PubMed Health⌕ Search

Biomedical subjects

T Sofuni

Publications and source records attributed to T Sofuni.

At least 19 recordsLinked to original sources
PubMed1992

Involvement of Cys69 residue in the catalytic mechanism of N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium. Sequence similarity at the amino acid level suggests a common catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms.

Acetyl-coenzyme A:N-hydroxyarylamine O-acetyltransferase is ubiquitous in species ranging from bacteria to mammals and is involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The nucleotide sequence of the gene that encodes O-acetyltransferase of Salmonella typhimurium was determined, and its deduced amino acid sequence was compared with those of arylamine N-acetyltransferases (EC 2.3.1.5) of higher organisms. The gene of S. typhimurium encoded a protein with a calculated molecular weight of 32,177. Chromosome DNA of S. typhimurium TA1538/1,8-DNP, an O-acetyltransferase-deficient strain, had a -1 frameshift mutation of CCC to CC at the coding region. To date, 11 genes encoding N-acetyltransferase have been cloned from human, rabbit, hamster, and chicken. The N-terminal region of O-acetyltransferase of S. typhimurium with about 170 amino acids showed 25-33% homology with the corresponding region of N-acetyltransferase of the higher organisms. Of the 5 cysteine residues of O-acetyltransferase of S. typhimurium, Cys69 was the only residue that was conserved in all N-acetyltransferases of the higher organisms. The amino acid sequence of Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. The mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and its corresponding cysteine residues of the higher organisms are essential for the enzyme activities as acetyl-coenzyme A-binding sites.

Acetyl Coenzyme A↗
PubMed1992

Effects of antioxidants on induction of micronuclei in rat peripheral blood reticulocytes by potassium bromate.

Micronucleus induction in male F344 rat peripheral blood by potassium bromate (KBrO3), a rat renal carcinogen, and its inhibition by several antioxidants were studied using the acridine orange supravital staining method. The frequency of micronucleated reticulocytes (MNRETs) peaked 32 h after a single i.p. treatment of rats with KBrO3 at a dose of 60 mg/kg. Co-treatment with glutathione (GSH) or cysteine (Cys) i.p. at doses of 800 mg/kg and 400 mg/kg, respectively, 30 min before and 30 min after the KBrO3 treatment significantly inhibited the micronucleus induction by KBrO3. Daily i.g. administration of vitamin C for 5 days at a dose of 200 mg/kg/day was also effective in protecting against micronucleus induction by KBrO3 given on the 4th day. However, co-treatment with superoxide dismutase in liposome-encapsulated form by i.p. injection at a dose of 18,000 U/kg 30 min before and 30 min after the KBrO3 application exerted no effect. The results indicate that antioxidants, especially sulfhydryl compounds, have protective potential against the clastogenicity of KBrO3, also suggesting that active oxygen species may play an important role in its clastogenicity.

Acridine Orange↗
PubMed1992

Micronucleated reticulocyte induction by ethylating agents in mice.

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.

Alkylating Agents↗
PubMed1992

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test.

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis (r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.

Acetates↗
PubMed1992

Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102.

The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells. The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains. Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent. Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique. It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102. These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions.

Animals↗
PubMed1992

Mouse bone marrow micronucleus test using flow cytometry.

At present, the micronucleus test is the most popular short-term assay for the in vivo detection of clastogens or spindle poisons. As micronuclei are rare events, it has been argued that the conventional microscopical analysis based on 500-2000 cells per animal does not provide enough sensitivity. In addition, the manual scoring of micronuclei is time-consuming and tedious. As an attempt to solve these problems, an automated method for the analysis of micronuclei in mouse bone marrow erythrocytes was developed using flow cytometry. Femoral bone marrow cells, from mice treated with known in vivo clastogens [mitomycin C (MMC): 0.5, 1 or 2 mg/kg body weight intraperitoneally; benzene: 1000, 2000 or 4000 mg/kg body weight orally] or vehicles for the clastogens (physiological saline, olive oil; for control animals), were fixed with 1% glutaraldehyde, suspended in 70% ethanol for storing, stained with 4',6-diamidino-2-phenylindole (0.5 microgram/ml) and analyzed (50,000 cells per sample) in an EPICS V flow cytometer using a Coherent 5W UV laser at 150-200 mV. The frequency of micronucleated erythrocytes (MNEs) was calculated from the cytometer, by a computer program involving model fitting to normal Gaussian distribution. Correlations between MNE frequencies obtained by manual microscopic observations (1000-2000 cells per animal) and by the flow cytometric measurements were good for both group results (means of 4-5 mice; linear correlation coefficient r = 0.89-0.998) and individual data (r = 0.82-0.96). Repeated experiments with MMC showed good reproducibility for the flow cytometric scoring of micronuclei.

Algorithms↗
PubMed1992

Flow cytometric micronucleus test with mouse peripheral erythrocytes.

Flow cytometric (FC) analysis was applied to micronucleus test with mouse peripheral blood erythrocytes. The method is based on the measurement of peak fluorescence (PFL) of sphered glutaraldehyde-fixed erythrocytes before and after staining with 4',6-diamidino-2-phenylindole (DAPI), in an EPICS V flow cytometer. The frequency of micronucleated erythrocytes (MNEs) is calculated by a computer program comparing PFL data obtained with and without DAPI. To evaluate the method, male ddY mice were treated with 6-mercaptopurine and benzene and blood was collected from tail vein at intervals of 4-7 days. Both microscopic and FC analysis showed a steady increase in the incidence of MNEs, reaching a plateau when about a month had passed from the start of the treatments. The effects of benzo[a]pyrene, mitomycin C,N-ethyl-N-nitrosourea, bromodichloromethane and potassium chromate (VI) were also studied with both the manual and FC assay in samples collected a week after five weekly treatments. The percentages of MNEs obtained manually and by the FC measurements showed good correlation, the former three chemicals being positive and the latter two negative or, in the FC analysis, difficult to classify. Because of the high number of cells examined (50,000/animal), the FC analysis was probably more sensitive than the manual method where only 2000 cells were scored per animal. This was further suggested by (i) steady time responses, also for individual animals, in the FC results on 6-mercaptopurine and benzene, (ii) overall reduced inter-individual variation in the FC measurements, and (iii) detection of MNE induction by mitomycin C at a lower dose level with the FC than the manual analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1992

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed.

Bacterial Proteins↗
PubMed1992

Combined use of several mitogens for mitotic stimulation to human lymphocytes.

Several different type of mitogens, such as phytohemagglutinin (PHA) including PHA-P, -M, -W, -C, -L and -E+L, pokeweed mitogen (PWM), Concanavalin A (Con-A), lipopolysaccharide (LPS), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were compared their mitotic stimulating effects to human lymphocytes in single use and also combined use of these mitogens. In the tests with single use of each mitogen, PHA was confirmed to be the most superior mitogen. Of several different types of PHA, PHA-C, -L and -E+L were not appropriate to stimulate human lymphocytes, though PHA-P, -M and -W induced sufficiently mitoses. The most appropriate concentration of PWM was 50 micrograms/ml, but the highest mitotic index reached at 5 days after the initiation of culture. Con-A did not induce sufficient mitoses, and LPS, WGA and SBA were inadequate to stimulate human lymphocytes in the single use. In the combined use of two mitogens, there was no reliable combination to enhance mitotic stimulation. When three different mitogens were used simultaneously, only one combination with PHA-M, PWM and SBA effectively enhanced the mitotic index when compared to the single use. In the confirmatory experiment for this combination carried out on 5 different donors, the enhanced effect to mitotic index was recognized in two out of three donors with low response to PHA, but the low level of mitotic indices was seen in remaining 2 donors with high response to PHA. This finding suggests that this combination may overcome a disadvantage in some of the low responders to PHA.

Drug Combinations↗
PubMed1991

Isolation of a menadione-resistant subclone from Chinese hamster lung (CHL) cells in culture.

Menadione-resistant subclones were selected from cultured Chinese hamster lung (CHL) cells which had been mutagenized with MNNG or ENU. The frequency of surviving colonies and the level of resistance were higher in mutagenized cells than in non-mutagenized cells. A subclone (designated MM1) was isolated from MNNG-treated cells and showed the highest level of resistance, 3 times higher than the parental CHL cells. The level of resistance was stable in non-selective medium over 3 months. The MM1 cells were also 2-3 times more resistant to other naphthoquinones. The activity of NADPH-cytochrome P-450 reductase, which is thought to play an important role in activation of menadione, was reduced in the MM1 cells to half that in the parental CHL cells. On the other hand, no differences between MM1 and CHL cells were found in the activity of superoxide dismutase and catalase which are assumed to defend against the cytotoxicity of menadione. Karyotype analyses indicated that one small chromosome was lost in the MM1 cells. The MM1 cells showed a 3-fold resistance to menadione in the chromosomal aberration test. The frequencies of chromosomal aberrations induced by adriamycin and mitomycin C which could be activated by NADPH-cytochrome P-450 reductase were almost the same in the MM1 and CHL cells, suggesting that the reductive activation of these compounds by this enzyme in microsomes may not be involved in the induction of chromosomal aberrations.

Animals↗
PubMed1991

Simulation study of the effects of multiple treatments in the mouse bone marrow micronucleus test.

The effect of multiple treatment with chemicals in the micronucleus test was evaluated by simulation involving an estimation of the additive accumulation of micronucleated polychromatic erythrocytes (MNPCEs) on the basis of time-response data available on single treatments with mitomycin C, 1-beta-D-arabinofuranosylcytosine, 6-mercaptopurine, and methotrexate. The frequency of MNPCEs calculated for different multiple treatment regimens by the model could predict the effects observed in real experiments. On the other hand, the effect of multiple treatments on bone marrow depression, expressed as a decrease in the frequency of polychromatic erythrocytes, was exponential according to both the simulation and actual data. These results suggest that although increasing the number of treatments may additively enhance the MNPCE response obtained with some agents it may, in the case of bone marrow-toxic chemicals and doses, make micronucleus analysis more time-consuming and even impossible due to the exponential decrease of analyzable cells, especially in the case of manual scoring.

Animals↗
PubMed1991

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells.

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.

Animals↗
PubMed1991

Mutagenicity of wood smoke condensates in the Salmonella/microsome assay.

Smoke condensates of woods used for food preservation and aromatization in Nigeria were tested for mutagenic activity using Salmonella typhimurium TA98 and TA100. The woods were: white mangrove (Avicennia nitida), red mangrove (Rhizophora racemosa), mahogany Khaya sp.), abura (Mitragyna ciliata), alstonia (Alstonia boonei) and black afara (Terminalia ivorensis). Cigarette tar was tested for comparison. The condensates induced dose-dependent increases in the number of His+ revertants mainly with S9 mix. With the exception of mahogany and cigarette smoke condensate, the smoke condensates induced more revertants/microgram condensate in TA100 than in TA98. The number of revertants/microgram condensate ranged between 0.04 and 0.9 for the wood smoke condensates and was 0.12 for the cigarette smoke in TA100. The range was between 0.1 and 0.30 for the wood smoke condensates and 0.18 revertants/microgram condensate for cigarette smoke condensate in TA98. Concentrations of 7 polycyclic aromatic hydrocarbons (PAHs) in the condensates were determined namely, pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[k]fluoranthene, benzo[b]chrysene, benzo[g,h,i]perylene and dibenzo[a,e]pyrene. The condensates contained varying concentrations of the individual PAHs and those with higher concentrations generally showed greater mutagenic activities. However, the order of mutagenic potency in the bacterial strains differed from the order of PAH concentrations, which were lower than the concentrations at which they are reported to induce mutations. When 6 of the PAHs were mixed in the concentrations in which they were found in the individual condensates, the mixtures did not induce mutation so that the contribution of the PAHs to the mutagenic activities of the condensates could not be determined.

Microsomes↗
PubMed1991

Decreased clastogenicity of dinitropyrenes in Chinese hamster lung (CHL) subclone cells with low NADPH-cytochrome P-450 reductase activity.

Clastogenic potentials of 1,3-, 1,6- and 1,8-dinitropyrenes (DNPs) were compared between Chinese hamster lung (CHL) cells and its subclone MM1 cells, which were recently isolated as menadione-resistant cells after treatment with MNNG. NADPH-cytochrome P-450 reductase activity of the MM1 cells decreased to 50% of that in the parental CHL cells. All 3 DNPs induced chromosomal aberrations without exogenous metabolic activation systems in the CHL cells. 1,6- and 1,8-DNP showed equivalent clastogenic potency: the maximum frequency of cells with chromosomal aberrations was about 50% for both chemicals. The clastogenic potential of 1,3-DNP was lower than that of 1,6- and 1,8-DNP: the maximum frequency of aberrant cells was 10%. In the MM1 cells, in contrast, the frequencies of aberrant cells decreased to about 30% of those observed for the parental CHL cells after treatment with 1,6- and 1,8-DNP, and to the same level as that of the concurrent control after treatment with 1,3-DNP. These results suggest a possibility that the reduced clastogenic effect of 3 DNPs in MM1 cells may correlate with the reduced activity of NADPH-cytochrome P-450 reductase which is thought to contribute to the metabolic conversion of these DNPs to their clastogenic forms in the CHL cells.

Animals↗
PubMed1991

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.

Amino Acid Sequence↗