS-independent separate and controllable expression in enterobacteria. Besides the matured forms of MucA', SamA' as well as MucB and SamB proteins we also expressed hybrid HisTag-MucA' and HisTag-SamA' proteins in which a synthetic 24 amino acid HisTag region replaces the natural 25 amino acid N-terminal leader present in the MucA and SamA precursors. In this study, we analyzed the effect of the mutagenesis proteins on the UV mutability of S. typhimurium YG5144. None of the proteins, if expressed alone, promoted UV mutagenesis. Different combinations of the proteins promoted mutagenesis to different extents in the order MucA' + MucB > SamA' + SamB > or = HisTag-MucA' + MucB > or = SamA' + MucB > MucA' + SamB > HisTag-SamA' + SamB. The mutagenesis enhancing potential of the combinations with MucB protein decreased as the expression of the proteins increased while the mutagenesis enhancing potential of the combinations with SamB protein increased together with the increase in the expression. The artificially expressed MucA' + MucB proteins were as active as their MucAB counterparts expressed from the plasmid pKM101 in promoting UV mutagenesis, but they were remarkably more efficient than their pKM101-born counterparts in promoting spontaneous mutagenesis. We conclude that the MucA'B and SamA'B proteins are partly interchangeable and the functionality of the resulting A' + B complex is largely dependent on the appropriate B-protein.

Amino Acid Sequence↗
PubMed1996

Organ variation in the mutagenicity of MeIQ in Big Blue lacI transgenic mice.

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which is a heterocyclic amine found in food and the potent mutagen in S. typhimurium TA98, was examined for its genotoxic potential using lacI transgenic mice (Big Blue, C57BL/6N lineage). Female mice, at 7 weeks of age, were given a diet containing 0.03% MeIQ for 1, 4 and 12 weeks, and mutant frequencies (MF) were analyzed in the bone marrow, liver, forestomach, colon and heart. The MF increased in a feeding period-dependent manner. Relative to untreated mice, the MF after a 12-week-feeding of MeIQ was 38 times higher in the colon, 5.8 times higher in the bone marrow, 4.6 times higher in the liver, and 2.6 times higher in the forestomach. No increase in MF was detected in the heart, where no tumors develop.

Animals↗
PubMed1996

IPCS harmonization of methods for the prediction and quantification of human carcinogenic/mutagenic hazard, and for indicating the probable mechanism of action of carcinogens.

A flow chart is presented as a recommended sequence of tests to predict the carcinogenic hazard, and to predict and quantify the mutagenic hazard to germ cells of chemicals to humans. Ten associated principles of testing for these endpoints are also suggested. These recommendations are the result of a meeting convened under the auspices of the International Programme on Chemical Safety (IPCS), as part of their project on 'Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals'. The meeting was held at Carshalton, Surrey, from 13-17 February 1995.

Animals↗
PubMed1996

Organ variation in the mutagenicity of dimethylnitrosamine in Big Blue mice.

Organ specificity in the lacI mutant frequency (MF) induced by dimethylnitrosamine (DMN) was analyzed in lung, liver, kidney, bone marrow, urinary bladder, and testis of Big Blue mice. Cell proliferative activity was also analyzed in some of these tissues by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Clastogenicity of DMN was concomitantly analyzed by the peripheral blood micronucleus assay with the same animals used for the lacI mutation assay. Five daily intraperitoneal (i.p.) treatments with DMN (1 mg/kg) increased MF in liver (6.2 x control), kidney (2.4 x control), and lung (2.1 x control). These are known target organs for DMN carcinogenesis. No MF increase was observed in nontarget organs studied, i.e., bone marrow, bladder, and testis. Single ip treatment with DMN also increased lacI MF in liver but the increases were smaller than in a 5-daily-treatment regimen. This result suggests that multiple dosing is more effective in the transgenic mutation assay. The enhancement of cell proliferation observed was in bronchial epithelia 7 days after treatment. No micronucleus induction in peripheral blood was observed 24 hours after 2 and 3 daily ip treatments with 1 mg/kg DMN. An increase in the incidence of micronucleated reticulocytes in peripheral blood was observed 48 hours after single ip treatment with 5 or 10 mg/kg DMN. The present study demonstrated organ-specific induction of gene mutations by DMN which suggests a relevance of this assay for the prediction of organ-specific carcinogenesis.

Animals↗
PubMed1996

Initial consideration for use of transgenic mutation assays in a regulatory submission.

As an initial consideration for use of TM assays in a regulatory submission, we focus here especially on the following questions: (1) What conditions would trigger the performance of a TM assay? (2) What data set is sufficient to support a negative result? and (3) What increase in mutation frequency is considered to be a positive result? In the first question, we discuss outcomes from traditional tests such as RM, CA, GM, and MN assays. In the second question, we propose a provisional data set including several items, i.e., experimental size [number of animals per group, number of plaques per animal (tissue)], tissue selection, route of administration, top dose level, number of dosings (single vs. multiple), sampling time, number of samples, dose levels, and positive and negative controls. Some statistical and biological considerations are needed for the third question.

Animals↗
PubMed1996

A new transgenic mouse mutagenesis test system using Spi- and 6-thioguanine selections.

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020-expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol-resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chloramphenicol and 6-thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild-type phages display Spi- (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi-. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 x 10(-5) in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol-resistant colonies per 7.5 micrograms bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi- mutation frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and sperm, respectively. No spontaneous Spi- mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma-ray-treated animals, however, induction of Spi- mutations was clearly observed in spleen, at frequencies of 1.4 x 10(-5) (5 Gy), 1.2 x 10(-5) (10 Gy), and 2.0 x 10(-5) (5O Gy). These results suggest that the new transgenic mouse "gpt delta" could be useful for the efficient detection of point mutations and deletion mutations in vivo.

Animals↗
PubMed1996

Detection of in vitro clastogens and spindle poisons by the mouse lymphoma assay using the microwell method: interim report of an international collaborative study.

Under the auspices of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer Association, a collaborative study of the mouse lymphoma assay (MLA) was conducted by 42 Japanese laboratories and seven overseas laboratories to clarify the performance of the MLA for the detection of in vitro clastogens and spindle poisons. Twenty-one chemicals that were positive in in vitro chromosomal aberration assays (CA) but negative in bacterial reverse mutation assays (BRM) were examined by the MLA using the microwell method. All chemicals were coded, and each chemical was tested by two or three laboratories. Positive responses were obtained with 14 chemicals: mitomycin C (an internal positive control), arsenic trioxide, cadmium sulphate, chlorendic acid, cytosine arabinoside, diethylstilbestrol, eugenol, 5-fluorouracil, griseofulvin, hexamethyl phosphoramide, hydroxyurea, methotrexate, monocrotaline and pentachloroethane. Two chemicals (benzene and chlorodibromomethane) showed positive responses in one of two laboratories and were judged probably positive chemicals. Three chemicals (bromodichloromethane, isophorone and tetrachloroethane) were inconclusive because of a marginal response in one laboratory and a negative response in the other. Urethane was judged probably negative because two laboratories out of three showed clear negative responses. Dideoxycytidine (DDC) was a clear negative chemical in this study. The present results showed that 75.0% of the test chemicals (15/20, excluding mitomycin C) were positive, 15.0% (3/20) were inconclusive, and 10.0% (2/20) were negative. This suggests that the MLA may detect a majority of CA-positive chemicals. The inconclusive chemicals, however, are critical for the judgement of the MLA potential to detect clastogens. The findings that DDC was clearly negative suggests that the MLA may not be able to detect some clastogens. To clarify these issues, we began the second phase of the collaborative study with other BRM-negative and CA-positive chemicals.

Animals↗
PubMed1996

Regionally-targeted mutagenesis by metabolically-activated steviol: DNA sequence analysis of steviol-induced mutants of guanine phosphoribosyltransferase (gpt) gene of Salmonella typhimurium TM677.

Steviol is the aglycone of stevioside, a non-caloric sugar substitute commonly used in Japan. Steviol strongly induces mutations at the guanine phosphoribosyltransferase gene (gpt) of Salmonella typhimurium TM677 when the metabolic activation system (S9 mix) is present. However, it is completely negative in the reverse mutation assays using Escherichia coli WP2uvrA/pKM101 or S.typhimurium TA strains. In order to characterize the mutations induced by metabolically-activated steviol, the chromosomal gpt alleles of 24 induced (ST clones) and 16 spontaneous mutants (SP clones) of S.typhimurium TM677 were sequenced and the mutation spectra were compared. About 40% of the mutations of ST clones (nine out of 24) were localized in the region between nucleotides 280 and 330 from the starting codon ATG, whereas no mutations of SP clones were found in that region. The mutations identified in the region included transitions (three clones), transversions (four clones), a duplication and a deletion. There were no other marked differences between ST and SP clones: base-change mutations were dominant over frameshifts and deletions (ST clones, 20 versus three; SP clones, 16 versus two) and base change mutations occurred more frequently at G:C pairs rather than at A:T pairs (ST clones, 15 versus five; SP clones, 12 versus four). The possibility that metabolically-activated steviol gives pause to DNA synthesis around nucleotide 280, thereby stimulating the duplication, deletion and untargeted mutagenesis in the defined region of the gpt gene is discussed.

Amino Acid Sequence↗
PubMed1996

Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays.

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Animals↗
PubMed1996

Molecular anatomy of human chromosome 9: comparative mapping of the immunoglobulin processed pseudogene C epsilon 3 (IGHEP2) in primates.

Karyotypic homology in relation to human chromosome 9 (HSA 9) was studied through comparative mapping of the immunoglobulin-processed pseudogene C epsilon 3 (IGHEP2) in primates. IGHEP2, which has been mapped to 9p24.2 --> p24.1 in the human genome, was assigned to PTR 11q34 (common chimpanzee), PPA 11q34 (pygmy chimpanzee), PPY 13q16 (orangutan), HLA 8qter (white-handed gibbon), HAG 8qter (agile gibbon), and MFU 14q22 (Japanese macaque) by fluorescence in situ hybridization. To verify the breakpoints of presumed pericentric inversions on the ancestral great ape chromosomes, three DNA markers on HSA 9, cCI9-37 (9q22.1 --> q22.2), cCI9-135 (9q22.32 --> q22.33), and cCI9-208 (9p13.3 --> p13.2), were also assigned to PTR/PPA 11p11 (cCI9-37 and 135), PTR/PPA 11q22 (cCI9-208), PPY 13q22 (cCI9-37 and 135), and PPY 13q12 (cCI9-208). These data more clearly define the position of the breakpoints of pericentric inversions that occurred in the human-chimp ancestral and chimpanzee ancestral chromosomes and support the hypothesis of HSA 9 genesis previously derived from banding analyses of HSA 9 and its homologs.

Animals↗