sodium-dodecyl-sulfate and thiazolyl-blue

The aim of this study was to improve the methodological procedure for the evaluation of sea urchin (Lytechinus variegatus) sperm sensitivity in MTT (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide) enzyme reduction assays with the formation of formazan (purple color) in the interior of viable cells. Assays were carried out with the reference toxicants sodium dodecyl sulfate (SDS), copper, zinc, cadmium and ammonium, using a sperm solution previously activated in sea water and a sperm solution prepared in sea water containing 400 μg L

Topics: Ammonium Compounds; Animals; Cell Survival; Colorimetry; Formazans; Lytechinus; Male; Metals, Heavy; Sodium Dodecyl Sulfate; Spermatozoa; Tetrazolium Salts; Thiazoles; Toxicity Tests; Water Pollutants, Chemical

Efforts to fully replace the in vivo Draize skin irritation test, according to the Directive 67/548/ECC or OECD TG 404, were reinforced with the seventh Amendment of the Cosmetic Directive and the REACh regulation. In 2007, the EpiSkin test method was scientifically validated and recognized as the stand alone method to discriminate skin irritants (R38) from non-irritants (no label) according to the definition of the EU risk phrases. An ECVAM performance standards (PS) document was defined to evaluate the accuracy and reliability of other analogous test methods (ECVAM SIVS, May 2007). The present test was designed to determine the reliability and relevance of the Reconstructed Human Epidermis (RHE) model commercialized by SkinEthic. The RHE skin irritation test method consisted to topically apply topically the test substances for 42min followed by a 42h post-incubation. The main selected endpoint was the cell viability (MTT reduction), with a threshold of 50% viability. The RHE test method showed a good intra and inter-laboratory reproducibilities in a multicentric study involving three independent laboratories. The SkinEthic RHE test method showed to be relevant and reliable with a sensitivity of 90% and a specificity of 80% (MTT only) and was not improved by integrating another endpoint such as IL-1alpha. The overall accuracy was 85% resulting in the recognition of the SkinEthic RHE test method, by the ECVAM Scientific Advisory Committee in November 2008, as a stand alone replacement test method for the Draize rabbit in vivo test, as a screen, or as part of a sequential testing strategy in a weight of evidence approach, for classifying non-irritant and irritant test substances, depending on country requirements.

Topics: Animal Testing Alternatives; Animals; Cell Survival; Cells, Cultured; Coloring Agents; Epidermis; Humans; Interleukin-1alpha; Irritants; Keratinocytes; Predictive Value of Tests; Rabbits; Reference Standards; Skin Irritancy Tests; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles; Toxicology

To avoid the need to use animals to test the skin irritancy potential of chemicals and cosmetics, it is important to establish an in vitro method based on the reconstructed human epidermal model. To evaluate skin irritancy efficiently and sensitively, we determined the gene expression induced by a topically-applied mild irritant sodium dodecyl sulfate (SDS) in a reconstructed human epidermal model LabCyte EPI-MODEL (LabCyte) using a DNA microarray carrying genes that were related to inflammation, immunity, stress and housekeeping. The expression and secretion of IL-1alpha in reconstructed human epidermal culture is known to be induced by irritation. We detected the induction of IL-1alpha expression and its secretion into the cell culture medium by treatment with 0.075% SDS for 18 h in LabCyte culture using DNA microarray, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and ELISA. DNA microarray analysis indicated that the expression of 10 of the 205 genes carried on the DNA microarray was significantly induced in a LabCyte culture by 0.05% or 0.075% SDS irritation for 18 h. RT-PCR analysis confirmed that SDS treatment significantly induced the expressions of interleukin-1 receptor antagonist (IL-1RN), FOS-like antigen 1 (FOSL1), heat shock 70 kDa protein 1A (HSPA1) and myeloid differentiation primary response gene (88) (MYD88), as well as the known marker genes for irritation IL-1beta and IL-8 in a LabCyte culture. Our results showed that a DNA microarray is a useful tool for efficiently evaluating mild skin irritation using a reconstructed human epidermal model.

Topics: DNA; Epidermal Cells; Epidermis; Gene Expression; HSP70 Heat-Shock Proteins; Humans; Irritants; Myeloid Differentiation Factor 88; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-fos; Receptors, Interleukin-1; Reverse Transcriptase Polymerase Chain Reaction; RNA; Skin; Sodium Dodecyl Sulfate; Tetrazolium Salts; Thiazoles; Tissue Culture Techniques

Toothpastes are thought to be of benefit to cleaning teeth but may also have the potential for soft tissue damage at least on the cellular level by inclusion of detergents in their formulation. The aim of this study was to observe the in vitro response of oral mucosa like cells to toothpaste detergents.. TERT-1 keratinocytes were exposed to varying concentrations of the detergents Adinol, Sodium Lauryl Sulphate, Tego Betain and Pluronic as well as PBS and culture medium. After 2-min exposure, cells were washed and incubated in fresh medium for 24 h. Cell death was then spectrophotometrically measured using an MTT assay.. Except for Pluronic, cell viability was markedly reduced for all detergents at all increasing concentrations when compared to the positive medium control. Cells treated with Pluronic were stimulated compared to medium alone.. These in vitro data suggest that some detergents may have the potential to cause soft tissue damage in the mouth. Although in vivo, saliva may neutralize such effects. The results for Pluronic suggest a possible oxidative stress response that bears further study.

Topics: Betaine; Cell Death; Cell Line; Cell Survival; Chemistry, Pharmaceutical; Coloring Agents; Detergents; Humans; Keratinocytes; Mouth Mucosa; Poloxamer; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles; Time Factors; Toothpastes

Surfactants represent one of the most common constituents in topical pharmaceutical and cosmetic applications or cleansers. Since adverse skin and ocular reactions can be caused by them, it is important to evaluate damaging effects. Amino acid-based surfactants deserve particular attention because of their low toxicity and environmental friendly properties. New lysine derivative surfactants associated with heavy and light counterions were tested. The ocular irritancy was assessed by hemolysis, and photohemolysis was employed to evaluate their phototoxicity. Cytotoxicity on HaCaT cells was determined by neutral red uptake and MTT assay to predict skin irritation. All lysine derivative surfactants were less hemolytic and thus less eye-irritating than the commercial surfactants used as model irritants. No phototoxic effects were found. All surfactants presented cytotoxic effects as demonstrated by decrease of neutral red uptake and reduction of MTT salt, with clear concentration-effect profiles. However, the rates of cytotoxicity on HaCaT for the new surfactants suggested that they were less cytotoxic and then, less skin-irritating than the reference ones; surfactants with heavy counterions were the less cytotoxic. The anionic surfactants investigated in the present work may constitute a promising class of surfactants given their low irritancy potential for pharmaceutical and cosmetic preparations.

Topics: Betaine; Cell Line; Cell Survival; Cetrimonium; Cetrimonium Compounds; Colorimetry; Erythrocytes; Hemolysis; Humans; Inhibitory Concentration 50; Irritants; Lysine; Molecular Structure; Neutral Red; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles

Lysine derivative surfactants are a class of amino acid-based surfactants synthesized as lecithin analogues that deserve particular attention because of their low toxicity and high biocompatibility. To complete the toxicological profile of these surfactants, IL-1 alpha production (cell-associated and release to the culture medium) was determined as an in vitro method for predicting skin irritation. In addition, an MTT assay was used as a viability marker in keratinocytes NCTC 2544. Keratinocytes are a biologically relevant target for developing in vitro techniques to assess skin irritants: moreover, they are the principal source of the proinflammatory cytokine IL-1 alpha in the epidermis. Lysine derivatives proved to be less potent in stimulating IL-1 alpha synthesis and induced a lower release of this cytokine into the culture medium when compared to the anionic surfactant sodium dodecyl sulfate. Due to their low irritancy potential, lysine-based surfactants may offer promising applications in pharmaceutical and cosmetic preparations.

Topics: Cell Line; Cell Survival; Cytokines; Humans; Interleukin-1alpha; Irritants; Keratinocytes; Lysine; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.

Topics: Amyloid; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Animals; Anions; Benzothiazoles; Biochemistry; Cell Line; Chromatography; Congo Red; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Light; Lysine; Magnetic Resonance Spectroscopy; Mice; Micelles; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Weight; Naphthalenesulfonates; Peptide Fragments; Peptides; Phenylalanine; Prions; Protein Structure, Secondary; Scattering, Radiation; Sodium Dodecyl Sulfate; Spectrophotometry; Static Electricity; Sulfonic Acids; Temperature; Tetrazolium Salts; Thiazoles; Time Factors

Reported parameters of the MTT assay vary widely, and reflect a need to optimise the assay for different cell types. The MTT assay conditions for the human B-lymphocyte-derived cell line WIL2NS were optimised for MTT incubation and formazan development. The optimised MTT assay was validated by examining the effects of the acaride amitraz on WIL2NS. In pH-buffered media in the absence of cells, MTT formed formazan spontaneously, and absorbance was proportional to both the initial concentration of MTT and the time of incubation at 37 degrees C. One milligram per millilitre MTT was toxic to WIL2NS cells, but the accuracy of the standard curve was reduced when only 0.2 mg/ml MTT was used. Twenty percent SDS in 0.2 M HCl was preferable to DMSO as a solvent for formazan. Exposure to 0.035% amitraz resulted in a significant reduction in WIL2NS cell numbers after only 2 h of exposure. It was concluded that 0.035% of amitraz has the potential to adversely affect lymphocytes in the systemic blood system in humans, and that an optimised MTT assay was obtained by incubating WIL2NS cells with 0.45 mg/ml MTT for 17 h, followed by addition of acidified SDS for 1 h.

Topics: Cell Line; Cell Survival; Coloring Agents; Freezing; Humans; Insecticides; Sodium Dodecyl Sulfate; Tetrazolium Salts; Thiazoles; Toluidines; Toxicity Tests

We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119-126)) and VVGGLGG (PrP(121-127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119-126) showed a helical conformational propensity, whereas PrP(121-127) adopted a more random coil structure. In 5% SDS, PrP(119-126) showed more alpha-helical content than in TFE solution, and PrP(121-127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a beta-sheet structure with a significant percentage of helical conformation and beta-turn structure in PrP(119-126) and PrP(121-127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119-126) were insoluble in SDS, and PrP(121-127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119-126) and PrP(121-127) formed stable monolayer's consisting of multimeric assemblages at the air-water interface. Monomeric PrP(119-126) was more toxic to astrocytes than the control Abeta peptide; however, the fibrillar form of PrP(119-126) was less toxic to astrocytes. PrP(121-127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119-126) and PrP(121-127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119-126) and PrP(121-127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119-126) and PrP(121-127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119-126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121-127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide.

Topics: Amyloid; Amyloid beta-Peptides; Animals; Astrocytes; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Disease Models, Animal; Lipids; Membrane Microdomains; Microscopy, Electron, Transmission; Peptide Fragments; Peptides; Prions; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrenes; Rats; Rats, Wistar; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Synaptosomes; Temperature; Tetrazolium Salts; Thiazoles

Amyloid precursor protein (APP) is the source of the neurotoxic amyloid beta (Abeta) peptide associated with Alzheimer's disease. Apolipoprotein A-I (apoA-I), a constituent of high-density lipoprotein complexes, was identified by a yeast two-hybrid system as a strong and specific binding partner of full-length APP (APPfl). This association between apoA-I and APPfl was localized to the extracellular domain of APP (APPextra). Furthermore, the interaction between apoA-I and APPfl was confirmed by coprecipitation using recombinant epitope-tagged APPextra and purified apoA-I. Several functional domains have been identified in APPextra, and we focused on a possible interaction between apoA-1 and the pathologically important Abeta peptide, because APPextra contains the nontransmembrane domain of Abeta. The binding between apoA-I and Abeta was saturable (K(d) = 6 nM), specific, and reversible. APPextra also competed with apoA-I for binding to Abeta. Direct evidence for this interaction was obtained by the formation of an SDS-resistant Abeta-apoA-I complex in polyacrylamide gels. Competitive experiments with apolipoprotein E (isoforms E2 and E4) showed that apoA-I had a higher binding affinity for Abeta. We also found that apoA-I inhibited the beta-sheet formation of Abeta with a mean inhibitory concentration close to that of alpha2-macroglobulin. Finally, we demonstrated that apoA-I attenuated Abeta-induced cytotoxicity. These results suggest apoA-I binds to at least one extracellular domain of APP and has a functional role in controlling Abeta aggregation and toxicity.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Apolipoprotein A-I; Enzyme-Linked Immunosorbent Assay; Extracellular Space; Humans; Mutagenesis, Site-Directed; Oxidation-Reduction; PC12 Cells; Protein Structure, Tertiary; Rats; Sodium Dodecyl Sulfate; Tetrazolium Salts; Thiazoles; Two-Hybrid System Techniques

The target of this research was to determine the cytotoxicity of sodium laurylsulfate on single-layer cultures of human fibroblasts, using two colorimetric methods (neutral red and MTT tests) and the evaluation of the pyruvic acid consumption by the cells. For the determination of the cytotoxicity by colorimetric tests, we have determined the absorbance at 540 nm using a spectrophotometer. Pyruvic acid, present in the culture medium, is the mitochondria's C3 energetic metabolite. So, a measure of the cell's consumption of pyruvic acid was developed. The reaction is as follows: Pyruvic acid + NADH --> Lactic acid + NAD+ and the enzyme employed is the LDH (lactate dehydrogenase). This method can be used to measure cytotoxicity, proliferation, and the cell's activation. The method is rapid, precise, and lacks any toxic byproduct. The absorbance was measured using a spectrophotometer at 340 nm. The consumption of pyruvic acid follows upon the fibroblast's growth. Sodium laurylsulfate cytotoxicity test after 24 h shows that the NR colorimetric test and the pyruvic acid consumption are correctly correlated (r = 0.91, alpha = 0.05). This dosage can be used to study the barrier properties of the corneocyte layer without destroying the artificial skin.

Topics: Cells, Cultured; Colorimetry; Fibroblasts; Humans; NAD; Neutral Red; Pyruvic Acid; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles

Topics: 1-Propanol; Cytotoxicity Tests, Immunologic; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Lymphocyte Activation; Sodium Dodecyl Sulfate; Solubility; Tetrazolium Salts; Thiazoles