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PubMed2003

Ozonation of an azo dye C.I. Remazol Black 5 and toxicological assessment of its oxidation products.

The effect of ozonation (20.5 mgl(-1)) on the degradation processes of an azo dye, Remazol Black 5 (RB5; CI) was studied. Conventional parameters such as chemical oxygen demand (COD), total organic carbon (TOC), pH, conductivity, colour removal, biodegradability (BOD(5/28)), and toxic potential of the dye and its degradation products were monitored during the process. The results obtained indicated that ozonation is a highly effective way to remove the colour of a corresponding dye solution. However, a considerable organic load still remained as indicated by high COD and TOC residues. The COD, TOC reductions were about 40% and 25% for 6 h ozonation of 2 gl(-1) RB5 aqueous solution. During the ozonation process the rapid decrease of pH and the sharp increase of conductivity indicated the formation of acidic by-products and small fragments and ions which were identified by high performance ion chromatography. The BOD28 data revealed that first by-products after partial ozonation (10-150 min) of RB5 were more biodegradable than the parent compound and ozonation can enhance the biodegradability of azo dyes. During the first 150 min of total 360 min of oxidation, the formation of first by-products with high toxic potential took place as it could be confirmed by two acute toxicity-screening tests, the bioluminescence test (Vibrio fischerii) and the neutral red cytotoxicity assay (rat hepatoma cells). The significant enhancement of microbial biodegradability after long-term ozonation could also be seen as a decrease of toxic intermediates in correlation with the ozonation time as indicated in BOD28 biological degradation test results.

Animals↗
PubMed1998

Increased cytotoxic sensitivity of cultured FHM fish cells by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine.

A previously described neutral red uptake inhibition assay on cultured fathead minnow (FHM) fish cells revealed a good correlation with fish toxicity data (Brandao et al., 1992) for a series of 50 test compounds, belonging to completely different chemical classes. The major drawback was the lower sensitivity of the cytotoxicity assay. Aiming at a higher sensitivity the assay was adapted by reducing the cell number, by a longer treatment period, and by simultaneous treatment with sodium dodecyl sulfate and buthionine sulfoximine (BSO). The fluorimetrically measured protein content was chosen as the endpoint. The endogenous glutathione (GSH) content was reduced by 54% of the test chemicals, confirming the importance of GSH in detoxification processes. Higher sensitivity was, at least partly, obtained by treating the FHM cells with BSO, reducing the GSH content to 22% of that in control cells. The cytotoxicity of the 50 chemicals was measured using the modified more sensitive assay. Although some exceptions were observed, this new assay is at least one order of magnitude more sensitive. Toxicity values comparable with fish toxicity data were obtained, making the assay sensitive enough to measure the toxicity of environmental water samples.

Animals↗
PubMed1979

Lymphocyte activation and the increase of nuclear birefringence.

Nuclear birefringence of neutral red or rivanol stained cell suspensions from rat spleen has been investigated. Polyclonal or monoclonal mitogens produced an increased birefringence of the nucleus following 30 min in vitro stimulation at 37 degrees C. The nuclear birefringence increased by 62.2% [p less than 0.001], when the cells were incubated in the supernatants of a previously phytohemaglutinin stimulated culture. Amantadine, a potent phytohemagglutinin inhibitor, was unable to prevent the effect of the supernatant, but heating for 1 h at 56 degrees C destroyed its activity. The results suggest that increase in nuclear birefringence is mediated by a soluble factor which is released in the course of lymphocyte activation. The nuclear birefringence of surviving cells from human spleens obtained within 6 to 24 h post mortem increases after in vitro stimulation.

Animals↗
PubMed1998

An in vitro biocompatibility evaluation of double-J stents.

OBJECTIVES: For several years, studies performed to estimate in vitro biocompatibility of urinary catheters have been carried out using permanent cell lines. However, for a rational design of the testing procedure, the cell culture model should depend on the material application. We assess the biocompatibility of 13 double-J stents using an in vitro model of normal human urothelial cells (HUC). This article aims to mimic in vitro, on HUC monolayers, the close contact existing in vivo between the urothelium and double-J stents and to evaluate the subsequent effect on these cells. METHODS: Fragments of each stent were deposited into the wells containing confluent HUC, with close contact maintained between the material and the cells. The same procedure with either no material or fragments of latex catheter was undertaken to provide the negative and positive controls, respectively. The contact was maintained for 1, 3, and 8 days. At the end of the incubation period, fragments of stent were removed and cell activity tests were performed (neutral red assay, MTT assay, and cell proliferation). RESULTS: One of the silicone stents is significantly deleterious on HUC as determined by three tests after 8 days of contact. For two copolymers, a tendency to increase cell proliferation was noted. Concerning polyurethanes, we observed significant decreases in HUC viability and cell metabolic activity for five stents after 8 days of contact. All seven polyurethane stents significantly inhibited cell proliferation. CONCLUSIONS: The HUC culture model may be of relevance for the screening of materials intended for use as double-J stents.

Biocompatible Materials↗
PubMed1993

Cytotoxicity of retrofill materials.

Dentin bonding agents reduce microleakage and enhance marginal adaption of composite resin restorations. These characteristics are advantages for their use as an endodontic retrofilling material. Because these materials will be in direct contact with vital tissues, their cytotoxic potential must be evaluated before clinical use. It was the purpose of this study to evaluate the cell cytotoxicity of amalgam, Caulk Universal Bond, Gluma, 35% HEMA, Morita Clearfil, Scotchbond 2, Super EBA, Tenure, and Tenure 5-4. VERO cells were grown in RPMI-1640 medium and cell monolayers were prepared by incubating 15 ml of the cell suspension in 60-mm culture dishes at 37 degrees C in 5% CO2. Twelve milliliters of a medium-agarose mixture containing 1% neutral red vital stain were overlayed onto the cell layer and allowed to solidify. The materials were directly exposed to the agarose overlays by inverting 6.0-mm diameter polypropylene capsules containing the cured and liquid sample materials either immediately (0 time) or after placement in phosphate-buffered saline with 1% gentamicin for 7, 15, or 30 days. Cytotoxicity was determined by measuring the zone of killed cells around the sample 24 h after placement on the agarose. Cytotoxicity was determined by measuring the zones of cell inhibition at 24 h and at 7, 15, and 30 days. Initially, all of the materials were found to be cytotoxic, except amalgam and the Tenure components. The dentin bonding primers showed a mean zone of inhibition of 13.2 mm and the cleansers a 40.0-mm zone. Amalgam demonstrated increasing cytotoxicity: 0.0 mm at 24 h to 12.0 mm at 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Etching, Dental↗
PubMedDate not supplied

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures.

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.

Adaptation, Physiological↗
PubMedDate not supplied

Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations.

Protein expression and lysosomal stability were observed in Mytilus edulis exposed to 0, 20, 40, 60, and 80 ppb copper sulfate for 24 h in a static exposure system. Haemolymph was removed from each mussel and analyzed for lysosomal damage using the Neutral Red retention assay expressed as the ratio of lysosomal size to cell area, the lysosomal destabilization ratio. Gill tissue from the same individuals was dissected, homogenized and the proteins extracted and then separated using two-dimensional gel electrophoresis. Two-dimensional protein maps from each concentration were analyzed using MELANIE II 2D gel analysis software (Biorad). Lysosomal damage increased with each dosage, with LDRs ranging from 0.18 +/- 0.01 at 0 ppb copper sulfate to 0.32 +/- 0.11 at 80 ppb copper sulfate. Orthogonal comparisons showed that the control, 20 and 40 ppb classes together differed significantly (P < 0.05) from the 60 and 80 ppb classes together. Specific protein expression signatures were identified at each dosage.

Animals↗
PubMedDate not supplied

Levels of cellular glutathione and metallothionein affect the toxicity of oxidative stressors in an established carp cell line.

The comparative toxicity of a variety of oxidative stressors was studied in the epithelioma papulosum cyprini line from carp using the neutral red cytotoxicity assay. LC50's decreased in the order t-butylhydroperoxide > hydrogen peroxide > diquat > paraquat. The cytotoxicity of hydrogen peroxide was significantly reduced when the cells were grown in L-15 medium rather than MEM and this could be attributed to elevated cellular glutathione and metallothionein levels and higher activities of GSH-dependent detoxification systems. The protective effect of metallothionein in radical scavenging was demonstrated by decreased toxicity of the redox-cycling toxicants, diquat and menadione after metallothionein levels had been pre-induced by Cd-exposure. This study demonstrates the relationship between toxic effects of oxidative stressors and expression of detoxification systems in fish.

Animals↗
PubMed2002

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments.

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.

Animal Diseases↗
PubMedDate not supplied

Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy).

A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments.

Animals↗
PubMed2000

Organotypic cultures of gingival cells: an epithelial model to assess putative local effects of orthodontic plate and occlusal splint materials under more tissue-like conditions.

This article explores whether organotypic cultures of immortalized gingival keratinocytes constitute a suitable model for assessing the epithelial cell compatibility of two groups of dental resins, each of them representing one group used in orthodontics and temporo-mandibular disorders (TMD) therapy under conditions more closely resembling the actual tissue situation. The resins were tested with the agar diffusion assay (ADA) in conventional monolayer and organotypic cultures. Compared to the control exhibiting a neutral red destaining index of 3, the index of 4 obtained after exposure of monolayers to one soft permanent resin (Durabase) indicated the presence of a non-lytic but physiologically active substance. In contrast, the adaptation of the ADA to organotypic cultures revealed no apparent lesions at the epithelial surface by performing scanning electron microscopy, while histoarchitecture indicated the development of stratified surface epithelia. This was substantiated by undamaged cells in the uppermost cell layers and by the preservation of cell-to-cell contacts. Furthermore, indirect immunofluorescence for Ki-67 and the cytokeratins ck 14 and ck4 revealed that cell proliferation and epithelial structure were maintained, while differentiation was enhanced, possibly increasing epithelial resistance. The results obtained from the organotypic cultures suggest that (i) cell-affecting effects of materials visible in monolayer cultures may not be seen in epithelia resembling that in vivo and that (ii) enhanced differentiation may be associated with increased stability of the epithelial cells. Thus, organotypic cultures of gingival cells constitute a tissue model allowing short-term tissue compatibility studies of dental materials and rendering a potential candidate also for long-term studies.

Acrylic Resins↗
PubMed2001

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use.

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Animals↗
PubMed2001

The influence of titania/hydroxyapatite composite coatings on in vitro osteoblasts behaviour.

The biocompatibility of titania/hydroxyapatite (TiO2 /HA) composite coatings, at different ratio obtained by sol-gel process, were investigated studying the behaviour of human MG63 osteoblast-like cells. The biocompatibility was evaluated by means of cytotoxicity and cytocompatibility tests. Cytotoxicity tests, i.e., neutral red (NR), MTT and kenacid blue (KB) assays, were performed to assess the influence of the material extracts on lysosomes, mitochondria and cell proliferation, respectively. Cell proliferation, some preliminary indications of cell morphology, alkaline phosphatase activity, collagen and osteocalcin production of MG63 cells, cultured directly onto TiO2/HA substrates, were evaluated. The results showed that these materials have no toxic effects. Cell growth and morphology were similar on all the materials tested: on the contrary, alkaline-phosphatase-specific activity and collagen production of osteoblasts cultured on TiO2/HA coatings were significantly higher than uncoated titanium and polystyrene of culture plate and were influenced by chemical composition of the coatings. In particular, TiO2/HA coating at 1:1 ratio (w/w) seems to stimulate more than others the expression of some differentiation markers of osteoblastic phenotype. TiO2/HA coatings resulted to be bioactive owing to the presence of hydroxyl groups detected on their surface that promote the calcium and phosphate precipitation and improve the interactions with osteoblastic cells.

Alkaline Phosphatase↗
PubMed2001

Study of a (trimethylenecarbonate-co-epsilon-caprolactone) polymer--part 2: in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide.

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and epsilon caprolactone (P(epsilonCL-TMC), 50: 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to EDI subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of epsilonCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of epsilonCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P(epsilonCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme (N-acetyl-beta-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on epsilonCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P(epsilonCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. epsilonCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair. El sevier Science Ltd. All rights reserved.

Animals↗
PubMed2002

In vitro testing of the potential for orthopedic bone cements to cause apoptosis of osteoblast-like cells.

The purpose of this study was to investigate in vitro the apoptosis- and/or necrosis-inducing potential of polymethylmethacrylate (PMMA)-based bone cements for prosthetic surgery. Four bone cements widely used in orthopedics were tested as extracts onto osteoblast-like MG-63 cells and for comparison, HL-60 cells, which are remarkably sensitive to apoptotic stimuli. Neutral red uptake (NRU) was used to measure cell viability while Hoechst 33258 staining was used to detect DNA content. Apoptosis was characterized using a BrdU-based ELISA assay for DNA fragmentation and examined by fluorescence microscopy using acridine orange and propidium iodide staining of nuclei. The generation of reactive oxygen species (ROS), which could mediate apoptosis, was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. After 24 h of challenge of the cells with the four cement extracts, the viability of either MG-63 or HL-60 cells was found to be unaltered, as recorded by NRU. Apoptotic cell death was induced by three cements in HL-60, whereas MG-63 cells were significantly affected by the four cements tested: the finding of DNA fragments both in the cytoplasm and supernatants of MG-63 after 24 h demonstrated that these cells underwent late-apoptosis secondary necrosis. Fluorescent staining of the nuclei confirmed the results obtained with the ELISA test. Oxygen free radicals were elicited by two cements in HL-60 cells, while MG-63 did not generate ROS in response to cements. This study helps to gain more insight into the mechanism of cell death induced by PMMA-based cements and suggests apoptosis of osteoblasts as a part of the tissue reaction around cemented prostheses.

Apoptosis↗
PubMed2002

Cytocompatibility study of organic matrix extracted from Caribbean coral porites astroides.

Since 1980, natural coral exoskeleton has been widely used as bone graft substitute. Despite numerous in vitro and in vivo studies. there is still a lack of knowledge concerning the organic matrix associated with coral exoskeleton (COM). In fact, some surgical interventions have failed and this has sometimes been attributed to the exoskeleton organic matrix. For others, only amino acids are present in the matrix after coral preparation for clinical use. The objective of this study was to extract the exoskeleton organic matrix to carry out biochemical analysis and study its specific cytocompatibility. Demineralized bone powder (DBP) was used as control. A decalcification process was used to extract COM and DBP. Protein, carbohydrate and glycosaminoglycan analysis was carried out in DBP and COM using classical staining methods. Human bone marrow cells were cultured in the presence of 20, 40, 80, 160 microg of COM or DBP for 24, 48 and 72 h. The methods used to analyze COM and DBP effects were scanning electron microscopy immunocytochemistry, manual cell count, and cyto-compatibility assay (Neutral red and MTT tests). Results showed that in spite of treating coral before clinical use, a COM was present in which GAG, protein and carbohydrate were found. The in vitro cytocompatibility of COM was confirmed for 20 and 40 microg values but was less pronounced for 80 and 160 microg levels.

Alkaline Phosphatase↗
PubMed2002

Physical and chemical processing for a human dura mater substitute.

OBJECT: Allogenic human fascia lata used in neurosurgery, as dura mater substitute, can be associated with a risk of viral and bacterial transmission. Chemical and physical procedures, developed to inactivate virus and bacteria, have been applied to fascia lata. The aim of this study consists in the evaluation of the biological properties of this treated graft. METHODS: Grafts were treated with solvent detergents, freeze-dried for conservation and gamma irradiated (25,000 Gy) for sterilization. The indirect toxicity evaluation was performed by extraction method, according to the International Standard Organization (ISO). First, the cytotoxic effect of each extracts incubated in the presence of human fibroblasts (WI38) was quantitatively assessed by measuring the cell growth, the viability (succinate dehydrogenase activity, MTT), the membrane integrity (uptake of the neutral red by viable cells, NR) as well as the release of lactate dehydrogenase in the culture medium. Second, confocal laser scanning microscopy (CLSM) was used to assess the direct contact between human primary fibroblasts and graft. CLSM was performed at days 3 and 7 after cells loading. RESULTS: No acute cytotoxicity was observed for chemically processed allografts. Cells loaded on the graft have demonstrated a good growth and spreading. CONCLUSIONS: Human fascia lata secured against conventional and non-conventional agents is a fully biocompatible alternative to the available dural graft materials.

Biocompatible Materials↗
PubMed2003

Hepatocyte performance on different crystallographic faces of rutile.

The influence of crystallographic orientation of polished rutile single crystal surfaces of the (100), (110) and (111) orientation on hepatocyte performance was tested in cell culture over 3 days. Cell adhesion was observed on the titanium dioxide surfaces and their performance was measured by means of cell number attached (protein mass), cell viability (neutral red assays) and metabolic activity (thiazolyl tetrazolium bromide assay). Titanium dioxide displays no cytotoxic effects on hepatocytes, and shows a performance in the range of standard collagen-coated tissue culture polystyrene dish. The number of hepatocytes adhered on the different rutile surfaces were not significantly different to those on dense rutile polycrystalline ceramic. These findings suggest that hepatocytes do not recognize the specific differences of differently orientated rutile crystal surfaces.

Animals↗