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PubMedDate not supplied

A test battery for the ecotoxicological evaluation of pentachlorophenol.

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective and quantitative test battery should be developed. The effects of pentachlorophenol were studied using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, bioluminiscence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction in the plant Allium cepa. The inhibition of cell proliferation and MTT reduction were investigated in Vero cells. Neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity were studied in the salmonid fish cell line RTG-2, derived from the gonad of rainbow trout. Pentachlorophenol was very toxic for all biota and cells. The system most sensitive to pentachlorophenol, was micronuclei induction in A. cepa, followed by D. magna immobilization, bioluminescence inhibition in V. fischeri bacteria at 60 min and cell proliferation inhibition of RTG-2 cells at 72 h. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity.

Animals↗
PubMedDate not supplied

Influence of cadmium speciation for the evaluation of in vitro cadmium toxicity on LLC-PK(1) cells.

The main objective of the present work was to assess the potentiality of in vitro models to improve our understanding of cadmium-induced toxicity, especially on epithelial renal cells. Indeed cadmium, a potent toxic metal, poses a serious environmental threat and the mechanisms of its renal toxicity need to be clarified. Cytotoxicity studies presented here were performed in a tubular proximal original established porcine kidney cell line (LLC-PK(1)). We have compared cytotoxicity induced by different chemical cadmium forms in LLC-PK(1) cells as a function of media cell culture pH and protein content. Cadmium stock solutions were prepared either by dissolving cadmium chloride or cadmium sulphate with increasing protein concentrations in the media cell culture. Its pH was monitored during experiments. Cytotoxicity was measured by neutral red uptake after 24 h of exposure. Dose-dependent cytotoxicity curves, calculated with REGTOX, were systematically correlated with pH and protein content. Experiments in vitro revealed that cadmium was dose-dependently toxic for LLC-PK(1) for concentrations ranging from 10(-4) to 10(-6) M. We have noticed a lack of influence of the media cell culture pH on the cadmium cytotoxicity. REGTOX determines closely the EC(50) values but EC(50)CdCl(2)>EC(50)CdSO(4) and cadmium have been assayed with an inductively coupled atomic emission spectrometer (ICP/AES) directly in the media cell culture and the cellular pellet.

Animal Testing Alternatives↗
PubMedDate not supplied

Comparative assessment of three in vitro exposure methods for combustion toxicity.

A comparative assessment of three approaches for the use of human cells in vitro to investigate combustion toxicity was conducted. These included one indirect and two direct (passive and dynamic) exposure methods. The indirect method used an impinger system in which culture medium was used to trap the toxicants, whilst the direct exposure involved the use of a Horizontal Harvard Navicyte Chamber at the air/liquid interface. The cytotoxic effects of thermal decomposition products were assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega) on a selection of human cells including: HepG2, A549 and skin fibroblasts. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. Polymethyl methacrylate (PMMA) was selected as a model polymer to study the cytotoxic effects of combustion products. NOAEC (no observable adverse effect concentration), IC10 (10% inhibitory concentration), IC50 (50% inhibitory concentration) and TLC (total lethal concentration) values were determined from dose response curves. Assessment using the NRU (neutral red uptake) and ATP (adenosine triphosphate) assays on human lung derived cells (A549) was also undertaken. Comparison between in vitro cytotoxicity results against published toxicity data for PMMA combustion and predicted LC50 (50% lethal concentration) values calculated from identified compounds using GCMS (gas chromatography mass spectrometry) was determined. The results suggested that the indirect exposure method did not appear to simulate closely exposure via inhalation, whilst exposure at the air/liquid interface by using the dynamic method proved to be a more representative method of human inhalation. This exposure method may be a potential system for in vitro cytotoxicity testing in combustion toxicity.

Adenosine Triphosphate↗
PubMedDate not supplied

A Genetically engineered cell-based system for detecting metabolism-mediated toxicity.

Xenobiotics undergoing bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue( reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.

3T3 Cells↗
PubMedDate not supplied

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.

Antioxidants↗
PubMedDate not supplied

Irritancy testing in cultured keratinocytes.

A differentiating keratinocyte cell line derived from explant cultures of rat sublingual epithelium has been used as a potential in vitro model for topical (skin) irritation of a range of detergents. The end points used to assess toxicity were acid phosphatase (AP) activity after 4 h of dosing and neutral red (NR) uptake and kenacid blue (KB) staining after 3 d to assess cell viability and number. The acid phosphatase activity increased to a sharp peak with increasing doses, then fell equally sharply for the anionic detergent sodium dodecyl sulfate (SDS) and cationic detergents TMABs (trimethylammonium bromides). With the Tweens (the least toxic group) there was no acid phosphatase peak or it appeared at the highest dose level used (1.0 mg/ml). The dose-response curves for NR uptake paralleled those for KB staining. With all of the three end points, it was apparent that the order of toxicity for the different groups was TMABs greater than SDS greater than Tweens, with a difference of one order of magnitude between consecutive groups when using NR and KB.

Acid Phosphatase↗
PubMed2005

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

Acetabularia↗
PubMed1979

Postmortal "vital" staining of the external eye.

Two hundred and two eyes from 112 individuals were vital-stained from 2 to 46 h after death. Intensity and extension of staining were studied in ten regions. The staining gradually progressed after death, also within non-exposed areas. It most often started anteriorly on the tarsus and finanally included the fornix. Microscopy revealed diffuse cell staining by rose bengal or trypan blue. The cell nucleus was most often more intensely stained than the cytoplasm. Up to five h after death, neutral red had only stained vacuoles in the cytoplasm. Later diffuse staining of cells occurred. Tetrazolium differed from the above dyes in that the pronounced staining seen immediately after death gradually decreased in the course of time. Microscopy disclosed stained inclusion bodies in the cytoplasm up to 18 h after death. Then cell staining was only seen as a rare exception in relation to extracellular dye granules. However, the mucous thread in the inferior fornix showed gradually increasing postmortal tetrazolium staining. The amount of mucus was found to be the same in dead persons as in the living. The characteristic appearance of the dead eye is due, among other things, to ruptures of the corneal epithelium (fluorescein-stained) and cell death, and not to drying up or coating by mucus.

Adult↗
PubMed1998

False positive results in cytotoxicity testing due to unexpectedly volatile compounds.

We investigated the cytotoxicity of different dental materials according to the study protocol adopted by our lab for the screening of new materials. Experimental parameters used in such testing are addressed mainly in documents EN 30993 "Biological evaluation of medical devices, Part 5: Tests for cytotoxicity: in vitro methods" and "Biological evaluation of medical devices, Part 12: Sample preparation and reference materials." Cells were cultured in microplates and challenged with aqueous extracts of the materials. The assay methods were neutral red- and propidium iodide-uptake assays, both indicative of cell viability and able to provide quantitative data. The observation of contrasting results for one material using the above-mentioned methods raised some concern about the assay system used. With further experimentation, it appeared that a sustained release of volatile substances still present in one extract exerted a toxic effect in neighboring cultures. It is concluded that in the microenvironment of a microplate the distribution of samples cannot be disregarded, as it may be responsible for toxicity cross-contamination. Moreover, the use of more than one single method has to be recommended in cytotoxicity testing, in order to avoid false positive results due to experimental artifacts.

Animals↗
PubMed2004

Metabolism-mediated cytotoxicity of ochratoxin A.

Ochratoxin A (OTA) is produced by various strains of Aspergillus and Penicillium and is a common contaminant of food commodities. OTA is metabolised by cytochrome P450 (CYP450) enzymes resulting in hydroxylated metabolites, 4R-OH-OTA and 4S-OH-OTA, and possibly in other minor metabolites including OTA-quinones. However, until now conflicting data have been presented regarding the role of biotransformation products in the adverse effects of OTA. Hence, the aim of this study was to further assess the metabolism-mediated cytotoxicity of OTA in an in vitro model encompassing NIH/3T3 cells, stably expressing the human CYP450 enzymes CYP2C9 and CYP3A4, respectively. In addition, modulation of the cellular glutathione (GSH) content was used to identify a role of GSH in OTA-induced cytotoxicity. Following exposure to OTA, cells expressing CYP2C9 showed a significant reduction in neutral red (NR) uptake but not in Alamar blue (AB) reduction, as compared to the control LNCX cells which do not express CYP450 enzymes. CYP3A4-expressing cells showed no difference in viability from control LNCX cells. When pre-treated with l-buthionine S,R-sulphoximine (BSO) to deplete GSH, CYP2C9-expressing cells showed also a loss of cell viability as compared to LNCX cells, although to a lesser extent as compared to non-depleted CYP2C9-expressing cells. Data presented in this study support previous findings, indicating that different biotransformation pathways contribute to the cytotoxicity induced by OTA.

Animals↗
PubMedDate not supplied

In vitro effects of lithium and nickel at different levels on Neuro-2a mouse neuroblastoma cells.

Lithium and nickel present low toxicity, but are able to cause alterations in different tissues. The toxic effects of lithium and nickel at different cellular levels were assessed using two inorganic chemical species: lithium chloride and nickel(II) chloride. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to both compounds for 24 h. The cytotoxic effects evaluated were cell proliferation by quantification of total protein content, cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage, and lysosomal hexosaminidase release. Metabolic markers were lactate dehydrogenase activity and mitochondrial succinate dehydrogenase activity. Lysosomal markers were relative neutral red uptake by lysosomes, and lysosomal hexosaminidase sphingolipid degradation activity. Acetylcholinesterase activity on intact cells was also quantified. Nickel was found to be 36 times more toxic than lithium to neuroblastoma cell proliferation (EC(50)= 0.29 and 10.5 mM, respectively), but the relative extent of other alterations differed. Lithium stimulated nearly all the indicators studied, particularly lactate dehydrogenase, mitochondrial succinate dehydrogenase and acetylcholinesterase activities, as well as hexosaminidase release. In contrast, nickel mainly stimulated hexosaminidase release and inhibited lactate dehydrogenase activity. The stabilization of the cytoplasmic membrane to lactate dehydrogenase leakage simultaneously with the secretion of lysosomal hexosaminidase for both compounds also shows that functional metabolic alterations produced by lithium and nickel are more important than cytoplasmic damage.

Acetylcholinesterase↗
PubMed2004

Phytosterols: lack of cytotoxicity but interference with beta-carotene uptake in Caco-2 cells in culture.

Ingestion of phytosterols has been shown to reduce plasma cholesterol in both animals and humans. The esterified forms of phytosterols are increasingly being incorporated into margarine and fat spreads, which are then marketed as functional foods. The aim was to assess the cytotoxicity and uptake of four phytosterols, beta-sitosterol, campesterol, stigmasterol and stigmastanol, in human intestinal cells in culture. Another aim was to determine if phytosterols would interfere with alpha-tocopherol or beta-carotene uptake by these cells. Human adenocarcinoma Caco-2 cells were supplemented for 24 h with increasing concentrations (0-12.5 microM) of each phytosterol. Cytotoxicity was assessed by neutral red uptake (NRU), lactate dehydrogenase release (LDH) and fluorescein diacetate/ethidium bromide (FDA/EtBr) assays. The phytosterols had no significant effects on Caco-2 cell viability assessed using LDH and FDA/EtBr assays. The highest concentrations of beta-sitosterol and campesterol tested (12.5 microM) resulted in decreased cell viability assessed using the NRU assay. All phytosterols were taken up by Caco-2 cells in culture. The results demonstrate a reduction in the uptake of beta-carotene when Caco-2 cells were supplemented with 20 microM beta-sitosterol. beta-Sitosterol did not interfere with alpha-tocopherol uptake by the cells. In conclusion, Caco-2 cells are a useful model system to study potential interactive effects of phytosterols with fat-soluble dietary components.

Anticholesteremic Agents↗
PubMed1999

Evaluation of in vitro toxicity of N,N-dimethyl-2-propen-1-amines isomers.

The trypanocidal activities of cis-3-(4'-bromo[1,1'-biphenyl]-4-yl)- 3-(phenyl)-N,N-dimethyl-2-propen-1-amine (Vb) and cis-3-(4'-bromo[1,1'-biphenyl]-4-yl)-3-(4-bromophenyl)-N,N-dimethyl-2- propen-1-anine (Vg) appeared 6.3 and 3.5 fold more active than the trans-isomers, respectively. Multi-endpoints for toxicity were also applied. Neutral red uptake (NRU), tetrazolium salt reduction (MTT), DNA content on V79 fibroblast cell culture and acute toxicity von E. coli were measured. The IC50 through DNA contents was lower for the cis-isomers in both series of compounds 5b: 7.8 microM and 5g: 5.2 microM). NRU values for derivative 5b in isomeric mixture shows the same value as the isolated isomers however, in the case of 5g a more significant toxicity of the cis-isomer was found. MTT values show that 5g is more toxic than 5b. In both cases, the acute toxicity of the trans-isomers was higher than that of the cis-isomers.

Amines↗
PubMed2000

Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots.

The most widespread type of mycorrhiza is the so-called vesicular-arbuscular mycorrhiza. In this endomycorrhiza, fungal hyphae penetrate plant cell walls in the root cortex. There they form densely branched arbuscules. Fungus and plant plasma membrane are separated by a common interfacial apoplast. The pH of the compartment between the symbionts is of pivotal importance for nutrient transfer. Histochemical experiments were conducted to check for an acidic nature of the interface in the model system Glomus versiforme (Karst.) Berch-Allium porrum L. Two chemically different acidotropic dyes (neutral red and LysoSensor Green DND-189) stained the arbuscules intensely. The staining of arbuscules could be eliminated by addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) or treatments leading to membrane rupture. Therefore, the staining of the arbuscules was based on the ion-trap mechanism, which indicates acidic, membrane-bound compartments. Microscopic examination of stained arbuscules at high optical resolution revealed a peripheral accumulation of the dye. Since plasmolysis rapidly destained the arbuscules, it is concluded that the dyes accumulate in the arbuscular interface, indicating the highly acidic nature of this compartment. The findings are discussed with respect to their relevance for the nutrient transfer in mycorrhizas. In addition, evidence for a discontinuity in the arbuscular interface between the stem and the branches of the arbuscule is given.

Allium↗
PubMed1991

In vitro staining of islets of Langerhans for fluorescence-activated cell sorting.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.

Animals↗
PubMed1980

Aqueous solutions of some basic dyes--their use in the cytochemical detection of DNA.

The paper contains results of staining DNA-aldehyde molecules with aqueous solutions of brilliant cresyl blue, thionin or neutral red, following Feulgen procedure and also reports on the use of aqueous solutions of these dyes, with primary amino group(s) in their molecules, for staining animal tissue nuclei after extraction of RNA with cold phosphoric acid. The pH of the dye solutions most suitable for optimum staining is 6.0. The time necessary for optimum staining of DNA-aldehydes and DNA-phosphate groups are 10 and 2 min respectively for tissues fixed in formalin, paraformaldehyde or Craf. Tissue fixed in Buin-fluid stain slower. The absorption curves of nuclei stained for DNA-aldehyde molecules and DNA-phosphate groups, stained with each of the three dyes are different from each other. The in vitro absorption curves of aqueous solutions of the three dyes have also been presented. Some implications of the results obtained are discussed.

Animals↗
PubMed2003

Effect of pH on uptake and photodynamic action of chlorin p6 on human colon and breast adenocarcinoma cell lines.

The effect of reducing the extracellular pH from 7.4 to 6.0 on the uptake and photosensitivity of chlorin p6, a potential photosensitising drug, has been investigated using two mammalian cell lines, human colon (Colo-205) and breast (MCF-7) adenocarcinoma cells. In Colo-205 cells, the uptake and phototoxicity of chlorin p6 was observed to increase as the pH of the incubation medium decreased. For light doses of up to [similar]6 kJ m(-2), although there was no evidence of mitochondrial damage, a significant reduction in Neutral Red uptake was observed, signifying damage to lysosomes. At higher light doses, significant mitochondrial damage was observed, accompanied by saturation of the lysosomal damage. This suggests light-induced relocalization of the photosensitizer from lysosomes to mitochondria. Furthermore, it was found that for a given light dose, lysosomes exhibit greater photosensitivity at lower pH. Since chlorin p6 is known to aggregate at pH 6.0, this observation suggests that the dye accumulation in these cells mainly takes place through endocytosis. In contrast, no significant variation in uptake, photosensitivity, and sites of photodamage was observed for MCF-7 cells at different extracellular pH. Additionally, the lower photosensitivity of lysosomes as compared to mitochondria in these cells suggests chlorin p6 is taken up through diffusion rather than endocytosis.

Adenocarcinoma↗
PubMed1999

A 63 kDa tumor necrosis factor inhibitor released from a human monocytic leukemia cell line, THP-1.

A human monocytic cell line, THP-1-S, was cultured in a serum-free medium. The effect of the culture supernatant of THP-1-S on the cytotoxicity of rTNF-alpha to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF-alpha when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of 125I-rTNF-alpha to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF-alpha and the inhibitory factor which formed during incubation with the culture supernatant from THP-1-S cells. However, the supernatant did not bind to or down-regulate the receptor for TNF-alpha on the cell surface of L-M-2d6 cells. This factor eluted with an apparent molecular mass of 63,000 Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF-alpha in serum-free culture conditions.

Cell Survival↗