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PubMed1987

Fathead minnow FHM cells for use in in vitro cytotoxicity assays of aquatic pollutants.

The suitability of the fathead minnow (FHM) epithelial cell line for use as the target (indicator) system in in vitro cytotoxicity assays was evaluated using several endpoints. The organometal diethyltin dichloride served as the representative test agent. The concentration of diethyltin dichloride which resulted in a midpoint toxicity was 3.5 microM in a 3-day cell growth assay, 3.8 microM in the 24-hr neutral red assay, and 16.5 microM in a 4-hr cell detachment assay. The neutral red assay was used to compare the relative sensitivities of the FHM cells (exposed at 34 degrees C) with those of bluegill sunfish (BF-2) cells, a fibroblastic cell culture (exposed at 26 degrees C), in the presence of different classes of test agents frequently occurring as aquatic pollutants. For both fish species the sequence of potencies of the test agents was in the order of organometals greater than pesticides approximately equal to polychlorinated biphenyls greater than polynuclear aromatic hydrocarbons greater than phenolics. Overall, the FHM cells were more sensitive than were the BF-2 cells. However, there was a better correlation between the in vitro cytotoxicity data for the BF-2 cell culture and LC50 data for bluegill sunfish than between similar data for the FHM cell line and fathead minnows.

Animals↗
PubMed2006

Effects of pyrene on mussels in different experimental conditions.

The toxicity of the polyaromatic hydrocarbon pyrene to Mytilus edulis and Mytilus galloprovincialis was investigated in experiments conducted in the United Kingdom for M. edulis and in Turkey for M. galloprovincialis. Experimental conditions of temperature and salinity were chosen to be appropriate to the ambient conditions in which the mussels typically live. The effect of different feeding regimes on pyrene bioaccumulation and toxicity was also investigated. Feeding rate and neutral red retention biomarker techniques were used for toxicity assessment. An experiment with M. edulis demonstrated that mussels exposed to pyrene accumulated increasing amounts of this compound throughout a 15-day exposure period and that accumulation increased in relation to exposure concentration and with increasing concentration of unicellular algal food material. However, in these experiments, which were protected from UV light, there was no clear relationship between pyrene concentration in tissues and feeding rate. A clear concentration-response relationship was observed between tissue concentration and neutral red retention for days 1 and 7 of the experiment, but this relationship was lost by day 15, with evidence of the cells recovering. A similar experiment was conducted with M. galloprovincialis at a single (high) pyrene concentration in darkness. The feeding rate of the exposed mussels was always lower than the feeding rate of the control mussels, although the difference was insignificant at 7-day exposure. A decreasing trend in health status of the mussels was indicated by the neutral red retention assay results after 7 and 15 days of exposure to pyrene. In a third experiment (with M. galloprovincialis), an illuminated algal chemostat system containing pyrene was inserted in the exposure system as the food source for the mussels. Both biomarker results showed conclusively that toxic effects correlated with pyrene bioaccumulation, though there was no clear evidence for light-enhanced toxicity. These experiments showed that experimental conditions such as salinity, temperature and feeding regime and perhaps the species of mussel used may influence whether pyrene is perceived as being a toxic molecule.

Animals↗
PubMed1989

Manipulating the intracellular environment of hippocampal slices: pH and high-energy phosphates.

The intracellular energetic environment of rat hippocampal slices was manipulated by bolstering ATP levels following the addition of adenosine to the incubation medium, or by manipulating intracellular pH. Addition of 8 mM adenosine to the incubation medium increased total tissue adenylate and ATP content, but did not prolong electrical function during anoxia. Further, it resulted in long-lasting alterations in normoxic evoked responses. Intracellular pH (pHi) was changed by manipulating the bicarbonate/CO2 ratio of the incubation medium, or by adding amiloride, a hydrogen/sodium antiport blocker. Estimates of intracellular pH using the creatine kinase equilibrium agree with those obtained by Neutral red scanning spectrophotometry in control conditions. However, only Neutral red indicated an acidification with amiloride treatment, while the creatine kinase equilibrium was preferentially affected by hypercapnia, suggesting the presence of at least two pH compartments in hippocampal brain slices. These manipulations cannot be carried out easily in vivo, and provide a means of determining the importance of metabolic changes on neural function during anoxia.

Action Potentials↗
PubMed1979

Cell differentiation during fruiting body formation in polysphondylium pallidum.

The spatial pattern of cellular differentiation was studied during fruiting body formation in Polysphondylium pallidum using 3 different staining methods: Calcofluor fluorescence (cellulose accumulation), neutral red (prestalk cells) and immunofluorescence (prespore cells). Neutral-red staining revealed the existence of a clear prestalk region which becomes evident during aggregation and continues throughout culmination. Immunofluorescent staining demonstrated that cells in the prestalk region gradually lose their presporeness (fluorescence) as they are transformed into differentiated stalk cells. Calcofluor staining revealed that stalk cell differentiation begins during the mid-aggregation phase and that the mode of formation of the main stalk and the side branches differs slightly in morphology. Calcofluor staining also demonstrated the development, during aggregation, of a thick cellulosic girdle with lateral tubular extensions which surround the aggregation streams. The above results are discussed in terms of our present knowledge about differentiation and morphogenesis in cellular slime moulds.

Dictyostelium↗
PubMed1984

A colorimetric assay for the assessment of cytotoxicity of yeasts.

A colorimetric assay for the quantitation of microbial cytotoxicity has been developed using cells from a monocyte-like human cell line (U937), epithelial cells (Hela), and fibroblast-like cells (Vero) as targets. The fraction of surviving cells was determined by their content of the dye neutral red which is retained only by live cells and can be quantitated photometrically after controlled lysis. The neutral red retention assay was at least as sensitive as the 51Cr-release assay; it was considerably less laborious, faster, and avoided handling of radioactivity. Among the different Candida species tested, the highest cytotoxicity was associated with C. albicans and C. tropicalis; a lower degree of cytotoxicity was exhibited by C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. Among the strains of a given fungal species cytotoxicity varied by up to 40%.

Animals↗
PubMed2001

Role of flavonoids in controlling the phototoxicity of Hypericum perforatum extracts.

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.

Cell Line↗
PubMed1975

Macrophages in bovine milk.

The high vacuolated mononuclear cells in milk from normal bovine mammary glands, usually designated epithelial cells, were examined for properties characteristic of macrophages. In vitro, these cells concentrated neutral red into cytoplasmic vacuoles, adhered rapidly to and spread on glass, and phagocytosed viable Streptococcus agalactiae and Staphylococcus aureus. Neutral red uptake was demonstrated in vivo, and the vacuolated mononuclear cells phagocytosed formalinkilled S aureus in vivo. These results suggest that these cells are macrophages.

Animals↗
PubMed2003

Bioelectrocatalysts: engineered oxidoreductase system for utilization of fumarate reductase in chemical synthesis, detection, and fuel cells.

Fumarate reductase was used as a model oxidoreductase to demonstrate continuous electrical cofactor reduction-oxidation during the bioelectrochemical synthesis and detection of chemicals. The enzyme preparation was immobilized onto a graphite felt electrode that was modified with carboxymethylcellulose (CMC). Nicotinamide adenine dinucleotide (NAD), neutral red, and fumarate reductase (which contained menaquinone) were covalently linked by peptide bonds to the CMC. The electron mediator neutral red allowed NAD and menaquinone to be recycled electrically during enzymatic chemical synthesis. Succinate detection by the bioelectrocatalyst was linear from 5 microM to 10 mM succinate. Fumarate synthesis using this bioelectrode was dependent on succinate utilization and resulted in proportional production of electricity and fumarate. Succinate synthesis using this bioelectrocatalyst was dependent on current and fumarate concentration. This bioelectrocatalyst system may enhance the utility of menaquinone- and/or pyridine nucleotide-linked oxidoreductases in diverse enzymatic fuel cells and sensors. It may also enhance the utility of oxidoreductase-based chemical synthesis systems because it eliminates the problem of cofactor recycling.

Actinobacillus↗
PubMed1994

Discrimination of the irritancy potential of surfactants in vitro by two cytotoxicity assays using normal human keratinocytes, HaCaT cells and 3T3 mouse fibroblasts: correlation with in vivo data from a soap chamber assay.

Cell cultures have been proposed as a promising model for local tolerance testing. This study evaluated the cytotoxic effects of surfactants on early passage normal human keratinocytes, transformed human keratinocytes (HaCaT cells) and Swiss 3T3 embryonic mouse fibroblasts. Cell membrane integrity, as assessed by the release of the vital dye neutral red, and cell proliferation, as assessed by measurement of the total protein content, were both affected in a dose-dependent manner in response to surfactant exposure. There was a close correlation between the dose-response characteristics for the three cell types. Two surfactants exhibited differential effects on membrane integrity and proliferation, and thus no significant correlation was found between the two endpoints. The irritation potential of the surfactants to human forearm skin in vivo was assessed in a soap chamber test using transepidermal water loss and skin redness as quantitative endpoints. A comparison between the responses in vivo and in vitro yielded the highest correlation for the neutral red release test on normal keratinocytes. The total protein test did not significantly correlate with the soap chamber assay for keratinocytes and HaCaT cells. These results suggest that cultured normal human keratinocytes may be predictive for the irritancy of various surfactants in man. Definite judgement, however, has still to be based on confirmation in human volunteers of larger groups of compounds with diverse physico-chemical properties.

3T3 Cells↗
PubMed2006

[Comparison the screening methods of the acute toxicity in vitro].

OBJECTIVE: To provide the proof for establishing the high throughput screening method of the acute toxicity by comparing the 4 screening methods of the acute toxicity in vitro. METHODS: HepG2 cells, plated in 96-well plates, were treated with 47 compounds and we detected the cytotoxicity using 4 methods and calculated the IC50, IC45, IC40, IC35, IC30, and these values were compared with the LD50. RESULTS: The IC45 from the neutral red method has the best relativity with the LD50. CONCLUSION: Plating HepG2 cells in 96-well plates, the cytotoxicity be detected using the neutral red method and the IC45 was calculated. It is considered to provide the proof for establishing the high throughput screening method of the acute toxicity in vitro.

Cell Survival↗
PubMed1997

Evaluation of the genotoxic and cytotoxic potential of mainstream whole smoke and smoke condensate from a cigarette containing a novel carbon filter.

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

Animals↗
PubMedDate not supplied

Evidence for delayed cytotoxicity effects following exposure of rat hepatoma-derived Fa32 cells: implications for predicting human acute toxicity.

The delayed cytotoxicity of the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The cells were treated for 24 h with the test chemicals, and were than further cultured for 5 days in normal culture medium. The cytotoxicity was measured by the neutral red uptake inhibition, and the results were quantified by determining the NI50del. This is the concentration of test compound required to decrease the neutral red uptake with 50% compared with control cells. The results were compared with the acute NI50, the corresponding value measured immediately after 24 h treatment of the cells. On a total of 44 chemicals, nine showed delayed cytotoxicity (NI50del lower than or equal to NI50), 11 a probably delayed, and 24 no delayed cytotoxicity (NI50del more than 1.5 x NI50). When the NI50del was compared with human toxicity, a correlation coefficient r2=0.761 was obtained. For the same series of 44 chemicals this correlation was clearly higher than that for human hepatoma-derived Hep G2 cells (r2=0.695). The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the delayed cytotoxicity assay on cultured Fa32 cells has the best prediction value so far obtained for the human toxicity.

Animals↗
PubMed1980

Purification of adrenal medullary chromaffin cells by density gradient centrifugation.

A method has been developed for the isolation of chromaffin cells from guinea pig adrenal glands. Crude suspensions of adrenal cells are prepared by the digestion of adrenal with collagenase. Chromaffin cells are then purified from these crude suspensions by isopycnic centrifugation through a 5-25% (w/v) gradient of metrizamide. More than 90% of the cells in the preparation are viable chromaffin cells, as judged by trypan blue exclusion and fluorescence histochemistry. A more convenient method for monitoring the purity of chromaffin cell preparations, using neutral red staining, is also described. Chromaffin cells are the primary neutral red staining cell in the adrenal gland, and more than 90% of the purified chromaffin cells stain with this dye. Purified chromaffin cells contain 400 +/- 50 nmol of epinephrine/mg protein (133 +/- 16 nmol/10(6) cells), and secrete epinephrine in response to acetylcholine. At a concentration of 100 microM, acetylcholine causes a 10- to 20-fold increase in the secretion of epinephrine from the cells. The method described in this paper is a useful procedure for the preparation of pure, functional chromaffin cells.

Adrenal Medulla↗
PubMed2004

Conjunctival proinflammatory and proapoptotic effects of latanoprost and preserved and unpreserved timolol: an ex vivo and in vitro study.

PURPOSE: To compare the toxicity of latanoprost and preserved and unpreserved timolol on conjunctival cells. Expression of inflammatory markers and MUC5AC-related mucin production were evaluated by impression cytology in a case-control ex vivo study. The proapoptotic effect of the same drugs was also evaluated in vitro in a conjunctival cell line and compared with that of benzalkonium chloride (BAC). METHODS: Impression cytology (IC) specimens were obtained from a series of normal subjects and from patients with glaucoma treated for at least 1 year with latanoprost eye drops or preserved or unpreserved timolol. All groups were comparable in age and duration of treatment. Expression of HLA-DR, intercellular adhesion molecule (ICAM)-1, and mucin was evaluated in a masked manner by flow cytometry. For the in vitro study, a human conjunctiva-derived cell line was treated with 0.02% BAC-containing latanoprost or timolol, unpreserved timolol, or 0.02% BAC alone for 15 minutes, followed or not by 4 or 24 hours of cell recovery in normal medium. Cell viability and chromatin condensation were evaluated using microplate cold light cytofluorometry with the neutral red and the Hoechst 33342 tests, respectively. The Hoechst-neutral red ratio was defined for the apoptosis assay, and cytoskeleton changes were assessed by confocal microscopy. RESULTS: No difference was found between normal eyes and those receiving unpreserved timolol. Preserved latanoprost and timolol significantly increased the inflammatory marker expression and decreased MUC5AC expression, but to a significantly higher extent in the preserved timolol group compared with latanoprost. In vitro, 0.02% BAC-containing timolol and latanoprost triggered conjunctival cell apoptosis-however, to a significantly lesser extent than did 0.02% BAC alone. Unpreserved timolol did not cause any cell toxicity. CONCLUSIONS: These ex vivo and in vitro studies demonstrate that BAC-containing latanoprost and timolol exhibit higher proinflammatory and proapoptotic effects on conjunctival cells than does unpreserved timolol. Latanoprost caused less toxicity, however, than preserved timolol, and both drugs were less toxic than BAC alone. These results suggest a potential protective effect of the prostaglandin analogue and to a lesser extent of timolol against the toxicity of BAC in conjunctival cells.

Adolescent↗
PubMed1989

Separation of pancreatic islets by fluorescence-activated sorting.

To allow clinical pancreatic islet transplantation, the yield and purity of islets must be improved. Intravital staining of islets with neutral red is a specific, nontoxic technique for labeling islets of various species. Using neutral red-stained rat islets, we investigated the known fluorescence absorbance and emission spectra in comparison with unstained exocrine tissue and have shown that stimulation with light of wavelength between 500 and 560 nm produces detectable emission greater than 610 nm, which is absent from unstained exocrine tissue. The PARTEC cell sorter is an inexpensive alternative to currently available fluorescence-activated cell sorters and has a sorting mechanism based on a piezoelectric valve. We made extensive modifications to this machine to allow passage of particles up to 300 micron diam. Using rat pancreas stained intravitally with neutral red and dispersed by intraductal collagenase technique, we have shown that islets can be accurately identified in a high-speed flow system and sorted to a purity of greater than 90% islet tissue. The islets remain intact and viable as determined by supravital staining and isogeneic transplantation to the kidney capsule site. These studies prove the feasibility of separating intact islets by fluorescence-activated sorting.

Animals↗
PubMed1984

Effect of dichloroflavan (BW683C) on the stability and uncoating of rhinovirus type 1B.

Dichloroflavan was found to stabilize rhinovirus 1B to inactivation by heat at 56 degrees C or by acid at pH 4.6 to 5.0. The effects of dichloroflavan, arildone and sodium dodecyl sulphate on uncoating of virus in M.HeLa cells were compared by examining the production of sub-viral particles separated on sucrose density gradients; the development of photoresistance by neutral red labelled virus. All three compounds significantly reduced but did not completely suppress the production of sub-viral particles. They also affected the uncoating of neutral red labelled virus, although dichloroflavan appeared to delay rather than prevent this process. A variant of rhinovirus 1B, isolated by growth in the presence of dichloroflavan, showed increased resistance to inhibition by arildone and SDS as well as dichloroflavan, suggesting a similar site of action for the three compounds.

Acetates↗
PubMed2004

Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation.

Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(l-arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 1 microM(-1) (hexadecyl trimethyl ammonium bromide) to 565 microM(-1) (12,12-l-arginine). Erythrocyte haemolysis also showed a clear concentration-response relationship, the 50% of haemolysis ranged from 37 microM(-1) (10,10-l-arginine) to 151 microM(-1) (sodium lauryl sulphate). Phototoxicity was performed with 12,12-l-acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential.

Animal Testing Alternatives↗
PubMed2002

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells.

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.

Bacterial Proteins↗