Takayuki Mokudai

Free radical formation from sonolysis of water in the presence of different gases

In the present study by applying electron spin resonance-spin trapping method, when a high frequency (1650 kHz) ultrasound was irradiated to water dissolved with different gas molecules (O2, N2, Ar, Ne, He, and H2) at 25°C of water bulk temperature, free radical generation pattern differed dependently on the dissolved gas molecules. Only •OH was detected in the O2-dissolved water sample, and the amount of the radical was much greater than that determined in any of other gas-dissolved water samples. One of the possible reasons to explain why the •H radical was not detected in the O2-dissolved water is that the •H reacts with O2 to form •OOH. However, no electron spin resonance signals related to the adduct of not only 5,5-dimethyl-1-pyrroline-N-oxide but 5-(2,2-Dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide and •OOH were observed. In the H2-dissolved water, only •H was detected, suggesting that H2 reduces or neutralizes •OH. In the N2-disolved water, both •OH and •H were detected at comparable level. In the water samples dissolved with rare gases (Ar, Ne, and He), the amount of •H was almost double as compared with that of •OH, and both •OH and •H yields increased in the order Ar > Ne > He.

In vitro and in vivo anti-Staphylococcus aureus activities of a new disinfection system utilizing photolysis of hydrogen peroxide

The present study aimed to evaluate in vitro and in vivo antibacterial activity of hydroxyl radical generation system by photolysis of H(2)O(2), which is a new disinfection system for the treatment of oral infection diseases such as periodontitis developed in our laboratory. Firstly, generation of the hydroxyl radical by the photolysis of H(2)O(2) in which 1 mol l(-1) H(2)O(2) was irradiated with a dual wavelength-light emitting diode (LED) at wavelengths of 400 and 465 nm was confirmed by applying an electron spin resonance-spin trapping technique. Secondly, the bactericidal effect of the system was examined under a similar condition in which Staphylococcus aureus suspended in 1 mol l(-1) H(2)O(2) was irradiated with LED light, resulting in substantial reduction of the colony forming unit (CFU) of the bacteria within a short time as 2 min. Finally, in vivo antibacterial effect of the photolysis of H(2)O(2) on a rat model of S. aureus infection was evaluated by a culture study. Since a significant reduction of recovered CFU of S. aureus was obtained, it is expected that in vitro antibacterial effect attributable to hydroxyl radicals generated by photolysis of H(2)O(2) could be well reflected in in vivo superficial bacterial infection.

Topical treatment of oral cavity and wounded skin with a new disinfection system utilizing photolysis of hydrogen peroxide in rats.

The present study aimed to evaluate the acute locally injurious property of hydroxyl radical generation system by photolysis of H(2)O(2), which is a new disinfection system for the treatment of periodontitis developed in our laboratory. Firstly, generation of the hydroxyl radical by a test device utilizing the photolysis of H(2)O(2) was confirmed by applying an electron spin resonance (ESR)-spin trapping technique. Secondly, the bactericidal effect of the device was examined under a simulant condition in which Staphylococcus aureus suspended in 1 M H(2)O(2) was irradiated with laser light emitted from the test device, resulting in substantial reduction of the colony forming unit of the bacteria within a short time as 2 min. Finally, acute topical effect of the disinfection system on rat oral mucosa and wounded skin was evaluated by histological examination. No abnormal findings were observed in the buccal mucosal region treated three times with 1 M H(2)O(2) and irradiation. Similarly, no abnormal findings were observed during the healing of skin treated with 1 M H(2)O(2) and irradiation immediately after wounding. Since topical treatment with the novel disinfection technique utilizing the photolysis of H(2)O(2) had no detrimental effect on the oral mucosa and the healing of full thickness skin wounds in rats, it is expected that the acute locally injurious property of the disinfection technique is low.

Fungicidal action of hydroxyl radicals generated by ultrasound in water.

It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection.

Metabolic alterations by indoxyl sulfate in skeletal muscle induce uremic sarcopenia in chronic kidney disease

Sarcopenia is associated with increased morbidity and mortality in chronic kidney disease (CKD). Pathogenic mechanism of skeletal muscle loss in CKD, which is defined as uremic sarcopenia, remains unclear. We found that causative pathological mechanism of uremic sarcopenia is metabolic alterations by uremic toxin indoxyl sulfate. Imaging mass spectrometry revealed indoxyl sulfate accumulated in muscle tissue of a mouse model of CKD. Comprehensive metabolomics revealed that indoxyl sulfate induces metabolic alterations such as upregulation of glycolysis, including pentose phosphate pathway acceleration as antioxidative stress response, via nuclear factor (erythroid-2-related factor)-2. The altered metabolic flow to excess antioxidative response resulted in downregulation of TCA cycle and its effected mitochondrial dysfunction and ATP shortage in muscle cells. In clinical research, a significant inverse association between plasma indoxyl sulfate and skeletal muscle mass in CKD patients was observed. Our results indicate that indoxyl sulfate is a pathogenic factor for sarcopenia in CKD.

Kinetic analysis of reactive oxygen species generated by the in vitro reconstituted NADPH oxidase and xanthine oxidase systems.

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (•)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (•)OH. In contrast, (•)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.

Presence of Hydrogen Peroxide, a Source of Hydroxyl Radicals, in Acid Electrolyzed Water

Background Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity. Methodology/Principal Findings The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity. Conclusions It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.

Microbial resistance in relation to catalase activity to oxidative stress induced by photolysis of hydrogen peroxide

The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H2O2) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H2O2 than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H2O2. Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H2O2 than that with weak catalase activity. Kinetic analysis using a modified Lineweaver‐Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H2O2. The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H2O2 can be alleviated by decomposition of H2O2 by catalase in microorganisms.

Bactericidal Action of Photo-Irradiated Aqueous Extracts from the Residue of Crushed Grapes from Winemaking.

Our previous studies revealed that photo-irradiation of polyphenols could exert bactericidal action via reactive oxygen species (ROS). In the present study, the photo-irradiation-induced bactericidal activity of the aqueous extract from the residue of crushed grapes from winemaking was investigated in relation to ROS formation. Staphylococcus aureus suspended in the extract was irradiated with LED light at 400 nm. This solution killed the bacteria, and a 3-4 log and a >5-log reduction of the viable counts were observed within 10 and 20 min, respectively. LED light irradiation alone also killed the bacteria, but the viable counts were 2-4 log higher than those of the photo-irradiated extract. In contrast, almost no change occurred in the suspension without LED irradiation. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of the photo-irradiated extract was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photo-irradiation of the extract. The present study suggests that polyphenolic compounds in the extract exert bactericidal activity via hydroxyl radical formation upon photo-irradiation.

Cytoprotective Effects of Grape Seed Extract on Human Gingival Fibroblasts in Relation to Its Antioxidant Potential

Cytoprotective effects of short-term treatment with grape seed extract (GSE) upon human gingival fibroblasts (hGFs) were evaluated in relation to its antioxidant properties and compared with those of a water-soluble analog of vitamin E: trolox (Tx). GSE and Tx showed comparable antioxidant potential in vitro against di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH; a stable radical), hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Pretreatment or concomitant treatment with GSE for 1 min protected hGFs from oxidative stressors, including H2O2, acid-electrolyzed water (AEW), and 1O2, and attenuated the intracellular formation of reactive oxygen species induced by H2O2 and AEW. Tx also reduced the H2O2- and AEW-induced intracellular formation of reactive oxygen species, but showed no cytoprotective effects on hGFs exposed to H2O2, AEW, or 1O2. These results suggest that the cytoprotective effects of GSE are likely exerted independently of its antioxidant potential.