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PubMed1994

Oxidative stress in the central nervous system: monitoring the metabolic response using the pentose phosphate pathway.

We propose that monitoring the activity of the pentose phosphate pathway (PPP) may provide an opportunity to obtain unique information regarding the metabolic response to oxidative stress since glutathione peroxidase activity is coupled, via glutathione reductase, to the PPP enzyme glucose-6-phosphate dehydrogenase. PPP activity was quantitated from data obtained from gas chromatography/mass spectrometry analysis of released lactate following metabolic degradation of (1,6-13C2,6,6-2H2)glucose. The feasibility of this approach for in vitro studies is shown using cultured rat 9L gliosarcoma cells, primary mixed cerebrocortical and primary astrocytic cultures and in vivo using intracerebral microdialysis. Exposure of 9L gliosarcoma cells to increasing concentrations of phenazine methosulfate, diamide and H2O2 correlated with increasing stimulation of the PPP, revealing the coupling of the PPP to the glutathione pathway. In all cultured cell types, the activity of the PPP was stimulated in a concentration-dependent fashion by exposure to H2O2. In primary mixed and purified astrocytic cultures, PPP activity was stimulated with H2O2 from 2.0 to 22.5 and from 5.9 to 66.7%, respectively. H2O2-induced neuronal injury was evident before saturation of the PPP occurred. H2O2 toxicity was attenuated when neurons were preincubated with the iron chelator, deferoxamine, and did not occur until saturation of the PPP. In vivo measurements of PPP activity in the conscious rat forebrain revealed basal levels of 4.5%, which was stimulated to 16.9 and 35.7% when 1 mM H2O2 and 500 microM phenazine methosulfate were added to the perfusion solution, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studied in vitro and with a model system of polyacrylamide films.

The applicability of phenazine methosulfate, 1-methoxyphenazine methosulfate, menadione, and meldola blue as exogenous electron carriers for the cytochemical staining of nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent dehydrogenases has been studied quantitatively with tetranitro BT in vitro and with a model system of polyacrylamide films incorporating either purified glucose-6-phosphate dehydrogenase or intact rat liver parenchymal cells. It was found that every assay in which a tetrazolium salt is used, whether or not an electron carrier is present, has to be carried out in darkness. Menadione did not appear to be useful, because electrons were not found to be transferred directly from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) to this compound. Phenazine methosulfate at higher concentrations and meldola blue at concentrations optimal for carrying electrons to tetrazolium salts yielded a high level of "nothing dehydrogenase" activity in cell-containing films, but no inhibition of enzymatic activity was found. Factors involved in the interference of oxygen with tetrazolium salt reduction are discussed. 1-Methoxyphenazine methosulfate did not stain cellular compounds and caused only a very low nothing dehydrogenase activity. The cytochemical demonstration of dehydrogenase activity was shown to be independent on the concentration of 1-methoxyphenazine methosulfate used (50-1000 microM). It is concluded that 1-methoxyphenazine methosulfate is the exogenous electron carrier of choice.

Acrylic Resins↗
PubMed2002

Superoxide scavenging by polyphenols: effect of conjugation and dimerization.

The superoxide anion scavenging capacity of two flavonols (quercetin and kaempferol) and some of their conjugates (quercetin-3-rhamnoglucoside, quercetin-3-sophoroside, quercetin-3-sulphate, quercetin-3-glucuronide, kaempferol-3-sophoroside, kaempferol-3-glucuronide) and of several hydroxycinnamic acids (caffeic acid, ferulic acid, 5-5 diferulic acid, 8-O-4 diferulic acid and 8-8 diferulic acid) were studied. Superoxide anions were generated non-enzymatically in a phenazine methosulphate-NADH system and assayed by reduction of nitro-blue tetrazolium. Among the flavonols examined, the most effective scavengers of superoxide anions were the sophoroside, glucuronide and rhamnoglucoside conjugates. Conversely, quercetin-3-sulphate and the flavonol aglycones, exhibited some pro-oxidant activity at the range of concentrations tested (0.5-10 microM). These results show that conjugation has a marked effect on the scavenging capacity of flavonols and that the type of conjugate at the 3-position determines the final superoxide scavenging capacity. Caffeic acid and ferulic acid showed no effect on the generation of superoxide anions by phenazine methosulphate-NADH. However, dimerization of ferulic acid enhanced the superoxide scavenging capacity of this hydroxycinnamic acid, but this depended on the type of linkage between the monomers. The order, from highest to lowest, of superoxide radical scavenging capacity for the dimers of ferulic acid was: 5-5-diferulic acid > 8-O-4-diferulic acid > 8-8-diferulic acid.

Dimerization↗
PubMed1983

Biochemical studies in the liver and muscle of patients with Zellweger syndrome.

Biochemical studies have been performed in muscle, liver, leukocytes, and fibroblasts from patients suffering from the Zellweger syndrome. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1-14C]2-oxoglutarate were strongly reduced in skeletal muscle homogenate. Oxygen consumption in isolated skeletal muscle mitochondria could only be stimulated by ADP in the presence of ascorbate and N,N,N1,N1-tetramethyl-p-phenylenediamine. Cytochrome contents in heart muscle and liver mitochondria were normal. A very low activity of succinate-ubiquinone oxidoreductase was found in liver homogenate of two patients. From the effect of 2-thenoyltrifluoroacetone on the succinate-phenazine methosulphate oxidoreductase activity, a nearly competitive inhibition with respect to phenazine methosulphate was demonstrated in contrast with a non-competitive inhibition in controls. Normal oxidation rate of [1-14C]pyruvate and [2-14C]pyruvate was found in leucocytes and fibroblasts. Lactate and pyruvate levels were normal in serum and cerebrospinal fluid and beta-hydroxybutyrate and acetoacetate levels were normal in blood. The ratios lactate/pyruvate and beta-hydroxybutyrate/acetoacetate were normal as well. These findings point to a defect in the electron transport chain at the succinate-ubiquinone oxidoreductase level. This defect might be related to the absence of peroxisomes in the cells of Zellweger patients.

Brain Diseases↗
PubMed1987

Cysteamine oxygenase: possible involvement of superoxide ion in the catalytic mechanism.

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.

1-Propanol↗
PubMed1999

Dimeric analogues of non-cationic tricyclic aromatic carboxamides are a new class of cytotoxic agents.

A series of tricyclic aromatic carboxamides, and their corresponding dimeric analogues, were prepared and their growth-inhibitory properties were evaluated in a series of cell lines. The dimeric compounds were prepared by reaction of the appropriate acids with carbonyl-1,1'-diimidazole, isolating the resulting imidazolides, and reacting these with a stoichiometric amount of the diamine. The monomeric carboxamides containing a (CH2)2NMe2 side chain had widely differing inhibitory potencies, with the known nitronaphthalimide (mitonafide) and acridine-4-carboxamide (DACA) being the most potent. The corresponding bis analogues, linked by a (CH2)3NMe(CH2)3 chain, were generally more potent, with the largest increases (dimer/monomer ratio 20- to 30-fold) seen for the nitronaphthalimides and the phenazines. Based on the intrinsic cytotoxicity of the monomers and the highest degree of increase in cytotoxicity on dimerization, the most interesting chromophores appear to be the acridine-4-carboxamide and phenazine-1-carboxamide. Both of these compounds showed significant growth delays (approximately 6 days) in an in vivo colon 38 tumour model in mice.

Amides↗
PubMed2000

Synthesis of tetracyclic benzodioxins as potential antitumour agents.

We report the synthesis of five new tetracyclic benzodioxins, from reaction of 1,2-naphthalenediol, 7,8-dihydroxyquinoline or 7,8-dihydroxyisoquinoline with isopropyl 2-chloro-3-nitrobenzoate. Mixtures of isomeric esters were obtained which were separated by conventional flash silica chromatography or preparative HPLC and fully characterized by NMR. Ester hydrolysis and amide formation afforded the target carboxamides 7-9, 11 and 12. The biological activities of the benzodioxins broadly paralleled those of the corresponding phenazines. A tetracyclic pyrido derivative was the most cytotoxic, with an IC50 of 0.11 microM in P388 leukaemia cell culture, but did not represent a significant improvement over a number of 9-substituted tricyclic phenazines.

Antineoplastic Agents↗
PubMed1989

L-pipecolic acid oxidation in the rabbit and cynomolgus monkey. Evidence for differing organellar locations and cofactor requirements in each species.

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.

Animals↗
PubMed1986

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c.

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.

Animals↗
PubMed1986

[Effect of trimetazidine on membrane changes induced by oxygen free radicals in human red cells].

It is well known that oxygen free radicals are generated in different ischaemic tissues and can mediate cell injury. Furthermore, a loss of intracellular potassium is involved in heart ischemia. We previously developed a method to study a possible connection between these two phenomena in human erythrocytes treated in vitro with an oxygen free radical generator: phenazine methosulfate. We showed that transport systems specific to potassium are poorly inhibited (sodium-potassium pump; sodium-potassium cotransport) or unaffected (Gardos effect, chlorine-dependent potassium transport); that the erythrocyte potassium loss is essentially due to an increase in passive potassium permeability resulting from lipid peroxidation and that alterations of transport pathways are enhanced by diethyldithiocarbamate, a superoxide dismutase inhibitor. On the other hand, a cardioprotective drug, trimetazidine, indicated in ischaemic heart disease, which has no calcium antagonist action or coronary vasodilator effect, was studied as a possible candidate for preventing such damage by free radicals. Red cells collected from healthy donors previously treated with trimetazidine were incubated in vitro with phenazine methosulfate and diethyldithiocarbamate. We observed significant decreases in oxygen free radical-dependent passive potassium permeability and lipid peroxidation. This strongly suggests that trimetazidine possesses a free radical scavenger activity which may explain its cardioprotective role.

Cell Membrane Permeability↗
PubMed1983

[Differences in the action of antimycin and 2-nonyl-4-hydroxyquinoline N-oxide on oxidation-reduction of mitochondrial cytochromes b].

The differences in the effects of two similar electron transfer inhibitors, antimycin and 2-nonyl-4-hydroxyquinoline N-oxide (HOQNO) on the reactions of cytochromes b are described. In the cyanide-inhibited submitochondrial particles of bovine heart, HOQNO strongly stimulates cytochrome b reduction by ascorbate in the presence of redox mediators, e. g. N,N,N',N'-tetramethylparaphenylene diamine, 2,6-dichlorophenolindophenol, diaminodurol and phenazine methosulfate; this effect can be reversed by antimycin. Addition of both inhibitors to the submitochondrial particles aerobically equilibrated with succinate/fumarate redox buffer at E = +54 mV in the presence of cyanide results in a similar reduction of cytochromes b, which in the case of antimycin is readily reversed by phenazine methosulfate but is resistant to this redox mediator in the presence of HOQNO. The latter causes additional reduction of cytochrome b562 in the argone atmosphere, the effect being reversed by antimycin. In the presence of HOQNO the anaerobic redox titration curve of cytochrome b562 is shifted towards a high potential region by 20-30 mV. An additional reduction of cytochromes b induced by HOQNO can be due to superposition of two effects, i. e. extra-reduction of cytochromes b566 and b562 requiring O2 and a true positive shift of E0 of cytochrome b562.

Animals↗
PubMedDate not supplied

Specific determination of alpha-tocopherol in food and feed by fluorometry. Part 1. Manual method.

The proposed fluorometric method for determining alpha-tocopherol is highly specific and sensitive, yet requires low-cost equipment available in any laboratory. It is robust and fairly fast (4 determinations in 100 min, sample preparation not included). It has been tested in parallel with a conventional thin layer chromatographic method on foods and feeds. The only necessary cleanup is the usual saponification. The unsaponifiable fraction can be extracted with ethyl ether or, preferably, with Extrelut columns. Isooctane is used as a carrier solvent. Reagents and their solvents are added to the isooctane solution before each successive reaction and are then eliminated by partition with water. The alpha-tocopherol (alpha-T) derivative always remains in isooctane. The first step is nitrosation and elimination of tocopherols and tocotrienols other than alpha-isomers. alpha-T is then oxidized to alpha-tocored (alpha-TR) with a mixture of sulfuric acid, ferric chloride, and iodine bromide. alpha-TR is then condensed to a new reagent: 4,5-dimethyl-o-phenylenediamine. The phenazine formed is strongly fluorescent. Iodine and bromine add to the double bonds of alpha-tocotrienol present and quench the fluorescence of its phenazine. A procedure for blank assays specifically inhibits the conversion of alpha-T to alpha-TR.

Animal Feed↗
PubMed1995

EPR characterization of an archaeal succinate dehydrogenase in the membrane-bound state.

The membrane-bound succinate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum was characterized by EPR spectroscopy and its functional properties were determined. The highest turnover values of succinate dehydrogenase activity were observed at pH 7.4, which is somewhat above the internal pH value of T. acidophilum. The temperature optimum of the reaction was determined as 78 degrees C and the Km value for succinate using phenazine methosulfate as the electron acceptor at 53 degrees C was 0.32 mM. The membrane-bound enzyme was able to reduce the artificial electron acceptors phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,6-dichloroindophenol. Succinate oxidation was coupled to oxygen consumption in a completely 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive manner. In the oxidized state, T. acidophilum membranes exhibited an almost isotropic EPR spectrum with g-values at gz = 2.017, gy = 2.000, and gx = 1.968 that were assigned to a [3Fe-4S]1+ cluster (S3). Upon reduction with succinate, the membranes displayed a spectrum characteristic of 2Fe-2S clusters (S1), with g-values at gz = 2.029, gy = 1.935, and gx = 1.915. In the dithionite-reduced state, additional resonances can be observed. An axial component, with g-values at gz = 2.057, gy = 1.917, and gx = 1.917 was assigned to a [4Fe-4S]1+ cluster. The saturation behaviour of the S1 cluster was strongly altered in the dithionite-reduced form, thus indicating spin-spin interaction between the S1 center and another paramagnetic center, possibly cluster S2. In both the succinate and the dithionite-reduced membranes, parallel-mode EPR spectra displayed a resonance at g = 14, which may be due to a transition of the S = 2 multiplet of the reduced 3Fe-4S cluster. Spin quantitation yielded a relative stoichiometry of cluster S1 to cluster S3 of 1:1. The results obtained by EPR spectroscopy indicated that the characteristic iron-sulfur cluster S1 [2Fe-2S], S2 [4Fe-4S], and S3 [3Fe-4S], were also present in this archaeal succinate dehydrogenase. EPR redox titrations of T. acidophilum membranes at pH 5.5 yielded a reduction potential of +60 +/- 20 mV for cluster S3 and of +68 +/- 20 mV for cluster S1. The axial [4Fe-4S]2+/1+ center had a reduction potential of -210 +/- 20 mV.

Dithionite↗
PubMed1993

Hyperbaric hyperoxia enhances the lethal effects of amphotericin B in Leishmania braziliensis panamensis.

Leishmania braziliensis panamensis promastigotes were exposed in vitro to amphotericin B (AmB), menadione, or phenazine methosulfate under normoxic conditions. Promastigotes were also exposed to hyperoxia alone (100% O2 at total pressures of 101.3 or 253.3 kPa), or combined with drugs. After incubation for 24 h at 27 degrees C, viable promastigotes were stained with fluorescein diacetate and counted using epifluorescence microscopy. Hyperbaric hyperoxia alone (PO2 = 229.3 kPa) was as effective as AmB alone (0.2 microM); both reduced the number of viable promastigotes to approximately 13% of the original inoculum. In addition, AmB in a hyperbaric hyperoxic environment killed more promastigotes (97% of the original inoculum) than AmB in normoxic (PO2 = 21.1 kPa) or hyperoxic conditions (PO2 = 91.7 kPa). Finally, AmB in hyperbaric hyperoxia killed significantly more (75%) promastigotes than hyperbaric hyperoxia alone. High oxygen tensions did not significantly alter the lethal effects of either menadione or phenazine methosulfate. In conclusion, the lethal effects of low dose AmB in Leishmania promastigotes were augmented by hyperbaric hyperoxia in vitro, but only at oxygen doses too high to be tolerated by human patients.

Amphotericin B↗
PubMed2002

Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex.

The metal-ligand complex, [Ru(bpy)2(dppz)]2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (Ru-BD), was used as a spectroscopic probe for studying nucleic acid dynamics. The Ru-BD complex displays a long lifetime of over 100 ns and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To further show the usefulness of this luminophore (Ru-BD) for probing DNA dynamics, we examined its intensity and anisotropy decays when intercalated into supercoiled and linear pTZ18U plasmids using frequency-domain fluorometry with a light-emitting diode (LED) as the modulated light source. Compared to the supercoiled plasmids with an average intensity decay time of 120.8 ns at 25 degrees C, we obtained somewhat longer lifetimes for the linear plasmids ((tau) = 141.4 ns at 25 degrees C), suggesting a more efficient shielding from water by the linear plasmids. The anisotropy decay data also showed longer rotational correlation times for the linear plasmids (495 and 35 ns at 25 degrees C) as compared to the supercoiled plasmids (412 and 27 ns at 25 degrees C). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the plasmids, respectively. The anisotropy values were quite similar, although the values of the supercoiled plasmids were slightly higher in both the steady-state and anisotropy decay measurements. These results indicate that Ru-BD can be applied in the study of both bending and torsional dynamics of nucleic acids.

DNA, Superhelical↗
PubMed2006

Dinuclear monointercalating RuII complexes that display high affinity binding to duplex and quadruplex DNA.

The DNA duplex binding properties of previously reported dinuclear Ru(II) complexes based on the ditopic ligands tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2'',3''-j]phenazine (tppz) and tetraazatetrapyrido[3,2-a:2'3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) are reported. Photophysical and biophysical studies indicate that, even at high ionic strengths, these complexes bind to duplex DNA, through intercalation, with affinities that are higher than any other monointercalating complex and are only equalled by DNA-threaded bisintercalating complexes. Additional studies at high ionic strengths using the 22-mer d(AG(3)[T(2)AG(3)](3)) [G3] human telomeric sequence reveal that the dinuclear tppz-based systems also bind with high affinity to quadruplex DNA. Furthermore, for these complexes, quadruplex binding is accompanied by a distinctive blue-shifted "light-switch" effect, characterized by higher emission enhancements than those observed in the analogous duplex effect. Calorimetry studies reveal that the thermodynamics of duplex and quadruplex binding is distinctly different, with the former being entirely entropically driven and the latter being both enthalpically and entropically favored.

DNA↗
PubMed1993

Assay of acyl-CoA dehydrogenase activity in frozen muscle biopsies: application to medium-chain acyl-CoA dehydrogenase deficiency.

The acyl-CoA dehydrogenases (ACDs) are mitochondrial enzymes that dehydrogenate acyl-coenzyme A esters of different chain lengths. Inherited deficiencies of these dehydrogenases are commonly associated with muscle weakness and lipid storage. Numerous assays including spectrophotometric, fluorometric, chemical, and radiochemical procedures have been used, but there is need for a rapid, reproducible assay for the different acyl-CoA dehydrogenases in small frozen samples of human muscle biopsies. We describe a comparative study of dye-linked spectrophotometric assays of the long, medium, and short chain acyl-CoA dehydrogenases in frozen rat and human muscle samples. An optimal procedure is described confirming the value of glass-glass homogenization and assay of a 600g supernatant. Higher activities for all acyl-CoA dehydrogenases, citrate synthase, and cytochrome c oxidase were obtained in rat in contrast to human. The substrate-linked dye reduction method was found superior to the ferricenium or electron transfer flavoprotein acceptor systems. Application of the phenazine ethosulfate-DCPIP-linked method to medium-chain acyl-CoA dehydrogenase (MCAD) was studied in detail and the effect of immunoprecipitation of MCAD allowed for the determination of substrate specificity and the degree of crossover between long-, medium-, and short-chain ACD activity following immunoprecipitation. Finally, a comparison of the specificity and validity of the assay in a patient with MCAD deficiency was performed.

Acyl-CoA Dehydrogenase↗
PubMed1993

The effect of NC-190, a novel antitumor compound, on the cell-cycle progression of HeLa S3 cells.

A novel antitumor compound, N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190), has potent antitumor activity against in vivo and in vitro tumor models. In this study, we evaluated the cell-cycle effect of NC-190 on cultured HeLa S3 cells using DNA/bromodeoxyuridine(BrdU) bivariate flow-cytometric analysis. Continuous treatment with NC-190 for 72 h inhibited the growth of cultured HeLa S3 cells in a concentration-dependent manner. Its 50% growth-inhibitory concentration (IC50) was 0.039 micrograms/ml (0.085 microM). The cell-cycle effects of NC-190 were dependent on the drug concentration and the treatment period. Continuous treatment with a low concentration (0.1 micrograms/ml) of NC-190 inhibited cell-cycle progression from the G2 to the M phase, resulting in G2 accumulation. With increasing concentration, NC-190 delayed cell-cycle traverse in the S and G2 phases. At a higher (10 micrograms/ml) concentration of NC-190, cell-cycle traverse was prevented in the G1, S, and G2 phases. Under such conditions, NC-190 increased the numbers of S0-phase cells (the cells with DNA content corresponding to that of S-phase cells, but without BrdU incorporation). Treatment for 2 h with NC-190 at 10 micrograms/ml induced the accumulation of cells in the G2 phase, and cell debris was observed at 48 and 72 h after drug treatment. During this time, the proportion of cells in the S0 phase increased up to 19.2%. The colcemid-induced mitotic cell accumulation was delayed by NC-190 at a concentration of 0.1 micrograms/ml at 4 h after the addition of the drugs. The addition of higher concentrations (1 and 10 micrograms/ml) of NC-190 inhibited the increase in the mitotic fraction completely. These results indicate that the mechanism involved in the G2 arrest and the increment of S0-phase cells caused by NC-190 is associated with this compound-induced cell death.

Antineoplastic Agents↗