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Biomedical subjects

Etsuo Niki

Publications and source records attributed to Etsuo Niki.

At least 37 records · Page 2Linked to original sources
PubMed2005

Cytotoxic effect of formaldehyde with free radicals via increment of cellular reactive oxygen species.

It is well known that formaldehyde (HCHO) and reactive oxygen species (ROS), such as free radicals, are cytotoxic as well as potentially carcinogenic. Although the individual effects of these reactants on cells have been investigated, the cytotoxicity exerted by the coexistence of HCHO and reactive radicals is poorly understood. The present study using Jurkat cells demonstrated that the coexistence of HCHO with water-soluble radical initiator, 2,2'-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) dramatically decreased cell viability, and that under such conditions scant cell death was observable induced by either of the reactants alone. Based on the results of phosphatidylserine exposure and caspase activation, this observed cell death, in fact, was apparently necrotic rather than apoptotic. To understand the mechanisms of the cell toxicity of HCHO and AIPH, we assessed two kinds of oxidative stress markers such as cellular glutathione (GSH) content and cellular ROS, and the DNA-protein cross-links, which formed as the result of HCHO treatment. A marked decrease in total cellular GSH was observed not only in the case of the coexistence conditions but also with AIPH alone. Dichlorodihydrofluorescein (DCF) assay revealed that cellular ROS were synergistically increased before cell death. The formation of DNA-protein cross-links was observed in the presence of HCHO and AIPH, and the extent was similar to HCHO alone. Co-incubation with semicarbazide, which inactivates HCHO, prevented this cell death induced by a combination of HCHO and AIPH. Semicarbazide also exhibited an inhibitory effect on the synergistic increment of cellular ROS and the formation of DNA-protein cross-links. These results suggest that the free radicals from AIPH induced GSH reduction, while HCHO resulted in the formation of DNA-protein cross-links, eventuating in a synergistic, incremental increase of cellular ROS and cell death brought about by this combination.

Azo Compounds↗
PubMed2005

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid.

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.

Animals↗
PubMed2005

Characterization of monochloramine toxicity on PC12 cells and protective effect of tocopherol via antioxidative function.

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH(2)Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 microM NH(2)Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH(2)Cl cytotoxicity, we examined the effect of various kinds of antioxidants including alpha-tocopherol (alpha-Toc) and beta-tocopherol (beta-Toc). These antioxidants exerted a protective effect against NH(2)Cl-induced cell death, and alpha-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 microM NH(2)Cl. The formation of reactive oxygen species (ROS) was also measured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an antioxidant, such as alpha-Toc, suppressed the increase in ROS. Interestingly, beta-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH(2)Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function.

Animals↗
PubMed2005

Closed-loop analysis of cardiovascular variability in rats under restraint stress.

Causal transfer function analysis was applied to the heart rate variability and blood pressure variability in normotensive male Sprague-Dawley rats those were measured before, during, and after acute restraint stress. The causal transfer gain (CTG) from systolic blood pressure (SBP) to RR interval (RRI) and CTG from RRI to SBP were estimated. The mean value of the CTG from SBP to RRI in the low-frequency (LF) band (0.27-0.74 Hz) was significantly lower during the restraint period than during the baseline period and remained significantly lower during the recovery period. The mean value of the CTG from RRI to SBP in the LF band, in contrast, was significantly higher during the restraint period than during the baseline period, and during the recovery period it tended to return relatively rapidly to the baseline level. Arterial pressure tended to increase only at the onset of the restraint stress and then to decline not only during the restraint period but also during the recovery period, finally leveling off significantly below the baseline level. These results suggest that restraint stress suppresses the baroreflex control gain not only during the restraint period but also during the recovery period and that this results in the arterial hypotension during the recovery period.

Animals↗
PubMed2005

Total hydroxyoctadecadienoic acid as a marker for lipid peroxidation in vivo.

An improved method for the measurement of lipid peroxidation in vivo has been recently developed, where total hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomer, (Z, E)-HODE/(E, E)-HODE, could be also measured. In the present study, in order to examine the effect of continuous, slow flux of free radicals in vivo, a water-soluble radical generator was administered to rats and mice and the amounts of HODE and 8-isoprostane in plasma and liver were measured. It was found that the administration of free radical-generating azo compound increased the level of HODE and decreased the (Z, E)-HODE/(E, E)-HODE ratio in both plasma and liver. The level of HODE was much higher than 8-isoprostane.

Amidines↗
PubMed2005

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL.

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.

Cell Count↗
PubMed2005

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase.

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.

Affinity Labels↗
PubMed2004

Simultaneous determination of nitrate and nitrite in biological fluids by capillary electrophoresis and preliminary study on their determination by microchip capillary electrophoresis.

In order to develop a highly sensitive and high-throughput screening method for nitrogen monoxide metabolites in biological fluids, we have investigated the simultaneous determination of nitrite and nitrate, using capillary electrophoresis and microchip capillary electrophoresis. In capillary zone electrophoresis, a running buffer based on human serum components with high ionic strength has been developed for the determination of nitrite and nitrate in human serum and human saliva. We obtained successful separation of nitrite and nitrate in the serum and the saliva within 7 min under optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and area were obtained by a standard addition method. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate in the serum were 2.6 and 1.5 microM, respectively. The values of the relative standard deviation of peak height for the serum with 9.2 microM nitrite and 20.9 microM nitrate were 5.7 and 4.1%, respectively. For on-site analysis with high-throughput screening, a microchip capillary electrophoresis method using a microchip made of quartz with a UV detector was developed. In this high-throughput format, using a running buffer with an electroosmotic flow modifier, the peaks of nitrite and nitrate in an artificial serum sample were obtained within 8 s. In high-resolution mode, using the buffer without electroosmotic flow modifier, the separation of nitrite and nitrate was obtained within 15 s. In high-resolution mode, using an artificial serum sample with 50 microM NO2- and 50 microM NO3-, the limits of detection (S/N = 3) of 41 microM for NO2- and 26 microM for NO3- were obtained. The method was applied to human serum and saliva. We obtained peaks due to nitrite and nitrate in 10-fold diluted saliva.

Electrochemistry↗
PubMed2004

Inhibition of plasma lipid peroxidation by anti-atherogenic antioxidant BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran.

BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, is a synthetic antioxidant which is now being developed as an anti-atherogenic drug. The antioxidant action of BO-653 against lipid peroxidation in rat plasma was studied and compared with its analogue BO-653M, 2,3-dihydro-5-hydroxy-4,6-di-methyl-2,2-dipentylbenzofuran, and vitamin E. BO-653 was readily incorporated into plasma by oral administration and it inhibited plasma lipid peroxidation more efficiently than vitamin E independent of the presence or absence of vitamin C. On the other hand, its analogue BO-653M having two methyl substituents in place of tert-butyl groups of BO-653 did not inhibit the lipid peroxidation in plasma as efficiently as BO-653, demonstrating clearly that the tert-butyl groups at the ortho-position play a key role in determining the antioxidant efficacy.

Animals↗
PubMed2004

p-Nitrotoluene causes hyperactivity in the rat.

It has not been known which endocrine disruptors exert their effects on neuronal functions, particularly leading to behavioral alterations. To address this, we examined the effects of p-nitrotoluene, an endocrine disruptor, on rat behavior and gene expression. Single intracisternal administration of p-nitrotoluene (ca. 10 microg) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. They were about 1.4-fold more active in the nocturnal phase after administration of p-nitrotoluene than control rats. Based on DNA array analyses, p-nitrotoluene decreased more than two-fold the levels of gene expression of the mesencephalic dopamine transporter at 8 weeks old. Thus, it was demonstrated for the first time that p-nitrotoluene definitely affected the developing brain, resulting in hyperactivity in the rat.

Animals↗
PubMed2004

Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells.

Mutation in DJ-1 gene is the cause of autosomal recessive Parkinson's disease, however, its physiological function remains unclear. The isoelectric point of DJ-1 shows an acidic shift after cells are treated with hydrogen peroxide. This suggests that DJ-1 is modified in response to oxidative stress. Here we report the structural characterization of an acidic isoform of DJ-1 using a proteomic approach with nanospray interface liquid chromatography-electrospray ionization/linear ion trap mass spectrometer. When human umbilical vein endothelial cells were exposed to hydrogen peroxide, all three cysteines in DJ-1 were oxidized to cysteine sulphonic acid. Although a small part of the Cys-46 and Cys-53 were oxidized, Cys-106 was oxidized completely at any hydrogen peroxide concentration used here. These results suggest that Cys-106 is the most sensitive among three cysteine residues to oxidative stress, and that DJ-1 function is regulated, in terms of the intracellular redox state, by oxidation of Cys-106.

Amino Acid Sequence↗
PubMed2004

Distribution of tocopherols and tocotrienols to rat ocular tissues after topical ophthalmic administration.

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. Chemically, tocopherols and tocotrienols are closely related; however, it has been observed that they have widely varying degrees of biological effectiveness. The present study has been carried out in an attempt to deepen our understanding of whether there is a significant difference in distribution between tocopherol and tocotrienol homologs to rat eye tissues. Rats were administered 5 microL of pure tocopherol or tocotrienol to each eye once a day for 4 d. Various tissues of the eyes were separated and analyzed for tocopherol and tocotrienol concentrations. The concentration of alpha-to-cotrienol increased markedly in every tissue to which it was administered; however, no significant increase was observed in the case of alpha-tocopherol. The intraocular penetration of gamma-tocopherol and gamma-tocotrienol did not differ significantly. Additionally, a significant increase in total vitamin E concentration was observed in ocular tissues, including crystalline lens, neural retina, and eye cup, with topical administration using a relatively small amount (5 microL) of vitamin E, whereas no significant increase was observed when the same amount of vitamin E was administered orally. Topical administration of tocotrienols is thus an effective way to increase ocular tissue vitamin E concentration.

Administration, Topical↗
PubMed2004

Dynamics of antioxidant action of vitamin E.

Vitamin E is the major lipophilic, radical-scavenging antioxidant in vivo and protects humans from the oxidative stress mediated by active oxygen and nitrogen species. The mechanisms of the inhibition of oxidation by vitamin E in vitro are now fairly well understood, but the dynamics of antioxidant action of vitamin E in vivo have not been well elucidated yet, primarily because of the inherent heterogeneity of biological systems. In this Account, the factors which determine the antioxidant capacity of vitamin E are discussed, and the importance of its localization and mobility in the membranes and lipoproteins is emphasized.

Antioxidants↗
PubMed2004

Detection of lipid peroxidation in vivo: total hydroxyoctadecadienoic acid and 7-hydroxycholesterol as oxidative stress marker.

It is important to assess the oxidative injury in vivo accurately and inclusively, as the oxidative stress induced by various oxidants in a random and destructive fashion is considered to play an important role in the pathophysiology of a number of human disorders and diseases. We have developed an improved method for the measurement of lipid peroxidation in vivo, where total hydroxyoctadecadienoic acids (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleate and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomers, (E,E)-HODE/(E,Z)-HODE, could be also measured. The plasma concentrations of total HODE were obtained as 76.5, 666 and 2225 nM for human, rat and mouse, respectively. It was found that HODE and FCOH could be measured satisfactorily by the present method from plasma, erythrocyte and urine of humans and experimental animals. It was also found that HODE in urine arose from both free and ester forms, while 8-iso-prostaglandin F2alpha was present primarily as a free acid form. As the concentrations of HODE were much higher than 8-iso-prostaglandin F2alpha, HODE may well be used as a good oxidative marker in vivo.

Adult↗
PubMed2004

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress.

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x 10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.

Adult↗
PubMed2004

Motor activity and gene expression in rats with neonatal 6-hydroxydopamine lesions.

A rat model of a hyperkinetic disorder was used to investigate the mechanisms underlying motor hyperactivity. Rats received an intracisternal injection of 6-hydroxydopamine on post-natal day 5. At 4 weeks of age, the animals showed significant motor hyperactivity during the dark phase, which was attenuated by methamphetamine injection. Gene expression profiling was carried out in the striatum and midbrain using a DNA macroarray. In the striatum at 4 weeks, there was increased gene expression of the NMDA receptor 1 and tachykinins, and decreased expression of a GABA transporter. At 8 weeks, expression of the NMDA receptor 1 in the striatum was attenuated, with enhanced expression of the glial glutamate/aspartate transporter. In the midbrain, a number of genes, including the GABA transporter gene, showed decreased expression at 4 weeks. At 8 weeks, gene expression was augmented for the dopamine transporter, D4 receptor, and several genes encoding peptides, such as tachykinins and their receptors. These results suggest that in the striatum the neurotransmitters glutamate, GABA and tachykinin may play crucial roles in motor hyperactivity during the juvenile period. Several classes of neurotransmitters, including dopamine and peptides, may be involved in compensatory mechanisms during early adulthood. These data may prompt further neurochemical investigations in hyperkinetic disorders.

Animals↗
PubMed2004

Dicyclohexylphthalate causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity.

Endocrine disruptors possibly exert effects on neuronal functions leading, in particular, to behavioural alterations. In this study, we examined the effects of dicyclohexylphthalate (DCHP), an endocrine disruptor, on rat behavioural and cellular responses. Single intracisternal administration of DCHP (0.87-87 nmol) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. It was about 1.4-fold more active in the nocturnal phase after administration of 87 nmol of DCHP than control rats (p < 0.001). The response had a tendency to be dose-dependent. Based on DNA macoarray analyses, DCHP down-regulated the levels of gene expression of the dopamine D4 receptor at 4 weeks old in both the midbrain and the striatum, and the dopamine transporter in the midbrain at 8 weeks old 1.7- to 2-fold. The gene expression of several subtypes of glutamate receptors was facilitated in the striatum at 4 weeks old and in the midbrain at 8 weeks old. Some normalization and/or compensatory changes seemed to occur in gene expression of GABA or glycine transmission. Furthermore, DCHP abolished immunoreactivity of tyrosine hydroxylase in the substantia nigra at 8 weeks of age, where TUNEL-positive cells were seen. We conclude that DCHP affected the developing rat brain, resulting in hyperactivity, probably as a result of degeneration of mesencephalic tyrosine hydroxylase rather than alteration of the level of gene expression.

Animals↗