Interaction of imidazolium-based lipids with phospholipid bilayer membranes of different complexity.

In recent years, alkylated imidazolium salts have been shown to affect lipid membranes and exhibit general cytotoxicity as well as significant anti-tumor activity. Here, we examined the interactions of a sterically demanding, biophysically unexplored imidazolium salt, 1,3-bis(2,6-diisopropylphenyl)-4,5-diundecylimidazolium bromide (C11IPr), on the physico-chemical properties of various model biomembrane systems. The results are compared with those for the smaller headgroup variant 1,3-dimethyl-4,5-diundecylimidazolium iodide (C11IMe). We studied the influence of these two lipid-based imidazolium salts at concentrations from 1 to about 10 mol% on model biomembrane systems of different complexity, including anionic heterogeneous raft membranes which are closer to natural membranes. Fluorescence spectroscopic, DSC, surface potential and FTIR measurements were carried out to reveal changes in membrane thermotropic phase behavior, lipid conformational order, fluidity and headgroup charge. Complementary AFM and confocal fluorescence microscopy measurements allowed us to detect changes in the lateral organization and membrane morphology. Both lipidated imidazolium salts increase the membrane fluidity and lead to a deterioration of the lateral domain structure of the membrane, in particular for C11IPr owing to its bulkier headgroup. Moreover, partitioning of the lipidated imidazolium salts into the lipid vesicles leads to marked changes in lateral organization, curvature and morphology of the lipid vesicles at high concentrations, with C11IPr having a more pronounced effect than C11IMe. Hence, these compounds seem to be vastly suitable for biochemical and biotechnological engineering, with high potentials for antimicrobial activity, drug delivery and gene transfer.

Bridging of membrane surfaces by annexin A2.

The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca2+ regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca2+ dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.

Towards quantification of toxicity of lithium ion battery electrolytes - development and validation of a liquid-liquid extraction GC-MS method for the determination of organic carbonates in cell culture materials.

A novel method based on liquid-liquid extraction with subsequent gas chromatography separation and mass spectrometric detection (GC-MS) for the quantification of organic carbonates in cell culture materials is presented. Method parameters including the choice of extraction solvent, of extraction method and of extraction time were optimised and the method was validated. The setup allowed for determination within a linear range of more than two orders of magnitude. The limits of detection (LODs) were between 0.0002 and 0.002 mmol/L and the repeatability precisions were in the range of 1.5-12.9%. It could be shown that no matrix effects were present and recovery rates between 98 and 104% were achieved. The methodology was applied to cell culture models incubated with commercial lithium ion battery (LIB) electrolytes to gain more insight into the potential toxic effects of these compounds. The stability of the organic carbonates in cell culture medium after incubation was studied. In a porcine model of the blood-cerebrospinal fluid (CSF) barrier, it could be shown that a transfer of organic carbonates into the brain facing compartment took place. Graphical abstract Schematic setup for the investigation of toxicity of lithium ion battery electrolytes.

The effects of gold nanoparticles functionalized with ß-amyloid specific peptides on an in vitro model of blood-brain barrier.

We studied the effect of gold nanoparticle (AuNP) size, surface charge, concentration and morphology on the integrity of the blood-brain barrier (BBB) in a well-established in vitro model set-up. We focused on the effect of peptide functionalized hollow gold nanospheres and gold nanorods, which selectively bind to amyloidogenic ß-amyloid structures. These AuNP conjugates have already been successfully tested as photothermal absorbers for potential application in Alzheimer's disease (AD) therapy in an in vitro set-up, but may exhibit a low passage through the BBB due to their overall negative charge. Our results show that: (i) small (1.4 nm) AuNPs strongly affects the BBB integrity, (ii) negative surface charge impedes BBB passage, and (iii) this charge effect caused by the peptide is compensated by covalent coupling to a polyethylene glycol ligand stabilizing the particles in diluted manner.

Influence of the Headgroup of Azolium-Based Lipids on Their Biophysical Properties and Cytotoxicity.

A series of (un-)charged NHC derivatives bearing two pentadecyl chains in the backbone was studied in detail to find cooperative effects between the membrane and the NHC derivative. The tendency to show lipid-like behavior is dependent on the properties of the NHC derivative headgroup, which can be modified on demand. The surface activity was investigated by film balance measurements, epifluorescence microscopy, and differential scanning calorimetry. Additionally the cytotoxicity was evaluated against different cell lines such as eukaryotic tumor cell lines. These novel lipid-like NHC derivatives offer a broad spectrum for biological applications.

Comparison of cellular effects of starch-coated SPIONs and poly(lactic-co-glycolic acid) matrix nanoparticles on human monocytes.

Within the last years, progress has been made in the knowledge of the properties of medically used nanoparticles and their toxic effects, but still, little is known about their influence on cellular processes of immune cells. The aim of our comparative study was to present the influence of two different nanoparticle types on subcellular processes of primary monocytes and the leukemic monocyte cell line MM6. We used core-shell starch-coated superparamagnetic iron oxide nanoparticles (SPIONs) and matrix poly(lactic-co-glycolic acid) (PLGA) nanoparticles for our experiments. In addition to typical biocompatibility testing like the detection of necrosis or secretion of interleukins (ILs), we investigated the impact of these nanoparticles on the actin cytoskeleton and the two voltage-gated potassium channels Kv1.3 and Kv7.1. Induction of necrosis was not seen for PLGA nanoparticles and SPIONs in primary monocytes and MM6 cells. Likewise, no alteration in secretion of IL-1ß and IL-10 was detected under the same experimental conditions. In contrast, IL-6 secretion was exclusively downregulated in primary monocytes after contact with both nanoparticles. Two-electrode voltage clamp experiments revealed that both nanoparticles reduce currents of the aforementioned potassium channels. The two nanoparticles differed significantly in their impact on the actin cytoskeleton, demonstrated via atomic force microscopy elasticity measurement and phalloidin staining. While SPIONs led to the disruption of the respective cytoskeleton, PLGA did not show any influence in both experimental setups. The difference in the effects on ion channels and the actin cytoskeleton suggests that nanoparticles affect these subcellular components via different pathways. Our data indicate that the alteration of the cytoskeleton and the effect on ion channels are new parameters that describe the influence of nanoparticles on cells. The results are highly relevant for medical application and further evaluation of nanomaterial biosafety.

ToF-SIMS and laser-SNMS analysis of Madin-Darby canine kidney II cells with silver nanoparticles using an argon cluster ion beam.

The use of nanoparticles is one of the fastest expanding fields in industrial as well as in medical applications, owing to their remarkable characteristics. Silver nanoparticles (AgNPs) are among the most-commercialized nanoparticles because of their antibacterial effects. Laser postionization secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry in combination with argon cluster ion sputtering was used for the first time to investigate the effects of AgNPs on Madin-Darby canine kidney (MDCK) II cells. Depth profiles and high-resolution three dimensional (3D) images of nanoparticles and organic compounds from cells were obtained using an Ar cluster ion beam for sputtering and Bi3 (+) primary ions for the analysis. The 3D distribution of AgNPs and other organic compounds in MDCK II cells could be readily detected with very high efficiency, sensitivity, and submicron lateral resolution. The argon cluster ion beam is well suited for the sputtering of biological samples. It enables a high sample removal rate along with low molecular degradation. The outer membrane, the cytoplasm, and the nuclei of the cells could be clearly visualized using the signals PO(+) and C3H8N(+) or CN(+) and C3H8N(+). The laser-SNMS images showed unambiguously that AgNPs are incorporated by MDCK II cells and often form silver aggregates with a diameter of a few micrometers, mainly close to the outside of the cell nuclei.

Anti-tumor activity and cytotoxicity in vitro of novel 4,5-dialkylimidazolium surfactants.

Natural monoalkylated imidazolium derivatives exhibit significant anti-tumor activity as well as general cytotoxicity. In the present study, we used a series of newly synthesized imidazolium derivatives bearing two alkyl chains in the backbone of the imidazolium core in 4- and 5-position and either dimethyl- or dibenzyl-substituents at 1- and 3-position. Their anti-tumor activity and cytotoxicity were determined in vitro using the lactate dehydrogenase (LDH) assay. The tumor cell line C6 from rat glioma, the non-tumor MDCK cell line (Madin-Darby canine kidney) as well as the mouse embryonic fibroblast cell line (NIH3T3) were used as cellular targets. Surface activity measurements were performed leading to the determination of their critical micelle concentration (CMC) of these new lipid analogues to evaluate the molecular mechanism of the observed cellular effects. We found that 4,5-dialkylation of the imidazole ring enhances the anti-tumor activity compared to simple 1-alkylated imidazoles. The corresponding C7 homologues are found to be the most potent compounds. Furthermore dibenzyl-substituted imidazolium surfactants exhibit higher surface activity and increased toxicity against tumor cells compared to dimethyl-substituted imidazolium surfactants. In summary the dibenzyl-derivative carrying the two C7 chains was found to exhibit a drastically increased anti-tumor activity especially compared to so far known monoalkylated species.

A Remarkably Simple Class of Imidazolium-Based Lipids and Their Biological Properties.

A series of imidazolium salts bearing two alkyl chains in the backbone of the imidazolium core were synthesized, resembling the structure of lipids. Their antibacterial activity and cytotoxicity were evaluated using Gram-positive and Gram-negative bacteria and eukaryotic cell lines including tumor cells. It is shown that the length of alkyl chains in the backbone is vital for the antibiofilm activities of these lipid-mimicking components. In addition to their biological activity, their surface activity and their membrane interactions are shown by film balance and quartz crystal microbalance (QCM) measurements. The structure-activity relationship indicates that the distinctive chemical structure contributes considerably to the biological activities of this novel class of lipids.

pH response and molecular recognition in a low molecular weight peptide hydrogel.

In this article we report the preparation and characterization of a peptide-based hydrogel, which possesses characteristic rheological properties, is pH responsive and can be functionalized at its thiol function. The tripeptide N-(fluorenyl-9-methoxycarbonyl)-L-Cys(acetamidomethyl)-L-His-L-Cys-OH 1 forms stable supramolecular aggregates in water leading to hydrogels above 1.5 wt%. Rheological analysis of the hydrogel revealed visco-elastic and shear thinning properties of samples containing 1.5 wt% of peptide 1. The hydrogel reversibly responds to pH changes. Below and above pH 6, electrostatic repulsion of the peptide results in a weakening of the three-dimensional gel network. Based on atomic force microscopy, small angle X-ray scattering and molecular dynamics simulations, it is proposed that the peptide assembles into nanostructures that tend to entangle at higher concentrations in water. The development of functional materials based on the peptide assemblies was possible via thiol-ene-click chemistry of the free thiol function at the C-terminal cysteine unit. As a proof of concept, the functionalization with adamantyl units to give 1-Ad was shown by molecular recognition of ß-cyclodextrin vesicles. These vesicles were used as supramolecular cross-linkers of the assemblies of peptide 1 mixed with peptide 1-Ad leading to gel networks at a reduced peptide concentration.

Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A.

Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently.

Cell adhesion behavior in 3D hydrogel scaffolds functionalized with D- or L-aminoacids.

Alginate hydrogels functionalized with D-, or L-penicillamine (D-, L-PEN-Alg) are used as new 3D scaffolds for cell adhesion studies. The cells recognize and show different adhesion properties in the respective 3D hydrogel scaffolds. C-6-glioma and endothelial cells show higher affinity to the D-PEN than to the L-PEN functionalized 3D alginate hydrogel scaffold. The cultivated cells are harvested from the hydrogel and are reused, for example, for cell growth experiments on 2D surfaces to prove their viability as well.

Ultra structure analysis of cell-cell interactions between pericytes and neutrophils in vitro.

Neutrophils' adhesion to the endothelium during inflammatory is a well-known processes. In contrast the interaction of neutrophils with cells of the neurovascular unit after they have been transmigrated into the brain is less clear. Recently, lymphocyte function-associated antigen-1 (LFA-1) dependent subendothelial crawling of neutrophils has been observed in vivo. This is mediated by intracellular adhesion molecule-1 (ICAM-1), which is expressed on the cell surface of pericytes. In our work we demonstrated in vitro a cell-cell interaction between porcine brain capillary pericytes (PBCPs) and neutrophils, with further characterization of the initial contact between these cells. PBCPs increase ICAM-1 protein expression in response to the cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, an increase in neutrophil adhesion to PBCPs was determined by immunofluorescence staining. By means of scanning force microscopy (SFM), we could additionally show that pericytes as well as neutrophils form cell extensions towards the neighboring cell. Interestingly, these extensions differ for different cell types.

Brain capillary pericytes contribute to the immune defense in response to cytokines or LPS in vitro.

The prevention of an inflammation in the brain is one of the most important goals the body has to achieve. As pericytes are located on the abluminal side of the capillaries in the brain, their role in fighting against invading pathogens has been investigated in some points, mostly in their ability to behave like macrophages. Here we studied the potential of pericytes to react as immune cells under inflammatory conditions, especially regarding the expression of the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), major histocompatibility complex II (MHC II) molecules, CD68, as well as the generation of reactive oxygen and nitrogen species (RONS), and their ability in phagocytosis. Quantitative real time PCR and western blot analysis showed that pericytes are able to increase the expression of typical inflammatory marker proteins after the stimulation with tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), interferon-gamma (IFN-γ), or lipopolysaccharides (LPS). Depending on the different specific pro-inflammatory factors pericytes changed the expression of alpha smooth muscle actin (αSMA), the most predominant pericyte marker. We conclude that the role of the pericytes within the immune system is regulated and fine-tuned by different cytokines strongly depending on the time when the cytokines are released and their concentration. The present results will help to understand the pericyte mediated defense mechanisms in the brain.

Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma.

Loss of folate receptor-α function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-α-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-α, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-α-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-α-positive exosomes. Loss of folate receptor-α-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-α-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-α-positive and -negative exosomes into mouse brains demonstrate folate receptor-α-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-α-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting.

Transepithelial permeability studies of flavan-3-ol-C-glucosides and procyanidin dimers and trimers across the Caco-2 cell monolayer.

In this study the permeability of two flavanol-C-glucosides (FCglcs) and five dimeric and trimeric flavan-3-ols, namely, procyanidins (PCs), was investigated with the human colon carcinoma cell line (Caco-2) model. These compounds can be found especially in cocoa, and they are of great interest due to their assumed beneficial health effects. Transepithelial electrical resistance (TEER) and capacitance were measured online with a CellZscope device prior to and during the whole experiment to guarantee the maintenance of the barrier properties. The transport experiments with pure, single compounds (50-300 µM) from apical to basolateral side showed slight permeation of PCs A2, B2, and B5 and cinnamtannin B1 (CB1) as well as (-)-catechin-6-C-glucoside (C6Cglc) and (-)-catechin-8-C-glucoside (C8Cglc) of about 0.02-0.2% after 24 h. Transport of PC C1 could not be detected. Inhibition of P-glycoprotein (Pgp) increased the permeation of PC B2 and CB1 to the basolateral side, which indicates that Pgp counteracts the transport of these compounds. Metabolites (epicatechin, 3'- and 4'-O-epicatechin) in very small amounts were detectable only for PC B2. These are the first data concerning the permeability of flavan-3-ol-C-glucosides across the Caco-2 cell monolayer.

Pericytes support neutrophil transmigration via interleukin-8 across a porcine co-culture model of the blood-brain barrier.

Transmigration of neutrophils across the blood-brain barrier (BBB) to an inflamed brain tissue is an important process during neuronal inflammation. The process of neutrophil activation as well as their way of rolling along the endothelium and their transmigration is quite well understood. Nevertheless, relatively little is known about the fate of neutrophils after they have transmigrated through the endothelium. The role of the other cells of the neurovascular unit in this process is also poorly understood. Here we studied the potential of pericytes to chemo-attract neutrophils under inflammatory conditions. Quantitative real time PCR, western blot analysis, and a chemotaxis assay showed that pericytes are able to chemo-attract neutrophils by interleukin-8 (IL-8) after stimulation with lipopolysaccharides (LPS), tumor necrosis factor-alpha (TNF-α), or interleukin-1beta (IL-1ß). Then, a co-culture model consisting of primary porcine brain capillary endothelial cells (PBCECs) and primary porcine brain capillary pericytes (PBCPs) was used to analyze neutrophil transmigration across the BBB. As a model for inflammation, LPS was used and the effects of the cytokines TNF-α, IL-1ß, and interferon-gamma (IFN-γ) were analyzed. In general, all stimulants apart from IFN-γ were able to increase transendothelial neutrophil migration. This effect was significantly reduced by a specific inhibitor of matrix metalloproteinases (MMPs)-2 and -9. MMP-2/-9 secretion is expected to decrease adhesion to pericytes and thus support the transmigration of neutrophils. Additionally, in an adhesion experiment, we showed that MMP-2/-9 inhibition significantly enhances the adhesion of neutrophils to pericytes.

In vitro intestinal bioavailability of arsenosugar metabolites and presystemic metabolism of thio-dimethylarsinic acid in Caco-2 cells.

Whereas inorganic arsenic is classified as a human carcinogen, risks to human health related to the presence of arsenosugars in marine food are still unclear. Since studies indicate that human inorganic arsenic metabolites contribute to inorganic arsenic induced carcinogenicity, a risk assessment for arsenosugars should also include a toxicological characterization of their respective metabolites. Here we assessed intestinal bioavailability of the human arsenosugar metabolites oxo-DMAA(V), thio-DMAA(V), oxo-DMAE(V), thio-DMAE(V) and thio-DMA(V) in relation to arsenite in the Caco-2 intestinal barrier model. Whereas arsenite and thio-DMA(V) caused barrier disruption at concentrations ≥10 µM, all other metabolites did not cause a barrier leakage, even when applied at 50 times higher concentrations than arsenite and thio-DMA(V). The transfer studies point to a strong intestinal bioavailability of thio-DMA(V) and thio-DMAE(V), whereas oxo-DMAA(V), thio-DMAA(V) and oxo-DMAE(V) passed the in vitro intestinal barrier only to a very small extent. Detailed influx and efflux studies indicate that arsenite and thio-DMA(V) cross the intestinal barrier most likely by passive diffusion (paracellular) and facilitated (transcellular) transport. LC-ICP-QMS based arsenic speciation studies during the transfer experiments demonstrate transfer of thio-DMA(V) itself across the intestinal barrier and suggest metabolism of thio-DMA(V) using the in vitro intestinal barrier model to its oxygen-analogue DMA(V). In the case of arsenite no metabolism was observed. In summary the two arsenosugar metabolites thio-DMA(V) and thio-DMAE(V) showed intestinal bioavailability similar to that of arsenite, and about 10-fold higher than that reported for arsenosugars (Leffers et al., Mol. Nutr. Food Res., 2013, DOI: 10.1002/mnfr.201200821) in the same in vitro model. Thus, a presystemic metabolism of arsenosugars might strongly impact arsenic intestinal bioavailability after arsenosugar intake and should therefore be considered when assessing the risks to human health related to the consumption of arsenosugar-containing food.

Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity.