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PubMed2004

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis.

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

3T3 Cells↗
PubMed1998

Chelation of intracellular calcium prevents mesangial cell proliferative responsiveness.

Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsiveness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was sufficient to elicit the same response. Rat primary mesangial cells (passages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine serum (FBS), then stimulated with 0.1 microM endothelin-1, 1.9 microM platelet-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 microM ionomycin, with or without the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was measured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofluorometry and in Fluo-3-loaded cells by confocal fluorescence laser microscopy. [3H]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was analyzed by Northern blot. All test agents stimulated a significant increase in cytosolic and nuclear calcium, which were both effectively inhibited with BAPTA. All agents stimulated a significant increase in [3H]-thymidine incorporation and enhanced c-fos mRNA expression (no detectable c-fos mRNA was observed in quiescent cells). BAPTA prevented the enhanced [3H]-thymidine incorporation stimulated by endothelin-1 and PDGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response to these agents. Phorbol ester induction of c-fos mRNA in the absence of raised cytosolic or nuclear calcium was also suppressed by BAPTA. Cell viability as measured by thiazolyl blue and trypan blue was not altered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.

Animals↗
PubMed2005

[Two-dimensional electrophoresis and mass spectrometry analysis on effects of trichloroethylene on protein of L-02 liver cells].

OBJECTIVE: To study the effects of trichloroethylene (TCE) on the protein in L-02 cells in vitro. METHODS: Thiazolyl blue and Trypan blue tests were used to investigate the cytotoxicity of TCE to L-02 liver cell. The 2-D electrophoresis was used to analyse the expression of proteins in L-02 liver cells. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS). RESULTS: When the concentration of TCE exceeded 30 micromol/L, there was distinct cytotoxicity to L-02 cell (P < 0.05). Selected 40 micromol/L to treat L-02 liver cells and analyze the differential proteome expression, the results showed that the expression level of 37 protein spots was up-regulated and 15 protein spots was down-regulated. And 15 proteins were identified by MALDI-TOF-TOF-MS. CONCLUSION: TCE can change the proteome expression of L-02 liver cell. It should provide the fundamental information to identify proteins related to TCE in further study.

Anesthetics, Inhalation↗
PubMed1984

The role of the lysosome in natural killing: inhibition by lysosomotropic vital dyes.

Recent evidence has indicated the importance of lysosomes in natural killing (NK). The lysosomotropic dye neutral red, at concentrations greater than 2 X 10(-5) M, inhibits NK activity of large granular lymphocytes (LGL). Inhibition is at the level of effector cell activity rather than target cell susceptibility. Neutral red inhibits NK activity without affecting the viability of the effector cell population or the response of lymphocytes to the mitogen, phytohaemagglutinin (PHA). While the mechanism of this inhibition is unknown, neutral red-treated peripheral blood lymphocytes (PBL) do not suppress untreated effector cell populations. Furthermore, neutral red does not influence the binding of effector cells to target cells. Since natural killing by neutral red-treated PBL recovers slowly upon incubation of the cells at 37 degrees, inhibition is not due to selective toxocity. Trypan blue, another frequently used lysosomotropic vital stain, has no effect when effector cells are pretreated with 10(-3) M dye for 30 min or less. However, effector cells first treated with neutral red lose red granular staining following treatment with trypan blue for as little as 10 min at room temperature. Natural killing by neutral red pretreated PBL also recovers more quickly following brief incubation with trypan blue. Treatment of effector cells with trypan blue and neutral red simultaneously prevents accumulation of neutral red in cytoplasmic granules and subsequent inhibition of NK.

Cytoplasmic Granules↗
PubMed1995

[Differential value of various vital stains of corneal endothelium].

Specular microscopy shows the integrity of the endothelial cell pattern, and vital staining methods allow the evaluation of endothelial viability. Dyes commonly used such as trypan blue enter the cells passively through the disturbed membrane. The uptake of fluorescein diacetate (FDA) is an active process. To compare the efficiency of the two methods we performed double stainings and examined the results. Pig cornea endothelium was stained by both trypan blue and FDA and vital and non-vital cells were compared. More non-vital cells were stained by FDA than by trypan blue, indicating that cells with membranes that are not permeable by trypan blue cannot metabolize FDA. This fact is important where estimating the endothelial viability of donor corneas.

Animals↗
PubMed1991

Prolonged hypothermic cardiac storage with University of Wisconsin solution. An assessment with human cell cultures.

Hypothermic storage of cardiac allografts is routinely used for transplantation but is associated with an increased mortality when ischemic times are greater than 4 hours. The ideal storage conditions (solution and temperature) could extend the current limits of cold ischemia. Human endothelial cells and ventricular myocytes were studied to screen various solutions and temperatures for organ preservation. Four solutions (modified Euro-Collins, phosphate-buffered saline, Stanford cardioplegia, and University of Wisconsin) were evaluated. Endothelial cells were evaluated after prolonged hypothermic storage consisting of 0 degree, 4 degrees, and 8 degrees C for 36 hours, and ventricular myocytes were stored at 0 degree and 8 degrees C for 24 hours. Cell viability was determined by morphology (10 dishes per group), and trypan blue exclusion (5 dishes per group) in addition to a cell adhesion assay (endothelial cells 5 dishes per group) and adenine nucleotide analysis with high-performance liquid chromatography techniques (ventricular myocytes 5 dishes per group). Endothelial cell morphology was best preserved by University of Wisconsin solution (p less than 0.001, chi 2) and at 0 degree C (p less than 0.01, chi 2). Endothelial cells stored with University of Wisconsin solution excluded trypan blue better (1.0% +/- 0.5% cells stained, p less than 0.001. Analysis of variance [ANOVA]). Cell adhesion was poorly protected with Stanford cardioplegia (p less than 0.001, ANOVA). Myocyte morphology was preserved best with University of Wisconsin solution at 0 degree C (p less than 0.001, chi 2). According to trypan blue staining, Euro-Collins and University of Wisconsin solutions were superior to Stanford cardioplegia or phosphate-buffered solutions (p less than 0.001, ANOVA). Temperature did not influence the trypan blue results. Adenosine triphosphate was maintained best with University of Wisconsin solution at 0 degree C (p less than 0.01, ANOVA). Myocytes were more sensitive to the effects of prolonged storage compared with endothelial cells by morphologic criteria and trypan blue staining characteristics, irrespective of the shorter preservation times. University of Wisconsin solution was the most effective solution tested. Colder temperatures (0 degree to 4 degrees C) provided better protection than 8 degrees C. Myocytes were more sensitive to prolonged preservation than endothelial cells. Furthermore, the technique used appears helpful as a model of prolonged hypothermic storage and could be expanded to assess other interventions.

Adenosine Diphosphate↗
PubMed1979

HLA--DR typing of frozen B lymphocytes.

In the complement dependent lymphocytotoxic microtechnique it was found that antibody-killed frozen B lymphocytes are sufficiently stained for reliable reading after 30 min of incubation with trypan blue, while the background of staining is only about 10%. During the next 30 min of incubation, however, the background of staining increases to about 30%, whereafter it remains constant for at least 24 h. Formaldehyde is able to stop the trypan blue uptake by killed or damaged lymphocytes completely. Consequently, if formaldehyde is added to the reactions 30 min after the trypan blue addition, the otherwise rapidly increasing background of staining is kept at an acceptable level of 10%, thus making HLA-DR typing of frozen stored B lymphocytes possible. The trypan blue staining seems rather independent of incubation conditions before the addition of the dye. Similar results were obtained with T lymphocytes.

B-Lymphocytes↗
PubMed1996

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli.

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P2Y-purinoceptor, which is activated by adenosine 5'-O-(2-thio-diphosphate) (ADP beta S), and a separate receptor for alpha, beta-methylene ATP (alpha,beta-MeATP). Effects of several as yet incompletely characterized P2-purinoceptor antagonists at these receptors were examined. The concentration-relaxation curve of ADP beta S was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulp honic acid (XAMR0721; at 1000 microM only), pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 microM only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADP beta S had slopes < 1. The concentration-relaxation curve of alpha,beta-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 microM). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against alpha,beta-MeATP had slopes > 1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against alpha,beta-MeATP than against ADP beta S. 2-Methylthio ATP (MeSATP; 1 microM) and ATP (100 microM) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides. The selective effect of XAMR0721 against alpha,beta-MeATP confirms the existence of two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P2Y-purinoceptor in turkey erythrocytes, was very weak at the P2Y-receptor of the taenia, thus supporting the existence of pharmacologic P2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas defrens, is the most selective P2X- (versus P2Y-) purinoceptor antagonists presently known, although its effect on the degradation of nucleotides must be kept in mind.

Adrenergic alpha-Agonists↗
PubMedDate not supplied

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells.

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.

Annexin A5↗
PubMed1981

Research on the production site of tumor necrosis factor (TNF).

Tumor Necrosis Factor (TNF) is an active component of serum taken from Corynebacterium parvum (C. parvum) infected mice treated with lipopolysaccharide (LPS). To locate the production site of TNF, we tried to block TNF production by using the following reagents; carrageenan, hydrocortisone, and trypan blue. Following the injection of a large dose of carrageenan, administered before C. parvum treatment, TNF production was completely blocked. However, when administered after C. parvum treatment but prior to LPS injection, no blockage was observed. Injecting hydrocortisone before the LPS injection also blocked TNF release. However, this treatment, when administered before the injection of C. parvum, had no observable influence on TNF production. A large dose of trypan blue, administered before the LPS injection, also blocked the release of TNF. A low dose of trypan blue resulted in only partial blockage of TNF production. A large dose of trypan blue, administered prior to C. parvum treatment, also showed partial blockage of TNF production. Macrophage-enriched peritoneal exudate cells (PEC), taken from mice infected with C. parvum, released TNF into the supernatant after stimulation with LPS. These results strongly suggest that the production site of TNF is located within the activated macrophage and deeply related with lysosome.

Animals↗
PubMed1983

Cytotoxicity of monofunctional alkylating agents. Methyl methanesulfonate and methyl-N'-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN1 and SN2 mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O6 position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect is potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS, MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMs kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.

Animals↗
PubMed1985

Comparative cytopathology of primary rat hepatocyte cultures exposed to aflatoxin B1, acetaminophen, and other hepatotoxins.

The cytomorphological alterations in primary monolayer cultures of Fischer 344 rat hepatocytes exposed continuously to lethal concentrations of various hepatotoxins were compared. Aflatoxin B1 (AFB1) at concentrations of 1 and 10 microM killed 67 and 97% hepatocytes, respectively, at 48 hr, as determined by release of lactate dehydrogenase (LDH) into culture medium, or by trypan blue uptake. AFB1 produced numerous radial finger-like projections (blebs) at attached margins of cytoplasm in almost all hepatocytes between 6 and 24 hr. Formation of blebs was dose dependent and preceded release of LDH and trypan blue uptake. Similar marginal blebs were produced by cycloheximide, 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, and senecionine. By comparison, acetaminophen, at equivalently lethal concentrations (4 or 16 mM) did not cause marginal blebs, but resulted in a similar time course of LDH release and trypan blue uptake. Carbon tetrachloride and bromobenzene also failed to produce blebs at lethal concentrations. Phalloidin and cytochalasin B rapidly produced smaller spherical blebs over the entire surface of all hepatocytes, distinct from the finger-like blebs produced only at the free attached margins of cells exposed to AFB1. Blebs and lethal injury by AFB1 were reduced by phenobarbitone or 3-methylcholanthrene pretreatments and by SKF-525-A in culture. The demonstration of morphological prelethal changes in hepatocytes injured by different classes of hepatotoxins in culture provides a means of differentiating the early biochemical mechanisms by which hepatotoxins and hepatocarcinogens lethally injure hepatocytes before membrane permeability to LDH and trypan blue is detectable.

Acetaminophen↗
PubMed1995

Liposomal entrapment of suramin(II): interaction of suramin with phospholipids of various chain lengths.

Previously, we reported that the entrapment of suramin in dipalmitoylphosphatidylcholine (DPPC, C16) multilamellar liposomes ranged from 25% to 65% and the addition of 30-50 mol% cholesterol (CHL) greatly reduced entrapment. Entrapment of small molecules similar to suramin, disodium 1,5-naphthalenedisulfonic acid (5.5%) and sodium 3-amino-2,7-naphthalenedisulfonic acid (1.2%), were very low. In the present study, the entrapment and interaction of suramin with dilauroylphosphatidylcholine (DLPC, C12), dimyristoylphosphatidylcholine (DMPC, C14), and distearoylphosphatidylcholine (DSPC, C18) liposomes was investigated. DLPC and DMPC showed 2-3-fold higher entrapment percentages (95.1% and 74.2%, respectively) than DPPC (37%). However, the entrapment with DSPC (29%) was about 25% lower than DPPC. Adding 50 mol% cholesterol greatly reduced suramin entrapment for all phospholipids. The entrapment of polysulfonated dyes such as Evans blue, Direct blue 1, or Trypan blue, which are structurally similar to suramin, was found to be in the same order of DLPC > DMPC > DPPC > DSPC. Differential scanning calorimetry of aqueous dispersions of DLPC and DMPC with suramin showed more apparent interaction than for DPPC and DSPC. These results suggest that a large portion of the associated suramin and other polysulfonated compounds results from binding to the surface of the phospholipid bilayer or intercalation into the liposomal bilayer. The phospholipid chain length effect on entrapment may be due to the lower net van der Waals interaction between hydrocarbon chains for shorter acyl chains which also increases the bilayer intermolecular spacing. Such effects could then increase the ability of suramin to interact with individual phospholipid molecules.

Azo Compounds↗
PubMed1991

Rat enterocyte injury by oxygen-dependent processes.

Neutrophils migrate to areas of inflammation and, when stimulated, produce O2-, H2O2, and other reactive O2 metabolites. To assess the effects of stimulated neutrophils on enterocytes, rat enterocytes were incubated with peripheral neutrophils. To assess cell viability, trypan blue exclusion and lactate dehydrogenase and protein release were measured. When 10(6) enterocytes/mL were incubated with 2.5 x 10(5) neutrophils/mL stimulated with phorbol myristate acetate, trypan blue exclusion decreased and lactate dehydrogenase and protein release increased. With the addition of 0.10 mg/mL of superoxide dismutase, trypan blue exclusion further decreased and lactate dehydrogenase and protein release increased. This suggests that H2O2- or H2O2/O2(-)-derived metabolites are more damaging to isolated enterocytes than O2-. To test this hypothesis, enterocytes were incubated with xanthine and increasing concentrations of xanthine oxidase in the presence and absence of superoxide dismutase. With increasing concentrations of xanthine oxidase, the cell number decreased and protein release increased. With the addition of superoxide dismutase, fewer cells were present, suggesting that cell lysis occurred. Protein release was further increased by the addition of superoxide dismutase. Enterocytes were then incubated with leucine and increasing concentrations of amino acid oxidase. With increasing concentrations of amino acid oxidase, trypan blue exclusion decreased and protein and lactate dehydrogenase release increased. These effects were ameliorated by the addition of 500 IU catalase/mL. These data suggest that O2- and H2O2, whether created by stimulated neutrophils or an enzyme-generating system, are damaging to isolated enterocytes. Superoxide dismutase did not offer enterocytes protection.

Animals↗
PubMed2003

Staining characteristics of preserved human amniotic membrane.

PURPOSE: Amniotic membrane is an ultra-thin cellophane-like membrane that is used in ocular surface reconstruction. We evaluated the staining characteristics of commonly available dyes on preserved human amniotic membrane to aid in handling of amniotic membrane during transplantation. METHODS: Five dyes, indocyanine green (2.5%, 1.0%, and 0.5%), fluorescein (0.25%), rose bengal (1%), lissamine green B (1%), and trypan blue (0.5%), were used to stain amniotic membrane. After staining, the specimens were observed under a dissecting microscope to evaluate for the uptake of the stains. Positively stained membranes were evaluated for the persistence of staining by placing them in 2 to 3 mL of balanced saline solution that was changed every 30 minutes over 6 hours. RESULTS: Preserved human amniotic membrane is stained by indocyanine green, rose bengal, lissamine green B, and trypan blue. Of these four dyes, only the membrane stained with 1% lissamine green B was free of stain after 120 minutes. Indocyanine green, rose bengal, and trypan blue continued to strongly stain the membrane after 24 hours. CONCLUSIONS: Indocyanine green, rose bengal, trypan blue, and lissamine green B all stain amniotic membrane. Lissamine green B appears to have advantages over the other dyes in that it will stain the membrane well, and in our model, dissipate in 120 minutes. Intraoperative staining with lissamine green B may be a simple and effective way to assist surgeons in the proper handling of amniotic membrane.

Amnion↗
PubMed1994

Oxygen free radical formation by rat hepatocytes during postanoxic reoxygenation: scavenging effect of albumin.

Free radical formation and reoxygenation injury were studied in rat hepatocytes perfused with Krebs-Henseleit bicarbonate buffer containing 1% or no albumin. After 2, 2.5, or 3 h of anoxia followed by 1 h reoxygenation in the absence of albumin, free radical formation assessed by low-level chemiluminescence and cell injury measured by lactate dehydrogenase (LDH) release and by trypan blue uptake increased proportionately. Chemiluminescence increased 4- to 7-fold, LDH release and trypan blue uptake increased 1.5- to 2-fold, compared with the end of anoxia. With 1% albumin, there was no increase in free radical formation during reoxygenation, and LDH release returned to control levels. There was a linear relation between the increase in chemiluminescence and the rise in LDH release (r2 = 0.83) and the increase in trypan blue uptake (r2 = 0.80), suggesting that free radical formation during reoxygenation is responsible for the cell injury. These experiments demonstrate that freshly isolated hepatocytes produce oxygen free radicals detectable by low-level chemiluminescence and that reoxygenation injury occurs after a relatively short period of anoxia (2-3 h). Albumin acts as a free radical scavenger, suppresses the release of reactive oxygen species, and significantly reduces reoxygenation injury.

Aerobiosis↗
PubMed1996

Trehalose-containing solutions enhance preservation of cultured endothelial cells.

BACKGROUND AND METHODS: We have developed two types of preservation solutions containing trehalose. ET-Kyoto solution (ET-K) is an extracellular type and IT-Kyoto solution (IT-K) is an intracellular type. In the present study we examined with in vitro assays the ability of ET-K, IT-K, Euro-Collins (EC), and University of Wisconsin (UW) solutions to preserve a murine endothelial cell line. The viability of cells stored in the solutions at 4 degrees C was determined by trypan blue exclusion and MTT assay. RESULTS: Trypan blue exclusion showed the viability after 48 hours of cold storage to be 49.5 +/- 4.7% (mean +/- standard error) in ET-K, 59.5 +/- 0.7% in IT-K, 29.2 +/- 2.5% in EC, and 55.3 +/- 7.6% in UW (ET-K or UW versus EC, p < 0.05; IT-K versus EC, p < 0.01). MTT assay absorbance values for cells after 48 hours of cold storage were 0.366 +/- 0.0066 (mean +/- standard error) in ET-K, 0.358 +/- 0.0044 in IT-K, 0.336 +/- 0.011 in EC, and 0.362 +/- 0.0019 in UW (ET-K or UW versus EC, p < 0.05). After 120 hours, absorbance values for cells were 0.303 +/- 0.0038 in ET-K, 0.269 +/- 0.0034 in IT-K, 0.186 +/- 0.011 in EC, and 0.265 +/- 0.0066 in UW (ET-K versus UW, p < 0.05; ET-K versus IT-K, p < 0.01; ET-K, IT-K or UW versus EC, p < 0.01). CONCLUSIONS: As far as the ability to preserve a murine endothelial cell line at a low temperature was concerned, the ET-K solution was superior to the UW solution, the IT-K solution and UW solution were equal, and the ET-K and IT-K solutions were superior to the EC solution.

Animals↗
PubMed1987

Differential effect of zinc on teratogen-induced inhibition of pinocytosis by cultured rat yolk sac.

Pinocytosis as measured by the uptake of 125I labelled PVP by the isolated cultured day 12 rat yolk sac was observed to be linear over a 4 h incubation period and to proceed at a rate of approximately 2.5 microliters/mg protein/h. Cadmium, anti-visceral yolk sac antibody (AVYS) and trypan blue all inhibited pinocytosis in a concentration-dependent fashion when added to the culture medium, although at low concentrations trypan blue was slightly stimulatory. The effect of zinc on the inhibition of pinocytosis by these three teratogens was studied. It was observed that zinc ameliorated the inhibitory effects of cadmium and AVYS, but had no effect on inhibition by trypan blue. These results indicate that the previously demonstrated protective action of zinc against cadmium-induced yolk sac dysfunction is not specific to that agent but extends to inhibition of pinocytosis by AVYS, and further suggest that, because of its refractoriness to zinc, trypan blue-induced inhibition of pinocytosis by yolk sac occurs by a mechanism different from that effected by cadmium and AVYS.

Animals↗