Development of an in vitro blood-brain barrier model - Cytotoxicity of mercury and aluminum

University of Tampere, Medical School, 33014 University of Tampere, Tampere, Finland.
Toxicology and Applied Pharmacology (Impact Factor: 3.71). 03/2004; 195(1):73-82. DOI: 10.1016/j.taap.2003.11.002
Source: PubMed


In this study, in vitro blood-brain barrier (BBB) models composed of two different cell types were compared. The aim of our study was to find an alternative human cell line that could be used in BBB models. Inorganic and organic mercury and aluminum were studied as model chemicals in the testing of the system. BBB models were composed of endothelial RBE4 cell line or retinal pigment epithelial (RPE) cell line ARPE-19 and neuronal SH-SY5Y cells as target cells. Glial U-373 MG cells were included in part of the tests to induce the formation of a tighter barrier. Millicell CM filter inserts were coated with rat-tail collagen, and RBE4 or ARPE-19 cells were placed on the filters at the density of 3.5-4 x 10(5) cells/filter. During culture, the state of confluency was microscopically observed and confirmed by the measurement of electrical resistance caused by the developing cell layer. The target cells, SH-SY5Y neuroblastoma cells, were plated on the bottom of cell culture wells at the density of 100000 cells/cm(2). In part of the studies, glial U-373 MG cells were placed on the under side of the membrane filter. When confluent filters with ARPE-19 or RBE4 cells were placed on top of the SH-SY5Y cells, different concentrations of mercuric chloride, methyl mercury chloride, and aluminum chloride were added into the filter cups along with a fluorescent tracer. Exposure time was 24 h, after which the cytotoxicity in the SH-SY5Y cell layer, as well as in the ARPE-19 or RBE4 cell layer, was evaluated by the luminescent measurement of total ATP. The leakage of the fluorescent tracer was also monitored. The results showed that both barrier cell types were induced by glial cells. Inorganic and organic mercury caused a leakage of the dye and cytotoxicity in SH-SY5Y cells. Especially, methyl mercury chloride could exert an effect on target cells before any profound cytotoxicity in barrier cells could be seen. Aluminum did not cause any leakage in the barrier cell layer, and even the highest concentration (1 mM) of aluminum did not cause any cytotoxicity in the SH-SY5Y cells. In conclusion, BBB models composed of RBE4 and ARPE-19 cells were able to distinguish between different toxicities, and ARPE-19 cells are thus promising candidates for studies of drug penetration through the blood-brain barrier.

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    • "First, the toxicity of 16 compounds was assessed on the BBB model and their permeability values were determined at a non toxic concentration. Previous studies have shown the possibility to combine an in vitro BBB model and CNS target cells in an integrated testing strategy (Tahti et al., 2003; Toimela et al., 2004). To explore the importance of BBB permeability in neurotoxicity assessment of the same compounds, their toxicity on SH-SY5Y after BBB transport have been compared to their direct toxicity. "
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    ABSTRACT: The combination of an in vitro BBB model (4d/24w) with a neuronal cell line (SH-SY5Y) provides a convenient approach to explore the importance of BBB permeability in neurotoxicity assessment of compounds. The toxicity of 16 compounds on SH-SY5Y cells was evaluated after 24h incubation with each compound and compared to their toxicity on SH-SY5Y after passage through the BBB model. Nine out of 16 compounds were found toxic after direct exposure at 100muM while only three still induced toxicity on SH-SY5Y cells after BBB transport. The BBB permeability values of each compound revealed that in the case of compounds that did not induce toxicity, the amount that crossed the BBB was not enough to exert a toxic effect on the neuronal cells. Since disrupting the BBB may also cause unwanted effect on brain cells, the BBB toxicity of these compounds have been assessed. Our results prompted the importance of BBB permeability assessment in neurotoxicity evaluation, as it allows a better estimation of the actual concentration at the target site.
    Full-text · Article · Apr 2009 · Toxicology in Vitro
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    • "These cells express an endothelial phenotype and, in the presence of astrocytes, exhibit differentiation characteristics of CNS endothelium (Roux et al., 1994; Begley et al., 1996; Rist et al., 1997). We and other groups have used RBE4 cells as a model of the BBB (Mroczkowska et al., 2000; Yang et al., 2001; Friedrich et al., 2001; Calhau et al., 2002; Reichel et al., 2002; Yang and Aschner, 2003; Toimela et al., 2004), and here we examine the effect of changes in temperature, mitochondrial function, pH, and Fe or Na concentration on the permeability and flux of Mn in this in vitro model of the BBB to shed further light on the putative transporter(s) associated with Mn uptake into the brain. "
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    ABSTRACT: Manganese (Mn), an essential nutrient, is neurotoxic at high levels and has been associated with the development of a parkinsonian syndrome termed manganism. Currently, the mechanisms responsible for transporting Mn across the blood-brain barrier (BBB) are unknown. By using rat brain endothelial 4 (RBE4) cell monolayers cultured in astrocyte-conditioned media (ACM), we examine the effects of temperature, energy, proton (pH), iron (Fe), and sodium (Na(+)) dependence on Mn transport. Our results suggest that Mn transport is temperature, energy, and pH dependent, but not Fe or Na(+) dependent. These data suggest that Mn transport across the BBB is an active process, but they also demonstrate that the presence of ACM in endothelial cell cultures decreases the permeability of these cells to Mn, reinforcing the use of ACM or astrocyte cocultures in studies examining metal transport across the BBB.
    Full-text · Article · Jul 2005 · Journal of Neuroscience Research
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    • "In addition, co-cultures of BBB endothelium with astrocytes express even more BBB-like properties. Electrical resistance in monolayers of endothelial cells (Rubin et al., 1991) and co-cultures with astrocytes (Hurst and Clark, 1998; Toimela et al., 2004) have been reported to be increased by the application of phosphodiesterase inhibitors together with membrane permeable cAMP analogues. However, the use of such systems may be questioned, particularly relating to the role of cAMP in the alteration of the cells apart from inducing tight junction formation. "
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    ABSTRACT: The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.
    Full-text · Article · Feb 2005 · NeuroToxicology
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