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PubMed1998

Photolysis and membrane lipid peroxidation of human erythrocytes by m-chloroperbenzoic acid.

The aim of the present work was to investigate the biological hazard of photooxidation products of m-chloroperbenzoic acid (mCPBA), as a novel photo-sensitizer, on lysis and membrane lipid peroxidation of human red blood cells (RBC). The photohemolysis activity of mCPBA oxidative products was concentration- and exposure time-dependent. Ten minutes photoexposure time and 100 micromol/L of mCPBA concentration were optimum to study the effect of generated superoxide (O2.-) and hydroxyl (.OH) radicals on RBC. The hemolytic effect of mCPBA was highly significantly inhibited by formate (as an .OH radical scavenger) compared with the partial inhibition effect of SOD-like Cu(II) complex (as O2.- radical Scavenger). The MDA value (an end product of membrane lipid peroxidation of RBC) induced by mCPBA was highly significantly decreased by formate. The generation of O2.- radicals by mCPBA was also confirmed by the partial hemolytic effect of phenazine methosulfate (PMS., O2.- radical generation). The data suggest the molecular mechanism of the oxygen radical species (O2.- and .OH through the photosensitization reaction of mCPBA and explain that hydroxyl radicals (.OH) play an active role in the photohemolysis process and peroxidation of membrane lipids of human erythrocytes.

Chlorobenzoates↗
PubMed1997

Electrogenic reduction of the primary electron donor P700+ in photosystem I by redox dyes.

The kinetics of reduction of the photo-oxidized primary electron donor P700+ by redox dyes N,N,N',N'-tetramethyl-p-phenylendiamine, 2,6-dichlorophenol-indophenol and phenazine methosulfate was studied in proteoliposomes containing Photosystem I complexes from cyanobacteria Synechocystis sp. PCC 6803 using direct electrometrical technique. In the presence of high concentrations of redox dyes, the fast generation of a membrane potential related to electron transfer between P700 and the terminal iron-sulfur clusters F(A)/F(B) was followed by a new electrogenic phase in the millisecond time domain, which contributes approximately 20% to the overall photoelectric response. This phase is ascribed to the vectorial transfer of an electron from the redox dye to the protein-embedded chlorophyll of P700+. Since the contribution of this electrogenic phase in the presence of artificial redox dyes is approximately equal to that of the phase observed earlier in the presence of cytochrome c6, it is likely that electrogenic reduction of P700+ in vivo occurs due to vectorial electron transfer within RC molecule rather than within the cytochrome c6-P700 complex.

2,6-Dichloroindophenol↗
PubMed1999

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability.

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.

Animals↗
PubMed2001

Human spermicidal activity of inorganic and organic oxidants.

OBJECTIVE: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. DESIGN: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. SETTING: Commercial and university-based laboratories. PATIENT(S): Semen samples obtained through a university-based andrology laboratory. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Quantitation of spermicidal activity of test oxidants. RESULT(S): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I(2)). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. CONCLUSION(S): Free I(2) formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I(2) may be beneficial in developing a novel new class of nondetergent-based spermicides.

Dose-Response Relationship, Drug↗
PubMed1982

Separation and determination of bile acids by high-performance liquid chromatography using immobilized 3 alpha-hydroxysteroid dehydrogenase and an electrochemical detector.

A high-performance liquid chromatographic method using an immobilized 3 alpha-hydroxysteroid dehydrogenase column and an electrochemical detector was developed for the determination of individual bile acids in serum and bile. Bile acids in the eluate from a Radial-Pak A column reacted with NAD in the enzyme column to generate NADH, which was monitored by a voltammetric detector after mixing with phenazine methosulphate solution. Each bile acid was measurable at the 20 pmole level at the highest sensitivity of the detector. The mean recoveries and reproducibilities of bile acids were 86.7-104.6% [coefficient of variation (C.V.) = 0.3-8.7%] within-assay and 83.8-103.4% (C.V. = 1.6-7.0%] between-assay.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents.

Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 microg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 microM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.

Anti-Inflammatory Agents↗
PubMed1996

The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

Animals↗
PubMed1997

Free radical scavenging activities of mushroom polysaccharide extracts.

The superoxide and hydroxyl radical scavenging activities of eight mushroom antitumor polysaccharide extracts were investigated using phenazin methosulphate-NADH-nitroblue tetrazolium system and ascorbic acid-Cu(2+)-cytochrome C system respectively. The results showed that six of eight mushroom polysaccharide extracts had superoxide and hydroxyl radical scavenging activities. The protein content of the polysaccharide extracts appeared to contribute a direct effect on free radical scavenging activity. However, none of the mushroom polysaccharide extracts had antioxidative activity as measured by detecting malondialdehyde (MDA) contents of liver microsomes.

Antineoplastic Agents↗
PubMed1998

Use of a soluble tetrazolium compound to assay metabolic activation of intact beta cells.

Although assessments of metabolic activation are central to studies of beta-cell function, available techniques are tedious, insensitive, and/or require cell disruption. We have investigated the use of a new water-soluble tetrazolium salt, MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4- sulfophenyl]-2H-tetrazolium, inner salt), in the presence of phenazine methosulfate (PMS), an intermediate electron acceptor that amplifies its signal (fluorescence at 490 nm). During static incubations of glucose-responsive (HIT-T15 or INS-1) dispersed beta cells with increasing glucose concentrations, there was a progressive increase in MTS reduction, with a maximum signal-to-noise (S/N) ratio of 24 with HIT-T15 cells and 10 with INS-1 cells. This was associated with, but not attributable to, parallel increases in insulin secretion. Pure mitochondrial fuels (alpha-ketoisocaproate [KIC], methyl pyruvate [MP], or L-glutamine [GLN] + L-leucine [LEU]) also increased the reduction of MTS in INS-1 cells (6.5-, 4.8-, and 14.4-fold, respectively), but generally less than glucose, suggesting a major role of glycolysis in the signal induced by glucose. Inhibitors of glucose metabolism (mannoheptulose [MH], lodoacetate [IA], or 2-deoxyglucose [2-DG]) markedly reduced the glucose-stimulated MTS signal. In comparison to another tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MTS assay provided a better S/N ratio with glucose or other nutrient secretagogues. Extant theory holds that activation of mitochondrial dehydrogenases by increments in Ca2+ influx couples glycolysis to mitochondrial oxidation of glucose-derived fuels. However, reduction of fuel-induced calcium influx (by Ca2+-free medium or diazoxide [DZX]) or direct stimulation of calcium influx (by 40 mmol/L K+) failed to significantly modulate the signal, arguing against this theory. We conclude that the MTS assay is a facile test that reflects the global metabolic function of insulin-secreting beta cells. Furthermore, since this assay does not require disruption of cells to solubilize the formazan product, and therefore also allows concomitant measurement of insulin secretion, it offers considerable advantages over earlier methods.

Animals↗
PubMed1988

The influence of calcium channel blockers on superoxide anions.

Four calcium channel blockers were tested from the point of view of their influence on enzymic lipid oxidation and on generation of superoxide anions. All the compounds were found to be antioxidants as tested by the inhibition of NADPH-stimulated malonaldehyde formation from lipids. IC50 values were 60 microM for nifedipine; 1.1 microM for verapamil; 1.4 microM for fendiline and 20.6 microM for diltiazem. Only nifedipine scavenged superoxide anions both in an enzymic (xanthine:xanthine oxidase) and non-enzymic (phenazine methosulphate:NADH) generating system. IC50 values for this inhibition were about 2.5 times higher than for inhibition of formation of malonaldehyde. Nifedipine inhibited also xanthine oxidase-mediated formation of uric acid.

Animals↗
PubMed2000

Oxidation and epimerization of epigallocatechin in banana fruits.

To examine the metabolism of proanthocyanidins in banana fruit, (-)-epigallocatechin was treated with the homogenate of the fruit flesh to yield (-)-gallocatechin and an oxidation product, 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)-2-hydroxy-1-propan one. The latter product is a stable form of a key intermediate in the oxidative metabolism of flavan-3-ols, and is also related to the biogenesis of A-type proanthocyanidins. In addition, treatment of the reaction mixture with o-phenylenediamine afforded monomeric and dimeric phenazine derivatives generated by condensation with the o-quinone form of the oxidation product.

Biotransformation↗
PubMed2002

Development of a quantitative assay method for 3 beta-hydroxy-delta 5-steroid dehydrogenase in the rat testis.

We investigated the first step of the sex steroid hormone biosynthesis pathway by assaying the activities of 3 beta-hydroxy-delta 5-steroid dehydrogenase, the rate-limiting enzyme in this pathway. We have developed a simple and rapid colorimetric assay for 3 beta-hydroxy-delta 5-steroid dehydrogenase in rat testis. The supernatant from rat testis tissue homogenates were used for the enzyme assay. The enzyme activity was determined by measuring the absorbance at 570nm which indicates the rate of conversion of pregnenolone into progesterone in the presence of NAD, using phenazine methosulfate and nitro blue tetrazolium as the color reagent. The activity of this enzyme ranged from 4.57+/-1.34 to 10.56+/-2.13 nmol/mg protein/min with a mean activity of 8.96+/-1.27 nmol/mg protein/min. The K(m) of the enzyme at an optimum pH of 7.25 was about 4.7+/-0.12 nM.

3-Hydroxysteroid Dehydrogenases↗
PubMed1977

Lactate dehydrogenase content related to cross-sectional area in muscle fibres.

Cross-sectional areas and lactate dehydrogenase activity were determined of separate muscle fibres of mouse rectus femoris muscle in both ventral and dorsal parts of the muscle. The lactate dehydrogenase activity was measured quantitatively by microscope photometry using a tetrazolium staining in absence or presence of either vitamin K3 or phenazine methosulfate. If the observations are restricted to a relatively small area of the muscle cross section, one can distinguish categorically narrow ("red") and broad ("white) fibres with average values for cross sectional area and LDH content that depend on the part of the muscle chosen (higher LDH and smaller area in ventral part). However, if these data from the different parts or from the whole muscle are added together, no categorical distinction between the fibre types is allowed. On the basis of the observations a categorical division of muscle fibres within relatively large muscle areas or in a whole muscle is rather dubious. A relation is suggested between the subjective impression of different muscle fibre types in a field of observation with the number of motorunits present in that field.

Animals↗
PubMed1997

Oxidative stress and the mobilisation of arachidonic acid in stimulated human platelets: role of hydroxyl radical.

Platelet functions, including eicosanoid biosynthesis, can be significantly altered by exposure to reactive oxygen species. We utilised the redox properties of the phenazine derivative, pyocyanin, to generate low micromolar levels of reactive oxygen species in order to investigate the metabolism of arachidonic acid by human platelets under oxidative stress. Eicosanoid production by platelets, pre-labelled with [3H]arachidonic acid (AA) and stimulated with the calcium ionophore A23187, was inhibited in the presence of pyocyanin. In contrast, platelets pre-treated with pyocyanin and concurrently exposed to A23187 and AA showed no evidence of inhibition. Analysis of the free label content of labelled, pyocyanin-treated platelets after stimulation revealed diminished levels of total free label and a corresponding increase in labelled phospholipid. Prior treatment with the antioxidants, superoxide dismutase, catalase or the hydroxyl radical scavenger, mannitol, before the addition of pyocyanin afforded protection against loss of eicosanoid production and restored AA release. We conclude that hydroxyl radicals inhibit one or more steps in the cascade leading to phospholipase A2 activation and release of arachidonic acid from platelet phospholipid stores.

Antioxidants↗
PubMed2003

Synthesis, crystal structure, and nuclease activity of planar mono-heterocyclic base copper(II) complexes.

A series of mononuclear copper(II) complexes having a 1:1 molar ratio of copper and the planar heterocyclic base like 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) are prepared from a reaction of copper(II) nitrate.trihydrate and the base (L) in ethanol or aqueous ethanol at different temperatures. The complexes [Cu(dpq)(NO(3))(2)] (2), [Cu(dpq)(NO(3))(H(2)O)(2)](NO(3)) (3), [Cu(dpq)(NO(3))(2)(H(2)O)(2)].2H(2)O (4.2H(2)O) and [Cu(dppz)(NO(3))(2)(H(2)O)].H(2)O (5.H(2)O) have been characterized by X-ray crystallography. The crystal structures show the presence of the heterocyclic base in the basal plane. The coordination geometries of the copper(II) centers are axially elongated square-pyramidal (4+1) in 2, 3 and 5, and octahedral (4+2) in 4. The nitrate anion in the coordination sphere displays unidentate and bidentate chelating bonding modes. The axial ligand is either H(2)O or NO(3) in these structures giving a Cu-L(ax) distance of approximately 2.4 A. The one-electron paramagnetic complexes (mu approximately 1.8 mu(B)) exhibit axial EPR spectra in DMF glass at 77 K giving g(parallel)>g( perpendicular ) with an A(parallel) value of approximately 170G indicating a [d(x)2(-y)2](1) ground state. The complexes are redox active and display a quasireversible cyclic voltammetric response for the Cu(II)/Cu(I) couple near 0.0 V vs. SCE giving an order of the E(1/2) values as 5(dppz)>2-4 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). The complexes bind to calf thymus DNA giving an order 5 (dppz)>2 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). An effect of the extended planar ring in dpq and dppz is observed in the DNA binding. The complexes show nuclease activity with pUC19 supercoiled DNA in DMF/Tris-HCl buffer containing NaCl in presence of mercaptopropanoic acid as a reducing agent. The extent of cleavage follows the order: [Cu(phen)(2)(H(2)O)](ClO(4))(2)>5>2 approximately 3 approximately 4>1. The bis-phen complex is a better cleaver of SC DNA than 1-5 having mono-heterocyclic base. Mechanistic investigations using distamycin reveal minor groove biding for the phen, dpq complexes, and a major groove binding for the dppz complex 5. The cleavage reactions are found to be inhibited in the presence of hydroxyl radical scavenger DMSO and the reactions are proposed to proceed via sugar hydrogen abstraction pathway. The ancillary ligand is found to have less effect in DNA binding but are of importance in DNA cleavage reactions.

Journal Article↗
PubMed2003

DNA interactions of new mixed-ligand complexes of cobalt(III) and nickel(II) that incorporate modified phenanthroline ligands.

Four new mixed-ligand complexes, namely [Co(phen)(2)(qdppz)](3+), [Ni(phen)(2)(qdppz)](2+), [Co(phen)(2)(dicnq)](3+) and [Ni(phen)(2)(dicnq)](2+) (phen=1,10-phenanthroline, qdppz=naptho[2,3-a]dipyrido[3,2-H:2',3'-f]phenazine-5,18-dione and dicnq=dicyanodipyrido quinoxaline), were synthesized and characterized by FAB-MS, UV/Vis, IR, 1H NMR, cyclic voltammetry and magnetic susceptibility methods. Absorption and viscometric titration as well as thermal denaturation studies revealed that each of these octahedral complexes is an avid binder of calf-thymus DNA. The apparent binding constants for the dicnq- and qdppz-bearing complexes are in the order of 10(4) and >10(6) M(-1), respectively. Based on the data obtained, an intercalative mode of DNA binding is suggested for these complexes. While both the investigated cobalt(III) complexes and also [Ni(phen)(2)(qdppz)](2+) affected the photocleavage of DNA (supercoiled pBR 322) upon irradiation by 360 nm light, the corresponding dicnq complex of nickel(II) was found to be ineffective under a similar set of experimental conditions. The physico-chemical properties as well as salient features involved in the DNA interactions of the cobalt(III) and nickel(II) complexes investigated here were compared with each other and also with the corresponding properties of the previously reported ruthenium(II) analogues.

Cobalt↗
PubMed1997

Synthesis and DNA binding studies of cobalt (III) mixed-polypyridyl complex.

The complex of Co(phen)2dppz3+ (where phen is ophenanthroline, and dppz is dipyrido [3, 2-a: 2', 3'-c] phenazine) has been synthesized. This complex was characterized by elemental analysis, molar conductivity, IR and 1H NMR spectroscopy. The interaction of the complex with calf thymus DNA has been studied using absorption and emission spectroscopy, DNA melting techniques, cyclic voltammetric, viscosity, and electrophoresis measurements. The compound shows absorption hypochromicity, fluorescence enhancement, DNA melting temperature, and the specific viscosity increased when binding to calf thymus DNA. CV measurement shows that the shifts in oxidation-reduction potential and change in peak current with addition of DNA. The complex is also shown to be more efficient photosensitisers for single strand breaks in plasmid DNA.

Cobalt↗
PubMed2002

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.

Animals↗