International Workshop on Standardisation of Genotoxicity Test Procedures. Summary of major conclusions.
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Animals↗
Biomedical subjects
T Sofuni
Publications and source records attributed to T Sofuni.
Report from working group on in vitro tests for chromosomal aberrations.
The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
Aneuploidy↗
Tissue-specific mutational spectra of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in the liver and bone marrow of lacI transgenic mice.
2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) is a food-borne heterocyclic amine, so clarification of its mutational spectrum is important for evaluation of its carcinogenic risk to humans. The mutational spectrum of MeIQ was investigated in the liver and bone marrow of transgenic mice carrying the lacI gene. By PCR--single-strand conformation polymorphism analysis and sequencing of the lacI gene, 81 and 61 mutations were identified in 80 and 59 mutants obtained from the liver and bone marrow respectively of three transgenic mice given food containing 300 p.p.m. MeIQ. In the liver, G-->T transversions were the most frequent, accounting for 46% of the total mutations, followed by G-->A transitions (25%). In the bone marrow, four types of mutations, G-->T transversions, G-->A transitions, complex mutations and one base deletions, each accounted for 21-23% of the total mutations. Of the total mutations, 10% were found at nucleotide 92 in the liver and at nucleotide 222 in the bone marrow. Analysis of 27 and 13 mutants from the liver and bone marrow respectively of control mice showed frequent G-->A transitions at CpG sites. These findings suggest a tissue-specific mechanism of mutagenesis.
Animals↗
Chromosome painting analysis of X-ray-induced aberrations in human lymphocytes in vitro.
Chromosomal rearrangements in human lymphocytes induced by X-rays (0, 0.5, 1.0 and 2.0 Gray) were analyzed using chromosome painting. DNA probes for human chromosomes 1, 3 or 4 alone, and a combination of 1 and 4, were used for analysis. The frequency of cells with rearrangements, i.e. reciprocal translocations, dicentrics, insertions, tricentrics and fragments, involving chromosome 4 increased with dose in both 48 and 72 h cultures. The number of translocations per cell also increased with dose at 48 and 72 h. Dicentrics increased with dose in 48 h but not in 72 h cultures. The estimated genomic frequency of aberrations per cell was comparable with results in banded cells. No difference was shown on the detection efficiency of chromosome rearrangements among the various DNA probes used. Since this technique does not necessarily require well-spread metaphases for analysis, it is possible to increase the number of analyzable metaphases compared with the banding technique. Chromosome painting is a simpler, more objective and more practical method for detecting chromosome rearrangements than conventional banding analyses.
Adult↗
Regional assignment of the human immunoglobulin processed pseudogene C epsilon 3 (IGHEP2) to 9p24.2-->p24.1 by fluorescence in situ hybridization.
The human immunoglobulin processed pseudogene C epsilon 3 (IGHEP2), which was assigned to chromosome 9 by somatic cell hybrid analysis, has not been regionally localized as yet. In this study, using fluorescence in situ hybridization (FISH) combined with conventional QFQ-, RBG- or GTG-banding, IGHEP2 was assigned to the p terminus region of chromosome 9, at band 9p24.2-->p24.1. This result suggests that the C epsilon 3 gene is a novel telomeric DNA marker useful not only for constructing the physical map of human chromosome 9 but also for cytogenetic analyses such as cryptic translocations. In addition, comparative mapping of this gene in other catarrhine primates would contribute to investigations of human and other primate karyotype evolution.
Cells, Cultured↗
Statistical analysis of data in mutagenicity assays: rodent micronucleus assay.
To evaluate chemical safety, many kinds of short-term mutagenicity assays are performed together with long-term assays in animals. Rationales and methodology for these assays have been well discussed and documented. No statistical method, however, has been singled out as the method of choice for the evaluation of mutagenicity assay data, although a number of reports on statistical methods to evaluate such data have been published. Among the mutagenicity assays, the micronucleus assay using mouse bone marrow erythropoietic cells have been widely used to assess cytogenetic activities of test chemicals. A statistical evaluation procedure for this assay is proposed herein, combining the use of historical control data and dose-response relationships. The probability of type I errors and the power of this method are compared with those of some other conventional methods by Monte Carlo simulation.
Animals↗
N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.
Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A 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 the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms.
Acetyltransferases↗
Japanese guidelines for mutagenicity testing. Ministry of Agriculture, Forestry and Fisheries; Ministry of Health and Welfare; Ministry of Labor.
Several Japanese agencies are required to perform mutagenicity tests according to regulatory guidelines. Although each agency's guidelines address a specific purpose, the experimental principles behind them are similar, and general methodological recommendations have been issued by the Ministry of Agriculture, Forestry, and Fisheries [1985]; Ministry of Health and Welfare [1990]; Ministry of Labor [1991]; and Ministry of Health and Welfare [1992]. Four major guidelines for mutagenicity testing in Japan and some amendments are briefly introduced. In addition, several procedures in Japanese guidelines that differ from those of other countries or organizations are discussed.
Animals↗
The two umuDC-like operons, samAB and umuDCST, in Salmonella typhimurium: the umuDCST operon may reduce UV-mutagenesis-promoting ability of the samAB operon.
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Cloning, Molecular↗
The concomitant detection of gene mutation and micronucleus induction by mitomycin C in vivo using lacZ transgenic mice.
A new assay system that can simultaneously provide gene mutagenicity and clastogenicity data in vivo is described. Transgenic mice (Muta Mouse) harboring the lacZ gene as a target for mutation analysis were injected intraperitoneally with mitomycin C (MMC), either once or on 5 successive days. Micronucleus assays were performed with small amounts of peripheral blood collected from a tail vessel. The spontaneous frequency of micronucleated reticulocytes was 0.42%. For the mutation analysis, DNA was extracted from bone marrow and liver cells at several harvest times. The lacZ gene was rescued by lambda packaging and infection of E. coli C (lac-), followed by plating on agarose plates containing X-gal. The spontaneous lacZ mutant frequencies were 37 and 29 x 10(-6) in bone marrow and liver, respectively. In the micronucleus assay, single treatments with 1.0 and 2.0 mg/kg of MMC induced micronuclei in 3.6 and 5.8% of reticulocytes, respectively, peaking 48 h after treatment. Muta Mouse sensitivity to micronucleus induction was similar to nontransgenic strains used routinely for the micronucleus test. On the other hand, single treatments with MMC at 1.0 and 2.0 mg/kg did not induce any significant increases in the frequency of lacZ- mutants in bone marrow or liver. N-Ethyl-N-nitrosourea, used at 100 mg/kg as a positive control, yielded a 5-fold increase in mutant frequency above untreated animals in bone marrow only. After 5-day treatments, MMC induced approximately a 2-fold increase in mutant frequency in bone marrow only for the sublethal dose of 2 mg/kg. Therefore, this study indicated that the strong clastogenic activity of MMC in bone marrow was not accompanied by significant gene mutagenic activity.
Animals↗
Detection of recombinational mutations in cultured human cells by Southern blot analysis with minisatellite DNA probes.
Using the human acute monocytic leukemia cell line, THP-1, a hypermutability of minisatellite loci was demonstrated in cell culture by Southern blot analysis with minisatellite DNA probes. DNA was isolated from 98 subclones and hybridized to a panel of minisatellite probes consisting of three multilocus minisatellite probes (ML probes) and seven locus-specific minisatellite probes (LS probes). The Southern blot patterns of the hybridized subclones were compared with those of the parental THP-1. Four mutated bands with two ML probes and two mutated bands with two LS probes were detected. The mutation frequency was estimated roughly at 0.1% based on the total number of bands analyzed, and it was much higher than that expected for other DNA regions. Four of these mutations were thought to be alterations of repetitions caused by insertion or deletion of tandem repeats, and one mutant lost a complete minisatellite allele. The nature of the sixth mutant was unclear. Because of the hypermutability of minisatellite DNA, Southern blot analysis using minisatellite DNA probes can be used as a mutation assay system directly based on the DNA.
Base Sequence↗
Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity.
Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium. The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S. typhimurium TA98 and TA100. The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains. These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.
Acetyltransferases↗
Chromosomal aberrations induced in vitro by 3,7- and 3,9-dinitrofluoranthene.
The chromosomal aberration test using a Chinese hamster cell line (CHL) was carried out with 3,7- and 3,9-dinitrofluoranthene (DNF) with and without exogenous metabolic activation (rat liver S9 mix). The highest dose tested was limited to 20 micrograms/ml because of the compounds' insolubility in dimethyl sulfoxide. Both DNFs induced chromosomal aberrations in the absence of S9 mix; the frequency was not very high. Results were reproducible, but without clear dose-response relationships. Neither DNF induced chromosomal aberrations in the presence of S9 mix. Both DNFs did not induce polyploid cells under any conditions.
Aneuploidy↗
Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes.
Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes. In this paper, we compared the sensitivities of YG1024 with those of S. typhimurium YG1012, which has about 4 times higher OAT activity than YG1024 but lacks plasmid pKM101. It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxy-acetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to YG1012. These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.
1-Naphthylamine↗
A new VNTR polymorphism at locus D1S340.
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Base Sequence↗
New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535.
A new method for gene disruption in Salmonella typhimurium was developed. The key steps of this method are to produce restriction fragments with compatible ends, preligate to produce concatemers, and then transform by electrotransformation. We developed and used this method to construct a mutant of S. typhimurium TA1535 in which the resident ada-like (adaST) gene was replaced with a kanamycin resistance gene to produce an adaST-deletion mutant derivative. The S. typhimurium adaST-deletion strain did not exhibit a higher level of mutability upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine than did its wild-type parent strain. However, it did exhibit a higher sensitivity with respect to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The ability of AdaST to function as a transcriptional activator is discussed.
Bacterial Proteins↗
[Significance of mutagenicity tests on safety evaluation of medicinal drugs].
As one of toxicological tests for safety evaluation of medicinal drugs, three representative mutagenicity tests, that is, reverse mutation test in bacteria, chromosomal aberration test with mammalian cells in culture and micronucleus test with rodents have been carried out. These three tests, especially two in vitro tests have been used for initial hazard identification. Further, the micronucleus test using experimental animals has offered significant information for in vivo safety evaluation. Based on recent collaborative studies with mouse micronucleus tests on human carcinogens and results from newly developed in vivo gene mutation assay using transgenic mice, the combination of both test systems seems to be powerful for in vivo safety evaluation.
Animals↗
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↗