Kiyomi Nishimaki

Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals

Acute oxidative stress induced by ischemia-reperfusion or inflammation causes serious damage to tissues, and persistent oxidative stress is accepted as one of the causes of many common diseases including cancer. We show here that hydrogen (H2) has potential as an antioxidant in preventive and therapeutic applications. We induced acute oxidative stress in cultured cells by three independent methods. H2 selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected cells; however, H2 did not react with other ROS, which possess physiological roles. We used an acute rat model in which oxidative stress damage was induced in the brain by focal ischemia and reperfusion. The inhalation of H2 gas markedly suppressed brain injury by buffering the effects of oxidative stress. Thus H2 can be used as an effective antioxidant therapy; owing to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage.

Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan.

To the Editor:As described in an editorial by Raymond (1997)xMolecular genetics of the glaucomas: mapping of the first five “GLC” loci. Raymond, V. Am J Hum Genet. 1997; 60: 272–277PubMedSee all ReferencesRaymond (1997), glaucoma is characterized by progressive excavation of the optic disk, with both loss of retinal nerve fiber and visual field defects. This disease is one of the most common causes of bilateral blindness, and it is estimated that by the year 2000 ∼66.8 million people worldwide will be affected by it (Quigley 1996xNumber of people with glaucoma worldwide. Quigley, HA. Br J Ophthalmol. 1996; 80: 389–393Crossref | PubMedSee all ReferencesQuigley 1996). Recently, the glaucoma geneGLC1A was shown to be identical to the trabecular meshwork–inducible glucocorticoid response (TIGR) gene (TIGR) (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). The TIGR gene was cloned by Polansky and colleagues (Nguyen et al. 1993xGlucocorticoid (GC) effects on HTM cells: molecular biology approaches. Nguyen, TD, Huang, W, Bloom, E, and Polansky, JR. : 331–343See all ReferencesNguyen et al. 1993; Polansky et al. 1997xCellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Polansky, JR, Fauss, DJ, Chen, P, Chen, H, Lutjen-Drecoll, E, Johnson, D, Kurtz, RM et al. Ophthalmologica. 1997; 211: 126–139Crossref | PubMedSee all ReferencesPolansky et al. 1997) and, also, was called “myocilin” (gene MYOC) when it was cloned by Kubota et al. (1997)xA novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping. Kubota, R, Noda, S, Wang, Y, Minoshima, S, Asakawa, S, Kudoh, J, Mashima, Y et al. Genomics. 1997; 41: 360–369Crossref | PubMed | Scopus (250)See all ReferencesKubota et al. (1997). Three different mutations in the gene were shown to be responsible for the development of primary open-angle glaucoma (POAG), the most common form of glaucoma (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). The prevalence of these mutations was reported to be 4.4% in familial POAG patients and 2.9% in unselected POAG patients (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). We investigated whether Japanese patients with familial POAG carry identical or other mutants on the same gene. As a result, two new mutations in the TIGR gene were found. The prevalence of mutations in the TIGR gene in Japanese patients with familial POAG was also investigated.Peripheral blood samples were collected, with informed consent, from 52 POAG patients of 50 pedigrees with a family history. The patients were diagnosed with POAG, by ocular and systemic examinations. The family history was obtained by direct interview with the patients. Subjects having at least one relative with POAG within the third degree of relationship were defined as belonging to a pedigree having familial POAG. They all had an elevated intraocular pressure (⩾22 mmHg), open-angle (Shaffer grade III or IV), visual-field loss characteristic of glaucoma, and glaucomatous optic-disk damage. Blood samples from five normal healthy volunteer were also obtained, as controls, with informed consent. Genomic DNA was purified from these blood samples by use of a QIAGEN QIAamp Blood Kit. A DNA fragment encoding a portion of the TIGR protein (amino acid residues 317–476, exon 3; GenBank accession number U85257-AF001620) was amplified, with samples of the purified genomic DNA used as templates, by PCR. The nucleotide sequences of primers used are 5′-ATACTGCCTAGGCCACTGGA (sense strand) and 5′-CATGCTGCTGTACTTATAGCGG (antisense strand). A 150-ng template was mixed with 10 μl of 10 × buffer, 8 μl of the deoxynucleotides mixture, 10 pmol of each primer, and 0.5 μl of Taq polymerase (AmpliTaq Gold; Perkin Elmer), to produce a 100−μl PCR mixture. The nucleotide sequences of both strands of the PCR products were directly determined with the terminator cycle–sequencing method, by use of fluorescent dideoxynucleotides and an automatic DNA sequencer (Applied Biosystems). Mutation was recognized by the approximately equal peak intensity of two fluorescent dyes at the mutation site. When a mutation was detected, the whole procedure of PCR and sequencing was repeated, and the existence of the mutation was confirmed.Of the 52 patients from the 50 pedigrees, 2 patients of one family (a father and a daughter [who was the proband]) carried a heterozygous C→T mutation at the second nucleotide position in the codon corresponding to the 370th amino acid residue of the TIGR protein, resulting in an amino acid change from proline to leucine (Pro370Leu) (figs. 1A1A and 22). The father was diagnosed with POAG at age 26 years old, the daughter at age 16 years. One other patient, from a different pedigree, had a heterozygous G→A mutation at the first nucleotide position in the codon corresponding to the 367th amino acid residue, resulting in an amino acid change from glycine to arginine (Gly367Arg) (fig. 1Bfig. 1B). She was diagnosed with POAG at age 45 years. Both mutations were different from those reported elsewhere (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997). In the pedigree with the Pro370Leu mutation, the mother and a sister of the proband were examined and proved to have neither symptoms of glaucoma nor mutations in the TIGR gene portion examined (fig. 2fig. 2). Therefore, the mutation was inherited in an autosomal dominant manner. The patient with the Gly367Arg mutation had a family history, in that at least her two aunts and five cousins had POAG; but we could not obtain blood samples from her relatives. The prevalence of the mutations in the TIGR gene was 4.0% (2/50 families), which was comparable to that reported in a previous study (Stone et al. 1997xIdentification of a gene that causes primary open angle glaucoma. Stone, EM, Fingert, JH, Alward, WLM, Nguyen, TD, Polansky, JR, Sunden, SLF, Nishimura, D et al. Science. 1997; 275: 668–670Crossref | PubMed | Scopus (975)See all ReferencesStone et al. 1997).Figure 1Chromatograms of nucleotide sequences from patients with mutations (A and B) and from a normal control (C). The double peak of cytosine (blue line) and thymine (red line) (A, red arrow) represents a heterozygous mutation in the codon corresponding to the 370th amino acid residue of the TIGR protein (Pro370Leu). The double peak of guanine (black line) and adenine (green line) (B, green arrow) represents a heterozygous mutation at the codon of 367th amino acid residue (Gly367Arg).View Large Image | View Hi-Res Image | Download PowerPoint SlideFigure 2Pedigree of patients with the Pro370Leu mutation. The proband is indicated by an arrow.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe present study has revealed that mutations in the TIGR gene are also responsible for familial POAG in Japan. The mutations in the third exon of the TIGR gene were found to be associated with ∼4% of Japanese familial POAG patients. The prevalence of the mutations in the gene was comparable in Japanese patients and in the population previously studied. The mutated sites, however, were different from those reported in the previous study, and no common mutations were found. Therefore, the distribution of the mutated sites in the TIGR gene in Japanese POAG may be different from those in other races. Known mutated sites in the TIGR-gene disease alleles are at the 364th, 367th, 368th, 370th, and 437th amino acid residues. The apparent focus of the reported mutations—around the 367th amino acid residue—indicates that the amino acid change around this position may play a critical role in the pathogenesis of POAG. The TIGR-protein product is overexpressed with glucocorticoid stimulation and is thought to contribute to steroid-responsive intraocular-pressure increase, by the obstruction of aqueous outflow (Nguyen et al. 1993xGlucocorticoid (GC) effects on HTM cells: molecular biology approaches. Nguyen, TD, Huang, W, Bloom, E, and Polansky, JR. : 331–343See all ReferencesNguyen et al. 1993; Polansky et al. 1997xCellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Polansky, JR, Fauss, DJ, Chen, P, Chen, H, Lutjen-Drecoll, E, Johnson, D, Kurtz, RM et al. Ophthalmologica. 1997; 211: 126–139Crossref | PubMedSee all ReferencesPolansky et al. 1997). Further investigations of the TIGR gene will reveal more information about the pathogenesis of POAG.

Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice.

Oxidative stress is implicated in atherogenesis; however most clinical trials with dietary antioxidants failed to show marked success in preventing atherosclerotic diseases. We have found that hydrogen (dihydrogen; H(2)) acts as an effective antioxidant to reduce oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K. Katsura, Y. Katayama, S, Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688-694]. Here, we investigated whether drinking H(2)-dissolved water at a saturated level (H(2)-water) ad libitum prevents arteriosclerosis using an apolipoprotein E knockout mouse (apoE(-/-)), a model of the spontaneous development of atherosclerosis. ApoE(-/-) mice drank H(2)-water ad libitum from 2 to 6 month old throughout the whole period. Atherosclerotic lesions were significantly reduced by ad libitum drinking of H(2)-water (p=0.0069) as judged by Oil-Red-O staining series of sections of aorta. The oxidative stress level of aorta was decreased. Accumulation of macrophages in atherosclerotic lesions was confirmed. Thus, consumption of H(2)-dissolved water has the potential to prevent arteriosclerosis.

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late‐onset Alzheimers disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2‐deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2‐deficient transfectants were highly vulnerable to exogenous 4‐hydroxy‐2‐nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2‐deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4‐hydroxy‐2‐nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.

Metabolic Remodeling Induced by Mitochondrial Aldehyde Stress Stimulates Tolerance to Oxidative Stress in the Heart

Rationale: Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress–associated cardiovascular disease. Objective: We investigated how the heart compensates for the accelerated accumulation of aldehydes. Methods and Results: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2&agr; phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress–resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.) Conclusions: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione–redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.

Molecular Hydrogen Improves Obesity and Diabetes by Inducing Hepatic FGF21 and Stimulating Energy Metabolism in db/db Mice

Recent extensive studies have revealed that molecular hydrogen (H2) has great potential for improving oxidative stress‐related diseases by inhaling H2 gas, injecting saline with dissolved H2, or drinking water with dissolved H2 (H2‐water); however, little is known about the dynamic movement of H2 in a body. First, we show that hepatic glycogen accumulates H2 after oral administration of H2‐water, explaining why consumption of even a small amount of H2 over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H2. Next, we examined the benefit of ad libitum drinking H2‐water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H2‐water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat‐diet‐induced fatty liver in wild‐type mice. Long‐term drinking H2‐water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H2‐water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H2‐water improves obesity and metabolic parameters at the molecular level, we examined gene‐expression profiles, and found enhanced expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which functions to enhance fatty acid and glucose expenditure. Indeed, H2 stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H2 in improving obesity, diabetes, and metabolic syndrome.

Prognostic significance of the immunohistochemical index of survivin in glioma: a comparative study with the MIB-1 index.

SummaryObjective: Survivin has been identified as a protein expressed in cancer cells and a member of the inhibitor-of-apoptosis protein family. Recent studies suggest that the expression of survivin increases during the G2/M phase of the cell cycle, and may be used in clinical prognosis. We examined whether survivin expression in human gliomas would be a correlative of prognosis. Methods: We prepared polyclonal anti-survivin serum to establish a survivin index for stained sections, using an immunohistochemical procedure, according to the method used for scoring MIB-1 index, and then stained 29 paraffin-embedded sections from surgical specimens of 29 patients who were classified into three grades of World Health Organization with the mean age of low grade astocytoma (grade II) being 34.7; anaplastic astrocytoma (grade III), 48.8; and glioblastoma multiform (grade IV), 58.4. Results: On staining with the anti-survivin antiserum, all specimens contained positive cells, but the survivin index was heterogeneous among grades. The mean percentage of immunoreactive cells in each specimen was 70.0 (SD 18.2) in grade II, 81.3 (16.5) in grade III, and 85.0 (13.6) in grade IV. Then we compared the survivin index to the MIB-1 index and found that in low-grade gliomas (grade II and III), the difference in survival times between the high and low survivin indexes was significant (P=0.007), whereas that between the high and low MIB-1 indexes was not significant (P=0.092).Conclusion: Survivin is more sensitive marker than MIB-1 for the evaluation of low-grade gliomas in that it helps to predict patient survival. Much larger glioma patient series are needed to validate the findings of our limited study.

A Trace Amount of the Human Pro-apoptotic Factor Bax Induces Bacterial Death Accompanied by Damage of DNA

An amount of human pro-apoptotic Bax as low as 0.01% of total protein was sufficient to cause cell death inEscherichia coli. The bacterial cell death was examined using a viable bacteria-specific fluorescence indicator system and loss of colony formation ability. Co-expression of anti-apoptotic Bcl-xL showed a modest inhibitory effect on the cell death caused by Bax. The trace amount of Bax elongated E. coliand accumulated monounsaturated fatty acids, suggesting an unusual metabolism of redox in the host. In fact, an increase of KCN-dependent O2 consumption accompanied the expression of Bax. At the same time, a fluorescent pH indicator showed the apparent accumulation of protons outside the cell, suggesting that the membrane is intact. Bax increased the level of superoxide anion as measured by the expression of superoxide-dependent promoter. Nicked DNA was significantly generated, and the frequency of mutations resistant to rifampicin was increased by 30-fold, depending upon the expression of Bax. It is proposed that trace amounts of Bax increase oxygen consumption, triggering generation of superoxide, which affects DNA, leading to bacterial death.

Real-Time Monitoring of Oxidative Stress in Live Mouse Skin

Oxidative stress is involved in many age-associated diseases, as well as in the aging process itself. The development of interventions to reduce oxidative stress is hampered by the absence of sensitive detection methods that can be used in live animals. We generated transgenic mice expressing ratiometric redox-sensitive green fluorescent protein (roGFP) in the cytosol or mitochondria of several tissues, including skin epidermal keratinocytes. Crossbreeding into hairless albino mice allowed noninvasive optical measurement of skin oxidative state. Topical application of hydrogen peroxide emulsion shifted the keratinocyte redox state toward oxidation within minutes and could be observed in real time by fluorescence ratio imaging. Exposing skin to 365 nm UVA radiation oxidized roGFP localized in keratinocyte mitochondria, but not when roGFP was localized in the cytosol. This suggests that significant amounts of the endogenous photosensitizers that mediate UVA-induced oxidative stress are located in the mitochondria. UVR is the major environmental cause of skin aging and UVA-mediated oxidative stress has been associated with the development of wrinkles in humans. Direct measurements of redox state in defined cell compartments of live animals should be a powerful and convenient tool for evaluating treatments that aim to modulate oxidative stress.

Molecular hydrogen regulates gene expression by modifying the free radical chain reaction-dependent generation of oxidized phospholipid mediators

We previously showed that H2 acts as a novel antioxidant to protect cells against oxidative stress. Subsequently, numerous studies have indicated the potential applications of H2 in therapeutic and preventive medicine. Moreover, H2 regulates various signal transduction pathways and the expression of many genes. However, the primary targets of H2 in the signal transduction pathways are unknown. Here, we attempted to determine how H2 regulates gene expression. In a pure chemical system, H2 gas (approximately 1%, v/v) suppressed the autoxidation of linoleic acid that proceeds by a free radical chain reaction, and pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC), one of the major phospholipids, was autoxidized in the presence or absence of H2. H2 modified the chemical production of the autoxidized phospholipid species in the cell-free system. Exposure of cultured cells to the H2-dependently autoxidized phospholipid species reduced Ca2+ signal transduction and mediated the expression of various genes as revealed by comprehensive microarray analysis. In the cultured cells, H2 suppressed free radical chain reaction-dependent peroxidation and recovered the increased cellular Ca2+, resulting in the regulation of Ca2+-dependent gene expression. Thus, H2 might regulate gene expression via the Ca2+ signal transduction pathway by modifying the free radical-dependent generation of oxidized phospholipid mediators.