Koji Yashiro

Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.

In the present study, we examined the effect of high fructose-induced metabolic syndrome (MetS) on tissue vitamin E and lipid peroxide (LPO) levels in rats. Feeding of a diet containing 60% fructose (HFD) to Wistar rats for 2, 4, and 6 wk caused week-dependent increases in HOMA-IR score and serum insulin, triglyceride, total cholesterol, and free fatty acid concentrations. Each week HFD feeding increased serum vitamin E concentration. Six-week HFD feeding reduced vitamin E status (the serum ratio of vitamin E/triglyceride+total cholesterol). Four- and 6-wk HFD feeding increased serum LPO concentration. Two-week HFD feeding increased liver, heart, kidney, and skeletal muscle (SM) vitamin E contents and decreased white adipose tissue (WAT) vitamin E content. Four- and 6-wk HFD feeding further reduced WAT vitamin E content without affecting the increased kidney and SM vitamin E contents. Six-week HFD feeding reduced the increased liver and heart vitamin E contents below the level of non-HFD feeding. Four-week HFD feeding increased heart and WAT LPO contents. Six-week HFD feeding increased liver LPO content and further increased heart and WAT LPO contents. Kidney and SM LPO contents remained unchanged. These results indicate that HFD-rats with early MetS have increased liver, kidney, heart, and SM vitamin E contents and decreased WAT vitamin E content under unchanged tissue LPO content and vitamin E status, while HFD-fed rats with progressed MetS have both decreased liver, heart, and WAT vitamin E contents under increased tissue LPO content and disrupted vitamin E status.

Four human hydroxysteroid dehydrogenases in the aldo-keto reductase (AKR) superfamily, AKR1C1-AKR1C4, are involved in the metabolism of steroids and other carbonyl compounds including drugs, and altered expression of AKRs (1C1, 1C2 and/or 1C3) is related to the pathogenesis of several extrahepatic cancers. Here, we report that unsaturated fatty acids (FAs) are potent competitive inhibitors of the AKR enzymes. The sensitivities to the FAs were different among the enzymes, especially between AKR1C1 and AKR1C2. The most potent inhibitors for AKR1C1, AKR1C2 and AKR1C4 were docosahexaenoic acid (K-i 0.77 A mu M), palmitoleic acid (K-i 0.41 A mu M) and linoleic acid (K-i 0.33 A mu M), respectively. AKR1C3 was the most sensitive to FA inhibition, showing low K-i values (0.23-0.29 A mu M) for oleic, linoleic, eicosapentaenoic and docosahexaenoic acids. Linoleic and oleic acids also inhibited AKR1C3-mediated metabolism of 9,10-phenanthrenequinone in colon DLD1 cells. Molecular docking and site-directed mutagenesis studies suggested upon FA binding to AKR1C1 and AKR1C3: (i) the carboxyl group of the FA binds to the oxyanion-binding site in the active site; (ii) the difference in FA sensitivity between AKR1C1 and AKR1C2 is due to their residue difference at position 54; (iii) Ser118, Phe306 and Phe311 of AKR1C3 are important for determining the inhibitory potency of FAs.

In the present study, we examined the protective effect of N,N'-dimethylthiourea (DMTU), a scavenger of hydroxyl radical (OH), against water-immersion restraint stress (WIRS)-induced gastric mucosal lesions in rats. When male Wistar rats fasted for 24 h were exposed to WIRS for 3 h, gastric mucosal lesions occurred with increases in the levels of gastric mucosal myeloperoxidase (MPO), an index of tissue neutrophil infiltration, pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin 1beta), lipid peroxide (LPO), and nitrite/nitrate (NOx), an index of nitric oxide synthesis, and decreases in the levels of gastric mucosal nonprotein SH and vitamin C and gastric adherent mucus. DMTU (1, 2.5, or 5 mmol/kg) administered orally at 0.5 h before the onset of WIRS reduced the severity of gastric mucosal lesions with attenuation of the changes in the levels of gastric mucosal MPO, pro-inflammatory cytokines, LPO, NOx, nonprotein SH, and vitamin C and gastric adherent mucus found at 3 h after the onset of WIRS in a dose-dependent manner. Serum levels of corticosterone and glucose, which are indices of stress responses, increased in rats exposed to WIRS for 3 h, but DMTU pre-administered at any dose had no effect on these increases. These results indicate that DMTU protects against WIRS-induced gastric mucosal lesions in rats by exerting its antioxidant action including OH scavenging and its anti-inflammatory action without affecting the stress response.

In this study, we examined whether compound 48/80 (C48/80), a mast cell degranulator, causes hepatic oxidative damage in rats. Serum and liver biochemical parameters were determined 0.5, 3 or 6 h after a single treatment with C48/80 (0.75 mg/kg). Serum histamine and serotonin levels increased 0.5 h after C48/80 treatment but diminished thereafter. Increases in serum vitamin C (VC) and transaminases and hepatic hydrogen peroxide, lipid peroxide, and myeloperoxidase levels and a decrease in hepatic reduced glutathione level occurred 0.5 h after C48/80 treatment and further proceeded at 3 h, but these changes diminished at 6 h. Serum lipid peroxide and hepatic VC levels increased 3 h after C48/80 treatment. Hepatic glycogen level decreased 0.5 h after C48/80 treatment and further decreased at 3 h. Pre-administered ketotifen diminished all these changes found at 3 h after treatment, while pre-administered. NPC 14686 diminished these changes except changes in serum histamine and serotonin levels. Hepatocellular apoptosis observed at 3 h after C48/80 treatment was attenuated by pre-administered ketotifen and NPC 14686. These results indicate that C48/80 causes oxidative damage by enhancing VC synthesis via reduced glutathione depletion-dependent glycogenolysis and lipid peroxidation through neutrophil infiltration following mast cell degranulation in rat livers.

The aim of this study was to clarify the effect of water-immersion restraint stress (WIRS) on tryptophan (Trp) catabolism through the kynurenine (Kyn) pathway in rat tissues. The tissues of rats subjected to 6 h of WIRS (+WIRS) had increased tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) activities and increased TDO and IDO1 (one of two IDO isozymes in mammals) mRNA expression levels, with decreased Trp and increased Kyn contents in the liver. +WIRS rats had unchanged TDO and IDO activities in the kidney, decreased TDO activity and unchanged IDO activity in the brain, and unchanged IDO activity in the lung and spleen, with increased Kyn content in all of these tissues. Pretreatment of stressed rats with RU486, a glucocorticoid antagonist, attenuated the increased TOD activity, but not the increased IDO activity, with partial recoveries of the decreased Trp and increased Kyn contents in the liver. These results indicate that WIRS enhances hepatic Trp catabolism by inducing both IDO1 and TDO in rats.

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC50 value of 4.2 mu M cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC50 values of 1.1 mu M. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the K-i values of 0.24 -1.1 mu M. These fatty acids, except for alpha-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10. (C) 2016 Elsevier Inc. All rights reserved.

We examined how dietary supplementation of vitamin E protects against liver oxidative damage in rats with water-immersion restraint stress (WIRS). Before WIRS exposure, rats received a normal diet (ND) or vitamin E-supplemented diet (VESD) (500 IU alpha-tocopherol/kg diet) at a mean dose of 15 g/animal/d for 4 wk. The two diet groups had serum transaminases and lactate dehydrogenase activities and adrenocorticotropic hormone, corticosterone, and glucose levels to a similar extent. VESD-fed rats had higher liver a-tocopherol concentrations and lower liver ascorbic acid, total coenzyme Q(9) (CoQ(9)), reduced CoQ(9), reduced CoQ(10), and lipid peroxide (LPO) concentrations than ND-fed rats. When the two diet groups were exposed to 6 h of WIRS, the serum liver cell damage index enzyme activities increased more greatly in ND-fed rats than in VESD-fed rats but the serum stress marker levels increased to a similar extent. The WIRS exposure caused no change in liver LPO concentration with the further increase in liver a-tocopherol concentration in VESD-fed rats but increased liver LPO concentration without changing liver alpha-tocopherol concentration in ND-fed rats. Upon the WIRS exposure, liver reduced glutathione concentration decreased with the further decrease in liver ascorbic acid concentration in VESD-fed rats and those concentrations decreased in ND-fed rats. The WIRS exposure recovered the decreased liver total CoQ(9) and reduced CoQ(9) concentrations in VESD-fed rats but decreased liver total CoQ(9), reduced CoQ(9), and reduced CoQ(10) concentrations in ND-fed rats. These results indicate that dietary vitamin E supplementation protects against liver oxidative damage without affecting the stress response in rats with WIRS.

We examined whether water-immersion restraint stress (WIRS) disrupts nonenzymatic antioxidant defense systems through ascorbic acid depletion in the adrenal gland of rats. Rats were exposed to WIRS for 0.5, 1.5, 3 or 6h. WIRS increased serum adrenocorticotropic hormone, corticosterone and glucose concentrations and adrenal corticosterone content at each time point. WIRS increased adrenal lipid peroxide content at 3 and 6h, and the increase was twofold higher than the unstressed level at 6h. WIRS decreased adrenal ascorbic acid content at each time point, and the decrease reached one-third of the unstressed level at 6h. WIRS increased adrenal reduced glutathione content at 0.5 and 6h but reduced that content to half of the unstressed level at 6h. WIRS increased adrenal -tocopherol content at 1.5h but returned that content to the unstressed level thereafter. When rats with 6h of WIRS was orally preadministered with l-ascorbic acid (250mg/kg), WIRS-induced changes in adrenal lipid peroxide, ascorbic acid and reduced glutathione contents were attenuated without any change in stress response. These results indicate that WIRS disrupts nonenzymatic antioxidant defense systems through rapid and continuous ascorbic acid depletion in the adrenal gland of rats. Copyright (c) 2012 John Wiley & Sons, Ltd.

We examined the effect of vitamin E depletion on liver oxidative damage in rats with water-immersion restraint stress (WIRS). Male Wistar rats were fed a normal diet (N) or vitamin E-depleted diet (VE-D) for 4 wk. N- and VE-D-fed rats were exposed to WIRS for 6 h. The activities of serum transaminases and lactate dehydrogenase and serum ascorbic acid concentration were similar in both diet groups. WIRS exposure increased these serum enzyme activities and the serum ascorbic acid concentration in both diet groups but the ratios of these increases were higher in VE-D-fed rats than in N-fed rats. Serum and liver α-tocopherol concentrations in VE-D-rats were approximately 50% and 30% of those in N-fed rats, respectively. WIRS exposure reduced liver α-tocopherol concentration in VE-Dfed rats, but not in N-fed rats. Liver ascorbic acid and reduced glutathione concentrations were higher in the VE-D-fed group than in the N-fed group. WIRS exposure reduced liver ascorbic acid and reduced glutathione concentrations in both diet groups. There were no differences in liver concentrations of coenzyme Q9 or coenzyme Q10 in the reduced form between the N- and VE-D-fed groups. WIRS exposure reduced liver concentrations of coenzyme Q9 and coenzyme Q10 in the reduced form in both diet groups. Liver lipid peroxide concentration was higher in the VE-D-fed group than in the N-fed group. WIRS exposure raised liver lipid peroxide concentration more in the VE-D-fed group than in the N-fed group. These results indicate that vitamin E depletion enhances liver oxidative damage in rats with WIRS.

Objectives: We examined whether a single exposure of rats to water-immersion restraint stress (WIRS) induces oxidative stress in the thymus and spleen. Methods: Vitamin E, ascorbic acid, reduced glutathione (GSH), and lipid peroxide (LPO) were assayed in the thymus and spleen of rats with and without 6 hours of WIRS. Results: In unstressed rats, vitamin E, ascorbic acid, GSH, and LPO levels were higher in the thymus than in the spleen. Thymic ascorbic acid level was lower in stressed rats than in unstressed rats. Splenic ascorbic acid level was similar in both groups. Thymic and splenic GSH levels were lower in stressed rats than in unstressed rats but the reduced amount of GSH was lower in the spleen than in the thymus. Thymic vitamin E level was lower in stressed than in unstressed rats. Splenic vitamin E level was higher in stressed rats than in unstressed rats. Thymic and splenic LPO levels were higher in stressed rats than in unstressed rats but the increased amount of LPO was higher in the thymus than in the spleen. Conclusion: It is indicated that a single expose of rats to WIRS induces oxidative stress more severely in the thymus than in the spleen.

We examined whether non-enzymatic antioxidant defense systems are disrupted in the brain of rats with water-immersion restraint stress. When rats were exposed to water-immersion restraint stress for 1.5, 3 or 6 h, the brain had decreased ascorbic acid and reduced glutathione contents and increased lipid peroxide and nitric oxide metabolites contents at 3 h and showed further changes in these components with a reduction of vitamin E content at 6 h. Increased serum levels of stress markers were found at 1.5, 3 or 6 h of WIRS. Oral pre-administration of L-ascorbic acid (1.5 mmol/kg) or vitamin E (0.5 mmol/kg) to rats with 6 h of water-immersion restraint stress attenuated the increases in lipid peroxide and nitric oxide metabolites contents and the decrease in vitamin E content in the brain. Pre-administered L-ascorbic acid attenuated the decreases in brain ascorbic acid and reduced glutathione contents at 6 h of water-immersion restraint stress, while pre-administered vitamin E enhanced the decreases in those contents. Pre-administered L-ascorbic acid or vitamin E did not affect the increased serum levels of stress markers in rats with 6 h of water-immersion restraint stress. These results indicate that water-immersion restraint stress causes disruption of nonenzymatic antioxidant defense systems through enhanced lipid peroxidation and nitric oxide generation in the brain of rats with water-immersion restraint stress.

Although CD57(+) lymphocytes are closely correlated with prognosis in various cancers, the role of subsets of CD57(+) cells in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) is unclear. In the present study, peripheral blood (PB) from HCV-related HCC patients was analyzed. Plasma cytokine levels and in vitro cytokine-producing capabilities were analyzed with enzyme-linked immunosorbent assays, and CD57(+) cell subsets were studied using a multi-color FACS system. Interferon (IFN)-gamma was undetectable in the plasma of patients with tumors at any stage, whereas the plasma levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10 and IL-18, but not that of IL-12, were significantly higher in stage IV patients compared to patients with earlier-stage tumors. In contrast, the IFN-gamma-producing capability of PB was highest in stage I patients and gradually decreased with tumor progression. The IL-10-, IL-18- and IL-12-proclucing capabilities of PB increased from stage I to III. However, PB-TNF-alpha, IL-10- and IL-18-producing capabilities were reduced in stage IV patients, probably due to repeated anti-cancer treatments. The percentage of CD4(+)CD57(+)alpha beta TCR+ cells (CD4(+)CD57(+) T cells) in peripheral blood lymphocytes (PBLs) increased with tumor progression. Moreover, the percentage of CD4(+)CD57(+) T cells in PBLs and the ratio of CD4(+)CD57(+) T cells to CD4(+)alpha beta TCR+ cells (CD4(+) T cells), but not that of CD4(+)CD57(+) T cells to CD57(+)alpha beta TCR+ cells (CD57(+) T cells), showed a significant inverse correlation with PB-IFN-gamma-producing capability. The present results suggest that an increase in CD4(+)CD57(+) T cells controls the capability of PB to produce the antitumor cytokine IFN-gamma and that PB-IFN-gamma production is impaired with HCC tumor progression.