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PubMedDate not supplied

Cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin B1 in the SF-9 insect cell line.

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.

Animals↗
PubMed1998

Hepatoprotective activity of thymoquinone in isolated rat hepatocytes.

Thymoquinone, the active constituent of Nigella sativa, was tested in isolated rat hepatocytes as a hepatoprotective agent against tert-butyl hydroperoxide (TBHP) toxicity. TBHP (2 mM) was used to produce oxidative injury in isolated rat hepatocytes and caused progressive depletion of intracellular glutathione (GSH), loss of cell viability as evidenced by trypan blue uptake and leakage of cytosolic enzymes, alanine transaminase (ALT) and aspartic transaminase (AST). Preincubation of hepatocytes with 1 mM of either thymoquinone or silybin, which is a known hepatoprotective agent, resulted in the protection of isolated hepatocytes against TBHP induced toxicity evidenced by decreased leakage of ALT and AST, and by decreased trypan blue uptake in comparison to TBHP treated hepatocytes. Both thymoquinone and silybin prevented TBHP induced depletion of GSH to the same extent. Although thymoquinone protected the liver enzymes leakage, the degree of protection was less than that caused by silybin.

Alanine Transaminase↗
PubMed1983

Role of polymorphonuclear leukocytes in the pulmonary clearance of arrested cancer cells.

Treatment of mice with trypan blue led to an accelerated clearance of 125I-5-iodo-2'-deoxyuridine-radiolabeled B16 melanoma cells arrested in the lungs of tumor-bearing mice and the numbers of pulmonary nodules developing after intravenous injection of nonradiolabeled melanoma cells was reduced. Although macrophage function was normal or depressed in trypan-blue-treated mice, there were increased numbers of circulating polymorphonuclear leukocytes which had increased cytotoxicity for B16 melanoma cells and which produced increased levels of toxic oxygen radical. These findings suggested that polymorphs were responsible for accelerated pulmonary clearance of arrested melanoma cells and this conclusion was supported by the decreased clearance of cancer cells from the lungs of mice treated with antipolymorphonuclear leukocyte antiserum. These studies expand the repertoire of effector cells responsible for determining the extent to which hematogenously disseminated cancer cells are retained in the pulmonary vasculature prior to their extravasation and secondary growth.

Animals↗
PubMed2003

Reaction of poly(acrylamide-co-vinylamine) with tresyl-PEG in the presence of PC12 cells.

Tresylation of an amine containing polymer film in the presence of PC12 cells did not result in a significant loss of cell viability, at least as assessed by trypan blue exclusion or MTT assay. PC12 cells were cultured atop reactive poly(acrylamide-co-vinyl amine) films or tissue culture polystyrene and exposed for 2 h to tresylated polyethylene glycol (TPEG) or unreactive hydrolyzed TPEG in 0.1M TES (N-tris hydroxymethyl-2-aminoethane sulfonic acid). The loss in trypan blue viability was limited ( approximately 80% retained), provided the TPEG concentration was 10 micromol/g or less. Similarly when microencapsulated PC12 cells (in a non-reactive polyacrylate hydrogel) were exposed to TPEG (10 micromol/g in 0.1M TES) the loss of MTT activity was small. The loss of vaibility was attributed to the toxicity of the tresyl leaving group and not the reaction itself. Thus, it may be possible to surface modify cell containing microcapsules, at least under limited conditions, in order to improve their biocompatibility without compromising the viability of the enclosed cells. This should lead to the development of new (reactive) polymers for microencapsulation since biocompatibility need not be a design consideration in the first instance.

Amines↗
PubMed1978

Evidence for quantitative variability of bacterial opsonic requirements.

We studied human serum opsonins by using combinations of heat inactivation and chelation to inhibit complement, adsorption to remove antibody, and trypan blue to inactivate the C3 receptor of human polymorphonuclear leukocytes. Streptococcus pneumoniae, serotype 25, required both complement and immunoglobulin for opsonization, even though that strain activated the alternative complement pathway. Both strains of Escherichia coli required antibody and complement, but varied in the degree of dependence on the C3 opsonin, since trypan blue moderately inhibited the killing of E. coli-1 and markedly inhibited the killing of E. coli-2. Serratia marcescens was opsonized in heat-inactivated serum (limited complement) or serum absorbed at 0 degrees C with S. marcescens (limited antibody), but depended on the C3 receptor in absorbed serum. S. marcescens activated the alternative pathway. Thus, opsonic requirements varied with the availability of opsonins. Requirements for bacterial opsonization vary with species and strains within species, perhaps reflecting quantitative relationships among alternative and classical pathway activation of C3, efficiency of adsorption of C3 or immunoglobulin G to bacterial surfaces, and efficiency of attachment of these ligands to polymorphonuclear leukocyte receptors. Furthermore, although not always sufficient for opsonization, the C3 opsonin (activated through either the classical or alternative pathway) appears necessary for effective phagocytosis and killing of all strains studied.

Antibodies, Bacterial↗
PubMed1999

Neuroprotective effects of riluzole: an electrophysiological and histological analysis in an in vitro model of ischemia.

The protective effects of riluzole against the neuronal damage caused by O2 and glucose deprivation (ischemia) was investigated in rat cortical slices by recording electrophysiologically the cortico-cortical field potential and by evaluating histologically the severity of neuronal death. Five minutes of ischemia determined an irreversible depression of the amplitude of the field potential. In addition, this insult caused a clear enhancement of the number of death cells that were specifically colored with trypan blue (a vital colorant which stains altered cells). We found that riluzole, which by itself depressed the synaptic transmission, neuroprotected when perfused 15-20 min before and during ischemia. In fact, due to the treatment with riluzole, the ischemia-induced irreversible depression of the field potential recovered and less cells were stained with trypan blue. These findings demonstrate that riluzole prevents neuronal death in an in vitro model of ischemia and suggest a therapeutic use of this drug in order to reduce the pathophysiological outcomes of stroke.

Animals↗
PubMed1993

Taurine protects against the cytotoxicity of hydrazine, 1,4-naphthoquinone and carbon tetrachloride in isolated rat hepatocytes.

Exposure of rat hepatocytes to hydrazine, carbon tetrachloride or 1,4-naphthoquinone results in cytotoxicity determined as uptake of Trypan blue and leakage of lactate dehydrogenase (LDH). After exposure of hepatocytes to hydrazine and 1,4-naphthoquinone, ATP was also measured and was found to be depleted. Addition of the beta-amino acid taurine to the hepatocyte incubation buffer partially protects the cells against the cytotoxicity of these three different cytotoxic compounds, as indicated by Trypan blue uptake and LDH leakage. Taurine also reduces the depletion of ATP caused by 1,4-naphthoquinone but not hydrazine. It is suggested that taurine may have a cytoprotective effect in vitro and may be a useful tool for the investigation of mechanisms of cytotoxicity.

Animals↗
PubMed1983

Effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: a light and electron microscopic study.

Using a ligation method, rat rectal epithelium was exposed to 2% sodium salicylate, and light and electron microscopic methods were used to assay for: 1) permeability of the epithelium to a marker dye, trypan blue, and 2) damage expressed in terms of disruption of the epithelial surface. Rectal mucosa was exposed to salicylate at pH 4.8, 7.0, and 9.0, and the effects of pretreatment with phlorizin were also studied. Results indicated that 2% sodium salicylate does very little damage to rectal epithelial cells at pH 7.0 while enhancing their permeability to trypan blue, an effect that is reversed upon washing out the sodium salicylate. The major cellular change induced by salicylate was a reduction in the length or distribution of glycocalyx filaments on microvilli of epithelial cells. It was also noted that pretreatment with phlorizin counteracted some of the effects of salicylate treatment.

Animals↗
PubMed2005

Comparative analysis of the cytotoxicity of homopolymeric amino acids.

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.

Amino Acids↗
PubMed1994

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture.

FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200 micrograms/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40 micrograms/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.

Cell Survival↗
PubMed2004

Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine.

The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.

Acetaminophen↗
PubMed2000

Superiority of mild hypothermic (20 degrees C) preservation for pancreatic microvasculature using the two-layer storage method.

Hypothermia causes vascular endothelial damage that leads to graft microcirculation disorder and eventually thrombosis after reperfusion. The two-layer cold storage method (TL) was previously demonstrated to supply oxygen to the pancreas graft and maintain high adenosine triphosphate tissue concentration. In this study, we evaluated whether mild hypothermic (20 degrees C) preservation using the TL method could reduce endothelial damage while maintaining parenchymal viability. Graft survival by 20 degrees C preservation was investigated using a dog segmental pancreas autotransplantation model (simple storage in University of Wisconsin solution (UW) for 5 and 8 hours or TL for 5, 8, 12, and 24 hrs. respectively). Subsequently, the grafts were preserved in four different conditions (4 and 20 degrees C UW. 4 and 20 degrees C TL) for 8 hours to evaluate microvascular endothelial damage. Trypan blue uptake of vascular endothelium and pancreatic tissue perfusion were evaluated. No graft preserved by 20 degrees C UW for 5 and 8 hours survived (0/7 and 0/4). In contrast, the graft survival rates by 20 degrees C TL for 5, 8, 12, and 24 hours were 100% (5/5), 80% (4/5), 20% (1/5), and 0% (0/4), respectively. In trypan blue uptake analysis, there were significant differences between 4 and 20 degrees C in both UW and TL (4 degrees C UW, 37% [n = 5) vs. 20 degrees C UW, 13% [n = 4] [p < 0.01]; 4 degrees C TL, 29% [n = 5] vs. 20 degrees C TL, 10% [n = 5] [p < 0.011). The perfusion values in 20 degrees C TL were significantly higher than those in other groups at least for up to 120 minutes after reperfusion (p < 0.01 ). In short-term pancreas preservation, mild hypothermic TL reduced vascular endothelial cell damage and ameliorated graft microcirculation while maintaining parenchymal viability. Mild hypothermic TL may lessen vascular complications in clinical pancreas transplantation when used for several-hour preservation.

Animals↗
PubMed1999

Duration of increased mucosal permeability of the urinary bladder after acute overdistension: an experimental study in rats.

The duration of damage to the mucosal barrier of the urinary bladder after overdistension was investigated in a rat model. Overdistension was induced for 3 h in 48 female Sprague-Dawley rats by forced diuresis and balloon obstruction of the bladder neck. In 24 rats 0.5 ml of 2% solution of Trypan blue in 0.9% NaCl solution was instilled into the bladder for 1 h at 0 h, 24 h, 48 h, 7 days and 21 days after overdistension. After dyeing, full-thickness samples were taken from the wall of the bladder dome and body immediately above the ureteral orifices for histological studies. Inflammatory reaction was investigated histologically without Trypan blue dyeing in 24 rats at 0 h, 24 h, 48 h, 7 days and 21 days after overdistension. At 0, 24 and 48 h after overdistension the bladder wall was deep blue throughout. The dome and body were similar. At 7 days there was only slight staining of the bladder surface urothelium and subjacent connective tissue, while at 21 days there was no longer any dye in the bladder wall or urothelium. Oedema reached its maximum at 48 h, and large numbers of inflammatory cells were seen in the submucosa at 48 h. These changes had normalized by 7 days. After overdistension urothelial integrity is destroyed for several days, making it possible for different substances in the urine to penetrate into the bladder wall. This renders questionable the use of bladder distension in the treatment of interstitial cystitis, as it may only increase leakage of the urothelium and accelerate inflammatory reaction in the bladder wall. However, in the present study of healthy rat bladders the integrity of the urothelium had recovered to a large extent after 1 week and completely after 3 weeks.

Animals↗
PubMed1990

[The hepatocyte of the fetal rat as an in vitro model system for testing the antioxidant activity of biological fluids].

In case of hepatocytes of the fetal rat at the end of the gestation (H.) the lipid-peroxidation was determined by measuring the malondialdehyde (MDA) and the vitality by means of the trypan-blue test. The H. with an incubation up to 120 minutes in PBS formed a high spontaneous value of MDA with a simultaneous loss of the cell activity (r = -0.89; p less than 0.05; n = 25). On the conditions of an experimentally generated activation of O2-radical-forming processes (addition of Fe2+/vit. C) concentration-depending increasing of the MDA-formation and trypan-blue reception of the H. were induced (p less than 0.01). The addition of vit. C reduced the MDA-formation and the vitality of the H. The results exclude a direct relation of the two parameters. The MDA-formation of the H. in vitro can be used for testing antioxidative effects of biological fluids.

Animals↗
PubMed1992

Viability measurements of hybridoma cells in suspension cultures.

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.

Adenosine Triphosphate↗
PubMed1999

Modulation of retinal pigment epithelial cell behavior by Agaricus bisporus lectin.

PURPOSE: To determine whether Agaricus bisporus lectin (ABL) binds retinal pigment epithelial cells (RPEs), to conduct a preliminary viability study of RPEs exposed to ABL, and to evaluate the effects of ABL on RPE proliferation and RPE-mediated matrix contraction in vitro. METHODS: Using cultured bovine RPEs, immunohistochemistry was used to study ABL binding. Morphologic and trypan blue exclusion techniques were used for toxicity studies. The effect of ABL on RPE proliferation was investigated by [methyl-3H]-thymidine incorporation. The effect of ABL on RPE-mediated matrix contraction was evaluated with RPE-populated three-dimensional collagen matrices. RESULTS: ABL bound to RPE cells. This binding was inhibited by asialomucin. No change in RPE morphology or trypan blue exclusion compared with controls was observed in RPEs incubated with 5 to 60 microg/ml ABL for 3 days. Twenty-four-hour incubations of RPEs with ABL significantly inhibited RPE proliferation in a dose-dependent way, 40 microg/ml ABL inhibited proliferation by 83% (SE 14, P<0.05). ABL showed a dose-dependent significant inhibition of RPE-mediated collagen matrix contraction over 3 days, with 93% inhibition compared with controls by 40 microg/ml lectin (P<0.05). The inhibitory effect of ABL on proliferation and gel contraction was partly reversible after eliminating ABL from the culture medium. CONCLUSIONS: Bovine RPE cells bind ABL, and preliminary evaluations suggest that levels of ABL that are nontoxic to the cells potently inhibit RPE proliferation and RPE-mediated matrix contraction. ABL deserves further investigation as a potential inhibitor of RPE proliferation and cell-mediated matrix contraction in anomalous reparative processes such as proliferative vitreoretinopathy and as a laboratory tool for RPE behavioral studies.

Animals↗
PubMed1999

Subzero nonfreezing storage of isolated rat hepatocytes in University of Wisconsin solution.

BACKGROUND: Various cryopreservation techniques have been investigated to elongate preservation time, however, most have failed to be clinically induced because of damage due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. We evaluated the superiority of subzero nonfreezing storage compared with conventional hypothermic storage using isolated rat hepatocytes stored in University of Wisconsin (UW) solution without cryoprotectants. METHODS: Hepatocytes of Wistar rats isolated by collagenase digestion were suspended in UW solution and divided into the following three groups: subzero nonfreezing group (-4 degrees C), zero nonfreezing group (0 degrees C), and control group (4 degrees C). They were stored for 48 hr at the temperatures indicated. After 24 and 48 hr of storage, we carried out a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and measured lactate dehydrogenase release, lactic acid, ATP content, and the ability of hepatocytes to synthesize urea. After 48 hr of storage, morphological differences between the control group and the subzero nonfreezing group were investigated by scanning and transmission electron microscopy. RESULTS: Significant improvements of the trypan blue exclusion test and ATP contents in the subzero nonfreezing group were observed. Lactic acid production was also significantly suppressed in the subzero nonfreezing group compared with that in the control group. The MTT assay value was significantly better at -4 degrees C than at 4 degrees C. The rate of urea synthesis at -4 degrees C was higher than that at 4 degrees C. Electron microscopy revealed that subzero nonfreezing delayed the lethal bleb-forming process of stored hepatocytes, which was followed by mitochondrial swelling, compared with the control group. CONCLUSIONS: Subzero nonfreezing storage (-4 degrees C) in UW solution could provide better preservability for isolated rat hepatocytes with protection against hypoxic cell injury compared with conventional hypothermic storage (4 degrees C).

Adenosine↗
PubMed2002

Vitality of epithelial cells after alcohol exposure during laser-assisted subepithelial keratectomy flap preparation.

PURPOSE: To evaluate the vitality of epithelial cells after various exposure times to 20% ethanol and epithelial flap preparation in laser-assisted subepithelial keratectomy (LASEK) using the trypan blue dye test. SETTING: University Eye Clinic Regensburg, Regensburg, Germany, and the Rayne Institute, Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom. METHODS: Five human cadaver eyes were exposed to 20% ethanol for 15, 30, 45, 60, and 120 seconds, respectively. After an epithelial flap (as in LASEK) was prepared, the flap was deliberately cut off. The flaps were soaked in a trypan blue 0.1% solution at 37 degrees C. After 3 washes with phosphate-buffered saline (PBS), the specimens were reincubated for 30 minutes in culture medium containing 10% fetal calf serum at 37 degrees C. After an additional wash with PBS, the cells were observed with a standard inverted light microscope. RESULTS: After 15- and 30-second exposure to 20% ethanol, most epithelial cells were vital. This changed substantially after 45 seconds, when vital and dead cells were approximately equal. Longer exposure times (60 seconds and 120 seconds) showed predominantly dead epithelial cells. CONCLUSIONS: Exposure to 20% ethanol should be 20 to 30 seconds as the number of vital epithelial cells rapidly decreased after that. Vitality of the epithelial flap is probably a crucial factor in the dampened wound response in LASEK compared to that in photorefractive keratectomy.

Cell Survival↗