Biomedical subjects

T Nohmi

Publications and source records attributed to T Nohmi.

At least 19 recordsLinked to original sources

PubMed2000

Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA.

Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324) and 2-H(310) proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced. The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations.

Adenine↗

PubMed2000

Recent advances in the protocols of transgenic mouse mutation assays.

Transgenic mutation assays were developed to detect gene mutations in multiple organs of mice or rats. The assays permit (1) quantitative measurements of mutation frequencies in all tissues/organs including germ cells and (2) molecular analysis of induced and spontaneous mutations by DNA sequencing analysis. The protocols of recently developed selections in the lambda phage-based transgenic mutation assays, i.e. cII, Spi(-) and 6-thioguanine selections, are described, and a data set of transgenic mutation assays, including those using Big Blue and Muta Mouse, is presented.

Animals↗

PubMed2000

Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines: expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S. typhimurium YG7108.

We developed a new Salmonella tester strain highly sensitive to promutagenic N-nitrosamines by introducing a plasmid carrying human cytochrome P450 2A6 (CYP2A6) and NADPH-cytochrome P450 reductase (OR) cDNA into the ada- and ogt-deficient strain YG7108. The YG7108 2A6/OR cells expressed high levels of CYP2A6 (77+/-8nmol/l) and OR (470+/-20 micromol cytochrome c reduced/min/l). The expressed CYP2A6 efficiently catalyzed coumarin 7-hydroxylation. N-Nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPhA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were mutagenic in the new strain in the absence of any exogenous activation system. The concentrations of promutagen that caused a two-fold increase in revertants were 7.1, 0.14, and 1.4 microM for NDEA, NMPhA, and NNK, respectively. YG7108 2A6/OR cells showed about 10- and 100-fold higher sensitivity to NDEA and NNK, respectively, than parental YG7108 cells assayed in the presence of rat liver S9 (final concentration, 21% (v/v)). Parental YG7108 cells did not detect NMPhA mutagenicity even in the presence of rat liver S9. We believe that this is the first demonstration that CYP2A6 is responsible for the metabolic activation of NMPhA. The established tester strain may be useful to predict human activation of N-nitrosamine promutagens.

Aryl Hydrocarbon Hydroxylases↗

PubMed2000

Mmh/Ogg1 gene inactivation results in accumulation of 8-hydroxyguanine in mice.

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine or 7, 8-dihydro-8-oxoguanine (8-OH-G). Products of the human MMH/OGG1 gene are known to catalyze in vitro the reactions repairing this DNA lesion. To analyze the function of Mmh in vivo, we generated a mouse line carrying a mutant Mmh allele by targeted gene disruption. Mmh homozygous mutant mice were found to have a physically normal appearance, but to have lost nicking activity in liver extracts for substrate DNA containing 8-OH-G, exhibiting a 3-fold increased accumulation of this adduct at 9 weeks of age compared with wild-type or heterozygous mice. Further elevation to 7-fold was observed in 14-week-old animals. Substantial increase of spontaneous mutation frequencies was clearly identified in Mmh mutant mice bearing transgenic gpt genes. These results indicate that exposure of DNA to endogenous oxidative species continuously produces the mutagenic adduct 8-OH-G in mice, and Mmh plays an essential role in repair of this DNA damage.

Animals↗

PubMed2000

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.

Alkylating Agents↗

PubMed2000

In vivo transgenic mutation assays.

Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control.

Animals↗

PubMed2000

Characterization of mutations induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the colon of gpt delta transgenic mouse: novel G:C deletions beside runs of identical bases.

Mutations induced by one of the typical dietary mutagens/carcinogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were characterized using gpt delta transgenic mice. This transgenic mouse model has two selection methods to efficiently detect different types of mutations, i.e. 6-thioguanine selection for point mutations and Spi(-) selection for deletions. The mice were fed with a diet containing 400 p.p.m. PhIP for 13 weeks and gpt and Spi(-) mutations were analyzed from the colon, where the highest mutant frequencies were detected. Concerning the types of gpt mutations from PhIP-treated mice, 81% were single base pair substitutions and G:C-->T:A transversions predominated; single base pair deletions at G:C base pairs were also observed. In untreated mice G:C-->A:T transitions predominated and >80% of these events involved 5'-CpG-3' sites. Concerning Spi(-) mutants from PhIP-treated mice, 76% were G:C base pair deletions and more than half of these events occurred in monotonic G or C run sequences. Interestingly, a novel type of frameshift motif, i.e. G:C base pair deletions beside run sequences, was observed. The most frequently observed mutation in this class was the 5'-TTTTTTG-3'-->5'-TTTTTT-3' event. These results suggest that PhIP induces point mutations, such as base substitutions and single base pair deletions, rather than larger deletions in vivo and that run sequences may play an important role in PhIP-induced G:C base pair deletions.

Animals↗

PubMed2000

Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity.

The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage. The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.

Adenosine Triphosphatases↗

PubMed2000

Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase.

A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established. The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of SALMONELLA: Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR. Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined. CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN. CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK. The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine. In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).

Aryl Hydrocarbon Hydroxylases↗

PubMed1999

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.

Animals↗

PubMed1999

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocylic amines.

A co-expression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-P450 reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella TA1538/ARO cells. The CYP and OAT expressed in the Salmonella cells showed catalytic activity. The TA1538/ARO strain exhibited high sensitivity to heterocyclic amines (HCAs) such as 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimenthylimidazo[4,5-f]quinoline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as compared with the parent Ames tester strain TA1538. It is suggested that the intracellular expression of drug-metabolising enzymes makes the Salmonella cells highly sensitive to the HCAs.

Catalysis↗

PubMed1999

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.

Acetyltransferases↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Preface

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Journal Article↗

PubMed1999

The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis.

In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.

Bacterial Proteins↗

PubMed1999

UVB-induced gpt mutations in the skin of gpt delta transgenic mice.

Ultraviolet light B (UVB)-induced mutagenesis was studied in gpt delta transgenic mice, which contain the lambdaEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m(2). At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m(2) UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB-induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5'-TC-3', 5'-CC-3', and 5'-T/C-CG-3'. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5'-TC-3' and 5'-CC-3' sites, but not at 5'-T/C-CG-3' site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB-irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB-induced mutations at the molecular level.

Animals↗

PubMed1999

Efficient detection of deletions induced by a single treatment of mitomycin C in transgenic mouse gpt delta using the Spi(-) selection.

Transgenic rodent mutation assays permit the detection and molecular analysis of various types of gene mutations, such as base changes and frameshifts, in a number of tissues. It is reported, however, that deletion mutations are not efficiently detected by the assays, in particular those using lambda phage shuttle vectors. Recently, a new transgenic mouse model, i.e., gpt delta, has been developed to selectively detect some types of deletions by Spi(-) selection. Spi(-) selection has an advantage over the other selections to preferentially identify deletions because only lambda phages deficient in both the red and gam gene functions are allowed to form phage plaques. In this study, we examined whether in vivo deletions induced by the treatment of mitomycin C (MMC) are detectable by the Spi(-) assay in the mouse model. The mice were treated with MMC (0.5, 1.0, 2.0, and 4.0 mg/kg, single intraperitoneal injection) and sacrificed 14 days after the dosing. The treatment at 4.0 mg/kg approximately doubled the mutant frequency of Spi(-) in the bone marrow, i.e., 2.52 x 10(-6) vs. 1.31 x 10(-6). The molecular analyses using polymerase chain reaction (PCR) and DNA sequencing indicated that seven Spi(-) mutants at 4.0 mg/kg group had deletions with molecular sizes from 0.8 kilo basepairs (kb) to 8.5 kb, whereas no such deletions were observed in the Spi(-) mutants in the control group. The results suggest that deletions induced by MMC in the bone marrow are efficiently detectable by Spi(-) selection and also that the molecular analyses are useful to evaluate the significance of a marginal increase in mutant frequency in the transgenic rodent mutation assays.

Animals↗