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Bacteriostasis of treated fabric was evaluated directly by a colony count made on the treated fabric after impregnation with seeded agar containing triphenyltetrazolium chloride and incubation in a horizontal, suspended position in a petri dish.
PURPOSE: There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations. EXPERIMENTAL DESIGN: Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation. RESULTS: Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism. CONCLUSION: These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.
AIM: To study the effects of recombinant human growth hormone (rhGH) on growth of human gastric cancer cell line in vitro. METHODS: Experiment was divided into control group, rhGH group, oxaliplatin (L-OHP) group and rhGH+L-OHP group. Cell inhibitory rate, cell cycle, cell proliferation index (PI) and DNA inhibitory rate of human gastric cancer line BGC823, at different concentrations of rhGH treatment were studied by cell culture, MTT assay and flow cytometry. RESULTS: The distinctly accelerated effects of rhGH on multiplication of BGC823 cell line were not found in vitro. There was no statistical significance between rhGH group and control group, or between rhGH+L-OHP group and L-OHP group (P>0.05). The cell growth curve did not rise. Cell inhibitory rate and cells arrested in G(0)-G(1) phase were obviously increased. Meanwhile, cells in S phase and PI were distinctly decreased and DNA inhibitory rate was obviously increased in rhGH+L-OHP group in comparison with control group and rhGH group, respectively (P<0.01). Cell inhibitory rate showed an increasing trend and PI showed a decreasing trend in rhGH+L-OHP group compared with L-OHP group. CONCLUSION: In vitro rhGH does not accelerate the multiplication of human gastric cancer cells. It may increase the therapeutic efficacy when it is used in combination with anticancer drugs.
The activity of superoxide dismutase (SOD) from bovine erythrocytes was measured by the inhibition of nitrotetrazolium blue reduction rate in superoxide anion radical generation systems--xanthine/xanthine oxidase of NADH/phenazine methasulfate. The enzyme activity increases in the presence of compounds acting as electron donors in radical-involving reactions and decreased in the presence of compounds possessing the properties of electron acceptors. Activation of SOD by electron donors and its inhibition by electron acceptors was dependent on the concentration of the above compounds. In the absence of SOD electron donors and acceptors did not change the rate of tetrazolium blue reduction by superoxide anion radicals. The role of the new type of SOD regulation for the enzyme functioning in the cell is discussed.
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A recently developed tetrazolium-based microculture assay was used to screen extracts of cultured cyanobacteria (blue-green algae) for inhibition of the cytopathic effects of the human immunodeficiency virus (HIV-1), which is implicated as a causative agent of AIDS. A number of extracts were found to be remarkably active against the AIDS virus. A new class of HIV-1-inhibitory compounds, the sulfonic acid-containing glycolipids, was discovered through the use of the microculture assay to guide the fractionation and purification process. The pure compounds were active against HIV-1 in cultured human lymphoblastoid CEM, MT-2, LDV-7, and C3-44 cell lines in the tetrazolium assay as well as in p24 viral protein and syncytium formation assays.
In order to enhance the sensitivity of detection of alkaline phosphatase immobilized on nitrocellulose, a homopolymer of 2-acrylamide 2-methylpropanesulfonate (poly-AMPS) and agarose were added to the reaction mixture using 5-bromochloroindoxyl phosphate and nitroblue tetrazolium. By addition of the above ingredients, the amount of the reaction product deposited on the nitrocellulose filters on which alkaline phosphatase had been dot-blotted was increased, and darkening of the background (un-dot-blotted area) was prevented. Reflective densitometry revealed that the addition of poly-AMPS and agarose increased the sensitivity of the detection approximately three times higher than without the use of these compounds.
The effect of nickel from soluble NiCl2 on Cu-Zn superoxide dismutase (SOD) activity, as well as on rate of nitro blue tetrazolium reduction, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel insoluble compounds, whose toxicity and carcinogenicity are well established. The physical and chemical nature of nickel compounds is one of the key determinations of its toxicity. Soluble nickel freely enter cells, but is just as readily excreted reducing the opportunity for production of lipid damage. Nickel from NiCl2 strongly activated SOD activity. In vitro addition of nickel chloride to a crude lung preparation altered the KM for SOD without changing the Vmax. Nickel chloride produced increased enzyme affinity to the substrate, because decreased (O2-) concentration that yields half-maximal velocity. The combination of nickel and SOD may contribute to stabilization of the particular conformation of SOD responsible for maximal catalytically activity.
Amburana cearensis a common tree found in Northeastern Brazil is widely used in folk medicine. The present work evaluated the cytotoxicity of kaempferol, isokaempferide, amburoside A and protocatechuic acid isolated from the ethanol extract of the trunk bark of A. cearensis. The compounds were tested for their cytotoxicity on the sea urchin egg development, hemolysis assay and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay using tumor cell lines. Isokaempferide and kaempferol, but not amburoside A and protocatechuic acid, inhibited the sea urchin egg development as well as tumor cell lines, but in this assay isokaempferide was more potent than kaempferol. Protocatechuic acid was the only compound able to induce hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of kaempferol and isokaempeferide was not related to membrane damage.
Efficient peroxidase substrates may have a critical role in the oxidation of secondary compounds by peroxidases. Hydrazines are often oxidized slowly by peroxidases due, in part, to hydrazine-dependent inactivation of these enzymes. Peroxidase-catalyzed oxidation of hydrazines may be dramatically affected by an efficient peroxidase substrate. We investigated this hypothesis in a model system using the well-known peroxidase substrate chlorpromazine (CPZ) and the hydrazine derivative isoniazid. CPZ stimulated isoniazid oxidation as measured by nitroblue tetrazolium (NBT) reduction and O2 consumption. The kinetics of isoniazid and CPZ oxidation by horseradish peroxidase (HRP) in the presence of both compounds suggested CPZ was acting as an electron transfer mediator between HRP and isoniazid. Indeed, CPZ.+, the product of CPZ oxidation by HRP, was able to oxidize isoniazid. The rate constant for this pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M-1 s-1 at pH 4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivation of HRP was observed. The inactivation process involved the formation of compound III followed by accumulation of irreversibly inactivated HRP. CPZ completely inhibited inactivation. Thus, by acting as a redox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient peroxidase substrates, such as phenol and tyrosine, were also able to dramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activation of isoniazid and other hydrazines. As such, these substrates may have a vital role in the pharmacological and toxicological properties of hydrazines and other compounds.
PURPOSE: Activator protein-1 (AP-1) has been implicated as playing important roles in apoptosis and cancer development. In this work, we studied several natural chemopreventive compounds for their potential chemopreventive properties in the modulation of AP-1 signaling pathway in HT-29 colon cancer cells. METHODS: The HT-29 cells were transfected with AP-1-luciferase reporter gene, and one of the stable clones (C-4) was used for subsequent experiments. The HT-29 C-4 cells were treated for 1 h with various natural chemopreventive agents and challenged with AP-1 stimulators such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or hydrogen peroxide (H2O2) for 6 h. The c-Jun N-terminal kinase (JNK) was examined to understand the effect of these compounds on the upstream signaling activator of AP-1. The protein expression level of endogenous cyclin D1, a gene that is under the control of AP-1, was also analyzed after treatments with the agents. In addition, cell death induced by these compounds was evaluated by MTS assay [3-(4.5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. RESULTS: TPA and H2O2 treatments strongly induced AP-1-luciferase activity as expected. Phenethyl isothiocyanate, sulforaphane, curcumin, and resveratrol increased AP-1-luciferase activity dose-dependently and then decreased at higher doses in the presence or absence of TPA. Allyl isothiocyanate and (-)-epigallocatechin-3-gallate (EGCG) increased AP-1-luciferase activity dose-dependently up to 50 and 100 microM. Other tea catechins and procyanidin dimers, however, had little or no effect on AP-1-luciferase activity. The JNK activity was induced by the isothiocyanates and EGCG. Most of the chemopreventive compounds induced cell death in a dose-dependent manner, with the exception of epicatechin (EC) and the procyanidins, which had little effect. The expression of endogenous cyclin D1 protein was well correlated with those of AP-1-luciferase assay. CONCLUSION: Taken together, these results suggest that natural chemopreventive compounds may have differential biological functions on the signal transduction pathways such as AP-1 in the intervention of colon cancer progression and carcinogenesis.
In this study, we evaluated the effects of three simple organochalcogenides (diphenyl diselenide, diphenyl ditelluride and diphenyl telluride) and ebselen on the glutamate-driven 45Ca2+ influx into chick embryonic retinal cells, as well as their effects on the excitotoxic injury in retina cells. None of the compounds tested interfered with basal 45Ca2+ uptake. Diphenyl diselenide and diphenyl ditelluride had no effects on glutamate-driven 45Ca2+ influx. Diphenyl telluride (100-400 microM) decreased and ebselen (100-400 microM) completely blocked the glutamate-driven 45Ca2+ influx (P < 0.01) into chick retinal explants. The assessment of neural injury was made spectrophotometrically by quantification of cellularly reduced MTT (3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) 24 h after the beginning of glutamate exposure (8 h). Ebselen had no effects on retinal MTT reduction when co-incubated with glutamate for 8 h. However, when ebselen (100 and 400 microM) was co-incubated for 8 h with glutamate and remained in the incubation media until MTT evaluation (24 h after the beginning of incubation), it protected retinal cells against the decrease in MTT reduction induced by glutamate. These data indicate that besides its capacity of interacting with Ca2+ channels, other mechanisms are involved in the neuroprotection afforded by ebselen in this work, possibly its antioxidant properties.
The effects of flavonoids on NADPH diaphorase activity were studied in vitro, and we found that the enzyme activity was markedly inhibited by quercetin. This inhibitory action was shown to be accompanied by an increase in the apparent Km value of the enzyme for the cofactor NADPH, with a decrease in the Vmax, and an increase in the apparent Km for the substrate nitro blue tetrazolium, without any significant change in the Vmax. These results indicate that quercetin may directly inhibit NADPH diaphorase, thus suggesting the possibility that this compound may be able to inhibit the production of nitric oxide in the brain.
We compared Barbeito-Lopez Trichrome stain with H-E, the basic fuchsin picric acid and Nitro Blue of Tetrazolium stains. The Barbeito-Lopez Trichrome stain was much more sensitive than the other stains for the diagnosis of early myocardial coagulation necrosis.
The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences.
The production of free radicals, superoxide anions (O2-), and hydrogen peroxide (H2O2) was histochemically investigated in human neutrophils that were stimulated by either phagocytosis or the calcium ionophore A23187. To demonstrate O2-, peripheral neutrophils from healthy donors were incubated at 37 degrees C in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A and/or A23187. To demonstrate H2O2, neutrophils pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeCl3 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for O2-) and cerium perhydroxide deposits (for H2O2) were restricted to the neutrophil-particle interface and on the inner surface of phagosomes. The remaining free surface of the plasma membrane was devoid of reaction products. In the case of neutrophils stimulated with A23187, the production of O2- and H2O2 was visualized over the whole surface of the plasma membrane. These histochemical reactions were inhibited by p-benzoquinone, superoxide dismutase, ferricytochrome c or catalase, and p-diazobenzenesulfonate (a membrane-impermeable protein denaturant). The results showed that human neutrophils produce free radicals exocellularly and that the site of production varies with different stimuli.
The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.