Biomedical subjects
T Sofuni
Publications and source records attributed to T Sofuni.
Spindle poisons induce allelic loss in mouse lymphoma cells through mitotic non-disjunction.
Aneuploidy is an important contributor to reproductive failure and tumor development. It arises spontaneously or as a result of exposure to aneugenic agents through non-disjunction. Two spindle poisons, colchicine (COL) and vinblastine (VBL) are mutagenic in the mouse lymphoma assay (MLA), a gene mutation assay that targets the heterozygous thymidine kinase (tk) gene on chromosome 11 in mouse lymphoma L5178Y tk+/- 3.7.2c cells. To investigate the mechanisms of spindle poison mutagenesis, we analyzed the COL- and VBL-induced TK mutants at the molecular and cytogenetic level. Loss of heterozygosity (LOH) analysis employing a microsatellite region within the tk locus revealed that almost all mutants had lost the functional tk allele. To determine the extent of the LOH, we further examined LOH mutants for heterozygosity at nine microsatellite loci spanning the entire chromosome 11. Interestingly, every microsatellite marker showed LOH in all COL- and VBL-induced LOH mutants, suggesting that these mutants were generated by loss of the whole chromosome 11 through mitotic non-disjunction. Chromosome painting analysis supported this hypothesis; there were no mutants showing structural changes such as deletions or translocations involving chromosome 11. In contrast, spontaneous TK mutants followed from point mutations, deletions and recombinational events as well as whole chromosome loss. Our present study indicates that spindle poisons induce mutations through mitotic non-disjunction without structural DNA changes and supports a possible mechanism in which a recessive mutation mediated by aneuploidy may develop tumors.
Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse).
Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.
In vivo genotoxicity of 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline in lacI transgenic (Big Blue) mice.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonella/microsome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic (Big Blue(R)) mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx (males) or 12 weeks of feeding in the diet (males and females). Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen (PCNA) immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx (100 mg/kg) did not increase mutant frequency (MF) in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx (300 ppm) in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound's mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed.
Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity.
In Japan, the Chemical Substances Control Law requires evaluation of the genotoxic potential of chemical substances semi-quantitatively by application of a ranking system. During the past 10 years under the law, 1049 new chemical substances were evaluated by a reverse mutation assay in bacteria (RMA) and a chromosome aberration test in cultured mammalian cells (CAT). Of them, 130 (12.4%) were positive in the RMA and 402 (38.3%) were positive in the CAT. Eighty (7.6%) were positive in both tests. Fifty (4.8%) were positive only in the RMA, 322 (30.7%) were positive only in the CAT, and 452 (43.1%) were positive in either the RMA or the CAT. Thus, the tests complement each other in detecting genotoxic substances in vitro. To explore the "threshold" concept, we compared the genotoxic responses of Salmonella typhimurium tester strains with and without DNA repair capacity. Recently constructed strains of TA1535 lacking O(6)-methylguanine DNA methyltransferase genes (ogt(ST) or ada(ST) and ogt(ST)) showed dose-related increases in the number of revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, dimethylnitrosamine, and ethylnitrosourea, while in the same dose ranges the parental strain TA1535 did not. This finding suggests that there is a threshold at which all DNA damage induced by low dose levels of genotoxic chemicals are repaired. That biological threshold seems to exist for both DNA and non-DNA targeting chemicals.
Summary of major conclusions from the International Workshop on Genotoxicity Test Procedures.
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Report from the In Vitro Micronucleus Assay Working Group.
At the Washington International Workshop on Genotoxicity Test Procedures (March 25-26, 1999), the current methodologies and data for the in vitro micronucleus test were reviewed. From this, guidelines for the conduct of specific aspects of the protocol were developed. Because there are a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at this time. Agreement was achieved on the following topics: Cells. The choice of cells is flexible, yet the choice of cell type should be justified and take into consideration doubling time, spontaneous frequency of micronuclei, and genetic background. Slide preparation. A fixation method that preserves the cytoplasm and cytoplasmic boundaries, and minimizes clumping should be used. Use of fluorescent DNA-specific dyes is encouraged for better detection of small micronuclei. Analysis. Micronuclei should have a diameter less than one-third of the main nucleus, and should be clearly distinguishable from the main nucleus. In the cytokinesis-block method, binucleated cells selected for analysis should have two clearly distinguishable main nuclei. Cells where the main nucleus(ei) is undergoing apoptosis should not be scored for micronuclei because the assumed micronuclei may have been the result of nuclear fragmentation during the apoptotic process. Toxicity. Cytotoxicity can be measured by various methods including cell growth, cell counts, nucleation (i.e., percent binucleated), division/proliferation index, confluence. A majority of the group recommended that the highest concentration should induce at least 50% cytotoxicity (by whatever measure is selected). Cytochalasin B. There is much debate regarding the use of cytochalasin B. For human lymphocytes, the use of cytochalasin B (6 microg/ml [lymphocytes cultured from whole blood cells] and 3-6 microg/ml [isolated lymphocyte cultures]) is recommended. For cell lines, because there were no definitive data showing a clear advantage or disadvantage of the use of cytochalasin B for a variety of chemicals, the majority opinion of the group was that at this time, the use of cytochalasin B for cell lines is considered optional. Further studies (many chemicals of a variety of potencies, tested both with and without cytochalasin B) are clearly needed to resolve this issue. Number of doses. At least three concentrations should be scored for micronuclei. Treatment/harvest times. At this time, there are not enough data to define the most appropriate treatment/harvest times. Following the principles of the in vitro metaphase assay (with or without metabolic activation), it was agreed that there was a need for a short treatment followed by a recovery time in the absence of test chemical, there was a need for a long treatment (maybe with and without recovery time), and ideally, treatment should cover cells in different cell cycle stages.
Requirement of wild-type p53 protein for maintenance of chromosomal integrity.
Chromosomal double-strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor-suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild-type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6-E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK-deficient mutants from TK6-E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage-fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK-deficient mutants from TK6-20C cells expressing wild-type p53. In wild-type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair.
Human monochromosome hybrid cell panel characterized by FISH in the JCRB/HSRRB.
The human monochromosome hybrid cell panel in the Japanese Collection of Research Bioresources (JCRB) consists of 23 mouse cell clones, each containing a different human chromosome (the Y chromosome is not yet included). The panel is currently distributed by the Human Science Research Resources Bank (HSRRB) in Osaka. In order to determine the state of the human chromosomes and to supply the information to investigators, we characterized the cells by fluorescence in-situ hybridization (FISH) with corresponding human chromosome-specific painting probes, and, in part, by reverse FISH with the hybrid total DNA hybridized onto human metaphase spreads. Here, we report the frequency of intact human chromosomes maintained in each hybrid and the retained subregions of corresponding human chromosomes with relative frequencies estimated by fluorescent intensity. We used specific painted patterns to classify each hybrid into tentative types with their frequencies showing the nature of each hybrid and the state of rearrangements. This characterization will provide valuable information to investigators using the panel.
Appropriate levels of cytotoxicity for genotoxicity tests using mammalian cells in vitro.
Among standard battery genotoxicity assays, the in vitro chromosome aberration test and the mouse lymphoma tk assay (MLA) yield about fourfold higher incidences of positive test results than the bacterial reverse mutation test or in vivo bone marrow tests. This is a result of experience with submissions of 335 new pharmaceuticals to the German Federal Institute for Drugs and Medical Devices. While all of the standard systems have their value in detecting relevant genotoxins, there is no supportive evidence for DNA reactivity for a considerable number of in vitro clastogens and MLA positives. In particular the clastogenic response of such compounds is often associated with high cytotoxicity. This may invoke the need to change the approach to test for clastogenicity in vitro. A combination of measures such as (1) a change in the upper limits of cytotoxicity that are currently given in International Conference on Harmonisation (ICH) and Organization for Economic Co-Operation & Economic Development (OECD) guidelines, (2) the creation of a common ground of understanding for interpretation of in vitro (positive) test results, and (3) lowering the upper limits of test compound concentration irrespective of cytotoxicity may prove useful to ensure a sufficient reliability of genotoxicity testing with mammalian cells in vitro.
A micronucleus assay in sea urchin embryos.
We have developed a micronucleus assay for use in sea urchin embryos. The embryos at the early blastula stage (about 256 cells) were exposed to genotoxic chemicals overnight until control embryos have reached the gastrula stage. Then all embryos were suspended in 1 M urea, dissociated by pipetting, and fixed with methanol:acetic acid (9:1). The preparations were air-dried and stained with acridine orange. The test chemicals (mitomycin C [MMC], vinblastine and 1-beta-D-arabinofuranosylcytosine [Ara C]) induced clear micronuclei dose-dependently. The maximum frequency induced with MMC was 2-3% in Clypeaster japonicus and 1-2% in Hemicentrotus pulcherrimus.
Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gpt delta transgenic mouse.
Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.
Procarbazine genotoxicity in the MutaMouse; strong clastogenicity and organ-specific induction of lacZ mutations.
Procarbazine, a drug used for cancer chemotherapy, is carcinogenic in rodent bioassays. We analyzed the mutagenicity of procarbazine in various organs and the clastogenicity of the drug in hematopoietic cells of the lacZ transgenic MutaMouse. This was part of the second collaborative study of the Mammalian Mutagenesis Study Group of the Japanese Environmental Mutagen Society on the transgenic mouse mutation assay. At 50 mg kg(-1), procarbazine induced micronuclei in hematopoietic cells, but it did not increase the lacZ mutant frequency (MF) in bone marrow. It was also negative in liver, testis, spleen, kidney, and lung. Five daily administrations of 150 mg kg(-1) yielded highly positive responses in the drug's target organs for carcinogenesis (lung, bone marrow, and spleen). Lower positive responses were detected in kidney, which is a minor target organ. Liver showed only a slight increase in lacZ MF and brain showed no increase. The testis MF more than doubled which suggest that procarbazine is mutagenic to germ cells. Thus, we demonstrated that procarbazine has a strong clastogenic effect in hematopoietic cells and is mutagenic in a variety organs after high dose treatment. The induced MF was especially high in procarbazine's target organs for carcinogenesis, which supports the relevance of the transgenic mouse mutation assay for the assessment of potential genotoxins in vivo.
New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption.
CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens.
Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.
We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465-470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice. In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5'-CpG-3' sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse.
Spi(-) selection: An efficient method to detect gamma-ray-induced deletions in transgenic mice.
Despite the importance of genome rearrangement in the etiology of cancer and human genetic disease, deletion mutations are poorly detectable by transgenic rodent mutagenicity tests. To facilitate the detection and molecular analysis of deletion mutations in vivo, we established a transgenic mouse model harboring a lambdaEG10 shuttle vector that includes the red and gam genes for Spi(-) (sensitive to P2 interference) selection [Nohmi et al. (1996] Environ. Mol. Mutagen. 28:465-470]. This selection has a great advantage over other genetic systems, because phage deletion mutants can be preferentially selected as Spi(-) plaques, which can then be subjected to molecular analysis. Here, we show nucleotide sequences of 41 junctions of deletion mutations induced by gamma-irradiation. Unlike spontaneous deletion mutants, more than half of the large deletions occurred between short homologous sequences from one to eight bp. The remaining junctions had no such homologous sequences. Intriguingly, two Spi(-) mutants had P (palindrome)-like nucleotide additions at the breakpoints, which are frequently observed in the coding junctions of V(D)J recombination, suggesting that broken DNA molecules with hairpin structures can be intermediates in the repair of radiation-induced double-strand breaks. We conclude that Spi(-) selection is useful for the efficient detection of deletion mutations in vivo and that most rearrangements induced by gamma-rays in mice are mediated by illegitimate recombination through DNA end-joining.
Toxicity study of a rubber antioxidant, mixture of 2-mercaptomethylbenzimidazoles, by repeated oral administration to rats.
2-Mercaptobenzimidazole (2-MBI), a rubber antioxidant, is known to exhibit potent antithyroid toxicity in rats and is a candidate as an environmental endocrine disrupter. 2-Mercaptomethylbenzimidazoles (a 1:1 mixture of 4-methyl and 5-methyl isomers, MMBIs), are also employed industrially as rubber antioxidants and are suspected to exert antithyroid toxicity such as 2-MBI. In this investigation, acute and subacute oral toxicity studies of MMBIs in Wistar rats were conducted. The clinical signs of acute oral toxicity were observed including decreased spontaneous movement, a paralytic gait, salivation and lacrimation, and adoption of prone and lateral positions. The LD50 was estimated to be 330 mg/kg. In the subacute oral toxicity study, male and female rats were treated with MMBIs by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times and micronuclei induction in bone marrow erythropoeitic cells, and histopathology were examined. Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MMBIs at doses of 20 and 100 mg/kg. Male rats administered 100 mg/kg MMBIs exhibited a 1.8-fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MMBIs/kg exhibited significant increases of liver and kidney but not thyroid weights, and serum cholesterol level. The antithyroid toxicity of MMBIs in rats was estimated to be one-tenth that of 2-MBI. No-observed-effect levels for male and female rats were found to be 4 and 20 mg/kg, respectively, in this subacute oral toxicity study.
ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.
The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.