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PubMed1997

Active oxygen generation as a possible mechanism of selenium toxicity.

Selenium plays an important role in scavenging active oxygen (AO) species as an essential constituent of glutathione peroxidase. On the other hand, several reports proposed a possible induction of toxic AO by selenium compounds in vitro. However, some of these experiments including ours, were revealed to conclude on the basis of experimental artifacts, and to have problems in the interpretation of data. Methods or principles so far used for the detection of AO species generated by selenium compound were measurement of chemiluminescence from lucigenin or luminol by AO species, the spectrophotometric analysis of reduction of ferricytochrome c or nitroblue tetrazolium (NBT) by superoxide anion (O2-), electron spin resonance (ESR) spectra using dimethylpyrroline oxide (DMPO) as a spin trapping agent, the deoxyribose decomposition by hydroxyl radical (HO.), the salicylate hydroxylation by HO., and the strand breakage of DNA by AO. Many of these methods together with their principles seem to have some defects which prevent clear conclusion as stated below. (i) Lucigenin was found to mediate the formation of O2- in the presence of selenite and reduced glutathione (GSH). Therefore, lucigenin is not a suitable reagent. (ii) Luminol may also mediate O2- generation in the presence of HO.. (iii) ferricytochrome c can be reduced to ferrocytochrome c in the mixture of selenite and GSH in the absence of oxygen. Moreover, the spectrophotometric method is interfered by turbidity of elemental selenium formed under some conditions in the reaction mixture containing selenite and GSH. (iv) NBT is also reduced by selenium compounds in the absence of O2. (v) ESR signals of AO species were obtained in the reaction mixture containing selenite and GSH, or in the solution of hydrogen selenide in the presence of O2. However, selenide decomposed spin adduct of DMPO with HO. (DMPO-OH). Therefore, the intensity of the signals is not quantitative. (vi) CuZn-SOD is not necessarily a good tool to prove the involvement of O2- because it enhanced HO. generation in the reaction mixture containing selenite and GSH. Thus, we would like to emphasize that carefully designed experiments are required to further identify the molecular species of active oxygen induced by selenium compounds.

Animals↗
PubMed1989

[Fluorescent formazans in flow cytometry. Studies of their oxygen sensitivity].

In order to supply the tetrazolium salt procedure to flow cytometry we have synthesized monotetrazolium salts which are converted into fluorescent formazans on reduction. In this preliminary study we investigated the "oxygen sensitivity" of four of our compounds and compared them to the commercially available nitrotetrazole blue (NBT). The amount of formazan produced by Ehrlich ascites tumour cells via the succinic dehydrogenase reaction during oxic and anoxic incubations was determined by elution technique. Under oxic conditions no NBT formazan but much cyano-di-chlorophenyl formazan (==CCPC) was generated. The yields of this formazan differed only slightly between oxic and anoxic incubations. Twice as much of the amount of the other three formazans cyano-ditolyl- (==CTC, Stellmach 1984), cyanodiphenyl- (==CPC), and cyano-dianisyl tetrazolium chloride (==CAC), were produced in nitrogen atmosphere as in oxygen. The amount of all formazans investigated increased both in oxic and in anoxic conditions if cyanide was present. All cyano-aryl formazans have the maximum of absorbance between 430 and 470 nm and fluoresce in the spectral range above 580 nm with CTC formazan being the brightest. The tetrazolium salts, i.e. in unreduced state, did not fluoresce in the visible part of the spectrum. The formazans could be applied to flow cytometry. After staining the nuclear DNA with the fluorochrome DAPI, we obtained detailed two-parametric distributions correlating the amount of formazan per cell with the DNA content. The results are a step to establish a method for automated identification of malignant cells.

Animals↗
PubMed1994

Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds.

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Borohydrides↗
PubMed2000

Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460.

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

ATP-Binding Cassette Transporters↗
PubMed1991

Chromogenic substrates for horseradish peroxidase.

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.

Alkaline Phosphatase↗
PubMed1991

Effects of the prolyl 4-hydroxylase proinhibitor HOE 077 on human and rat hepatocytes in primary culture.

Alterations in extracellular matrix occur in many chronic liver diseases leading to the formation of hepatic fibrosis. We have studied the effects of the putative hepatoselective fibrosuppressive compound HOE 077, a proinhibitor of prolyl 4-hydroxylase, on normal adult human and rat hepatocytes in primary culture. In human hepatocyte cultures, the cytotoxicity of HOE 077 was assessed after a 20-h treatment at concentrations ranging from 0.125 to 2 mg/ml of medium. No significant change was found in cell morphology, neutral red uptake, red oil staining, lactate dehydrogenase release, tetrazolium salt reduction, ethoxyresorufin O-deethylase activity and protein synthesis; however, HOE 077 slightly decreased DNA synthesis at 2 mg/ml. In rat hepatocyte cultures, the cytotoxicity of the compound was assessed by testing the same parameters after a daily exposure of cultures for 2 days or 4 days, at concentrations ranging from 0.25 to 4.5 mg/ml of medium. Whatever the concentration, the compound had no obvious morphological effect. However, hepatocytes were less spread at the concentration of 4.5 mg/ml. HOE 077 at 2 mg/ml slightly decreased neutral red uptake but was without obvious effect on protein synthesis after 2 days. By contrast, on day 4, protein synthesis was markedly reduced in hepatocyte cultures exposed to HOE 077 at 4.5 mg/ml. Hydroxyproline content determination in media from 4-day-old hepatocyte cultures incubated with HOE 077 at 0.5 to 4.5 mg/ml, showed a dose-dependent decrease in the hydroxyproline/proline ratio in acetic acid soluble material. By indirect immunoperoxidase, intracellular collagen IV was found to be inhibited in hepatocyte cultures after 4 days of exposure to 4.5 mg/ml HOE 077.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

The nitroblue tetrazolium reduction test in workers exposed to benzene and its homologues.

In 106 workers aged 18 to 56 years, occupationally exposed to organic solvents of paints and varnishes containing benzene, toluene and xylene for 1 to 122 months, the spontaneous and the stimulated reduction of nitroblue tetrazolium (NBT) in neutrophils was estimated. In all the subjects studied, both the spontaneous and the stimulated reduction of NBT was increased. However, after longer occupational exposure to benzene and its homologues the stimulated NBT reduction decreases. The negative correlation between the number of NBT-positive stimulated neutrophils and the exposure time was noted. The stimulated NBT test may be of value for industrial health control in the estimation of the toxic effects of the above compounds on the neutrophil system.

Adult↗
PubMed2001

Strategies for neuroprotection against L-trans-2,4-pyrrolidine dicarboxylate-induced neuronal damage during energy impairment in vitro.

Increased levels of extracellular excitatory amino acids and failure of energy metabolism are two conditions associated with brain ischemia. In the present study we have combined the simultaneous inhibition of glutamate uptake and mitochondrial electron transport chain to simulate neuronal damage associated with brain ischemia. Results show that cerebellar granule neurons are not vulnerable to transient glutamate uptake inhibition by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) despite the increase in the extracellular concentration of glutamate, unless they are simultaneously exposed to the mitochondrial toxins 3-nitropropionic acid (3-NP) or sodium azide. Cell damage was assessed by light microscopy observation, by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and by the fluorescent markers for live and dead cells, calcein and ethidium homodimer, respectively. The protective effect of alternative energy substrates, such as pyruvate, acetoacetate, and beta-hydroxybutyrate against PDC-induced neuronal death during 3-NP exposure was studied and compared to the effects of the antioxidant vitamin E, the spin trapper alpha-phenyl-N-tert-butylnitrone (PBN), voltage-dependent calcium channel antagonists, and glutamate receptor antagonists. Results show that neuronal damage can be efficiently prevented in the presence of pyruvate and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, whereas the non-NMDA receptor antagonist NBQX, acetoacetate, vitamin E, and PBN showed partial protection. In contrast, beta-hydroxybutyrate and voltage-dependent calcium channels blockers did not show any protective effect at the concentrations tested.

Animals↗
PubMed2005

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance.

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.

Alitretinoin↗
PubMed2006

Role of glutathione in determining the differential sensitivity between the cortical and cerebellar regions towards mercury-induced oxidative stress.

Certain discrete areas of the CNS exhibit enhanced sensitivity towards MeHg. To determine whether GSH is responsible for this particular sensitivity, we investigated its role in MeHg-induced oxidative insult in primary neuronal and astroglial cell cultures of both cerebellar and cortical origins. For this purpose, ROS and GSH were measured with the fluorescent indicators, CMH(2)DCFDA and MCB. Cell associated-MeHg was measured with (14)C-radiolabeled MeHg. The intracellular GSH content was modified by pretreatment with NAC or DEM. For each of the dependent variables (ROS, GSH, and MTT), there was an overall significant effect of cellular origin, MeHg and pretreatment in all the cell cultures. A trend towards significant interaction between originxMeHgxpretreatment was observed only for the dependent variable, ROS (astrocytes p=0.056; neurons p=0.000). For GSH, a significant interaction between originxMeHg was observed only in astrocytes (p=0.030). The cerebellar cell cultures were more vulnerable (astrocytes(mean)=223.77; neurons(mean)=138.06) to ROS than the cortical cell cultures (astrocytes(mean)=125.18; neurons(mean)=107.91) for each of the tested treatments. The cell associated-MeHg increased when treated with DEM, and the cerebellar cultures varied significantly from the cortical cultures. Non-significant interactions between originxMeHgxpretreatment for GSH did not explain the significant interactions responsible for the increased amount of ROS produced in these cultures. In summary, although GSH modulation influences MeHg-induced toxicity, the difference in the content of GSH in cortical and cerebellar cultures fails to account for the increased ROS production in cerebellar cultures. Hence, different approaches for the future studies regarding the mechanisms behind selectivity of MeHg have been discussed.

Animals↗
PubMed1999

L-DOPA-quinone inactivates tryptophan hydroxylase and converts the enzyme to a redox-cycling quinoprotein.

Tryptophan hydroxylase, the initial and rate limiting enzyme in the biosynthesis of serotonin (5-HT), is inactivated by the quinone of L-DOPA. L-DOPA itself has no effect on enzyme activity. The inactivation of tryptophan hydroxylase could be prevented by glutathione (GSH), dithiothreitol, cysteine, and ascorbic acid but not by scavengers of hydrogen peroxide (catalase), hydroxyl radical (DMSO), or superoxide (superoxide dismutase). All cysteinyl residues within tryptophan hydroxylase are modified after treatment with L-DOPA-quinone as revealed by loss of DTNB-reactivity and formation of cysteinyl-DOPA residues. L-DOPA-quinone also converts tryptophan hydroxylase to a redox-cycling quinoprotein. These results suggest a possible mechanism of 5-HT neuronal damage in Parkinson's Disease by a redox-cycling quinoprotein.

Antioxidants↗
PubMed2004

Mononuclear and dinuclear monoperoxovanadium(v) complexes with a hetero ligand. 1.(1) Self-decomposition reaction, detection of reactive oxygen species, and oxidizing ability.

A mononuclear peroxovanadium(V) complex with histamine-N,N-diacetate (histada), K[VO(O(2))(histada)], and a dinuclear peroxovanadium(V) complex with 2-oxo-1,3-diaminopropane-N,N,N',N'-tetraacetate (dpot), Cs(3)[(VO)(2)(O(2))(2)(dpot)], were prepared and characterized. The self-decomposition reaction was examined for these peroxovanadium(V) complexes as well as for K[VO(O(2))(cmhist)] (cmhist = N-carboxymethylhistidinate). The reaction profiles depicted by the absorbance change in the UV-vis spectrum show a sigmoid shape with an induction period. The induction period is reduced by the addition of acid, fluoride, thiocyanate, VO(2+), VO(2)(+), and trolox compared to the solution containing perchlorate. On the other hand, the induction period was elongated by the addition of chloride, bromide, and 2-tert-butyl-p-cresol. These behaviors are discussed on the basis of a radical chain mechanism. The self-decomposition reactions have also been followed by the (1)H and (51)V NMR and EPR spectra. These spectral studies as well as the UV-vis spectral study indicate that vanadium(V) is partly reduced to vanadium(IV) in the self-decomposition process. The histada complex yields a mixed-valence dinuclear complex in a concentrated solution, and the dpot complex yields a mixed-valence tetranuclear complex. The reduction of vanadium ion suggests that the peroxo ligand may act as a reducing agent. In order to know the fate of the peroxo ligand, we tried to detect superoxide anion and hydroxyl radical, which were anticipated to be produced in the self-decomposition process. The formation of superoxide anion was spectrophotometrically confirmed using two independent methods, including the reduction of cytochrome c and the reduction of sodium 4-[3-(iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1). The formation of hydroxyl radical was confirmed by an EPR spin trapping technique. The oxidizing abilities of the peroxovanadium(V) complexes toward bovine serum albumin (BSA) were also evaluated. In the protein carbonyl assay, it was found that the total amount of protein carbonyl in BSA was increased by the reaction with the peroxovanadium complexes in the concentration-dependent manner. In addition, the oxidation of sulfhydryl group in BSA induced by the peroxovanadium complexes was confirmed.

Animals↗
PubMed2003

Triphenyl tin benzimidazolethiol, a novel antitumor agent, induces mitochondrial-mediated apoptosis in human cervical cancer cells via suppression of HPV-18 encoded E6.

Here we report the effect of TPT-benzimidazolethiol, a novel anti-tumor agent developed by our group, on the apoptotic pathway of human cervical carcinoma cells. Treatment of HeLa cells with TPT-benzimidazolethiol arrests the cell cycle at G0/G1 phase and transcriptionally downregulates HPV-encoded E6, restoring p53 expression from E6 suppression. Increased p53 accumulation up-regulates p21/waf and ultimately induces apoptosis. The effect of TPT-benzimidazolethiol is far more potent in inducing apoptosis than cisplatin. Treatment with TPT-benzimidazolethiol in HeLa cells is accompanied by the up-regulation of Bak at the transcriptional level, resulting in the release of cytochrome c and Smac/DIABLO from mitochondria to cytosol and, subsequently, the activation of procaspase-9, -3 and PARP, suggesting that TPT-benzimidazolethiol induced-apoptosis signaling is by an intrinsic mitochondrial pathway. Taken together, we propose that TPT-benzimidazolethiol could has the potential to be developed into a new therapeutic agent for treating HPV-associated cervical neoplasia.

Antineoplastic Agents↗
PubMed2004

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition.

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.

Animals↗
PubMed2004

The involvement of oxidative stress in bovine herpesvirus type 4-mediated apoptosis.

Bovine herpesvirus type 4 (BHV-4) belongs to the gamma-2-herpesviruses of the Gammaherpesvirinae subfamily. BHV-4 has a worldwide distribution and has been isolated in a variety of clinical diseases as well as from healthy cattle. In this report we demonstrate that BHV-4 induces apoptosis in MDBK cells. In the early phases of apoptosis, cells show an increase in the intracellular level of reactive oxygen species, which is indicative of oxidative stress. This precedes DNA fragmentation, a hallmark typical of apoptosis. Cells were protected from apoptosis only by certain antioxidants (butylated hydroxyanisole and ebselen), whereas N-acetylcysteine turned out to be ineffective. Antioxidants that protected cells from apoptosis prevented oxidative stress but failed to block virus growth. These observations suggest that oxidative stress may be a crucial event in the sequence leading to apoptotic cell death but apoptosis is not required for the multiplication of BHV-4.

Acetylcysteine↗