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This paper examines whether the COVID-19-induced employment shocks are associated with increases in suicides and safety net use in the second and third quarters of 2020. We exploit plausibly exogenous regional variation in the magnitude of the employment shocks in Japan and adopt a difference-in-differences research design to examine and control for possible confounders. Our preferred point estimates suggest that a one-percentage-point increase in the unemployment rate in the second quarter of 2020 is associated with, approximately, an additional 0.52 suicides, 28 unemployment benefit recipients, 88 recipients of a temporary loan program, and 10 recipients of public assistance per 100,000 population per month. A simple calculation based on these estimates suggests that if a region experienced a one-percentage-point increase in the unemployment rate caused by the COVID-19 crisis in the second quarter of 2020, which is roughly equivalent to the third-highest regional employment shock, this would be associated with 37.4%, 60.5%, and 26.5% increases in the total, female, and male suicide rates respectively in July 2020 compared with July 2019. Our baseline findings are robust to several different model specifications, although we do not assert that our research design perfectly solves the problem of estimation bias.

This paper examines whether the COVID-19-induced employment shock has increased suicides and the utilization of means-tested poverty alleviation programs in the first eight months of the COVID-19 crisis. We exploit plausibly exogenous regional variation in the magnitude of the employment shock in Japan and adopt a difference-in-differences (DID) research design to identify the employment-shock impact. Our preferred point estimates suggest that a one-percentage-point decrease in the employment rate in the second quarter of 2020 resulted in an additional 0.14-0.44 male suicides per one hundred thousand male population in June 2020 and an additional 5.3-7.9 Public Assistance recipients per one hundred thousand population in August 2020. The impacts on female suicides and other poverty alleviation programs are not precisely estimated, but there is suggestive evidence that the same employment shock has also increased the caseloads of the other poverty alleviation programs. We also examine the impact of the increase in the unemployment rate and find a positive impact on the poverty alleviation programs but not on suicides.

Although a public long-term care (LTC) program is a potentially important factor for the labor supply of female informal caregivers, there are only a handful of individual-level studies on this topic and the macro-level impacts of LTC programs are still largely unknown. Exploiting the introduction of nationwide long-term care insurance (LTCI) in Japan and utilizing a synthetic control method, we examine how LTCI introduction has altered the trends of public expenditures on in-kind benefits for the elderly, public health expenditure, and female labor force participation. The estimation results using the panel data of OECD countries (1980–2013) suggest that LTCI introduction substantially increased the in-kind benefits for the elderly by around one percentage point of GDP 10 years after LTCI introduction, but we do not find a positive effect on the labor force participation for middle-aged women. The fact that we do not observe any positive LTCI effects on middle-aged female labor force participation on a macro level implies that positive LTCI effects on female labor supply observed in some previous microlevel studies may be cancelled out by some other factors or are small enough to be detected under a general-equilibrium setting.

Human MTH1 protein hydrolyzes oxidized purine nucleotides 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), 2-OH-dATP or their ribo-forms to their monophosphates, thus minimizing replicational and transcriptional errors both in the nuclei and mitochondria. MTH1 suppresses mitochondrial dysfunction and cell death caused by H(2)O(2). Furthermore, MTH1 suppresses the transient increase in 8-oxoguanine in mitochondrial DNA in the dopaminergic nerve terminals in mouse striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, and it protects the nerve terminals. We previously reported that a novel MTH1 allele with a single nucleotide polymorphism (SNP) in its exon 2c segment encodes the fourth MTH1 isoform, namely, MTH1a (p26), in addition to the three known isoforms, MTH1b (p22), c (p21), and d (p18). Another SNP located in exon 4 of the MTH1 gene, which is closely linked to the SNP in exon 2c, substitutes the Val83 residue in MTH1d with Met83. We herein show that all MTH1 isoforms efficiently hydrolyzed 2-OH-dATP and 8-oxo-dGTP. The amino terminal region of MTH1a functioned as a mitochondrial targeting signal when it was expressed in the HeLa cells as a fusion protein with enhanced green fluorescent protein. The cellular fractionation revealed that MTH1a(Met83) was localized in the mitochondria to the same extent as was MTH1d(Val83). However, the mitochondrial translocation of MTH1d(Met83) was less efficient than that of MTH1d(Val83).

Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.

To counteract oxidative damage in nucleic acids, mammalian cells are equipped with several defense mechanisms. We herein review that MTH1, MUTYH and OGG1 play important roles in mammalian cells avoiding an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. MTH1 efficiently hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP and 2-hydroxy (OH)-dATP, to the monophosphates, thus avoiding the incorporation of such oxidized nucleotides into the nuclear and mitochondrial genomes. OGG1 excises 8-oxoG in DNA as a DNA glycosylase and thus minimizes the accumulation of 8-oxoG in the cellular genomes. MUTYH excises adenine opposite 8-oxoG, and thus suppresses 8-oxoG-induced mutagenesis. MUTYH also possesses a 2-OH-A DNA glycosylase activity for excising 2-OH-A incorporated into the cellular genomes. Increased susceptibilities to spontaneous carcinogenesis of the liver, lung or intestine were observed in MTH1-, OGG1- and MUTYH-null mice, respectively. The increased occurrence of lung tumors in OGG1-null mice was abolished by the concomitant disruption of the Mth1 gene, indicating that an increased accumulation of 8-oxoG and/or 2-OH-A might cause cell death. Furthermore, these defense mechanisms also likely play an important role in neuroprotection.

In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.

During ischemia-reperfusion (I/R) injury in the rat kidney, apoptosis was observed in the distal tubules of the cortico-medullary region and outer medulla (OM) while severe necrosis was seen in the proximal straight tubules of the OM. The majority of these changes disappeared within 2 weeks. We examined the contents of 8-oxo-2'-deoxyguanosine (8-oxo-dG), which is a major type of oxidative damage in DNA, in the rat kidney during I/R injury, and also investigated the expression level of the OGG1 gene encoding the 8-oxoguanine DNA glycosylase. High-performance liquid chromatography with an MS/MS analysis of the nuclear DNA revealed an immediate accumulation of 8-oxo-dG in the nuclear DNA prepared from the cortex and OM of the kidney 1h after I/R, and an immunohistochemical analysis demonstrated the immediate accumulation of 8-oxo-dG in the nuclei of renal tubular cells both in the cortex and OM. A delayed increase of cytoplasmic staining with anti-8-oxo-dG was observed only in the cortico-medulla and OM, where the cytoplasmic staining in the proximal tubular cells is higher than in the distal tubular cells. The level of cytoplasmic staining representing 8-oxo-dG in mitochondrial DNA, peaked at 6h after I/R and preceded the necrosis of proximal tubular cells in the OM. An RNase protection assay showed a high level of OGG1 mRNA in the normal kidney, and the level decreased within 3h only in the OM, and increased thereafter 1-7 days of I/R both in the cortex and OM. In situ hybridization showed higher levels of OGG1 mRNA expression in the renal tubules in the OM than in the cortex of the normal kidney, which decreased rapidly within 3h of I/R. Thus, the accumulation of 8-oxo-dG in the mitochondrial DNA rather than in nuclear DNA is likely to be involved in the pathogenic responses such as necrosis of renal tubular cells during I/R injury of the kidney, together with an altered level of OGG1 expression.

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-dGTP, 8-oxo-dATP, 2-hydroxy-dATP, and 2-hydroxy rATP to monophosphates, and thus avoids errors caused by their misincorporation during DNA replication or transcription, which may result in carcinogenesis or neurodegeneration. This substrate specificity for oxidized purine nucleoside triphosphates was investigated by mutation analyses based on the sequence comparison with the Escherichia coli homolog, MutT, which hydrolyzes only 8-oxo-dGTP and 8-oxo-rGTP but not oxidized forms of dATP or ATP. Neither a replacement of the phosphohydrolase module of MTH1 with that of MutT nor deletions of the C-terminal region of MTH1, which is unique for MTH1, altered the substrate specificity of MTH1. In contrast, the substitution of residues at position Trp-117 and Asp-119 of MTH1, which showed apparent chemical shift perturbations with 8-oxo-dGDP in NMR analyses but are not conserved in MutT, affected the substrate specificity. Trp-117 is essential for MTH1 to recognize both 8-oxo-dGTP and 2-hydroxy-dATP, whereas Asp-119 is only essential for recognizing 2-hydroxy-dATP, thus suggesting that origins of the substrate-binding pockets for MTH1 and MutT are different.

Spontaneous germline mutations generate genetic diversity in populations of sexually reproductive organisms and are thus regarded as a driving force of evolution. However, the cause and mechanism remain unclear. 8-oxoguanine (8-oxoG) is a candidate molecule that causes germline mutations, because it makes DNA more prone to mutation and is constantly generated by reactive oxygen species in vivo. We show here that endogenous 8-oxoG caused de novo spontaneous and heritable G to T mutations in mice, which occurred at different stages in the germ cell lineage and were distributed throughout the chromosomes. Using exome analyses covering 40.9 Mb of mouse transcribed regions, we found increased frequencies of G to T mutations at a rate of 2 × 10−7 mutations/base/generation in offspring of Mth1/Ogg1/Mutyh triple knockout (TOY-KO) mice, which accumulate 8-oxoG in the nuclear DNA of gonadal cells. The roles of MTH1, OGG1 and MUTYH are specific for the prevention of 8-oxoG-induced mutation and 99% of the mutations observed in TOY-KO mice were G to T transversions caused by 8-oxoG; therefore, we concluded that 8-oxoG is a causative molecule for spontaneous and inheritable mutations of the germ lineage cells.

Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2'-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37-->Gly59). By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T-->C:G transversion mutation in a mutT(-) mutator strain. For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function. Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function. As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.

Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples. Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump. We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996). In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA. Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861. In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed. Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities. Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

The enzyme 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxo-dGTP into DNA. We investigated expression of MTH1 gene encoding 8-oxo-dGTPase. Large amounts of MTH1 mRNA were present in thymus and testis, embryonic tissues, and certain cell lines. In peripheral blood lymphocytes, the level of MTH1 mRNA was significantly increased after concomitant treatment with phytohemagglutinin and interleukin-2. Analyses of the 5' regions of the MTH1 transcripts revealed that 7 types of MTH1 mRNAs, which may be produced by transcription initiation at different sites and/or alternative splicing. The MTH1 gene consists of 5 major exons, some of which are composed of differentially processed segments. All types of MTH1 mRNAs carry the entire coding region, and may be functional. Three ATG initiation codons in-frame were found in the 5' regions of some of the MTH1 mRNAs. There is a polymorphic alteration at the 5' splicing site (GT to GC) located in exon 2, an event which affects splicing patterns of the MTH1 transcript. Allele frequency of this polymorphism is about 20% among healthy volunteers.

Cysteine residue 69 of the Escherichia coli Ada transcription factor, which accepts a methyl group from methylphosphotriester in methylated DNA, was substituted by each of 19 other amino acids. Only the mutant Ada (C69H), carrying a histidine substitution of Cys69, exhibited a limited degree of transactivating potential for the ada promoter in E. coli cells although the mutant protein was completely devoid of methylphosphotriester-DNA methyltransferase activity. Using a multicopy plasmid system for the expression of Ada protein, we have shown that Ada C69H has a transactivating capacity equivalent to that of wild-type Ada protein in the absence of an alkylating agent. This indicates that the zinc-binding capacity of histidine at residue 69 is likely to be sufficient for Ada to recognize and bind to the ada promoter. Furthermore, transactivation of the ada promoter by Ada C69H was enhanced up to 6-fold by treatment with methylating agents. An additional substitution was made with alanine in Ada C69H, replacing Cys321, the site for acceptance of a methyl group from O6-methylguanine and O4-methylthymine residues in DNA, with alanine. This renders the protein completely inactive as a methyltransferase but this derivative is constitutively active as a transactivator for the ada promoter. Therefore, acquisition of a methyl group at Cys321 apparently enhances the transactivating capacity of Ada protein on the ada promoter. We propose that the transcription-regulating function of Ada protein is under dual control by methylation of cysteine residues at positions 69 and 321; the former enhances DNA binding, while the latter enhances the transactivating capacity of the protein.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.

8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues. Human cells contain enzyme activity, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA. We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence. We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence. The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity. When the human cDNA was expressed in E. coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity. In such cells, the frequency of spontaneous mutation was greatly reduced. We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals.

Expression of the alkA gene in Escherichia coli is controlled by Ada protein, which binds to a specific region of the alkA promoter and enhances further binding of RNA polymerase holoenzyme to the complex. To determine the sequence recognized by the Ada protein, we introduced various base substitutions into the promoter region of alkA and examined their effects on expression of the gene, both in vivo and in vitro. Base changes within the sequence AAAGCAAA, located between positions -41 and -34 from the transcription initiation site, greatly decreased the frequencies of initiation of transcription. In footprinting experiments, the region containing this sequence was protected by the Ada protein and base changes within this sequence led to failure of binding of Ada protein to the promoter. It is likely that the Ada protein recognizes the AAAGCAAA sequence in the alkA promoter and binds to the region containing the sequence, thereby allowing ready access of RNA polymerase to the promoter. There are considerable differences between the mechanisms of action of Ada protein on the promoters of alkA and ada, even though the expression of both genes is positively regulated by Ada protein.

Human MutT homolog (hMTH1) hydrolyzes oxidized purine nucleoside triphosphates to monophosphates, thereby avoiding incorporation of such oxidized purines into DNA or RNA. We examined whether hMTH1 prevents cellular dysfunction induced by sodium nitroprusside, a spontaneous NO donor. Exposure to sodium nitroprusside caused an 8-oxoguanine (8-oxoG) buildup in DNA of proliferating MTH1-null cells which underwent mitochondrial degeneration and subsequently died. Quiescent MTH1-null cells also died with 8-oxoG buildup but only when the buildup affected mitochondrial and not nuclear DNA. In both proliferative and quiescent conditions, the accumulation of 8-oxoG in DNA and cell death was effectively prevented by hMTH1. Knockdown of MUTYH in quiescent MTH1-null cells significantly prevented the cell death, suggesting that 8-oxoG incorporated into mitochondrial DNA is a main cause of this form of cell death. To verify this possibility, an artificially modified hMTH1, namely mTP-EGFP-hMTH1, which localizes exclusively in mitochondria, was expressed in MTH1-null cells. mTP-EGFP-hMTH1 selectively prevented buildup of 8-oxoG in mitochondrial but not nuclear DNA after exposure of proliferating cells to sodium nitroprusside, and also efficiently prevented cell death. We thus concluded that exposure of cells to sodium nitroprusside causes oxidation of mitochondrial deoxynucleotide pools, and that buildup of oxidized bases in mitochondrial DNA initiates cell death.

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.

Accumulation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister–Kyoto rats (WKY), aged 5–32 weeks. HPLC–MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1 mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.

8-Oxoguanine (8-oxoG), a major spontaneous form of oxidative DNA damage, is considered to be a natural cause of genomic diversity in organisms because of its mutagenic potential. The steady-state level of 8-oxoG in the nuclear genome of a human cell has been estimated to be several residues per 106 guanines. In the present study, to clarify the genome-wide distribution of 8-oxoG in the steady state, we performed fluorescence in situ detection of 8-oxoG on human metaphase chromosomes using a monoclonal antibody. Multiple dot-like signals were observed on each metaphase chromosome. We then mapped the position of the signal at megabase resolution referring to the cytogenetically identified chromosomal band, and demonstrated that 8-oxoG is unevenly distributed in the normal human genome and that the distribution pattern is conserved among different individuals. Moreover, we found that regions with a high frequency of recombination and single nucleotide polymorphisms (SNPs) are preferentially located within chromosomal regions with a high density of 8-oxoG. Our findings suggest that 8-oxoG is one of the main causes of frequent recombinations and SNPs in the human genome, which largely contribute to the genomic diversity in human beings.

Enhanced oxidative stress has been implicated in the excitotoxicity of the CNS, and 8-oxo-7,8-dihydro-guanine (8-oxoG), a major type of oxidative damage in nucleic acids, was reported to be accumulated in the rat hippocampus after kainate administration. We herein showed that the 8-oxoG levels in mitochondrial DNA and cellular RNA increased significantly in the CA3 subregion of the mouse hippocampus 6–12 h after kainate administration but returned to basal levels within a few days. Laser-scanning confocal microscopy revealed the 8-oxoG accumulation in mitochondrial DNA to be remarkable in CA3 microglia, whereas that in nuclear DNA or cellular RNA was also detected in the CA3 pyramidal cells and astrocytes. 8-oxoG accumulation in cellular DNA or RNA should be suppressed by MutT homolog 1 (MTH1) with 8-oxo-dGTPase (8-oxo-7,8-dihydro-2′-deoxyguanosine triphosphatase) activity and 8-oxoG-DNA glycosylase 1 (OGG1) with 8-oxoG DNA glycosylase activity. We thus examined the expression level of MTH1 and OGG1 in the mouse hippocampus after kainate administration. TheMth1mRNA level decreased soon after kainate administration and then quickly recovered beyond the basal level, and a continuously increased MTH1 protein level was observed, whereas theOgg1mRNA level remained constant. MTH1-null and wild-type mice exhibited a similar degree of CA3 neuron loss after kainate administration; however, the 8-oxoG levels that accumulated in mitochondrial DNA and cellular RNA in the CA3 microglia significantly increased in the MTH1-null mice in comparison with wild-type mice, thus demonstrating that MTH1 efficiently suppresses the accumulation of 8-oxoG in both cellular DNA and RNA in the hippocampus, especially in microglia, caused by excitotoxicity.

We previously reported that 8-oxoguanine (8-oxoG) accumulates in the cytoplasm of dopamine neurons in the substantia nigra of patients with Parkinson's disease and the expression of MTH1 carrying an oxidized purine nucleoside triphosphatase activity increases in these neurons, thus suggesting that oxidative damage in nucleic acids is involved in dopamine neuron loss. In the present study, we found that levels of 8-oxoG in cellular DNA and RNA increased in the mouse nigrostriatal system during the tyrosine hydroxylase (TH)-positive dopamine neuron loss induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MTH1-null mice exhibited a greater accumulation of 8-oxoG in mitochondrial DNA accompanied by a more significant decrease in TH and dopamine transporter immunoreactivities in the striatum after MPTP administration, than in wild-type mice. We thus demonstrated that MTH1 protects the dopamine neurons from oxidative damage in the nucleic acids, especially in the mitochondrial DNA of striatal nerve terminals of dopamine neurons.

In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

Using Mth1 and Ogg1 knockout mice, we evaluated the roles of these enzymes to prevent tumorigenesis and the accumulation of 8-oxoguanine (8-oxoG) in DNA. We found that lung adenoma/carcinoma spontaneously developed in Ogg1 knockout mice approximately 1.5 years after birth in which 8-oxoG was found to accumulate in their genomes. The mean number of tumors/mouse was 0.71 for the Ogg1 knockout mice, which was five times higher than that observed in wild-type mice (0.14). Although the accumulation of 8-oxoG was also confirmed in the Ogg1, Mth1 double knockout mice, we found no tumor in the lungs of these mice. This observation suggests that Mth1 gene disruption resulted in a suppression of the tumorigenesis caused by an Ogg1 deficiency.

The mitochondrial respiratory chain inevitably produces reactive oxygen species as byproducts of aerobic ATP synthesis. Mitochondrial DNA (mtDNA), which is located close to the respiratory chain, is reported to contain much more 8-oxoguanine (8-oxoG), an oxidatively modified guanine base, than nuclear DNA. Despite such a high amount of 8-oxoG in mtDNA (1-2 8-oxoG/10(4) G), mtDNA is barely cleaved by an 8-oxoG DNA glycosylase or MutM, which specifically excises 8-oxoG from a C:8-oxoG pair. We find here that about half of human mtDNA molecules are cleaved by another 8-oxoG-recognizing enzyme, an adenine DNA glycosylase or MutY, which excises adenine from an A:8-oxoG pair. The cleavage sites are mapped to adenines. The calculated number of MutY-sensitive sites in mtDNA is approximately 1.4/10(4) G. This value roughly corresponds with the electrochemically measured amount of 8-oxoG in mtDNA (2.2/10(4) G), raising the possibility that 8-oxoG mainly accumulates as an A:8-oxoG pair.

Expression of the MRP1 gene encoding the GS-X pump and of the gamma-GCSh gene encoding the heavy (catalytic) subunit of the gamma-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like gamma-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1, 4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably gamma-GCSh-transfected cell lines down-regulated endogenous MRP1 and gamma-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and gamma-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and gamma-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and gamma-GCSh expression.

8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is produced by active oxygen species in the nucleotide pool of the cell and can be incorporated into cellular DNA. Human cells contain enzyme activity that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of mutations, caused by misincorporation. When the cDNA for human 8-oxo-dGTPase was expressed in Escherichia coli mutT- mutant cells devoid of self 8-oxo-dGTPase activity, the elevated level of spontaneous A:T to C:G mutation frequency reverted to normal. We isolated the genomic sequence encoding the enzyme and named the gene MTH1 (for mutT human homologue). This gene is composed of at least 4 exons, spans approximately 9 kb, and is located on human chromosome 7p22.

The rat α2u globulin gene family is composed of about 25 members with closely related structure. Several of these genes are transcriptionally active in the liver of adult males. We isolated about 50 independent genomic clones from the library and determined complete nucleotide sequences of two. Although they were alike and revealed structural features prerequisite for active genes, one cloned gene was interrupted in the 6th intron with about 900 bp 5′ truncated and 3′-terminal triplicated form of LINE 1 sequence flanked by 16 bp complete direct repeat originating from the 6th intron. Within the inserted LINE 1 an excellent splicing acceptor homologue with a precedent lariat junction motif and multiple polyadenylation signals was identified. Thus, the possible production of a chimeric RNA, the last exon of which would be replaced by a part of LINE 1, has to be considered.

Adenosine-, guanosine-, cytidine- and uridine-5'-di(tri)-phosphate-3'-diphosphates were enzymatically prepared by the use of Streptomyces adephospholyticus ATP nucleotide pyrophospho-kinase (E.C. 2.7.6.4). Their regulatory effects were then investigated on translation of rat liver, rabbit globin and silkworm pupae chorion mRNAs by the wheat germ lysate system in vitro. They were found to exert base-specific and mRNA species-specific stimulatory or inhibitory effects, respectively. In particular, cytidine 5'-diphosphate-3'-diphosphate stimulated chorion mRNA translation 3-fold. The significance and limitations of the findings were discussed in relation to natural occurrence and possible regulatory functions of the nucleotides in eucaryotes.

The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and assigned the coding region of a transforming protein, p21, to the sequence. Examination of nucleotide sequence, taken together with the result of analysis of the ras mRNAs (1), has revealed that p21 is encoded from a continuous coding region starting with the 5' proximal initiation codon but not a processed protein. However, there were found several differences between the sequence published by Dhar et. al. (2) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacement of glycine by alanine at position 122 of p21. The evidences are presented with some of actual gel autoradiographs.