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PubMed1997

Analyzing cytotoxic T lymphocyte activity: a simple and reliable flow cytometry-based assay.

A flow cytometry-based assay for analyzing cytotoxic T lymphocyte (CTL) activity is presented. This new approach is characterized by easy handling, the generation of highly reproducible data sets and is not dependent on the use of radioactivity. Before exposure to primed CTL effector cells the target cells were labeled with the green fluorescent dye DiO18(3) which is incorporated stably into the cell membrane. After a 4-h incubation period, samples were counterstained with the red fluorescent nuclear dye propidium iodide in order to permit discrimination between live and dead cells within both cell populations. The assay has been used to quantitate CTL effector activity against allogeneic lymphoblasts. Results derived from this novel flow cytometry assay show an excellent correlation (r = 0.988) with data obtained using the standard 51chromium release assay. An additional advantage of the assay is that freshly prepared splenocytes may be used as target cells because culturing and activation of target cells is no longer required. The results demonstrate that the fluorescent dyes DiO18(3) and propidium iodide in combination with flow cytometry permit accurate analysis of cytotoxic T cell activity.

Animals↗
PubMed1986

The effect of phenothiazines upon maintenance of membrane integrity in the cultured myocardial cell.

The cultured myocardial cell provides a defined model for examining factors which are responsible for maintaining cellular viability and sarcolemmal integrity. Our data indicates that the spontaneous loss of myocyte membrane integrity is a calcium-dependent process and thus provides a method for examining the mechanism through which calcium exerts this effect. Antimyosin antibody staining and propidium iodide uptake were used to quantitate membrane integrity. The integrity of the cell membrane was inversely related to the calcium concentration in the culture medium. This loss of membrane integrity was calmodulin-dependent as demonstrated by the following: phenothiazines (trifluoperazine greater than chlorpromazine greater than promethazine) and structurally dissimilar calmodulin-inhibitors prevented the formation of sarcolemmal defects at concentrations similar to those known to inhibit calmodulin; phenothiazines and calcium demonstrated a competitive interaction with respect to this effect on membrane integrity. Electron microscopy confirmed the integrity of the sarcolemma of the cells exposed to high phenothiazine concentrations although metabolic alterations occurred in these cells as evidenced by an increased membrane permeability to the low molecular weight probe propidium iodide, degenerative changes in the fine structure of the mitochondria, the accumulation of autophagic vacuoles in the cytoplasm and the loss of contractile ability. These findings indicate that calmodulin inhibitory compounds are capable of preserving the membrane integrity of cardiac myocytes, interfering with a calcium-dependent process that is associated with the spontaneous attrition of these cells in culture. Significant intracellular alterations appear at high doses of these agents even while the sarcolemma is free of gross defects.

Animals↗
PubMed2002

Methamphetamine induces apoptosis in an immortalized rat striatal cell line by activating the mitochondrial cell death pathway.

Methamphetamine is a neurotoxic drug of abuse known to cause cell death both in vitro and in vivo. Nevertheless, the molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that methamphetamine-induced apoptosis is associated with early (2 h) overexpression of bax, decreases of mitochondrial membrane potential and oxygen consumption as well as release of cytochrome c from mitochondria. In addition, activated caspase-9 was detected at 4 h post-METH exposure. Cell death was detectable by annexin V and propidium iodide staining after 8 h of methamphetamine exposure. At that time, the majority of the cells were stained by annexin V alone, with some cells being stained for both annexin V and propidium iodide. Moreover, cleavage of caspase-3, poly (ADP-ribose) polymerase and DNA fragmentation-related factor 45 was detected at 8 h post drug treatment. These results indicate that methamphetamine-induced apoptotic cell death results from early overexpression of bax, reduction of mitochondrial respiration and membrane potential and release of mitochondrial cytochrome c with subsequent activation of the caspase cascade.

Animals↗
PubMed1998

Simultaneous measurement of cell cycle and apoptotic cell death.

Understanding the dynamics of cell death in conjunction with those of cell cycle can be illuminating in the investigation of various cellular behaviors. Robust assays for measuring such parameters are invaluable. Many assays of apoptosis and/or cell cycle use flow cytometry. This chapter describes two different assays to measure apoptosis and cell cycle simultaneously using flow cytometry. The first involves the use of terminal transferase (the "TUNEL" assay) together with propidium iodide for identification of cell cycle. The second uses fluorescently labeled annexin V, together with propidium iodide as an indicator of cell membrane integrity; and additionally Hoechst 33342 for determination of cell cycle. Each assay has positive and negative attributes. The terminal transferase assay is performed using fixed cells and is therefore useful in the analysis of samples collected over time. The annexin V assay is performed using unfixed cells, and thus provides information regarding membrane integrity. Other practical aspects of both assays are discussed.

Animals↗
PubMed1999

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry.

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.

Acrosome↗
PubMed1999

LIVE/DEAD BacLight : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water.

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.

Chlorine↗
PubMed2001

Decrease of cyclin D1 in the human lung adenocarcinoma cell line A-427 by 7-hydroxycoumarin.

Coumarin in vivo has antitumor activity in various types of cancer. In vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.

Adenocarcinoma↗
PubMed2001

Hyperoxia- and hypoxia-mediated activation of polymorphonuclear leukocytes: a comparison of cord and adult venous blood.

BACKGROUND: Among the most prominent changes occurring in newborn infants is the exposure of tissues and blood cells to increased oxygen tension. This increase is even more pronounced in neonatal resuscitation using 100% oxygen, currently recommended in the published guidelines. OBJECTIVE: To analyse the response of neonatal and adult polymorphonuclear neutrophils (PMN) to high or low oxygen tension in vitro. MATERIALS: Neonatal cord blood and adult venous blood without previous contact to ambient air was exposed to 0, 21, or 100% oxygen for 30 min followed by incubation for up to 24 h. METHODS: Flow cytometry was used to assess PMN activation as indicated by downregulation of L-selectin expression. Cell viability was quantified by the amount of propidium iodide uptake. RESULTS: In adult PMN, L-selectin downregulation was greatly accelerated by hypoxia (PO2=27.2+/-3.4 mmHg) compared with both normoxia (PO2=71.0+/-11.0 mmHg) or hyperoxia (PO2=653.2+/-9.4) (P<0.05). In contrast, hyperoxia was the most potent stimulus for cord blood PMN, compared with both normoxia and hypoxia (P<0.05). Evidence of necrosis as indicated by positive staining for propidium iodide was similar in cord blood (10 h: 5.83% in oxygen) and in adult blood (10 h: 6.45% in oxygen). No differences were found between exposure to hypoxia, normoxia, or hyperoxia. CONCLUSION: Oxygen exposure of neonatal PMN leads to a more pronounced activation as compared with adult cells. Exposure towards high concentrations of oxygen may contribute to inflammatory processes during early neonatal life.

Adult↗
PubMed2002

Cyclopentenone prostaglandins induce caspase activation and apoptosis in dendritic cells by a PPAR-gamma-independent mechanism: regulation by inflammatory and T cell-derived stimuli.

OBJECTIVE: Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC. MATERIALS AND METHODS: Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR. RESULTS: Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability. CONCLUSIONS: Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity.

Fas (APO-1/CD95) is a cell surface receptor, initially identified in lymphoid cells, but more recently detected in the central nervous system under pathologic conditions. Ligation of the fas receptor by fas ligand or by agonist antibodies induces apoptotic cell death in most fas-expressing cells. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we detected fas expression on astrocytes but not on neurons. Such expression differs from our previous results using cultures of human adult central nervous system-derived cells, which demonstrated fas expression on oligodendrocytes but not on astrocytes; the oligodendrocytes were susceptible to cell death via this pathway. Using multiple assays of cell death, including nuclear propidium iodide and TUNEL staining to detect nuclear-directed injury, cytofluorometric propidium iodide inclusion, and lactate dehydrogenase release to detect membrane-directed injury, we found that fas ligation, however, did not induce cell death in the cultured fetal astrocytes. Cytokines that augmented (gamma-interferon) or inhibited (interleukin-4) fetal astrocyte proliferation did not alter fas expression or resistance to fas ligation. Cells obtained immediately ex vivo from human fetal but not from adult central nervous system tissue expressed fas; such expression was restricted to astrocytes as assessed by dual-stain immunohistochemistry. The fetal central nervous system cells did not express fas ligand. Our findings indicate that fas expression on central nervous system cells may reflect their state of maturity; expression may not, however, always be coupled to susceptibility to cell death via this pathway.

Adult↗
PubMed2001

Chondrocyte necrosis and apoptosis in impact damaged articular cartilage.

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.

Animals↗
PubMed2003

Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca(2+) homeostasis, and cytokine gene expression.

Mercury is a highly toxic heavy metal; exposure to mercury in humans and animals causes damage in several organs or systems including the immune system. To characterize the toxicity of mercury in the immune cells, the cytotoxic effects of inorganic mercury were studied in two distinct lymphoma lines, the murine T lymphoma (EL4) and B lymphoma (A20) cells. Mercury concentration-dependently decreased cell viability, membrane integrity, and proliferation in both EL4 and A20 cells. Mercury increased the reactive oxygen species (ROS) production in both EL4 and A20 cells, and pretreatment with antioxidants reversed mercury-induced ROS generation. Pretreatment of cells with antioxidants N-acetylcysteine (NAC) and silymarin decreased mercury-induced lactate dehydrogenase (LDH) release in both types of cells; however, Ca(2+) channel blocker lanthanum (La(2+)) decreased it only in A20 cells. The mode of cytotoxicity was a mixture of both apoptosis and necrosis. Mercury-induced apoptosis and necrosis in the two cell lines were indicated by staining with Hoechst 33258, propidium iodide, and co-staining with annexin V and propidium iodide. Both mercury-induced apoptosis and necrosis were attenuated by antioxidants. Mercury increased gene expression of IL-4 and TNFalpha in EL4 cells; these cytokines were not expressed in A20 cells. Data suggested different pathways of mercury-induced cytotoxicity in T and B lymphoma cells and involvement of ROS, Ca(2+) homeostasis, and inflammatory cytokine gene expression.

Animals↗
PubMed2000

Energetics of DNA intercalation reactions.

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.

Animals↗
PubMed1985

Flexibility of the molecular forms of acetylcholinesterase measured with steady-state and time-correlated fluorescence polarization spectroscopy.

Steady-state and time-correlated fluorescence polarizations have been examined for selected complexes and covalent conjugates of the 11S and (17 + 13)S forms of Torpedo acetylcholinesterase. The 11S form exists as a tetramer of apparently identical subunits, whereas the (17 + 13)S forms contain two or three sets of tetramers disulfide-linked to an elongated collagen-like tail unit. Pyrenebutyl methylphosphonofluoridate and (dansylsulfonamido)pentyl methylphosphonofluoridate were conjugated at the active center serine whereas propidium was employed as a fluorescent ligand for the spatially removed peripheral anionic site. Steady-state polarization of the pyrenebutyl conjugates indicates rotational correlation times of approximately 400 ns for the 11S species and greater than 1100 ns for the (17 + 13)S species. Hence, the tail unit severely restricts rotational motion of the catalytic subunits. Time-correlated fluorescence polarization analysis of the 11S species indicates multiple rotational correlation times. Anisotropy decay of the propidium complex (tau = 6 ns) occurs in exponential manner with a rotational correlation time of approximately 150 ns, while covalent adducts at the active center exhibit rotational correlation times greater than or equal to 300 ns. Anisotropy decay of the (dansylsulfonamido)pentyl conjugate (tau = 16 ns) appears exponential with a correlation time of approximately 320 ns, whereas decay of the pyrenebutyl conjugate (tau = 100 ns) is described by two correlation times, phi S = 18 ns and phi L = 320 ns, of small (15%) and large (85%) amplitudes, respectively. Two limiting models have been considered to explain the results.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1999

Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect.

Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions were simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed KS values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.

Acetylcholinesterase↗
PubMed2001

Hydration changes for DNA intercalation reactions.

The hydration changes that accompany the DNA binding of five intercalators (ethidium, propidium, proflavine, daunomycin, and 7-aminoactinomycin D) were measured by the osmotic stress method with use of the osmolytes betaine, sucrose, and triethylene glycol. Water uptake was found to accompany complex formation for all intercalators except ethidium. The difference in the number of bound water molecules between the complex and the free reactants (Deltan(w)) was different for each intercalator. The values found for Deltan(w) were the following: propidium, +6; daunomycin, +18; proflavine, +30; and 7-aminoactinomycin D, +32. For ethidium binding to DNA a value of Deltan(w) = +0.25(+/-0.3) was found, indicating that within experimental error no water was released or taken up upon complex formation. Intercalation association constants measured in D2O were found to increase relative to values measured in H2O for all compounds except ethidium. A positive correlation between the ratio of binding constants (K(D2O)/K(H2O)) and Deltan(w) was found. These combined studies identify water as an important thermodynamic participant in the formation of certain intercalation complexes.

DNA↗
PubMed1998

Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line.

PURPOSE: An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/toxicity by pharmaceuticals METHODS: Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitro cytotoxicity tests, 3-(4,5-dimethlthiazol-2-yl)-205-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. RESULTS: Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine approximately equal to dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2 EDTA)>polyvinyl alcohol (PVA) > methylparaben; and for CDs :alpha- CD > dimethyl beta-cyclodextrin (DM-beta-CD) > sulfobutyl ether beta-cyclodextrin ((SBE)7m-beta-CD approximately equal to hydroxypropyl-beta-cyclodextrin (HP-beta-CD) > lambda CD. In consideration of the in vivo clinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure time (30 and 60 min). CONCLUSIONS: Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivo coreal toxicity of ocularly applied compounds.

Cell Line, Transformed↗
PubMed1997

Pharmacological evidence that the activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells during chemical hypoxia.

1. In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium-iodide positive dead cells. 2. Extracellular Na+ removal, which activates the Na(+)-Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. 3. During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent. 4. The blockade of the Na(+)-Ca2+ exchanger by the amiloride derivative 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia. 5. The inhibition of the Na(+)-Ca2+ antiporter enhanced chemical hypoxia-induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na(+)-Ca2+ antiporter during chemical hypoxia can lead to increased cell damage. 6. Collectively, these results suggest that activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

Amiloride↗