Maria Engelke

Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, Laurdan generalized polarization and carboxyfluorescein leakage studies

The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.

Carotenoid incorporation into natural membranes from artificial carriers: liposomes and β-cyclodextrins

Liposomes and beta-cyclodextrin (beta-CD) have been used as carriers for the incorporation of three dietary carotenoids (beta-carotene (BC), lutein (LUT) and canthaxanthin (CTX)) into plasma, mitochondrial, microsomal and nuclear membrane fractions from pig liver cells or the retinal epithelial cell line D407. The uptake dynamics of the carotenoids from the carriers to the organelle membranes and their incorporation yield (IY) was followed by incubations at pH 7.4 for up to 3 h. The mean IYs saturated between 0.1 and 0.9 after 10-30 min of incubation, depending on membrane characteristics (cholesterol to phospholipid ratio) and carotenoid specificity. Mitochondrial membranes (more fluid) favour the incorporation of BC (non-polar), while plasma membranes (more rigid) facilitate the incorporation of lutein, the most polar carotenoid. A high susceptibility of BC to degradation in the microsomal suspension was observed by parallel incubations with/without 2,6-di-t-buthyl-p-cresol (BHT) as antioxidant additive. The beta-CD carrier showed to be more effective for the incorporation of lutein while BC was incorporated equally into natural membranes either from liposomes or from cyclodextrins. The presence of cytosol in the incubation mixture had no significant effects on the carotenoid incorporations.

Effect of inhalation anaesthetics on the phase behaviour, permeability and order of phosphatidylcholine bilayers

We have used differential scanning calorimetry and fluorescence anisotropy measurements to investigate the effect of five inhalation anaesthetics of diverse chemical structure (halothane, enflurane, n-pentane, chloroform and diethylether) on the phase behaviour of liposomes prepared from dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), respectively. The incorporation of these anaesthetics induced a decrease of the phase transition temperature and/or a broadening of the phase transition peak depending on the transverse localisation of the investigated anaesthetic. At high anaesthetic concentrations we observed the disappearance of the pretransition peak and the appearance of a shoulder on the main phase transition peak due to the domain formation of the anaesthetics. An anaesthetic induced carboxyfluorescein efflux from the vesicle lumen was completed within a few minutes after the addition of the anaesthetics, probably resulting from a transient formation of membrane holes. All results are discussed with regard to the physicochemical properties of the anaesthetics applied.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 test

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.

Staurosporine‐induced apoptosis in human cornea epithelial cells in vitro

Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV‐40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes.

Perturbation of artificial and biological membranes by organic compounds of aliphatic, alicyclic and aromatic structure

Aliphatic, alicyclic and aromatic hydrocarbons interact with biological membranes. Until now little has been known about their mode of interaction with the membrane bilayer and membrane integral proteins with toxic effects to cells. The lipid theory hypothesis explains the toxic effects by the organic solvent-induced disorder in the lipid bilayer, which indirectly affects the function of membrane-embedded proteins. The extent of bilayer perturbations is ascribed to the solvent accumulation in the bilayer, which is related only to the lipophilicity of the molecule, independent of the chemical structure. In this study the fluidizing effects of aliphatic, alicyclic and aromatic hydrocarbons were compared. Membrane fluidity changes were estimated from the pyrene excimer formation, using pyrene and pyrene derivatives to label specifically the localization of solvent molecules in the transverse plane of the bilayer. Liposomal, microsomal and synaptosomal membrane preparations were evaluated because proteins and cholesterol, as natural membrane components, increase the bilayer order and reduce the organic solvent membrane/buffer partition. In the concentration range investigated, only the aromatic solvents disorder the lipid bilayer, with the greatest perturbation in the centre of the bilayer. These results are related to structural properties of the organic solvents investigated.

Heterogeneity of microsomal membrane fluidity: evaluation using intrinsic tryptophan energy transfer to pyrene probes

Membrane fluidity measurements based on excimer formation of pyrene and pyrene derivatives as a measure of lateral diffusion yield a decreased fluidity in the presence of proteins [1-3]. It was the aim of our study to investigate whether the reduced excimer formation is due to a rigidifying effect of proteins on the whole membrane or if the fluorophore mobility is mainly hindered in the immediate protein environment. Resonance energy transfer in microsomal membranes between intrinsic tryptophan residues and pyrene were used to study the excimer formation rate in the vicinity of proteins. The excimer-to-monomer fluorescence ratio at excitation via resonance energy transfer is lower than that observed for the direct excitation. The results suggest that, because of a reduced fluidity in the neighbourhood of proteins, pyrene and pyrene fatty acids do not diffuse homogeneously in the membrane plane. A fluidity gradient exists from the membrane proteins to the bulk lipid.

A human hemi-cornea model for eye irritation testing: Quality control of production, reliability and predictive capacity

We have developed a 3-dimensional human hemi-cornea which comprises an immortalized epithelial cell line and keratocytes embedded in a collagen stroma. In the present study, we have used MTT reduction of the whole tissue to clarify whether the production of this complex 3-D-model is transferable into other laboratories and whether these tissues can be constructed reproducibly. Our results demonstrate the reproducible production of the hemi-cornea model according to standard operation procedures using 15 independent batches of reconstructed hemi-cornea models in two independent laboratories each. Furthermore, the hemi-cornea tissues have been treated with 20 chemicals of different eye-irritating potential under blind conditions to assess the performance and limitations of our test system comparing three different prediction models. The most suitable prediction model revealed an overall in vitro-in vivo concordance of 80% and 70% in the participating laboratories, respectively, and an inter-laboratory concordance of 80%. Sensitivity of the test was 77% and specificity was between 57% and 86% to discriminate classified from non-classified chemicals. We conclude that additional physiologically relevant endpoints in both epithelium and stroma have to be developed for the reliable prediction of all GHS classes of eye irritation in one stand alone test system.

A miniaturized solid contact test with Arthrobacter globiformis for the assessment of the environmental impact of silver nanoparticles.

Silver nanoparticles (AgNPs) are widely applied for their antibacterial activity. Their increasing use in consumer products implies that they will find their way into the environment via wastewater-treatment plants. The aim of the present study was to compare the ecotoxicological impact of 2 differently designed AgNPs using the solid contact test for the bacterial strain Arthrobacter globiformis. In addition, a miniaturized version of this test system was established, which requires only small-sized samples because AgNPs are produced in small quantities during the design level. The results demonstrate that the solid contact test can be performed in 24-well microplates and that the miniaturized test system fulfills the validity criterion. Soils spiked with AgNPs showed a concentration-dependent reduction of Arthrobacter dehydrogenase activity for both AgNPs and Ag ions (Ag(+)). The toxic effect of the investigated AgNPs on the bacterial viability differed by 1 order of magnitude and can be related to the release of dissolved Ag(+). The release of dissolved Ag(+) can be attributed to particle size and surface area or to the fact that AgNPs are in either metallic or oxide form. Environ