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PubMed2000

Ebselen protection against hydrogen peroxide-induced cytotoxicity and DNA damage in HL-60 cells.

AIM: To study the protective effect of ebselen (Ebs) on hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage in human leukemia cell line HL-60. METHODS: The inhibitory effect of H2O2 on cell growth was determined using the tetrazolium dye colorimetric test, and the lipid peroxidation was estimated by malondialdehyde (MDA) formation. DNA damage was detected using single cell gel electrophoresis, and intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA). RESULTS: H2O2 (100 mumol.L-1) suppressed the growth of HL-60 cells and the addition of Ebs (1-20 mumol.L-1) reduced the suppression in a concentration-dependent manner. Furthermore, Ebs also displayed a concentration-dependent reduction of MDA formation in H2O2-treated cells, at the concentration of 20 mumol.L-1 the inhibitory rate was 56.4%. Ebs was able to reduce the ROS formation and DNA damage caused by H2O2 in a concentration-dependent manner. CONCLUSION: Ebs has a strong protective ability against the cytotoxicity and DNA damage caused by reactive oxygen species (ROS).

Antioxidants↗

PubMed2005

The evaluation of cytotoxic activity of planar pentadentate ligand 2',2"'-(2,6-pyridindiyldiethylidyne) dioxamohydrazide dihydrate (H2l x 2H2O) and its metal coordination complexes; pitfalls in the use of the MTT-assay.

In this study we have investigated, for the first time to our knowledge, the antineoplastic activity of a planar pentadentate ligand (H2L.2H2O = 2', 2'''-(2,6-pyridindiyldiethylidyne)dioxamohydrazide dihydrate) and some of its metal coordination complexes [Cu(L)(H2O)].H2O, [Cu(HL)(H2O)]Cl04, [Co(L)(H20)2] 6H20, [Co(H2L)(H2O)(MeOH)](ClO4)2 and [Fe(L)(H2O)2]ClO4-3H2O, as well as of inorganic salts CuCl2 2H20, CoCl2 6H2O and FeCl3.6H2O of corresponding metal ions. The antiproliferative activity of these compounds was examined in a human melanoma cell line FemX with exposure time of 48 hours by performing two cytotoxicity tests: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sulforhodamine B (SRB) assay. Among these substances, the ligand H2L.2H2O expressed the greatest antineoplastic activity IC50 = 45.40 microM, while the IC50 of others could not be determined by SRB assay in the examined range of concentrations due to their low activity. FeCl3.6H2O showed stimulatory activity. We have found remarkable discrepancies between the results obtained by MTT assay and SRB assay that influence IC50 value as well as other measures of cytotoxicity, which led to the conlusion of uncertainty of using the MTT assay in evaluation of antineoplastic activity of organometalic complexes and inorganic metal salts.

Cell Line, Tumor↗

PubMed1998

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures.

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures. 2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 microM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors. 3. Daily treatment with nitric oxide synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), led to the stimulation of cell proliferation by 44+/-5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect. 5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-gamma and uncoupling protein-1, which are upregulated during differentiation. 6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100-1000 microM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-gamma and uncoupling protein-1.

Adipose Tissue, Brown↗

PubMed2006

Calcium salicylate-mediated apoptosis in human HT-1080 fibrosarcoma cells.

Salicylates are novel biologically active compounds that exhibit multiple therapeutic activities. The anti-cancer effectiveness of calcium salicylate has been investigated on human HT-1080 fibrosarcoma cell lines at relatively low concentrations (predominantly 0.4 mM) compared to those previously reported. Although low calcium salicylate concentrations did not retard tumour growth progression significantly, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and time-lapse assays, its cytotoxic characteristics were proven to be prominent by various morphological and immunocytological techniques. The results here demonstrate evidence for approximately 25% apoptosis after treatment with calcium salicylate, which up-regulatd the expression of p53, p21 and Bax, and down-regulated Bcl-2 in HT-1080 cells.

Antineoplastic Agents↗

PubMed2005

In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells.

Hundreds of mycotoxins are known to date and many of them are of great interest with regard to human and animal health since they are detected frequently in plant-derived products. Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions. Considering this coincident production, it is very likely, that humans and animals are always exposed to mixtures rather than to individual compounds. Therefore, future risk assessments should consider mixture toxicity data. This is particularly true for ochratoxin A (OTA), ochratoxin B (OTB), citrinin (CIT) and occasionally for patulin (PAT) as they are all produced by a number of Penicillium and Aspergillus species. Therefore, these four toxins were chosen to study the interactive effects in vitro, using the well-established porcine renal cell line LLC-PK1 and the MTT reduction test as a cytotoxicity endpoint. By application of a step-wise approach to test combination toxicity, using various full factorial as well as a central composite experimental designs, the interactive (synergistic) cytotoxic effects of the these four toxins were assessed. The results obtained in this study confirm a potential for interactive (synergistic) effects of CIT and OTA and possibly other mycotoxins in cells of renal origin.

Algorithms↗

PubMed1999

The angiotensin II AT1 receptor antagonist candesartan at antihypertensive plasma concentrations reduces damage induced by ischemia-reperfusion.

The aim of this study was to investigate if the angiotensin II AT1 receptor antagonist candesartan in antihypertensive plasma concentrations improves myocardial function and limits infarct size in anesthetized pigs. Animals were subjected to 45 min of regional ischemia and 240 min of reperfusion. Starting 60 min before ischemia, two groups of pigs (n = 6 in each) received either candesartan (25 micrograms/kg bolus followed by a continuous infusion at a rate of 14 micrograms/kg/h) or the corresponding volume of vehicle throughout the study period. Left ventricular systolic segment shortening (%SS) was measured by sonomicrometry, and infarct size was determined by triphenyl tetrazolium chloride staining. The plasma concentration of candesartan during the experiment was between 100 and 150 nmol/L, which was considered to be within the therapeutic range. Neither candesartan nor vehicle affected hemodynamics or coronary blood flow prior to ischemia. Compared to vehicle, candesartan improved recovery of %SS in the ischemic area. At 240 min of reperfusion, the %SS was significantly higher in pigs given candesartan than in pigs given vehicle (7.1 +/- 0.87% vs-1 +/- 1.79%; p < 0.01). In both groups the area at risk was approximately 20% of the left ventricle. Infarct size as a percentage of the area at risk was significantly smaller in the candesartan group than in the vehicle group (46 +/- 3.0 vs 73 +/- 3.6%; p < 0.01). The results suggest that angiotensin II AT1 receptor blockade, obtained in antihypertensive plasma concentrations, supports myocardial functional recovery and limits infarct size.

Anesthesia↗

PubMed1998

Interference with cellular energy metabolism reduces kynurenic acid formation in rat brain slices: reversal by lactate and pyruvate.

This study was designed to investigate the role of cellular energy metabolism in the de novo formation of the endogenous excitatory amino acid receptor antagonist, kynurenic acid. Using rat cortical tissue slices, the roles of glucose transport, glycolysis, tricarboxylic acid cycle intermediates and oxidative phosphorylation were studied. Inhibition of glucose utilization resulted in quantitatively similar decreases in kynurenine uptake, kynurenic acid production and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, a marker of mitochondrial activity. The end product of glycolysis, pyruvate, as well as lactate, attenuated all three deficits. Pyruvate also significantly increased kynurenic acid formation in normal brain slices without affecting kynurenine uptake. Oxaloacetate and alpha-ketoglutarate (tricarboxylic acid cycle intermediates) were the only compounds tested which were capable of duplicating the effects of pyruvate, indicating that 2-oxoacids can stimulate kynurenic acid synthesis by acting as aminoacceptors in the enzymatic transamination of kynurenine. When the mitochondrial electron transport chain was blocked by specific inhibitors, coincubation with succinate restored the rate of MTT formazan formation to normal (except in the case of 3-nitropropionic acid), yet failed to prevent the resulting reduction in kynurenic acid synthesis. Conversely, pyruvate increased kynurenic acid production in the presence of all inhibitors (except cyanide), but did not attenuate the reduction in kynurenine uptake and MTT formazan formation. Taken together, these results demonstrate that interference with cellular energy metabolism causes mechanistically diverse, pronounced reductions in the cerebral neosynthesis of kynurenic acid, and that 2-oxoacids and lactate can effectively reverse most of these detrimental effects.

Animals↗

PubMedDate not supplied

Cell responses to xenobiotics: comparison of MCF7 multi-drug- and mussel blood cell multi-xenobiotic-defense mechanisms.

Multi-drug resistance (MDR) in MCF7 breast cancer cells and multi-xenobiotic resistance (MXR) in mussel (Mytilus edulis) blood cells (MBC) are well known mechanisms that contribute to the decrease in intracellular concentrations of many unrelated but cytotoxic compounds. In the present work, we have carried out comparative investigations of the MDR/MXR protective mechanisms using a rapid colorimetric assay for cell viability and calcein accumulation for MDR/MXR activities. These studies were performed using cultured MCF7 and MBC before and after in vitro exposure to xenobiotics. Our results indicate that a 5-day exposure to doxorubicin or vincristine decreased calcein accumulation in MBC which is consistent with an induction of multi-xenobiotic resistance. The increase in calcein accumulation provoked by 1-h treatment with 50 microM verapamil was much lower in MBC when compared to the P-glycoprotein overexpressing MCF7 cell line. We conclude that such microplate assays could be used in primary cultures of MBC to estimate the effects of various chemicals on MXR activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Demonstration of nitric oxide synthase (NOS) in marmosets by NADPH diaphorase (NADPH-d) histochemistry and NOS immunoreactivity.

Since species interdiversity often prevents the extrapolation of laboratory rodent data to man and similar problems may exist for nitric oxide synthase (NOS), NADPH-d activity and immunohistochemistry of NOS were investigated in the New World monkey Callithrix jacchus (marmoset), which has been shown to be close to the human situation in many respects. Using the NADPHd reaction with beta-NADPH and nitroblue tetrazolium (NBT) on acetone-chloroform pretreated cryosections, NBT formazan was found in many neural and non-neural (e.g. diverse epithelia, striated muscle fibers, vascular endothelium) cells in numerous tissues and organs. Prefixation with formaldehyde lowered the number of NADPH-d active sites and the amount of formazan with the exception of neuronal NADPH-d as did incubation of fresh or acetone-chloroform-pretreated sections for NADPH-d in the presence of 0.5% formaldehyde. When 1% formaldehyde or 0.5 mM permanganate were used significant amounts of formazan appeared only in central and peripheral neurons, vasal endothelial cells, small intestinal enterocytes, plasma membrane region of striated muscle fibers as well as arteriolar cells in the kidney; except for enterocytes, these observations were confirmed by NOS-immunohistochemistry which revealed in addition reactive cells in the thymus and intestinal lamina propria.

Acetone↗

PubMed1992

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.

Alkaline Phosphatase↗

PubMed1993

Chemosensitivity testing of human mouth carcinoma cell line.

The chemosensitivity of KB cells derived from oral epidermal carcinoma to various antitumor agents was analyzed using the MTT[3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H tetrazolium bromide] assay. Optical density (OD) for MTT assay was measured with dual wavelengths. The chemosensitivity of the drugs was evaluated by the 50% OD (OD50) of each drug concentration in the control group. Five platinum (Pt) drugs and 3 anthracycline (AC) drugs were used in this study. The chemosensitivity differed among the 5 Pt drugs. No significant difference was observed among the 3 AC drugs. A linear increase in OD corresponding to an increase in number of cells was observed. When 0.1 M sodium succinate (S.S.) was added to 0.4% MTT, the sensitivity increased five-fold compared to the control group without S.S. The MTT assay is a precise, rapid, easy and inexpensive experimental system useful for evaluation of antitumor drug sensitivity on tumor cell lines.

Antibiotics, Antineoplastic↗

PubMed2000

Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes.

Methylation has been considered to be the primary detoxication pathway of inorganic arsenic. Inorganic arsenic is methylated by many, but not all animal species, to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), and dimethylarsinic acid (DMA(V)). The As(V) derivatives have been assumed to produce low toxicity, but the relative toxicity of MMA(III) remains unknown. In vitro toxicities of arsenate, arsenite, MMA(V), MMA(III), and DMA(V) were determined in Chang human hepatocytes. Leakage of lactate dehydrogenase (LDH) and intracellular potassium (K(+)) and mitochondrial metabolism of the tetrazolium salt XTT were used to assess cytotoxicity due to arsenic exposure. The mean LC50 based on LDH assays in phosphate media was 6 microM for MMA(III) and 68 microM for arsenite. Using the assay for K(+) leakage in phosphate media, the mean LC50 was 6.3 microM for MMA(III) and 19.8 microM for arsenite. The mean LC50 based on the XTT assay in phosphate media was 13.6 microM for MMA(III) and 164 microM for arsenite. The results of the three cytotoxicity assays (LDH, K(+), and XTT) reveal the following order of toxicity in Chang human hepatocytes: MMA(III) > arsenite > arsenate > MMA(V) = DMA(V). Data demonstrate that MMA(III), an intermediate in inorganic arsenic methylation, is highly toxic and again raises the question as to whether methylation of inorganic arsenic is a detoxication process.

Arsenates↗

PubMed1990

Repeated, in vivo observation of frog neuromuscular junctions: remodelling involves concurrent growth and retraction.

The fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide was used as a vital stain to study remodelling of motor nerve terminals in sartorius muscles of living frogs (Rana pipiens). Identified terminals were observed twice in vivo at intervals of 87-192 days. After the second observation, muscles were fixed and stained with the nitroblue tetrazolium method for nerve terminals and with cholinesterase stain. Observations were made of 243 junctions in 26 frogs. Most nerve terminals grew during the observation interval, with an average increase in total terminal length of 29%. This growth involved substantial remodelling. Within single junctions, the change in size was the net result of differing degrees of growth or shrinkage in individual nerve terminal branches. At least one new terminal branch appeared in 25% of the junctions. Terminal retraction was also common, with branch shortening seen in 60% of junctions and the complete disappearance of a branch in 12%. In one case the original axonal input retracted completely and the junction was partially reinnervated by a terminal sprout from a junction on an adjacent fibre. Some discrepancies between histological and in vivo observations of remodelling were noted. These observations confirm that frog neuromuscular junctions are highly dynamic synapses, subject to profound structural remodelling throughout adult life.

Animals↗

PubMed2006

Varying the unsaturation in N4,N9-dioctadecanoyl spermines: nonviral lipopolyamine vectors for more efficient plasmid DNA formulation.

PURPOSE: The aim of the study is to analyze the effect of varying the degree of unsaturation in synthesized N4,N9-dioctadecanoyl spermines on DNA condensation and then to compare their transfection efficiency in cell culture. METHODS: The N4,N9-di-C18 lipopolyamines-saturated (stearoyl), C9-cis- (oleoyl), and C9,12-di-cis- (linoleoyl)-were synthesized from the naturally occurring polyamine spermine. The ability of these novel compounds to condense DNA and form nanoparticles was studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in several primary skin cells (FEK4, FCP4, FCP5, FCP7, and FCP8) and in an immortalized cancer cell line (HtTA) and was compared with the commercially available nonliposomal transfection formulation Transfectam (dioctadecylamidoglycyl spermine), which also contains two saturated C18 lipid chains. RESULTS: N4,N9-Dilinoleoyl spermine (C18, di-cis-9,12) is efficient at circular plasmid DNA (pEGFP) condensation and gives the most effective transfection in a series of primary skin cells and cancer cell lines at low charge ratios of 5.5 (+/-ammonium/phosphate). CONCLUSIONS: The dienoic fatty acyl spermine conjugate N4,N9-dilinoleoyl spermine efficiently condenses DNA and achieves the highest transfection levels among the studied lipopolyamines in cultured cells.

Cell Line↗

PubMed1999

Scavenging effect of benzophenones on the oxidative stress of skeletal muscle cells.

Benzophenone is an ultraviolet (UV)-absorbing agent that has been used in industry and medicine for more than 30 years. Consumers of cosmetics and sunscreens containing UV-absorbers are exposed to benzophenones on a daily basis, owing to the widespread use of these compounds. However, the efficacy of these compounds as scavengers of oxidative stress is still not well established. In the present study, we investigate the antioxidative capacity of six sunscreen benzophenone compounds. A primary myoblast culture was mixed in vitro with 100 microM menadione. The cytotoxic effect by menadione-induced oxidative stress was monitored by the lucigenin- or luminol-amplified chemiluminescence, methylthiotetrazole (MTT) assay, and the antioxidative effects of various benzophenone compounds were evaluated. The results showed that the addition of menadione can induce oxidative stress on myoblasts by superoxide and hydrogen peroxide production, which can be eradicated by superoxide dismutase (SOD) and catalase, respectively, in a dose-dependent mode. The catalase has a protective effect on the cytotoxicity induced by menadione as measured by the MTT assay, while the SOD does not. The selected benzophenones also have a significant scavenging effect on the menadione-induced cell death on the myoblasts. The ortho-dihydroxyl structure and other hydroxy groups in the same ring have a stronger scavenging effect on the superoxide anion on myoblasts; thus, a stable penoxy radical may be formed. The mechanism of this effect remains to be clarified.

Acridines↗

PubMed2002

Which dosing scheme is suitable for the taxanes? An in vitro model.

The discovery and development of the taxane class of antitumor compounds represent significant advances in the treatment of patients with a variety of malignancies. These drugs are effectively used in the treatment of breast cancer. In this study we evaluated the efficacy of fractionated usage of both paclitaxel and docetaxel as a single agent in the breast cancer cell line MCF-7. It has been shown that the cytotoxic effect of paclitaxel was increased when the divided IC50 concentrations were used sequentially and in contrast to paclitaxel, cytotoxic effect of docetaxel was decreased with the same schema and the single dose of IC50 concentration was optimal. The cause of the difference between the cytotoxic effects of two agents with this schedule is obscure. Demonstrating mechanisms, which are responsible for these differences, will be important for more rational use of taxoids and to provide basis for the following clinical trials.

Antineoplastic Agents, Phytogenic↗

PubMed1997

[Compared study on Fura-2/AM assay and MTT assay for screening multidrug resistant modulators].

To explore the difference of screening results of reversing multidrug resistance (MDR) by modulators between Fura-2/AM assay and MTT assay, 25 compounds which have active structure were studied for MDR reversal activity with both methods. The fold of MDR reversal was shown to have remarkable relation with the amount of Fura-2 accumulation (Y = -3.66 + 17.5X, gamma = 0.86, P < 0.01). On the other hand, Fura-2/AM assay has several advantages as compared with MTT assay. Fura-2/AM assay needs shorter time (4 h) than MTT assay (96 h), and the MTT assay needs more steps than the Fura-2/AM assay. Furthermore, Fura-2/AM assay was more reliable than MTT assay for screening MDR modulators because MTT assay was dependent on the viable cells, while Fura-2/AM assay was dependent on the function of P-gp. The results suggest that Fura-2/AM assay may replace MTT assay in the screening of MDR modulators on a large scale.

Bepridil↗

PubMed1981

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase.

This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitates outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performed in situ.

Acid Phosphatase↗