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PubMed1994

Heavy metal-induced cytotoxicity to cultured human epidermal keratinocytes and effects of antioxidants.

Human epidermal keratinocytes which have been cultured were treated with the heavy metal ions of cadmium, mercury, copper and zinc. Cytotoxicity was measured either by protein estimation or by using the neutral red assay. Antioxidants were added in order to find out whether heavy metal-induced cytotoxicity is related to oxidative stress. All metals used showed considerable cytotoxic effects within 24 h in moderate concentrations. None of the antioxidants vitamin E (alpha-tocopherol), pyrogallol, propyl gallate, BHT or ebselen showed any protective or preventive effect. This indicates that oxidative stress may not be involved in the cytotoxicity induced by heavy metals in human epidermal keratinocytes. The cells used are, however, a valuable tool to study mechanisms of cytotoxicity.

Antioxidants↗
PubMed1993

Use of human renal proximal tubule cell cultures for studying foscarnet-induced nephrotoxicity in vitro.

Foscarnet is an antiviral agent used for the treatment of cytomegalovirus retinitis and acyclovir-resistant herpes simplex virus infections in AIDS patients. Renal impairment has been reported for many patients treated with foscarnet. We have studied the effects of foscarnet on the viability (estimated by neutral red inclusion) and ultrastructure of cultures of human renal proximal tubule cells (HRPTC) isolated from the kidneys of five cadavers and cultured. The degree of foscarnet-induced toxicity was dose dependent and varied among the HRPTC cultures. The data obtained by using the in vitro system of HRPTC mimic the data of the clinical trials in that there is a dose-dependent individual variation among human cases in response to foscarnet treatment. Thus, these cultures are extremely well-suited to investigations of the mechanism of toxicity at the subcellular level.

Adolescent↗
PubMed2006

The FRAME alternatives laboratory database. 1. In vitro basal cytotoxicity determined by the Kenacid blue total protein assay.

A database of over 280 chemicals has been compiled by using a mouse 3T3-L1 fibroblast-like cell line in exponential growth, exposed to chemicals for 72 hours in a 96-well tissue culture plate format, and determining cell number via the Kenacid blue (KB) assay for total protein. Ranking the chemicals according to their basal cytotoxicity, expressed as the concentration (mM) that inhibits increase in total cellular protein over 72 hours by 50% (the ID50 value) shows a wide range of ID50 values, from 0.00003 mM to 10,096 mM. This information includes the results for MEIC chemicals 1-50, and we have now added basal cytotoxicity data for 23 of the next 25 MEIC chemicals. When the neutral red uptake (NRU) assay was performed with the same cell cultures, before the KB assay, very similar indications of basal cytotoxicity were obtained. Comparisons between the results with 3T3-L1 cells and with a human fibroblast-like cell line, BCL-D1 showed a significant difference in order of magnitude of the ID50 value for only 5 of 52 chemicals. However, there was a difference in ID50 value of more than one order of magnitude for 8 of 24 chemicals tested with an undifferentiated teratocarcinoma cell line, F9.

3T3 Cells↗
PubMed2000

Population growth responses of Tetrahymena shanghaiensis in exposure to rare earth elements.

This article presents the population growth responses of Tetrahymena shanghaiensis s1 in exposure to rare earth elements (REEs). Both the light REEs (La, Sm) and the heavy REEs (Y, Gd) were investigated with 24- and 96-hr population growth assays to evaluate their aquatic toxicity. Four end points, cell count, frequency of neutral red (NR) uptake, total protein, and nucleic acid content were employed in the 24-h assay, and a population growth curve was plotted in the 96-h assay. The results of 24- and 96-h assays suggest a dual effect of REEs on T. shanghaiensis of the stimulated growth at low concentrations and the toxicity at higher ones. In the promoted growth of T. shanghaiensis, however, the cell density increased with an increased growth rate and the protein and nucleic acid content per 10(4) cells undertook no remarkable changes, which suggests possible cell growth control by REE.

Algorithms↗
PubMed1985

Ca2+ buffer sites in intact bovine rod outer segments: introduction to a novel optical probe to measure ionic permeabilities in suspensions of small particles.

The nature of the Ca2+ buffer sites in intact rod outer segments isolated from bovine retinas (ROS) was investigated. The predominant Ca2+ buffer in intact ROS was found to be negatively charged groups confined to the surface of the disk membranes. Accordingly, Ca2+ buffering in ROS was strongly influenced by the electrostatic surface potential. The concentration of Ca2+ buffer sites was about 30 mM, 80% of which were located at the membrane surface in the intradiskal space. A comparison with observations in model systems suggests that phosphatidylserine is the major Ca2+ buffer site in ROS. Protons and alkali cations could replace Ca2+ as mobile counterions for the fixed negatively charged groups. At physiological ionic strength, the total number of these diffusible, but osmotically inactive, counterions was as large as the number of osmotically active cations in ROS. The surface potential is dependent on the concentration of cations in ROS and can be measured with the optical dye neutral red. Addition of cations to the external solution led to the release of the internally bound dye as the cations crossed the outer membrane. The chemical and spectral properties of the dye enable its use as a real-time indicator of cation transport across the outer envelope of small particles in suspension. In this study, the dye method is illustrated by the use of well-defined ionophores in intact ROS and in liposomes. In the companion paper this method is used to describe the cation permeabilities native to ROS.

Animals↗
PubMed1992

Characterization of HeLa cell vacuoles induced by Helicobacter pylori broth culture supernatant.

Helicobacter pylori broth culture supernatants induce eukaryotic cell vacuolation in vitro, a phenomenon that has been attributed to cytotoxic activity. We sought to characterize further the vacuolation of HeLa cells that occurs in response to H pylori culture supernatant. Nascent vacuoles were detectable by electron microscopy after 90 minutes of incubation with H pylori supernatant and were not associated with any identifiable organelle. After 6 days of incubation with H pylori supernatant, vacuoles were membrane-bound structures filled with electron-dense debris, which resembled secondary lysosomes. Acid phosphatase activity was detected within the vacuoles. The vacuoles induced by H pylori supernatant were then compared with vacuoles induced by trimethylamine, a weak base known to induce lysosomal swelling. Neutral red dye rapidly entered the vacuoles induced by either H pylori supernatant or trimethylamine, and both types of vacuoles were reversible. Compared with trimethylamine-induced vacuoles, the vacuoles induced by H pylori supernatant were larger and typically lacked a limiting membrane. In the early stages of formation, vacuoles induced by trimethylamine were labeled by lucifer yellow, a pinocytotic marker, whereas H pylori cytotoxin-induced vacuoles were not. These data suggest that trimethylamine-induced vacuoles arise directly from endocytic compartments, whereas H pylori cytotoxin induces vacuole formation via an autophagic mechanism.

Acid Phosphatase↗
PubMed2006

Effects of 3-methoxy-2(5H)-furanone-containing extracts from Narthecium ossifragum (L.) Huds. on renal tubular cells in vitro.

Narthecium ossifragum, a perennial herb of the lily family, causes toxic renal tubular necrosis in several ruminant species. 3-Methoxy-2(5H)-furanone (3M2F) has been identified as a nephrotoxin present in N. ossifragum extracts. We studied effects of three different 3M2F-containing fractions isolated from N. ossifragum and synthetic 3M2F on the porcine kidney cell line LLC-PK1. In some of the experiments, we included the glioma cell lines U251 and BT4Cn to compare the effects of the toxin on LLC-PK1 cells to the effect on these cell lines. The synthetic 3M2F was shown to be only mildly toxic, and the most purified fraction from N. ossifragum showed the highest degree of toxicity in our studies. When monolayer cultures were exposed to increasing amounts of 3M2F-containing extract, a dose-dependent increase in cell death was observed. Similarly, reduced neutral red uptake and 3H-thymidine uptake (DNA synthesis) was observed. There was increased apoptotic activity in the LLC-PK1 cells with increasing concentration of 3M2F-containing extract. Multicellular three-dimensional spheroids from LLC-PK1 cells stopped fluid transport, showed degenerative changes and collapsed totally 6 h after extract exposure. Our findings indicate junctional damage, reduced cellular endocytosis and DNA-synthesis as well as induction of apoptosis as possible mechanisms for the acute tubular necrosis observed in ruminant species.

Animals↗
PubMed1998

Cell vacuolization induced by Helicobacter pylori VacA toxin: cell line sensitivity and quantitative estimation.

A major virulence factor released by Helicobacter pylori is a protein toxin, termed VacA, which induces the formation of large intracellular vacuoles characterised by a lumenal acidic pH. Consequently they accumulate membrane permeable weak bases. The increase in neutral red uptake by intoxicated cells is the only known in vitro procedure to estimate quantitatively the activity of VacA. With the goal to standardize this assay, several parameters were evaluated: cell type, serum concentration, cell density and toxin concentration. Among the different cell types tested, HeLa cells were found to be the most sensitive to VacA. Results show that several factors contribute to VacA activity and that optimal vacuolation is achieved at non-confluent cell density, in the presence of low serum concentrations.

Bacterial Proteins↗
PubMed2005

Vestibular afferent innervation in the vestibular efferent nucleus of rats.

To delineate the vestibular afferent innervation in the vestibular efferent nucleus in the brainstem, neurobiotin or biotinylated dextran amine was injected into the superior Scarpa's ganglion of Sprague-Dawley rats. The locations of vestibular efferent neurons in the brainstem were identified by neutral red or choline acetyltransferase staining. Of the three pairs of vestibular efferent nuclei, labeled fibers and bouton-like endings were found only within the dorsolateral vestibular efferent nucleus on the ipsilateral side. Labeled afferent terminals with bouton-like varicosities were observed in the vicinity of cell bodies or dendrites of these efferent neurons. Our findings suggest that vestibular primary afferents may exert direct influence on vestibular efferent neurons, constituting an ipsilateral close-loop arrangement in the central vestibular system.

Animals↗
PubMed1983

A colorimetric assay for the measurement of the sensitivity of herpes simplex viruses to antiviral agents.

A quantitative colorimetric method for measuring the inhibition of viral cytopathic effects has been adapted to the assay of antiviral compounds. Drug-treated, virus-infected cultures in microtiter plates were stained with the vital dye neutral red and the amount of dye incorporated was determined in a multichannel spectrophotometer. The technique required smaller volumes of reagents, was more easily automated than the standard plaque reduction assay and had good reproducibility. Standard conditions of 30 infectious units of challenge virus and 72-h incubation were judged to be optimal. Median inhibitory concentrations (ID50) for a number of compounds were approximately tenfold higher in the dye-uptake assay compared with the plaque reduction assay, possibly related to the higher multiplicity of infection required to give the desired level of cytopathic effect in the microtiter method.

Antiviral Agents↗
PubMed2002

On the interaction of polyacrylic acid as a conditioning agent with bovine enamel.

In the present paper, the effects of polyacrylic acid (PAA) conditioning on the morphology and chemistry of bovine enamel surface and the resulting interfacial reactions are being investigated using photometric, microscopic (SEM, AFM), electron spectroscopic (XPS) and staining methods (neutral red dye). The results are compared to two reference surfaces obtained by simple grinding and by etching with a phosphoric acid solution. It is shown that PAA conditioning leads to the leaching of calcium and phosphorus ions, to the smoothening of the surface and probably to the formation of a polymeric film at the surface. A mechanism by which a preliminary PAA conditioning of the enamel leads to the reported higher bonding strength between enamel and glass ionomer cements is proposed.

Acid Etching, Dental↗
PubMed2003

Thalictrum minus cell cultures and ABC-like transporter.

Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

An electrogenic proton pump associated with the Golgi apparatus of mouse liver driven by NADH and ATP.

Golgi-apparatus membranes, isolated from mouse liver, pump protons inwards, when supplied with NADH or ATP. The acidification of Golgi-apparatus cisternae and vesicles was detected with neutral red, a permeant dye, as a difference in absorbance at 550 nm minus that at 600 nm. The maximum rates detected with NADH and ATP were between 0.0006-0.0009 and 0.0030-0.0050 delta OD units/mg of protein/min, respectively, at pH 7.5. The outside buffer used was a bovine serum albumin suspension. The acidification of Golgi apparatus was inhibited from 45 to 100% by ionophores and from 22 to 100% by uncouplers. The results implicate both ATP and a redox system coupled to NADH oxidation in the acidification of Golgi-apparatus membranes.

Adenosine Triphosphate↗
PubMed1984

Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone.

The spectrophotometric indicators neutral red and safranine were used to determine the relative H+/e- ratios of proton uptake from the mitochondrial matrix, and the q+/e- ratios of electrical charge translocation, during oxidation of beta-OH-butyrate and succinate by ferricyanide in rat liver mitochondria. With beta-OH-butyrate both ratios were higher than with succinate by a factor close to 3.0. Since there is full agreement that H+/e- of proton uptake and q+/e- of charge translocation are both equal to unity for oxidation of succinate (or ubiquinol) by ferricytochrome c, the corresponding ratios for oxidation of NADH by ubiquinone and cytochrome c are near 2.0 and 3.0, respectively.

Animals↗
PubMed2001

Cytotoxicity of derivatives from dehydrocrotonin on V79 cells and Escherichia coli.

New derivatives from dehydrocrotonin (DHC, compound I), with the same anti-ulcerogenic properties but less toxicity were synthesised by reducing the cyclohexenone moiety of DHC with NaBH(4) (compound II), by reducing the cyclohexenone and lactone moieties with LiAlH(4) (compound III) and by transforming the lactone moiety into an amide (compound IV) using dimethylamine. The cytotoxicity of these derivatives from DHC was assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), tetrazolium reduction (MTT) and neutral red uptake (NRU). IC(50) values of 540 and 350 microM were obtained for compound II in the NRU and NAC tests, respectively. Compound III was less toxic than the other DHC derivatives (IC(50)=1800 microM) on V79 cells based on NAC assay. Compound IV showed an IC(50) ranging from 350 to 600 microM based on the three endpoints evaluated. The three compounds were less toxic on V79 cells than DHC. DHC, compounds II, III and IV did not change the respiration rate of Escherichia coli on the acute toxicity assay.

Animals↗
PubMed1981

Synthesis and properties of acrylamide-substituted base pair specific dyes for deoxyribonucleic acid template mediated synthesis of dye polymers.

We have tried to construct synthetic polymers for sequence-specific recognition and complexation of longer deoxynucleotide sequences. For this purpose, we developed a method of template-directed polymerization of basic pair specific DNA ligands such as basic dyes. The template-directed polymerization consists in a copolymerization of various dyes of different specificities staying simultaneously in a binding equilibrium with DNA. In the present paper, we describe the synthesis and the properties of base pair specific monomers especially designed for performance of radical chain polymerization reactions in aqueous medium at room temperature. Different acrylamide derivatives of well-known dyes, such as AT-specific malachite green and GC-specific phenyl neutral red, were synthesized and studied for their ability of base pair specific complex formation with DNA of different base composition. Partition equilibrium dialysis and dye titration agarose gel electrophoresis were used to ensure for several dyes that they may be incorporated into different polymers via copolymerization of their acrylamido derivatives with various small base-unspecific monomers without substantial change of their binding parameters. Furthermore, we demonstrate that acrylamide and other small acrylamide derivatives can be used as building blocks for the synthesis of polymeric links between base pair specific monomers. The results and their consequences for template polymerization reactions are discussed.

Acrylamides↗
PubMed1998

Propylene glycol-mediated cell injury in a primary culture of human proximal tubule cells.

Propylene glycol (propane-1,2-diol; PD) is a widely used solvent for intravenous drugs. Clinical studies have reported serious side effects, including the development of renal insufficiency in patients receiving PD as drug vehicle. Despite such clinical reports, the data on the toxicity of PD in isolated renal cells are limited. Using primary cultured human proximal tubule (HPT) cells as an in vitro model, we have previously shown the acute toxic effects of PD in HPT cells (Morshed et al., Fundam. Appl. Toxicol. 23, 38-43, 1994). Since most cases of clinical toxicity are noted after prolonged administration of PD, the current studies were designed to investigate the toxicity of repeated exposure of PD in HPT cells. The onset of toxicity was determined using 10-50 mM racemic, sinister, and rectus PD (rac-, S-, and R-PD, respectively) for periods up to 6 days. Cytotoxicity was noted by decreases in thymidine incorporation, in mitochondrial metabolic activity, and in lysosomal accumulation of neutral red. Exposure of HPT cells to 50 mM PD produced toxic responses, while at 10 mM, responses were not significantly greater than those of osmotic controls. The toxicity was caused by a PD-specific mechanism and by a secondary mechanism without any enantiomeric specificity. The HPT cell toxicity was associated with a 35% increase in cellular thiobarbituric acid-reactive substances and a 20% decrease in glutathione. These findings suggest the development of a mild, subacute toxicity in normally proliferating HPT cells at concentrations that could be achieved in human plasma when PD is used as a drug vehicle.

Cell Division↗
PubMed2003

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.

Bacterial Proteins↗