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PubMed1996

Immunomodulatory effect of some amphiphilic detergents on the human promyelocytic HL-60 cells.

The effect of two anionic (sodium dodecyl sulfate (SDS) and sodium laurate), two cationic (didodecyldimethylammonium bromide (DDAB) and [(1-methyldodecyl)-trimethylammonium bromide (2-ATDBr)] and a nonionic [(1-methyldodecyl)-dimethyl-amine N-oxide (2-ATDNO)] amphiphilic detergents on the metabolic activation of phagocytes derived from human promyelocytic leukemia cell line HL-60 was investigated. SDS in the concentrations of 10(-4) M significantly stimulated the INT-reduction and superoxide production by HL-60 cells which were preincubated with phorbol 12-myristate 13-acetate (PMA) for a macrophage-like differentiation. This effect was not significant in the case of cells differentiated along monocyte-like and granulocyte-like pathways. A similar influence was observed by sodium laurate, but this effect was significant (P < 0.01) by INT-reduction only. Concentrations higher than 5 x 10(-5) M DDAB, 2-ATDBr and 2-ATDNO inhibited INT-reduction and superoxide generation in HL-60 cells differentiated along all three ways. Our results indicated that differentiated HL-60 cells may be used for in vitro testing of immunotoxic effects on respiratory burst in phagocytes.

Detergents↗
PubMed1995

Prevention and reduction of neural damage in ischemic strokes by w-(N,N'-diethylamino)-n-alkyl-3,4,5-trimethoxybenzoate compounds.

Experimental ischemic stroke was induced by an occlusion of middle cerebral artery and common carotid artery of rats for 60 min. The occlusion was then released and the extent of neural damage was measured by the estimation of necrosis areas where the dead neurons lost their ability to take up triphenyl tetrazolium chloride. w-(N,N'-Diethylamino)-n-alkyl-3,4,5-trimethoxybenzoate (TMB) compounds are calcium antagonists capable of reducing intracellular free calcium through inhibition of calcium release from the sarcoplasmic reticulum. These compounds were found to prevent and/or reduce neural damage effectively. When the animals were pretreated with TMB-2, TMB-5 and TMB-8, 1 hr before ischemia, the areas of neural necrosis were reduced almost completely by 95%, 99% and 92%, respectively. When these TMB compounds were given at 0 time and 1 hr after ischemia, the prevention of necrosis was almost complete also. However, when TMB-2, TMB-5 and TMB-8 were administered 6 hr after ischemia, the necrosis areas were reduced less effectively by 83%, 78% and 91%, respectively. TMB compounds were also found to increase cAMP and to reduce intracellular free calcium concentration. These results indicate that TMB compounds are effective calcium antagonists to prevent/treat ischemic stroke by reducing the intracellular free calcium through reduction of calcium release from sarcoplasmic reticulum.

Animals↗
PubMed1980

Synthesis of some newer formazans and tetrazolium salts and their effect on Ranikhet disease virus and the vaccinia virus.

Synthesis and biological evaluation of a series of formazans and their corresponding tetrazolium salts are described. Several 1,3,5-trisubstituted formazans were prepared by condensing 4-acetoxy-3-methoxybenzaldehydephenylhydrazone and 2,4-dinitrophenylhydrazone of 2-benzimidazolecarboxaldehyde, with an appropriate phenyldiazonium salt. The formazans derived from 4-acetoxy-3-methoxybenzaldehydephenylhydrazone were further oxidized to their corresponding cyclization products, 2,3,5-trisubstituted tetrazolium salts. Oxidation of 1-aryl-3-(2'-benzimidazolyl)-5-(5-(2',4'-dinitrophenyl) formazans to their corresponding tetrazolium salt did not meet with success. Both the highly coloured formazans and water-soluble tetrazolium salts were screened for their antiviral, activity against vaccinia virus and Ranikhet disease virus in a stationary culture of chorioallantoic membrane of chick embryo. Two of the compounds were found to exhibit significant activity (87% and 83%) against the Ranikhet disease virus. However no activity against vaccinia virus could be observed.

Antiviral Agents↗
PubMed1981

Induction of differentiation of cultured human and mouse myeloid leukemia cells by alkyl-lysophospholipids.

Alkyl-lysophospholipids are synthetic analogs of naturally occurring lysophospholipids. The effects of these compounds on cell proliferation and differentiation of cultured human (HL-60) and mouse (M1) myeloid leukemia cells were studied. Both cell lines were induced to differentiate into morphologically and functionally mature granulocytes and macrophages by incubation with a wide variety of these compounds. Some alkyl-lysophospholipids induced differentiation (judged morphologically and by the appearance of abilities to reduce nitro blue tetrazolium, to phagocytize latex particles, and to induce lysozyme activity) of both the cells lines at concentrations of 1 microgram/ml. However, these compounds did not affect colony formation of normal mouse bone marrow cells even at a higher concentration, 20 microgram/ml. These results suggest that alkyl-lysophospholipids induce cell differentiation of myeloid leukemia cells without affecting proliferation and differentiation of normal bone marrow cells. Thus, these compounds could be useful in therapy of myeloid leukemia.

Animals↗
PubMed1978

Oxygen and the production of formazan from neotetrazolium chloride.

Electrons, generated from dehydrogenase reactions, are transferred to oxygen in preference to neotetrazolium chloride. In model systems in solution the presence of a small amount of oxygen drastically reduces the rate of formazan production. The rate of reaction in tissue sections has been followed using scanning and integrating microdensitometry. As in solution, electrons are transferred preferentially to oxygen. However, oxygen seems unable to diffuse through the incubation medium and thus the supply of oxygen at the site of the enzyme activity becomes exhausted; the time taken to use up the oxygen will depend on the rate of the enzyme activity. It is only then that electrons are passed to the tetrazolium salt and formazan is precipitated.

Animals↗
PubMed2001

Antioxidant capacity of phenolic and related compounds: correlation among electrochemical, visible spectroscopy methods and structure-antioxidant activity.

We investigated the antioxidant activity of phenylpropionic acids--caffeic (CAF), ferulic (FER), para-coumaric (COU) and cinnamic (CIN)--and phenolic acids and related compounds--gallic (GAL), methyl gallate (meGAL), vanillic (VAN) and gentisic (GEN)--using visible spectroscopy, inhibition of nitroblue tetrazolium (NBT) reduction, and electrochemical methods including cyclic voltammetry and potentiometry. In the spectroscopic assays, only CAF, GAL and meGAL were able to inhibit NBT reduction. The same compounds showed the lowest oxidation potentials (Epa) and the highest redox potentials deltaE) in the cyclic voltammetric and potentiometric studies, respectively. In addition, it was observed that the greater the number of hydroxyls linked to the aromatic ring, the greater was the antioxidant activity of the analysed compounds. The correlations of Spermann--used to compare the methods between themselves and the methods with the relationship structure-antioxidant activity--were r = -0.9762 for the cyclic voltammetric-potentiometric methods. r = 0.8333 for the inhibition of NBT reduction-potentiometric methods and r = -0.8095 for the inhibition of NBT reduction-cyclic voltammetric methods. The correlations for cyclic voltammetric, potentiometric and inhibition of NBT reduction methods-number of hydroxyls linked to the aromatic ring were r = -0.9636, 0.9636 and 0.9142, respectively. These findings indicate that the electrochemical methods together with spectroscopic studies are a good tool to evaluate the antioxidant activity of substances.

Antioxidants↗
PubMed2006

Induction of differentiation and apoptosis in three human leukemia cell lines by a new compound from Dendrostellera lessertii.

It has previously been shown that Dendrostellera lessertii (Thymelaeaceae) has strong anticancer activity. In this study, the antileukemic activity of another new compound from the same plant extract is reported. Promyelocytic (NB4 and HL-60) and erythroleukemia (K562) cells were cultured in the presence of various concentrations of the new compound (0.5-3.0 mug/ml) for 3 d. The cell numbers were then determined by trypan blue exclusion test. The new compound inhibited growth and proliferation of NB4, HL-60 and K562 with IC50 values of 1.5, 2.0 and 2.5 mg/ml, respectively. We also found that the new compound inhibited cell proliferation in a dose- and time-dependent manner. At low concentrations and after 48 h of treatment, approximately 50%-70% of NB4 and HL-60 cells were differentiated to monocyte/macrophage lineage and approximately 30%-40% of the treated K562 cells were differentiated in the megakaryocytic lineage, as evidenced by morphological changes and nitro blue tetrazolium reduction assays. Results of Hoechst 33258 staining also indicated that the new compound induced NB4 and HL-60 cell apoptosis at their respective IC50 values after 72 h of treatment. Based on the present data, the new compound seems a good candidate for further evaluation as an effective chemotherapeutic agent acting through induction of differentiation and apoptosis.

Antineoplastic Agents↗
PubMed2004

Cell surface oxygen consumption by mitochondrial gene knockout cells.

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

Aerobiosis↗
PubMed1993

NADPH-oxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnaea stagnalis.

Macrophage-like defense cells (hemocytes) of the pond snail Lymnaea stagnalis generate reactive oxygen intermediates (ROIs) upon contact with non-self, following kinetics similar to those of ROI production by mammalian leukocytes during respiratory burst. In this study, several inhibitors of NADPH-oxidase, the key enzyme of the respiratory burst in mammalian phagocytes, were tested for their effect on oxidative activities [as demonstrated by nitroblue tetrazolium (NBT) reduction and luminol-dependent chemiluminescence (LDCL)] of phagocytosing snail hemocytes. In the presence of diphenylene iodonium, zymosan-stimulated hemocytes of L. stagnalis failed to reduce NBT and showed a markedly reduced LDCL response. Also, compounds that prevent assembly of functional NADPH-oxidase complexes in activated mammalian cells were effective; preincubation of hemocytes with 1,4-naphthoquinone inhibited the LDCL response and NBT reduction upon phagocytic stimulation. Furthermore, coincubation but not preincubation with five different catechol-like phenols inhibited oxidative activities of zymosan-stimulated hemocytes. These results imply similarities in composition and regulation of the ROI-generating mechanisms of both mammalian and snail defense cells. It is postulated that in L. stagnalis hemocytes, (1) NADPH-oxidase activity is responsible for ROI production, (2) an active NADPH-oxidase enzyme complex has to be assembled from putative cytosolic and membrane-associated components, and (3) continuous replacement of active NADPH-oxidase enzyme complexes is necessary to sustain respiratory burst-like oxidative activities during interactions with non-self.

Animals↗
PubMed2002

Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing.

The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40. Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity. Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium. Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results. However, the LDH test seemed less reliable due to great variation. The use of serum was found to result in lower toxicity of the compounds in both tests. The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA. The human cell line is a promising in vitro alternative in oculotoxicity testing.

Animals↗
PubMed1991

Poly(amidoamine)s with potential as drug carriers: degradation and cellular toxicity.

Poly(amidoamine)s were synthesized by polyaddition reaction: to bis-acryloylpiperazine of piperazine (1), or N,N'-bis(2-hydroxyethyl)ethylenediamine (2), and to 2,2-bis(acrylamido)acetic acid of piperazine (3). Compound 2 was also end-capped with 4-hydroxythiophenol, thus introducing a terminal moiety suitable for radio-iodination using the chloramine T method (4). Such polymers behave as bases in aqueous solution, and their net average charge alters considerably as the pH changes from 7.4 to 5.5. This results in a change in polymer conformation which may prove useful in the design of polymeric drug delivery systems. However, their suitability for use in the organism will depend on polymer toxicity and also on their rate of biodegradation. Here we studied the biological properties of the above poly(amidoamine)s with a view to optimizing the synthesis of novel drug carriers. The general cytotoxicity of compounds 1, 2, 3, and 4 was examined in vitro using two human cell lines, hepatoma (HepG2) and a lymphoblastoid leukaemia (CCRF). Several different methods [the tetrazolium (MTT) test, [3H]leucine or [3H]thymidine incorporation, or counting cell numbers] were used to measure cell viability. Compounds 1, 2, and 4 were much less toxic to both cell lines than equivalent concentrations of the polycationic poly-L-lysine, and in no case did viability fall below 50% (concentrations up to 2 mg/ml). Although compound 2 was not markedly toxic to HepG2 cells, concentration-dependent toxicity was observed against CCRF cells. In this case, the polymer concentration decreasing viability by 59% (ID50) was approximately 50 micrograms/ml for compound 2 compared with an ID50 of approximately 10 micrograms/ml for poly-L-lysine. The rate of hydrolytic degradation of compound 2 was examined using viscometric measurements and gel permeation chromatography (GPC). After incubation at pH 7.5 and 8.0 for 24 h, polymer intrinsic viscosity was decreased by approximately 50% and GPC elution profiles showed a simultaneous increase in polymer retention time, indicating a fall in molecular weight. Hydrolytic degradation progressed much more slowly at pH 5.5. Compound 4 was also incubated with a mixture of isolated rat liver lysosomal enzymes (tritosomes) at pH 5.5, but no increase in the rate of degradation was observed.

Biocompatible Materials↗
PubMed1993

Effect of 1,25,28-trihydroxyvitamin D2 and 1,24,25-trihydroxyvitamin D3 on intestinal calbindin-D9K mRNA and protein: is there a correlation with intestinal calcium transport?

Although analogs and metabolites of vitamin D have been tested for their calciotropic activity, very little information has been available concerning the effects of these compounds on gene expression. In this study one analog of vitamin D, 1,25,28-trihydroxyvitamin D2 [1,25,28-(OH)3D2], and one metabolite, 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3], were tested for their effect on intestinal calbindin-D9K mRNA and protein as well as for their effect on intestinal calcium absorption and bone calcium mobilization. These compounds were also evaluated for their ability to compete for rat intestinal 1,25-(OH)2D3 receptor sites and to induce differentiation of human leukemia (HL-60) cells as indicated by reduction of nitro blue tetrazolium. In vivo studies involved intrajugular injection of 12.5 ng 1,25-(OH)2D3 or test compound to vitamin D-deficient rats and sacrifice after 18 h. 1,25,28-Trihydroxyvitamin D2 had no effect on intestinal calcium absorption, bone calcium mobilization, or intestinal calbindin-D9K protein and mRNA. Competitive binding to 1,25-(OH)2D3 receptors was 0.8% of that observed using 1,25-(OH)2D3. However, 20- and 40-fold higher doses of 1,25,28-(OH)3D2 (250 and 500 ng) resulted in significant inductions in calbindin-D9K protein and mRNA (3.5 to 7.4-fold), although doses as high as 800 ng were found to have no effect on intestinal calcium absorption or bone calcium mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

25-Hydroxyvitamin D 2↗
PubMed2003

Gastrointestinal toxicity, antiinflammatory activity, and superoxide dismutase activity of copper and zinc complexes of the antiinflammatory drug indomethacin.

Gastrointestinal (GI) toxicity is one of the major problems associated with antiinflammatory drugs. The complexation of the powerful antiinflammatory drug (IndoH) by metal ions, as a means of reducing GI toxicity, has been studied. The in vitro superoxide dismutase (SOD) activity, in vivo antiinflammatory activity, and gastrointestinal ulcerogenic properties of IndoH, [Cu2(Indo)4(DMF)2], and [Zn2(Indo)4(DMA)2] are reported. No SOD activity was observed for IndoH or [Zn2(Indo)4(DMA)2], but [Cu2(Indo)4(DMF)2] inhibited the reduction of nitroblue tetrazolium (NBT) at an IC50 value of 0.23 microM. All three compounds exhibited antiinflammatory activity in male Sprague-Dawley rats at an equivalent Indo dose of 10 mg/kg following oral administration of the drugs in 2% CMC solution. The severity of the toxicity (macroscopic ulcerations) in the stomach following oral dosing with [Zn2(Indo)4(DMF)2] was not significantly lower than that induced by IndoH (P = 0.78). Gastric ulcerations induced by [Cu2(Indo)4(DMF)2] were significantly lower than those induced by IndoH or [Zn2(Indo)4(DMA)2] (P = 0.0012 and P = 0.0175, respectively) but significantly greater than the control (P = 0.0013). The intestinal ulcerations induced by [Cu2(Indo)4(DMF)2] or [Zn2(Indo)4(DMA)2] were approximately 15 times lower than those of IndoH. A further indicator of gastrointestinal toxicity, caecal haemoglobin, increased in the following order: control < [Cu2(Indo)4(DMF)2] < [Zn2(Indo)4(DMA)2] < IndoH.[Cu2(Indo)4(DMF)2] exhibited the most promising results of the Indo complexes assayed, in that it exhibited SOD activity and the lowest gastrointestinal damage while also exhibiting antiinflammatory activity that was comparable to that for IndoH. Low-temperature EPR analyses also showed that the formulation used for [Cu2(Indo)4(DMF)2] administration was crucial to the integrity of the complex.

Animals↗
PubMed1998

Growth inhibition of prostate cell lines in vitro by phyto-oestrogens.

OBJECTIVE: To assess a range of phyto-oestrogens as moderators of growth and metabolism in several prostate cell lines. MATERIALS AND METHODS: Four prostate cell lines (PNT-1/A, PNT-2, PC-3 and DU145) were challenged with different doses of five phyto-oestrogens (biochanin A, daidzein, genistein, genistin and nordihydroguaiaretic acid) over 3 days in culture. Cell proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and metabolic activity by cleavage of a tetrazolium salt (XTT). RESULTS: Growth and metabolism were inhibited with all compounds and cell lines (e.g. the dose for 50% inhibition of proliferation of PC-3 cells by genistein was 38 micromol/L); differences in the patterns of results suggested that different mechanisms operated, but there was no evidence for any synergistic activity on the inhibition of cell proliferation. CONCLUSION: These results offer further support for the hypothesized role of phyto-oestrogens as dietary protectors against prostatic cancer.

Cell Division↗
PubMed1984

A new method for the cytochemical staining of cells immobilized in agarose.

A quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.

Animals↗