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PubMed2006

Comparative study of in vitro cytotoxicity of povidone-iodine in solution, in ointment or in a liposomal formulation (Repithel) and selected antiseptics.

The cytotoxicity of povidone-iodine in Repithel, Betaisodona ointment and Betaisodona solution was investigated using CHO-K1 cells. To estimate the vitality of test cells after 30 min contact time using vital dye neutral red, the following IC(50) were determined: 16-18% Repithel, 8-9% Betaisodona ointment and 1.8-2% Betaisodona solution; using MTT for detecting vitality, the IC(50) were: 5-10% Repithel, 1.3-2.5% Betaisodona ointment and 0.6-1.3% Betaisodona solution. Therefore, the first attack of the antiseptic agent iodine to mammalian cells is carried out on enzymes, perhaps by oxidation, followed by membrane attack. Murine fibroblasts were used to compare the cytotoxic impact of povidone-iodine with those of chlorhexidine digluconate (CHex), octenidine dihydrochloride (Oct) and polyhexamethylene biguanide (PHMB). On the base of molecular concentration, povidone-iodine is more than 20 times better tolerated by L929 cells than CHex, Oct or PHMB. Moreover, after 30 min contact of L929 cells with povidone-iodine, there is a temporarily cytotoxic reaction, which leads after 24 h culture to an unexpected revitalisation of murine fibroblasts. This phenomenon was not detected using CHex, Oct or PHMB. Povidone-iodine seems to be the most tolerated antiseptic in comparison with CHex, Oct or PHMB.

Administration, Topical↗
PubMedDate not supplied

Inhibition of fibroblast and smooth muscle cell proliferation and migration in vitro by a novel aminochromone U-67154.

Medial smooth muscle cell migration and neointimal proliferation are primary contributors to the delayed restenosis that occurs after percutaneous transluminal coronary angioplasty. In this study, we describe the antiproliferative and antichemotactic properties of U-67154, the parent compound of a series of novel aminochromones, determined using in vitro fibroblast and smooth muscle cell culture systems. U-67154 inhibited the induction of DNA synthesis in confluent BALB/c 3T3 fibroblasts and early-passage rat aortic smooth muscle cells by several different growth factors in a concentration-dependent manner. U-67154 similarly inhibited the proliferation of these cells stimulated by serum. Growth-factor-induced chemotaxis of fibroblasts and early-passage rat aortic smooth muscle cells also was inhibited by U-67154 in a concentration-dependent manner. The IC50s for all of these functions were similar (between 120 and 200 microM). Such antiproliferative and antichemotactic effects did not result from cytotoxicity (as measured by lactate dehydrogenase release, neutral red uptake or nonspecific inhibition of protein synthesis). Most important, inhibition of long-term proliferation of fibroblasts and early-passage smooth muscle cells by U-67154 was fully reversible upon removal of the drug. Thus, U-67154 represents a class of novel, noncytotoxic compounds that may prove useful in the treatment of proliferative disorders such as delayed restenosis after percutaneous transluminal coronary angioplasty.

3T3 Cells↗
PubMed1991

In vitro methods for the assessment of primary local effects of topically applied preparations.

Three 'alternative' methods for the screening of the primary irritation potentials of topically applied preparations and raw materials are presented as a test battery and their in vitro endpoints are compared with in vivo data. The first method is the intermediate test on the chorioallantoic membrane of hens' fertilized eggs, which is generally proposed for the prediction of the irritation potential of chemicals. Relevant test parameters, such as the time-dependent appearance of hemorrhages, vascular lysis and coagulation, are used to classify the test substances. Secondly, cytotoxicity tests with Balb/c 3T3 fibroblasts are used to characterize the influence of test materials on cellular homeostasis and viability, which is quantified by the dose-dependent uptake of Neutral red. As a second parameter changes in the cellular protein level can be easily measured by subsequent staining with Kenacid blue. Finally, the third approach to in vitro evaluation is presented in the form of the red blood cell assay, a rapid photometric assay which permits distinguishing basically between damage to the membrane and to proteins as endpoints which correlate with lesions observed on the conjunctiva, iris and cornea in the eye and the acute inflammatory responses evoked during epicutaneous irritancy testing. Changes in the optical behavior of oxyhemoglobin are used as the inherent indicator for monitoring both endpoints and their interrelations. These methods are proposed and can be used advantageously for testing newly developed topical preparations and their ingredients in view of their local irritation potential without further use of animal testing. Moreover, they can also be used for screening chemicals.

Administration, Topical↗
PubMed1995

Acetaminophen-induced toxicity to human epidermoid cell line A431 and hepatoblastoma cell line Hep G2, in vitro, is diminished by silymarin.

The skin and liver may be targets for cytotoxicity induced by oxidative drug metabolites. We used human epidermoid A431 cells and human hepatoblastoma Hep G2 cells as the experimental model. The aim of the study was to investigate and evaluate the effect of silymarin on acetaminophen (APAP)-induced toxicity under controlled conditions. Silymarin is known to be a potent antioxidant that diminishes toxicity induced by a variety of other hepatotoxins (e.g. Amanita phaloides, algae's toxins, carbon tetrachloride). Glutathione (GSH) depletion was enhanced by adding to the medium buthionine sulfoximine [L-buthionine-(S,R)-sulfoximine, BSO]. Cells were incubated with high-concentration 5-20 mM APAP or alpha-(minimum essential medium for 2-24 h to evaluate the drug's ability to reduce cytoviability. Viability was then quantitated by metabolism of the tetrazolium dyes (MTT) and neutral red (NR). Cytoviability was 100% for controls. For Hep G2 treated for 24 h with 20 mM, APAP viability was 56.0% by MTT and 62.5% by NR. BSO-treated cells showed an enhanced cytotoxicity, determined by both assays. Administration of 0.5 mM silymarin reduced cytotoxicity significantly. In A431 cells, treatment with 20 mM APAP reduced viability by 57% (MTT) and 69% (NR) versus control (100%). BSO further decreased viability. Since incubation with silymarin showed significant protection against APAP toxicity, it can be considered a cytoprotective agent in this in vitro model of drug toxicity. GSH concentrations in both cell lines decrease significantly after exposure to 20 mM APAP, or 0.5 mM versus control (p < 0.05), and increased (p < 0.001) if incubated with APAP and silymarin. The protective effect could be through mitochondrial membrane stabilization and/or an increase in available GSH.

Acetaminophen↗
PubMed1997

Biphasic effects of minoxidil on the proliferation and differentiation of normal human keratinocytes.

Minoxidil is the most used drug with proved effects in the treatment of androgenetic alopecia (AGA), but little is known about its pharmacological activity and target cells in hair follicles. As AGA is characterized by follicle atrophy, accelerated hair cycles and hair fiber thinning, we postulated that keratinocyte proliferation/differentiation is affected and we tested Minoxidil's effects on those parameters. Normal human keratinocytes (NHK) of follicular or epidermal origin were cultured in the presence of Minoxidil (0, 0.1, 1, 10, 100, 1,000 microM) during 5-8 days in various media (high-/low-calcium content, with or without serum). Proliferation was assessed by mitochondrial dehydrogenase activity (XTT), BrdU incorporation, lysosome numeration (neutral red incorporation) and total protein dosage. Drug-induced cytotoxicity was measured by lactate dehydrogenase release in culture supernatant, and pro-differentiating effects were evaluated by relative involucrin expression (ELISA dosage). On this basis, we showed that Minoxidil had biphasic effects on the proliferation and differentiation of NHK: Minoxidil stimulated NHK proliferation at micromolar doses, while antiproliferative, pro-differentiative and partially cytotoxic effects were observed with millimolar concentrations. We can hypothesize that Minoxidil hypertrichotic activity in vivo is possibly mediated by the maintenance of proliferative potential in follicular keratinocytes precociously committed to differentiation.

Cell Differentiation↗
PubMed2004

7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.

OBJECTIVE: Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms. METHODS AND RESULTS: Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage-inducible protein 153) at the mRNA but not at the protein level. 7-KC-treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs. CONCLUSIONS: 7-KC activates the ubiquitin-proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3 evocating autophagic processes.

Aorta↗
PubMed1996

A cytotoxic electronegative LDL subfraction is present in human plasma.

By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.

Adult↗
PubMed1986

Blood flow through cerebral collateral vessels in hypertensive and normotensive rats.

We tested the hypothesis that blood flow through cerebral collateral vessels is lower in stroke-prone spontaneously hypertensive rats (SHRSP) than in normotensive Wistar-Kyoto rats (WKY) after occlusion of the middle cerebral artery and during maximal vasodilatation. Cerebral blood flow, measured with microspheres, was similar in adult male SHRSP and WKY under control conditions. In both strains, occlusion of the middle cerebral artery reduced blood flow and vascular conductance to the territory of the occluded artery, as compared with homologous tissue on the side contralateral to the occlusion. The territory distal to the site of occlusion was identified by intravital demarcation with neutral red dye. In both strains, vasodilatation produced by seizures after occlusion of the middle cerebral artery produced minimal increases in blood flow to the territory of the occluded artery. Blood flow and vascular conductance to the territory of the occluded MCA were significantly lower in SHRSP than in WKY (p less than 0.05) after occlusion and during seizure after occlusion. We conclude that after occlusion of the middle cerebral artery, there is less blood flow through cerebral collateral vessels in SHRSP than in WKY. We speculate that the lower blood flow through collateral vessels in SHRSP may be related to structural differences in those vessels. Thus, the tendency toward infarction after occlusion of the middle cerebral artery in SHRSP may be related, at least in part, to a more limited dilator reserve of cerebral collateral vessels in SHRSP.

Animals↗
PubMed1990

Impairment of metabolic recovery with increasing periods of middle cerebral artery occlusion in rats.

We examined the consequences of reflow on metabolic recovery following increasing periods of focal ischemia. The middle cerebral artery of 21 Sprague-Dawley rats was occluded with a snare ligature for 1, 2, or 6 hours followed by 5, 4, or 0 hours of reflow, respectively (seven rats in each group). All animals were injected with neutral red for visual confirmation that the affected regions were reperfused. The brains were frozen in situ, and the concentrations of adenosine triphosphate, phosphocreatine, glycogen, and lactate were determined in those areas corresponding to the normally perfused medial ipsilateral cortex, the perifocal region, and the ischemic focus. Values for the 6 hours' occlusion with no reflow group served as a control to demonstrate restoration of metabolite concentrations. In both groups with reflow, the levels of high-energy phosphates were greater than control, but this effect of reflow was primarily significant for the group with 1 hour's occlusion (p less than 0.05). The levels of glycogen and lactate provided additional evidence that the extent of metabolite restoration was graded; following 2 hours of occlusion, metabolite recovery was compromised (p less than 0.05). Our data strongly support the concept that the window of opportunity for effective treatment of focal ischemia by reperfusion is narrow (of short duration).

Adenosine Triphosphate↗
PubMed2000

Biocompatibility and performance in vitro of a hemostatic gelatin sponge.

The biocompatibility of a hemostatic gelatin sponge (Cutanplast Standard) was evaluated in vitro. Cytotoxicity was assessed by neutral red uptake and amido black staining tests; genotoxicity was assayed using the Ames test, Sister Chromatides Exchanges (SCE) and chromosomal aberrations. The ability of the hemostatic gelatin sponge to induce platelet adhesion and release reaction was also determined. The undiluted extract of the test material was found to be cytotoxic, but cell viability was not affected by 1:2 and 1:10 diluted extract. The same extract was found to be non-genotoxic using the three assays for genotoxicity. A significant decrease of platelet number, as well as a significant release of platelet factor 4 was found to be caused by the solid material. In conclusion, Cutanplast Standard is neither cytotoxic nor genotoxic, while inducing platelet adhesion and release reaction when challenged with blood.

Absorbable Implants↗
PubMed2006

Synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) poly(ethylene glycol) tri-block co-polymers modified with collagen: a model surface suitable for cell interaction.

This study focused on the synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) tri-block co-polymer (PEG-PDLLG-PEG), and its modification with type-I collagen. To this aim, a PEG-PDLLG-PEG tri-block co-polymer was synthesized in two steps by reacting poly(ethylene glycol)bis(carboxymethyl)ether with thionyl chloride to obtain an acyl-halide-terminated poly(ethylene glycol) and subsequently coupling this compound to hydroxyl-terminated poly(D,L-lactide-co-glycolide) (PDLLG). The new carboxyl endgroups of PEG-PDLLG-PEG were subsequently reacted with N-hydroxysuccinimide (NHS) in the presence of the hetero-bifunctional cross-linking agent dicyclohexylcarbodiimide (DCC) in order to activate the co-polymer for coupling with collagen. PEG-PDLLG-PEG and its activated form PEG-PDLLG-NHS were characterized by Fourier transform infrared (FT-IR) and 1H-NMR spectroscopy. Molecular weights of the polymeric products were determined by SEC. Type-I collagen in phosphate buffer was reacted with PEG-PDLLG-NHS. The resultant product, PEG-PDLLG-Col, was characterized by FT-IR. This biopolymer was used for preparation of a suitable surface for cell growth experiment. To measure the degree of cell proliferation, the films prepared with PDLLG, PEG-PDLLG-NHS and PEG-PDLLG-Col were seeded with L929 mouse fibroblasts. Cell growth was followed by SEM photography and quantitated by the neutral red uptake assay. It was shown that the attachment of collagen significantly increased the number of cells on the co-polymers.

Animals↗
PubMed1976

Characterization of immunocompetent cells recovered from the respiratory tract and tracheobronchial lymph node of normal guinea pigs.

To gain insight into the structure of the lung's immune system, the identity and function of immunocompetent cells in the broncho-alveolar air spaces and tracheobronchial lymph nodes of normal guinea pigs were examined. Guinea pig thymus-derived (T) lymphocytes (mediators of cellular immunity) were identified by their ability to form rosettes with rabbit erythrocytes (E rosettes). Bone marrow-derived (B) lymphocytes (mediators of humoral immunity) were identified by their ability to form rosettes with sheep erythrocytes sensitized with antibody and complement (EAC rosettes) and by the presence of surface immunoglobulin, detected by direct immunofluorescence. Total lung lavage yielded 14+/-5.0 X 10(6) (mean +/- SD) cells. There were 68+/-5 per cent cells of the monocyte-macrophage series as judged by morphology, the ingestion of latex particles, and the uptake of neutral red; 20+/-9 per cent were eosinophils, and 12+/-5 per cent were lymphocytes. Stable populations of T and B lymphocytes were recovered from guinea pig airways and tracheobronchial lymph nodes. T cells represented 76 per cent of lymphocytes from the airways and 64 per cent of lymphocytes in tracheobronchial lymph node; B cells equalled 14 per cent and 30 per cent, respectively. The functional potential of T cells present in the lung and tracheobronchial lymph node was demonstrated by their proliferative response to phytohemagglutinin. These results indicated that, although macrophages are the predominant cell type, viable T and B lymphocytes are present in the guinea pig broncho-alveolar air space. The composition of these cells is comparable to what has been observed in normal human lungs and provides preliminary evidence that the guinea pig may be a useful model for humans. This report also describes a method using 2 vital stains whereby macrophages, lymphocytes, and rosetted lymphocytes can be distinguished in unseparated cell suspensions prepared from bronchial aspirates.

Animals↗
PubMed1984

Processing of lung lavage fluid causes variability in bronchoalveolar cell count.

To study the effects on cell counts of different ways of processing bronchoalveolar lavage (BAL) fluid, we performed 77 BAL in 19 healthy control subjects and in 58 patients with sarcoidosis. We investigated the role of readers, hemocytometers, mode of identification of macrophages, cell washing, and speed of cytocentrifugation. No significant effect of reader or hemocytometer was observed. The percentage of macrophages, determined as large cells stained with neutral red in a Malassez hemocytometer (63.9 +/- 24.6%, mean +/- SD) was lower than the percentage of macrophages determined by May Grunwald Giemsa staining (76.3 +/- 19.2%, p less than 10(-9)). Cell counts decreased 34% after 2 washings (p less than 0.001), and more lymphocytes were counted after cytocentrifugation at 90 g (33.2 +/- 25.3%) than at 23 g (27.7 +/- 22.1%). We conclude that bronchoalveolar cell counts vary with changes in processing lung lavage fluid and that this variability should be considered when using BAL cell counts.

Bronchi↗
PubMed2003

Role of PPAR-gamma ligands in neuroprotection against glutamate-induced cytotoxicity in retinal ganglion cells.

PURPOSE: The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is the target of the insulin sensitizing thiazolidinediones (TZDs), a class of drugs used in the treatment of type 2 diabetes mellitus. Glaucoma and other retinal disorders are some of the major complications in diabetes. In the present study, the role that PPAR-gamma ligands play in protecting retinal ganglion cells (RGC-5) against glutamate insult was explored. METHODS: Transformed rat RGC (RGC-5 cells) and two PPAR-gamma agonists, 15-deoxy-D(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone were used. RGC-5 cells were incubated with either of the PPAR-gamma ligands and were exposed to either L-glutamic acid or buthionine sulfoximine (BSO). Cell viability was determined with the neutral red dye uptake assay. Levels of PPAR-gamma receptor proteins were monitored by immunoblot analysis. RESULTS: Glutamate treatment resulted in RGC-5 cell death, and both 15d-PGJ2 and troglitazone protected the RGC-5 cells from glutamate cytotoxicity. The neuroprotective concentrations of both compounds ranged from approximately 1 to 5 micro M. Troglitazone further protected against BSO toxicity, whereas 15d-PGJ2 did not. Glutamate treatment appears to exert its cytotoxicity through oxidative damage, because pretreatment of RGC-5 cells with the antioxidants N-acetyl cysteine (NAC) and thiourea resulted in the reversal of glutamate cytotoxicity. Furthermore, the glutamate effect was not reversed by pretreatment with MK801 or DL-threo-betabenzyloxyaspartate (DL-TBOA), suggesting that glutamate cytotoxicity is not mediated through the NMDA receptor and/or glutamate transporter, respectively. Levels of PPAR-gamma receptor protein did not show any appreciable change in response to glutamate exposure, with or without 15d-PGJ2 or troglitazone. CONCLUSIONS: Two PPAR-gamma ligands, 15d-PGJ2 and troglitazone, protect RGC-5, an established transformed rat retinal ganglion cell line, against glutamate cytotoxicity. The neuroprotective effects of the two compounds appear to be mediated through an antioxidant rather than a PPAR-gamma-dependent pathway. These results suggest that PPAR-gamma agonists, in addition to improving insulin sensitivity, may also provide a valuable antioxidant benefit that could prove valuable in targeting ocular complications including glaucoma.

Animals↗
PubMed2005

In vitro study of inflammatory potential and toxicity profile of latanoprost, travoprost, and bimatoprost in conjunctiva-derived epithelial cells.

PURPOSE: Conjunctiva-derived epithelial cells were used to investigate, in vitro, the expression of various inflammation-associated markers known to be overexpressed in patients with glaucoma after contact with the three major commercially available eye drops containing prostaglandin analogues. The impact on cellular viability and apoptosis in the same cell line was evaluated, to address the possible proinflammatory and/or toxic origin of the most frequent clinical impairments induced by prostanoids (i.e., conjunctival hyperemia). METHODS: Conjunctiva-derived cells were treated in vitro with the commercial solutions of latanoprost, travoprost, bimatoprost, prostaglandin (PG)F2alpha, tumor necrosis factor (TNF)-alpha, and different concentrations of benzalkonium chloride (BAC). Expressions of three inflammation- and immune-related markers, intercellular adhesion molecule (ICAM)-1, platelet-endothelial cell adhesion molecule (PECAM)-1 and HLA DR, were evaluated with flow cytometry after 24 to 72 hours of contact at low, subtoxic concentrations. Toxicological tests were also performed with cold-light cytofluorometry, in which cellular viability and apoptosis were evaluated with the neutral red and Hoechst/propidium iodide tests, respectively. RESULTS: TNFalpha induced or stimulated expression of the three inflammatory markers, whereas the PGF2alpha, latanoprost, travoprost, and bimatoprost solutions did not induce an increase in these markers and even produced a marked reduction of ICAM-1 and PECAM-1 expression in those solutions most concentrated in BAC, thus suggesting a toxic phenomenon in cellular membranes induced by the preservative rather than the medication itself. Cytotoxic assays confirmed this hypothesis and showed significant toxicity with prostaglandin analogues after prolonged contact, proportional to the concentration of BAC in the solution and similar to that of the corresponding concentration of BAC alone, bimatoprost having both the least concentration of BAC and the least cytotoxic in these experimental conditions. CONCLUSIONS: The comparison of latanoprost, travoprost, and bimatoprost, in their commercial formulations, showed that none of them appeared to induce direct stimulation of the inflammatory pathways involving adhesion molecules or class II antigens, although these markers have been found ex vivo in conjunctival specimens from patients treated with prostaglandins. In fact, their toxicity was mild and seemed to be primarily related to the concentration of BAC, their common preservative, which may be the major factor responsible for long-term ocular surface reactions in patients receiving topical prostaglandins, but most likely is not a factor in early and transient conjunctival hyperemia.

Amides↗
PubMed2005

In vitro comparison of cytoprotective and antioxidative effects of latanoprost, travoprost, and bimatoprost on conjunctiva-derived epithelial cells.

PURPOSE: In a previous study, it was demonstrated that in vitro in a human conjunctiva-derived cell line, latanoprost in its commercial presentation appeared to be less toxic than the benzalkonium chloride (BAC) it contains as a preservative. Through a microplate cytometry technique, the investigation was furthered by study of whether the three commercially available antiglaucoma prostaglandin analogs could protect the same cell line in vitro against BAC toxicity and whether an antioxidative mechanism could be involved in such prostaglandin effects. METHODS: Human conjunctiva-derived epithelial cells from the Chang cell line were exposed to three prostaglandins in their commercial presentation (latanoprost, travoprost, and bimatoprost) and to three concentrations of BAC (0.02%, 0.015%, and 0.005%), corresponding to the concentrations contained in the three prostaglandin eyedrops. Each solution was diluted to 1/10 and was applied for 30 minutes Cellular membrane integrity, cytosolic H2O2, cytosolic O2*- and apoptosis were evaluated using neutral red, H2DCF-DA, hydroethidine, and Yopro-1 probes, respectively. RESULTS: Cellular viability decreased as BAC concentration increased, but it was accompanied by concentration-dependent toxicity. Toxicity of latanoprost and travoprost commercial solutions was statistically significantly lower than their respective BAC concentrations (P < 0.01), whereas bimatoprost induced no significant effects. There was a statistically significant decrease in H2O2 detection with cells exposed to latanoprost (P < 0.01) and travoprost (P < 0.01) and a lower detection of O2*- with cells exposed to latanoprost (P < 0.01) compared with the corresponding BAC concentration alone. The Yopro-1 test showed a BAC-induced apoptotic effect that increased with its concentration. Latanoprost and travoprost produced proapoptotic effects compared with control (P < 0.01), but these were lower than their respective preservative concentrations (statistically significant difference; P < 0.01). CONCLUSIONS: Latanoprost and travoprost were responsible for significant protective effects against BAC toxicity on conjunctiva-derived epithelial cells in vitro, probably related to their antioxidative properties. The low toxicity of the bimatoprost solution did not reveal a possible antioxidative effect. Reduced reactive oxygen species production could be the main mechanism by which prostaglandin analogs protect epithelial cells from the proapoptotic effects of BAC. Further studies will be useful to confirm this hypothesis.

Amides↗
PubMed2006

Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis.

PURPOSE: To investigate in vitro whether eye toxicity is attributable to the preservative or the fluoroquinolone used in ophthalmic formulations. METHODS: Corneal and conjunctival cell lines were incubated with preserved (benzalkonium chloride [BAC]) or unpreserved ofloxacin solutions for 15 minutes. Several concentrations of BAC were also tested (0.0025%-0.01%). Membrane integrity, reactive oxygen species, and superoxide anion production were assessed with the neutral red test, the 2',7'-dichlorofluorescein diacetate test, and the dihydroethidium test, respectively. P2X7 cell death receptor activation was evaluated using the YO-PRO-1 assay and apoptosis (chromatin condensation and translocation of phosphatidylserine) using the Hoechst 33342 and annexin V-FITC dyes. Tests were performed with microplate cytofluorometry, inverted fluorescence microscopy, and flow cytometry. RESULTS: The preserved solution and all tested BAC concentrations induced a significant decrease in membrane integrity, unlike the unpreserved ofloxacin. Reactive oxygen species and superoxide anion productions observed for all solutions were significantly higher for the preserved ofloxacin and BAC solutions, which also induced apoptosis (chromatin condensation and translocation of phosphatidylserine) through P2X7 pore opening, whereas unpreserved ofloxacin did not. CONCLUSIONS: The cytotoxicity observed with fluoroquinolone eye drops seems to be caused mainly by the preservative, which induced P2X7 cell death receptor activation associated with oxidative stress and apoptosis. Therefore, it is recommended that fluoroquinolone be used in preservative-free formulations.

Animals↗
PubMed2001

Dynamin is involved in human epithelial cell vacuolation caused by the Helicobacter pylori-produced cytotoxin VacA.

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. To elucidate the molecular mechanism of vacuole formation by VacA, we examined the participation of dynamin, a GTPase functioning in intracellular vesicle formation, in human HeLa cells. Immunocytochemistry revealed that endogenous dynamin was localized to vacuoles induced by VacA. In cells transiently transfected with a GTPase-defective (dominant-negative) dynamin mutant, VacA failed to induce vacuolation. In contrast, VacA did induce vacuolation in cells transiently transfected with wild-type dynamin. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with the dominant-negative dynamin mutant. In contrast, uptake was markedly enhanced in cells stably transfected with wild-type dynamin. Moreover, VacA cytopathic effects on the viability of HeLa cells were inhibited in cells stably transfected with dominant-negative dynamin-1. Sequential immunocytochemical observation confirmed that expression of dominant-negative dynamin did not affect VacA attachment to or internalization into HeLa cells. We suggest that dynamin is involved in the intracellular vacuolation induced by VacA.

Bacterial Proteins↗