[Show abstract][Hide abstract] ABSTRACT: In this study, atmospheric nonequilibrium plasmas were generated with six gas species using a multi-gas plasma jet. Singlet oxygen, OH radicals, H radicals, and NO radicals, in reaction with a solution interface, were measured using electron spin resonance. Carbon dioxide plasma generated the largest amount (90 µM) of singlet oxygen at 30 s, and argon-containing vapor gas plasma generated the largest amount (210 µM) of OH radicals. Among the monatomic gas species, nitrogen plasma generated the largest amount (130 µM) of OH radicals. In addition, H radicals were generated with argon, helium, and nitrogen plasmas. NO radicals were generated with nitrogen–oxygen plasma, and the largest amount of NO radicals was generated at a 1:1 volume ratio. These measurement results of the reactive species generated by individual gas plasmas demonstrate the production processes of reactive species.
[Show abstract][Hide abstract] ABSTRACT: Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.2% accuracy (range, 73.9–87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.
Biochemical and Biophysical Research Communications 01/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro cytotoxicity of tafluprost, which is the most recently developed anti-glaucoma prostaglandin (PG) analog, in ocular surface cells is addressed in comparison with other PG analogs. Irrespective of cell lines and models, the cytotoxicity of anti-glaucoma PG eyedrops was primarily related to the concentration of benzalkonium chloride (BAK) contained in the eyedrops as a preservative. Accordingly, preservative-free tafluprost was apparently less cytotoxic than BAK-preserved PG analogs. Furthermore, our study for cytotoxicity assays on ocular cells, conducted by comprehensive investigations covering a variety of concentrations and treatment times, which is termed the cell viability score (CVS) system, demonstrated that 0.001% BAK-preserved tafluprost was not cytotoxic, and suggested that tafluprost may even reduce the cytotoxic effect of BAK. It has been reported that adverse reactions associated with tafluprost in healthy human volunteers and patients with glaucoma include conjunctival hyperemia, eyelid pigmentation, eyelash bristles, and deepening of upper eyelid sulcus. Nonetheless, most clinical studies have demonstrated that not only preservative-free tafluprost but also BAK-preserved tafluprost is well tolerated and safe in patients with glaucoma and ocular hypertension.
[Show abstract][Hide abstract] ABSTRACT: To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.
Analytical and Bioanalytical Chemistry 11/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60 minutes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ⩾ 50% of control. The CVS40/80 was calculated as follows: CVS40/80 = (number of measurements for viability values > 80%) - (number of measurements for viability values < 40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher,%CVS50 and%CVS40/80 became 0 and less than -90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower,%CVS50 became 100. In the case of%CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the%CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.
Regulatory Toxicology and Pharmacology 04/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The reactions of three α-oxoaldehydes (methylglyoxal, glyoxal, and pyruvic acid) with hydroxyl radicals generated by sonolysis of water were investigated using an electron spin resonance (electron paramagnetic resonance) spin-trapping method, and their reaction kinetics were investigated. It is apparent from our experimental results that methylglyoxal exhibits the highest reactivity of the three α-oxoaldehydes. These α-oxoaldehydes can react with hydroxyl radicals faster than other well-known antioxidants can. The reactivity of hydroxyl radicals is higher than that of hydrogen peroxides.
Journal of Clinical Biochemistry and Nutrition 03/2013; 52(2):128-32. · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate a new denture-cleaning device using hydroxyl radicals generated from photolysis of hydrogen peroxide (H2O2). Electron spin resonance analysis demonstrated that the yield of hydroxyl radicals increased with the concentration of H2O2 and light irradiation time. Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant S aureus were killed within 10 minutes with a > 5-log reduction when treated with photolysis of 500 mM H2O2; Candida albicans was killed within 30 minutes with a > 4-log reduction with photolysis of 1,000 mM H2O2. The clinical test demonstrated that the device could effectively reduce microorganisms in denture plaque by approximately 7-log order within 20 minutes.
The International journal of prosthodontics 07/2012; 25(4):376-80. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the cytotoxicity of antibiotic eyedrops to ocular surface cells using a semi-quantitative method, a range of commercially available antibiotic eyedrops were assessed by using three corneal cell lines and one conjunctival cell line. All antibiotic solutions were free of benzalkonium chloride. Cell viability was determined by the MTT assay and neutral red assay following the exposure of cells to the undiluted, 2- and 10-fold diluted drugs for 10, 30, and 60 min. Toxicity was compared using % cell viability score (%CVS) . The tested eyedrops and values of %CVS50 and %CVS40/80 were Bestron(®) (cefmenoxime, 100, 94) , Panimycin(®) (dibekacin, 86, 58) , Noflo(®) (norfloxacin, 90, 50) , Cravit(®) (levofloxacin, 86, 46) , Tosfulo(®) (tosufloxacin, 57, -3) , and Vigamox(®) (moxifloxacin, 57, -6) . Cell viability markedly increased after dilution. For instance, cell viability assayed by MTT was > 80% for all the measurements in antibiotics diluted 10-fold, and the rate of the measurements showing > 80% cell viability decreased to 43% (31 out of 72 measurements) in the solutions diluted 2-fold. Of the drugs tested, Bestron(®) containing cefmenoxime showed the weakest toxicity. Vigamox(®) containing moxifloxacin and Tosuflo(®) containing tosufloxacin were more toxic when compared with the other antibiotics. CVS was useful for the comparison of the cytotoxicity of the drugs.
[Show abstract][Hide abstract] ABSTRACT: We investigated the virucidal activity of commercially available alcohol-based hand rub products against coxsackievirus A7, B5, feline calicivirus F9, and human adenovirus type 3, type 7, type 8 using susceptible cell lines, Vero cells, CRFK cells, and A549 cells. Fifteen tested hand rub products were ethanol (EtOH) for disinfection (Japanese Pharmacopoeia Grade), two EtOH-based products, one povidone iode-containing product, one alkyldiaminoethylglycine hydrochloride-containing product, six benzalkonium chloride (BAK)-containing products, and four chlorohexidine gluconate (CHG)-containing products. Some active ingredients (BAK, benzetonium chloride, and CHG) were diluted with EtOH to make 0.5% and 0.2% solutions. Virus inactivation rates were calculated after contact with each hand rub product for 10 or 60 seconds. Of the hand rub products tested, only the povidone iode-based product showed antiviral activity superior to that of EtOH against all the strains. EtOH solutions of active ingredients (0.2% and 0.5%) also showed decreased antiviral activity. In conclusion, antiviral activity of all the commercially available alcohol-based hand rub products except that containing povidone idode was dependent on their active ingredients. The povidone idode-containing hand rub product kept its effectiveness even after the dilution with EtOH. Although alcohol-based hand rub products are convenient and suitable for the control of some microbes, they are not generally recommended for the control of viral infections.
[Show abstract][Hide abstract] ABSTRACT: The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2). Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H(2)O(2) 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 H(2)O(2). The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H(2)O(2) can be alleviated by decomposition of H(2)O(2) by catalase in microorganisms.
Microbiology and Immunology 01/2012; 56(1):48-55. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anti-inflammatory eyedrops are often used in the treatment of corneal epithelial disorders. In the present study, we evaluated the cytotoxicity of six anti-inflammatory eyedrops in four ocular surface cell lines.
The cytotoxicity of six commercially available anti-inflammatory ophthalmic solutions (ie, diclofenac, bromfenac, pranoprofen, betamethasone, and fluoromethorone) was assessed in three corneal cell lines and one conjunctival cell line. Cell viability was determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and neutral red assays after exposing the cells to 10, 30, and 60 minutes of onefold, twofold, and tenfold dilutions of the drugs. Cytotoxicity was compared using the cell viability score (CVS), an integrated cytotoxic parameter that takes various factors into account, such as dilution by tear fluid or concentration by evaporation, drug exposure time, and ocular surface cell type.
Based on the CVS scores, the order of the anti-inflammatory eyedrops tested from least to most cytotoxic, with the active ingredient %CVS(50), and %CVS(40/80) for each solution given in parentheses, was as follows: Rinderon(®) (betamethasone, 100%, 100%) >0.02% Flumethoron(®) (fluoromethorone, 68%, 22%) = 0.1% Flumethoron(®) (fluoromethorone, 76%, 22%) >Bronuck(®) (0.1% bromfenac, 53%, -8%) = Diclod(®) (0.1% diclofenac, 44%, -15%) = Niflan(®) (pranoprofen, 50%, -19%). Rinderon(®) exhibited the least toxicity of all the anti-inflammatory eyedrops tested. Eyedrops containing non-steroidal anti-inflammatory drugs exhibited greater cytotoxicity than those containing steroids with benzalkonium at comparable concentrations. Concentration was the most significant factor affecting cell viability.
The cytotoxicity of the anti-inflammatory eyedrops evaluated in the present study depended on both the pharmaceutical components and preservatives. The CVS is a concise indicator of drug cytotoxicity.
[Show abstract][Hide abstract] ABSTRACT: We investigated the bactericidal effects and cytotoxicity of an ortho-phthalaldehyde product in comparison with those of its predecessor glutaraldehyde products. Bactericidal effects ware examined on Mycobacterium terrae, a standard organism used for investigating the bactericidal effect of high-level disinfectants. Cytotoxicity as determined by the MTT assay was examined by using four cell lines. The colony forming test, a method to examine residual toxicity, and the evaporation test, a newly developed method to examine the toxicity of the evaporated ingredients, were performed. Test solutions were 2.25% and 3.5% glutaraldehyde (GA) products and a 0.55% ortho-phthalaldehyde (OPA) product, and glutaraldehyde itself. All the disinfectants showed sufficient bactericidal effects on M. terrae. Meanwhile, the OPA product was less toxic than GA products and GA itself to all the cell lines tested. The colony forming test showed that GA products and GA itself exerted residual cytotoxicity more potently than did the OPA product. The evaporation test showed that GA products and GA itself exerted cytotoxicity via evaporation more potently than did the OPA product. In conclusion, OPA appears to be less cytotoxic than GA even though bactericidal effects were comparable. This may be due to the lower concentration of the active ingredient (ortho-phthalaldehyde) in the OPA product.
[Show abstract][Hide abstract] ABSTRACT: Human oral polymorphonuclear cells (OPMNs) play an important role in the defence of oral cavity from bacteria by releasing reactive oxygen species (ROS). The purpose of the study is to determine ROS generated by OPMNs collected from healthy volunteers without any dental problems by applying a luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt-dependent chemiluminescence (CL) response in combination with radical and ROS scavengers. In the CL response induced by OPMNs primed with phorbol 12-myristate 13-acetate, 0.23 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, 12.5 U/ml of super oxide dismutase (SOD) as a superoxide anion (O(2)(·-)) scavenger, and 0.96 M dimethyl sulfoxide (DMSO) as a hydroxyl radical ((·)OH) scavenger inhibited the response by approximately 90%, 70%, and 60%, respectively. The inhibitory effects were obtained in a range of concentrations where viability of the OPMNs exposed to DMPO, SOD, and DMSO were more than 70%. Electron spin resonance-spin trapping analysis confirmed that at least O(2)(·-) and (·)OH were generated via primed OPMNs. Furthermore, the addition of both 12.5 U/ml of SOD and 0.96 M DMSO inhibited the CL response by more than 90%, which was in accordance with the inhibition rate obtained by the addition of DMPO. Therefore, it is suggested that around 90% of the CL response is induced by free radicals, and at least around 70% of the radicals are SOD-inhibitable, meaning that they are originally derived from O(2)(·-). In addition, some of the (·)OH are generated independently of O(2)(·-) because if all of the (·)OH were formed through dismutation of O(2)(·-), only 70% of the CL response would be inhibited by the combination of SOD and DMSO as was inhibited by SOD alone. The present study suggests that OPMNs in health individuals have an ability to generate free radicals, which consist mainly of O(2)(·-), (·)OH and possibly an intermediate ROS derived from O(2)(·-) and (·)OH.
Archives of oral biology 11/2011; 57(6):636-41. · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the cytotoxicity of six ingredients used in eyedrops with regard to four corneoconjunctival cell lines. Cells were treated with the undiluted solution, and 2-fold, and 10-fold dilutions of each solution for 10, 30, and 60 min and cell viability was measured with the neutral red assay and the MTT assay. The degree of toxicity was based on the cell viability score (CVS). The CVS50 was determined by the number of measurements with a viability ≥ 50% of control. The CVS40/80 was calculated as follows: CVS40/80 = (number of measurements with a viability value >80%) - (number of measurements with a viability value <40%). Results were expressed as % of total measurements (%CVS). The results of each ingredient for %CVS50, and %CVS40/80 were 0.01% benzalkonium chloride (51, -13), 1% boric acid (100, 99), 0.4% methyl paraoxybenzoate (100, 100), 0.4% propyl paraoxybenzoate (100, 100), 1.0% polysorbate 80 (68, 18), and 0.5% chlorobutanol (100, 100). The use of benzalkonium chloride led to apparently low cell viability compared to the other five solutions.
[Show abstract][Hide abstract] ABSTRACT: 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymer is a multifunctional agent with antiadhesive, antithrombogenetic, and strong hydrating properties. MPC polymer-containing eye drops are the first such ophthalmic product to be commercially available; they contain approximately 0.1% Lipidure-PMB (copolymer of MPC and butyl methacrylate; NOF Corporation, Tokyo, Japan). This study evaluated the cytotoxicity of this new product toward ocular surface cells.
SIRC (rabbit cornea), BCE C/D-1b (bovine cornea), RC-1 (rabbit cornea), and Chang (human conjunctiva) cell lines were tested in this study. Cell viability was measured using both the MTT assay and the neutral red test in cells treated for 10, 30, or 60 min with MPC-containing eye drops and 4 commercial ophthalmic solutions containing sodium hyaluronate (SH) at various doses (undiluted, twofold diluted, and tenfold diluted). Cell viability scores were calculated. Cell viability was analyzed using ANOVA and the Dunnett test.
After treatment with the MPC eye drops, cell viability rates were maintained at over 80% irrespective of the cell lines, dilution rates, exposure times, and assays, and were similar to those of the clinically approved artificial tear products other than Hyalein 0.1%, although some significant differences were found.
The MPC eye drops were tolerable to ocular surface cells, and comparable to single doses of clinically approved drugs containing sodium hyaluronate.
Japanese Journal of Ophthalmology 08/2011; 55(5):541-6. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the cytotoxicity of anti-allergic eye drops for human corneal endothelial cells (HCEC) and commercially available ocular surface cells. A primary HCEC culture was derived from human eye bank specimens. SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells) were obtained commercially. The WST-1 assay was used to measure HCEC viability, and the viability of other cells was measured using the MTT assay. Cells were treated with 7 commercially available anti-allergic eye drops for 48 h and cell viability was measured and calculated as a percentage of control. The degree of toxicity for each eye-drop solution was based on the cell viability score (CVS). HCECs treated with a 1000-fold dilution of the eye-drop solution had a viability score of 67% for Rizaben and ≥80% for the other solutions with Zepelin being the least toxic. Cell viability in response to eye-drop solutions preserved with benzalkonium chloride (BAK) was dependent on the concentration of the drug solution and exposure time. Treatment of ocular surface cells with a 20-fold dilution of the eye-drop solution resulted in the following order of cell viability as determined by their CVS: Zepelin > Ketas = Zaditen ≥ Tramelas PF = Patanol ≥ Rizaben ≥ Livostin. This order was similar to that observed for HCECs, and cell viability was found to be concentration-dependent. Based on the penetration of the drug into eye tissues, HCECs are only likely to be pharmaceutically damaging in rare cases. Epithelial cell viability depends primarily on the concentration of BAK rather than on the action of the active component in the eye-drop solution. CVS values were useful for comparison of toxicity.
Journal of oleo science 01/2011; 60(3):139-44. · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.
[Show abstract][Hide abstract] ABSTRACT: The relationship between the amount of hydroxyl radicals generated by photolysis of H(2)O(2) and bactericidal activity was examined. H(2)O(2) (1 M) was irradiated with laser light at a wavelength of 405 nm to generate hydroxyl radicals. Electron spin resonance spin trapping analysis showed that the amount of hydroxyl radicals produced increased with the irradiation time. Four species of pathogenic oral bacteria, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Enterococcus faecalis, were used in the bactericidal assay. S. mutans in a model biofilm was also examined. Laser irradiation of suspensions in 1 M H(2)O(2) resulted in a >99.99% reduction of the viable counts of each of the test species within 3 min of treatment. Treatment of S. mutans in a biofilm resulted in a >99.999% reduction of viable counts within 3 min. Other results demonstrated that the bactericidal activity was dependent on the amount of hydroxyl radicals generated. Treatment of bacteria with 200 to 300 μM hydroxyl radicals would result in reductions of viable counts of >99.99%.
Antimicrobial Agents and Chemotherapy 10/2010; 54(12):5086-91. · 4.57 Impact Factor