Shinkatsu Morisawa

Kinetic analysis of superoxide anion radical-scavenging and hydroxyl radical-scavenging activities of platinum nanoparticles

There are few reports on the physiological effects of metal nanoparticles (nps), especially with respect to their functions as scavengers for superoxide anion radical (O2(.-)) and hydroxyl radical (.OH). We tried to detect the scavenging activity of Pt nps using a hypoxanthine-xanthine oxidase system for O2(.-) and using a Fenton and a UV/H2O2 system for .OH. Electron spin resonance analysis revealed that 2 nm particle size Pt nps have the ability to scavenge O2(.-) and .OH. The calculated rate constant for the O2(.-)-scavenging reaction was 5.03 +/- 0.03 x 10(7) M (-1) s (-1). However, the analysis of the Fenton and UV/H 2O 2 system in the presence of Pt nps suggested that the .OH-scavenging reaction cannot be determined in both systems. Among particle sizes tested from 1 to 5 nm, 1 nm Pt nps showed the highest O2(.-)-scavenging ability. Almost no cytotoxicity was observed even after adherent cells (TIG-1, HeLa, HepG2, WI-38, and MRC-5) were exposed to Pt nps at concentrations as high as 50 mg/L. Pt nps scavenged intrinsically generated reactive oxygen species (ROS) in HeLa cells. Additionally, Pt nps significantly reduced the levels of intracellular O2(.-) generated by UVA irradiation and subsequently protected HeLa cells from ROS damage-induced cell death. These findings suggest that Pt nps may be a new type of antioxidant capable of circumventing the paradoxical effects of conventional antioxidants.

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Molecular Hydrogen Reduces LPS-Induced Neuroinflammation and Promotes Recovery from Sickness Behaviour in Mice

Molecular hydrogen has been shown to have neuroprotective effects in mouse models of acute neurodegeneration. The effect was suggested to be mediated by its free-radical scavenger properties. However, it has been shown recently that molecular hydrogen alters gene expression and protein phosphorylation. The aim of this study was to test whether chronic ad libitum consumption of molecular hydrogen-enriched electrochemically reduced water (H-ERW) improves the outcome of lipopolysaccharide (LPS)-induced neuroinflammation. Seven days after the initiation of H-ERW treatment, C57Bl/6 mice received a single injection of LPS (0.33 mg/kg i.p.) or an equivalent volume of vehicle. The LPS-induced sickness behaviour was assessed 2 h after the injection, and recovery was assessed by monitoring the spontaneous locomotor activity in the homecage for 72 h after the administration of LPS. The mice were killed in the acute or recovery phase, and the expression of pro- and antiinflammatory cytokines in the hippocampus was assessed by real-time PCR. We found that molecular hydrogen reduces the LPS-induced sickness behaviour and promotes recovery. These effects are associated with a shift towards anti-inflammatory gene expression profile at baseline (downregulation of TNF- α and upregulation of IL-10). In addition, molecular hydrogen increases the amplitude, but shortens the duration and promotes the extinction of neuroinflammation. Consistently, molecular hydrogen modulates the activation and gene expression in a similar fashion in immortalized murine microglia (BV-2 cell line), suggesting that the effects observed in vivo may involve the modulation of microglial activation. Taken together, our data point to the regulation of cytokine expression being an additional critical mechanism underlying the beneficial effects of molecular hydrogen.

Electrolysed-reduced water dialysate improves T-cell damage in end-stage renal disease patients with chronic haemodialysis

BACKGROUND T-cell damage by increased oxidative stress in end-stage renal disease (ESRD) patients undergoing chronic haemodialysis (HD) led to the increased T-cell apoptosis and the alteration of surface markers and Th1/Th2 ratio in CD4(+) T lymphocytes. Antioxidant electrolysed-reduced water (ERW) was used as the dialysate in ESRD patients undergoing chronic HD to test for improved oxidative stress-related T-cell apoptosis, alterations of surface markers and intracellular cytokine profile. METHODS We evaluated apoptosis formation by annexin V, CD25-related surface markers, and cytokine ratio of Th1/Th2 in CD4(+) T lymphocytes and Tc1/Tc2 in CD8(+) T lymphocytes of 42 ESRD patients haemodialysed with ERW for 1 year. RESULTS In comparison to 12 healthy individuals, the ESRD patients had more T-cell apoptosis and less CD3(+), CD4(+) and CD8(+) T cells and CD25/CD69/CD94/CD3(+) phenotypes at baseline. Lower intracellular IL-2 and IFN-gamma levels in the Th1/CD4(+) and Tc1/CD8(+) cells and higher intracellular IL-4, IL-6 and IL-10 levels in the Th2/CD4(+) and Tc2/CD8(+) cells were also noted in the ESRD patients. After a 1-year ERW treatment, the patients had a decrease in T-cell apoptosis and increases in CD3(+), CD4(+) and CD8(+) cell numbers and CD25/CD69/CD94/CD3(+) phenotypes in the T cells. The intracellular IL-2 and IFN-gamma levels in the Th1/Tc1 cells significantly (P < 0.05) increased and the intracellular IL-4, IL-6 and IL-10 levels in the Th2/Tc2 cells decreased. Furthermore, the Th1/Th2 and Tc1/Tc2 cytokine ratios were improved toward a normal status. CONCLUSION One-year ERW treatment effectively ameliorated T-cell apoptosis, altered CD25-related surface markers and intracellular cytokine profile in the HD patients.

Anti-Oxidative Water Improves Diabetes

Electrolyzed reduced water and natural waters such as Hita Tenryosui water in Japan, Nordenau water in Germany and Tracote water in Mexico, which are known to improve various diseases, were all and- oxidative waters which could scavenge intracellular reactive oxygen species. The and-oxidative waters stimulated not only the glucose uptake of rat myotube L6 cells, but also the secretion of insulin from a pancreatic beta cell line HIT-T15. The anti-oxidative waters improved the damage in the sugar tolerance test of type 2 diabetes model mice (db/db mice). A clinical investigation demonstrated that Nordenau water could significantly improve the diabetes mellitus.

Suppressive Effect of Electrolyzed-Reduced Water on the Growth of Cancer Cells and Microorganisms

We investigated the suppressive effect of electrolyzed reduced water (ERW) on the growth of not only various human cancer cells but also microorganisms such as gram-negative Escherichia coli. ERW suppressed the growth of cancer cells, especially in soft-agar culture. The suppressive effect of ERW on the growth of cancer cells depended upon cell types and malignancy of cancer cells and the production methods of ERW. We assumed that scavenging intracellular reactive oxygen species (ROS) by ERW resulted in impairing the tumor phenotypes such as rapid proliferation and anchorage-independent growth without affecting serious damage to normal cells. We also found that ERW exhibited weak microbicidal effect, especially in low cell densities of microorganisms. It may contribute to prevent the rot of food or improve the intestinal microflora to prevent abnormal fermentation.

Telomere Shortening in Cancer Cells by Electrolyzed-Reduced Water

Electroly-reduced water (ERW) which is produced near cathode during electrolysis of water scavenges reactive oxygen species and protects DNA from oxidative damage (Shirahata et al., 1997). Most of cancer cells exhibit high telomerase activity to elongate telomere length, insuring their immortality. Here we found that cultivation of human lung adenocarciomaA549, human uterine cancer HeLaand human normal fibroblast TIG-1 cells were growth inhibited in medium containing ERW and drastic morphological changes occurred in A549 and HeLa cancer cells but not in TIG-1 cells. Telomerase activity did not change but telomere length became shorter depending upon cell division in medium containing ERW. Telomere binding activities of telomere binding proteins in cancer cells decreased in medium containing ERW, suggesting that ERW inhibit the binding of’ telomerase to telomere region via telomere binding proteins, resulting in the shortening of telomerelength.

SUPPRESSION OF CELL GROWTH BY PLATINUM NANOCOLLOIDS AS SCAVENGERS AGAINST REACTIVE OXYGEN SPECIES

Electrolysed-reduced water (ERW) contained Platinum nanocolloids (PtNCs) of 1–10 nm, suggesting that PtNCs in ERW functioned as active hydrogen donors and scavenge intracellular reactive oxygen species (ROS). Electron paramagnetic resonance (EPR) analysis revealed that synthesized PtNCs of about 2 nm scavenged superoxide anion radicals and DPPH radicals. Synthesized PtNCs scavenged intracellular ROS and suppressed the growth of human leukemia K562 cells.

Electrolyzed Reduced Water Induces Differentiation in K-562 Human Leukemia Cells

Electrolyzed reduced water (ERW) is known to scavenge reactive oxygen species (ROS) which is related to malignant tumor phenotypes. We attempted to clarify the effect of ERW on tumor phenotypes of K562 human leukemia cells. Treatment of K562 cells with ERW resulted in growth arrest, morphological changes, and expression of CD41, a cell marker of megakaryocyte differentiation. However, the morphology of N-acetylcysteine (NAC)-treated cells was rather similar to that of non-treated control K562 cells. These results suggested that ERW, but not NAC can differentiate K562 cells into megakaryocytes. The induction of megakaryocytes from K562 cells by ERW was preceded by a rapid rise in the activity of MEK (MAP kinase /extra-cellular regulated kinases) that leads to sustained activation of ERK (extra-cellular regulated kinases; MAPK). However, In NAC-treated K562 cells, ERK activation was only transient. The different persistency of ERK activation induced by ERW and NAC might affect the cell fate.

Electrolyzed Reduced Water Prolongs Caenorhabditis elegans’ Lifespan

Electrolyzed reduced water (ERW) has been reported to scavenge intracellular reactive oxygen species (ROS) and improve oxidative stress-related diseases. A new culture method using water medium for Caenorhabditis elegans (C.elegans) was developed in order to elucidate the effects of ERW on the lifespan of nematode. ERW significantly extended the lifespan of C. elegans and alleviated the ROS Level in nematode in water medium, but not in conventional S-medium. These results suggested that the nematode lifespan was elongated at least in part by ROS-scavenging action of ERW.