Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood-brain barrier.

There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood-brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds.

Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2).

Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Evaluation of a proposed in vitro test strategy using neuronal and non-neuronal cell systems for detecting neurotoxicity.

The European Commission White Paper, "Strategy for a future chemicals policy" (EC, 2001) is estimated to require the testing of approximately 30,000 "existing" chemicals by 2012. Recommended in vitro tests require validation. As the White Paper (EC, 2001) requires neurotoxic data, this study evaluated an in vitro testing strategy for predicting in vivo neurotoxicity. The sensitivities of differentiated PC12 cells and primary cerebellum granule cells (CGC) were compared to undifferentiated PC12 cells which can indicate basal cytotoxicity. Cytotoxicants and neurotoxicants selected for testing covered a range of mechanisms and potencies. Neurotoxicants were not distinguished from cytotoxicants despite significantly different cell system responses using all endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton modifications. For all chemicals tested, neuronal-like cell systems were generally less sensitive than undifferentiated PC12 cells. Acute oral rodent LD(50) values correlated with cytotoxicity IC(50) values for the respective chemicals tested in each cell system. This study concluded that although simple non-specific assays are required to distinguish basal cytotoxicity from specific neurotoxicity by using different cell systems with different states of neuronal differentiation, further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants.

Highly purified lipoteichoic acid from gram-positive bacteria induces in vitro blood-brain barrier disruption through glia activation: role of pro-inflammatory cytokines and nitric oxide.

The co-culture of bovine brain capillary endothelial cells and rat primary glial cells was established as an in vitro blood-brain barrier model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid and muramyl dipeptide induced injury of blood-brain barrier structure and function. We found that highly purified lipoteichoic acid disrupted blood-brain barrier integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance and high permeability to fluorescein isothiocyanate-inulin observed in the presence of lipoteichoic acid-activated glial cells were potentiated by muramyl dipeptide and could be reversed only when glial cells were activated by lipoteichoic acid at 10 microg/ml but not with a higher lipoteichoic acid concentration (30 microg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction proteins occludin and claudin after lipoteichoic acid treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by lipoteichoic acid. Lipoteichoic acid-activated glial cells produced nitric oxide and pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) that contributed to lipoteichoic acid-induced blood-brain barrier disruption, since the direct treatment of the endothelial monolayer with tumor necrosis factor-alpha or interleukin-1beta increased blood-brain barrier permeability, whereas the pre-treatment of lipoteichoic acid-activated glial cells with antibodies against these two cytokines blocked lipoteichoic acid effects. Additionally, nitric oxide was also involved in blood-brain barrier damage, since the nitric oxide donor itself (diethylenetriamine-nitric oxide adduct) increased blood-brain barrier permeability and inducible nitric oxide synthase inhibitor (1400W) partially reversed lipoteichoic acid-induced trans-endothelial electrical resistance decrease.

Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity.

Glia undergo inflammatory activation in most CNS pathologies and are capable of killing cocultured neurons. We investigated the mechanisms of this inflammatory neurodegeneration using a mixed culture of neurons, microglia, and astrocytes, either when the astrocytes were activated directly with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or LPS/IFN-gamma-activated microglia were added to mixed neuronal cultures. In either case, activated glia caused 75-100% necrotic cell death within 48 hr, which was completely prevented by inhibitors of inducible nitric oxide synthase (iNOS) (aminoguanidine or 1400W). Activated astrocytes or microglia produced nitric oxide (NO) (steady-state level approximately 0.5 microm), which immediately inhibited the cellular respiration of cocultured neurons, as did authentic NO. NO donors also decreased ATP levels and stimulated lactate production by neurons, consistent with NO-induced respiratory inhibition. NO donors or a specific respiratory inhibitor caused rapid (<1 min) release of glutamate from neuronal and neuronal-astrocytic cultures and subsequent neuronal death that was blocked by an antagonist of NMDA receptor (MK-801). MK-801 also blocked neuronal death induced by activated glia. High oxygen also prevented NO-induced neuronal death, consistent with death being induced by NO inhibition of cytochrome c oxidation in competition with oxygen. Thus activated glia kill neurons via NO from iNOS, which inhibits neuronal respiration resulting in glutamate release and subsequent excitotoxicity. This may contribute to neuronal cell death in inflammatory, infectious, ischemic, and neurodegenerative diseases.

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria.

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.