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Background: Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-beta seems to play an important role in the regulation of central inflammation. In addition, PPAR-beta agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-beta agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-gamma and LPS.Methods: Aggregating brain cells cultures were prepared from embryonal rat brain, and used to study the inflammatory responses triggered by IFN-gamma and LPS and by antibody-mediated demyelination induced by antibodies directed against myelin-oligodendrocyte glycoprotein (MOG). The effects of GW 501516 on cellular responses were characterized by the quantification of the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), inducible NO synthase (i-NOS), PPAR-beta, PPAR-gamma, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and high molecular weight neurofilament protein (NF-H). GFAP expression was also examined by immunocytochemistry, and microglial cells were visualized by isolectin B4 (IB4) and ED1 labeling.Results: GW 501516 decreased the IFN-gamma-induced up-regulation of TNF-alpha and iNOS in accord with the proposed anti-inflammatory effects of this PPAR-beta agonist. However, it increased IL-6 m-RNA expression. In demyelinating cultures, reactivity of both microglial cells and astrocytes was observed, while the expression of the inflammatory cytokines and iNOS remained unaffected. Furthermore, GW 501516 did not protect against the demyelination-induced changes in gene expression.Conclusion: Although GW 501516 showed anti-inflammatory activity, it did not protect against antibody-mediated demyelination. This suggests that the protective effects of PPAR-beta agonists observed in vivo can be attributed to their anti-inflammatory properties rather than to a direct protective or trophic effect on oligodendrocytes. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology., Competing Interests: Declaration of competing interest The authors declare no competing interests. JG and RGV are co-founders of Evercyte. RGV and ZM are employees of Evercyte., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study., Objective: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays., Methods: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures., Results: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic-related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity., Conclusions: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals., International Registered Report Identifier (irrid): DERR1-10.2196/50300., (©Nancy B Hopf, Laura Suter-Dick, Jörg Huwyler, Myriam Borgatta, Lucie Hegg, David Pamies, Hélène Paschoud, Ramya Deepthi Puligilla, Elena Reale, Sophie Werner, Marie-Gabrielle Zurich. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 18.01.2024.)

For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625-15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription-polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentration-dependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodarone-induced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro . In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nunes, Proença, Ambrosini, Pamies, Thomas, Kramer and Zurich.)

Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1-10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes., (© 2022. The Author(s).)

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases. The differences observed between human and rodent oligodendrocytes make animals inadequate for modeling these diseases. Although developing human in vitro models for oligodendrocytes and myelinated axons has been a great challenge, 3D cell cultures derived from iPSC are now available and able to partially reproduce the myelination process. We have previously developed a human iPSC-derived 3D brain organoid model (also called BrainSpheres) that contains a high percentage of myelinated axons and is highly reproducible. Here, we have further refined this technology by applying multiple readouts to study myelination disruption. Myelin was assessed by quantifying immunostaining/confocal microscopy of co-localized myelin basic protein (MBP) with neurofilament proteins as well as proteolipid protein 1 (PLP1). Levels of PLP1 were also assessed by Western blot. We identified compounds capable of inducing developmental neurotoxicity by disrupting myelin in a systematic review to evaluate the relevance of our BrainSphere model for the study of the myelination/demyelination processes. Results demonstrated that the positive reference compound (cuprizone) and two of the three potential myelin disruptors tested (Bisphenol A, Tris(1,3-dichloro-2-propyl) phosphate, but not methyl mercury) decreased myelination, while ibuprofen (negative control) had no effect. Here, we define a methodology that allows quantification of myelin disruption and provides reference compounds for chemical-induced myelin disruption.

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1β (IL1β) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Glioblastoma is a very aggressive primary brain tumor in adults, with very low survival rates and no curative treatments. The high failure rate of drug development for this cancer is linked to the high-cost, time-consuming, and inefficient models used to study the disease. Advances in stem cell and in vitro cultures technologies are promising, however, and here we present the advantages and limitations of available organotypic culture models and discuss their possible applications for studying glioblastoma., Competing Interests: T.H. and D.P. are named inventors on a patent by Johns Hopkins University on the production of mini-brains (also called BrainSpheres), which is licensed to AxoSim, New Orleans, LA, USA. They consult AxoSim, and T.H. is a shareholder., (© 2020 The Author(s).)

Selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat depression during pregnancy. Various concerns have been raised about the possible effects of these drugs on fetal development. Current developmental neurotoxicity (DNT) testing conducted in rodents is expensive, time-consuming, and does not necessarily represent human pathophysiology. A human, in vitro testing battery to cover key events of brain development, could potentially overcome these challenges. In this study, we assess the DNT of paroxetine-a widely used SSRI which has shown contradictory evidence regarding effects on human brain development using a versatile, organotypic human induced pluripotent stem cell (iPSC)-derived brain model (BrainSpheres). At therapeutic blood concentrations, which lie between 20 and 60 ng/ml, Paroxetine led to an 80% decrease in the expression of synaptic markers, a 60% decrease in neurite outgrowth and a 40-75% decrease in the overall oligodendrocyte cell population, compared to controls. These results were consistently shown in two different iPSC lines and indicate that relevant therapeutic concentrations of Paroxetine induce brain cell development abnormalities which could lead to adverse effects., (Copyright © 2020 Zhong, Harris, Smirnova, Zufferey, Sá, Baldino Russo, Baleeiro Beltrao Braga, Chesnut, Zurich, Hogberg, Hartung and Pamies.)

Trimethyltin is an organometallic compound, described to be neurotoxic and to trigger neuroinflammation and oxidative stress. Previous studies associated TMT with the perturbation of mitochondrial function, or neurotransmission. However, the mechanisms of toxicity may differ depending on the duration of exposure and on the stage of maturation of brain cells. This study aim at elucidating whether the toxicity pathways triggered by a known neurotoxicant (TMT) differs depending on cell maturation stage or duration of exposure. To this end omics profiling of immature and differentiated 3D rat brain cell cultures exposed for 24 h or 10 days (10-d) to 0.5 and 1 μM of TMT was performed to better understand the underlying mechanisms of TMT associated toxicity. Proteomics identified 55 and 17 proteins affected by acute TMT treatment in immature and differentiated cultures respectively, while 10-day treatment altered 96 proteins in immature cultures versus 353 in differentiated. The results suggest different sensitivity to TMT depending on treatment duration and cell maturation. In accordance with known TMT mechanisms oxidative stress and neuroinflammation was observed after 10-d treatment at both maturation stages, whereas the neuroinflammatory process was more prominent in differentiated cultures than in the immature, no development-dependent difference could be detected for oxidative stress or synaptic neurodegeneration. Pathway analysis revealed that both vesicular trafficking and the synaptic machinery were strongly affected by 10-d TMT treatment in both maturation stages, as was GABAergic and glutamatergic neurotransmission. This study shows that omics approaches combined with pathway analysis constitutes an improved tool-set in elucidating toxicity mechanisms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A paradigm shift is occurring in toxicology following the report of the National Research Council of the USA National Academies entitled "Toxicity testing in the 21st Century: a vision and strategy". This new vision encourages the use of in vitro and in silico models for toxicity testing. In the goal to identify new reliable markers of toxicity, the responsiveness of different genes to various drugs (amiodarone: 0.312-2.5 [Formula: see text]; cyclosporine A: 0.25-2 [Formula: see text]; chlorpromazine: 0.625-10 [Formula: see text]; diazepam: 1-8 [Formula: see text]; carbamazepine: 6.25-50 [Formula: see text]) is studied in 3D aggregate brain cell cultures. Genes' responsiveness is quantified and ranked according to the Lowest Observed Effect Concentration (LOEC), which is estimated by reverse regression under a log-logistic model assumption. In contrast to approaches where LOEC is identified by the first observed concentration level at which the response is significantly different from a control, the model-based approach allows a principled estimation of the LOEC and of its uncertainty. The Box-Cox transform both sides approach is adopted to deal with heteroscedastic and/or non-normal residuals, while estimates from repeated experiments are summarized by a meta-analytic approach. Different inferential procedures to estimate the Box-Cox coefficient, and to obtain confidence intervals for the log-logistic curve parameters and the LOEC, are explored. A simulation study is performed to compare coverage properties and estimation errors for each approach. Application to the toxicological data identifies the genes Cort, Bdnf, and Nov as good candidates for in vitro biomarkers of toxicity.

In this manuscript, which appeared in ALTEX 35 , 306-352 ( doi:10.14573/altex.1712081 ), the Acknowledgements should read: This work was supported by the Doerenkamp-Zbinden Foundation, EFSA, the BMBF, JPI-NutriCog-Selenius, and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

The aim of the present study was to establish the developmental profile of metabolic changes of 3D aggregating brain cell cultures by 1 H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. The histotypic 3D brain aggregate, containing all brain cell types, is an excellent model for mechanistic studies including OMICS analysis; however, their metabolic profile has not been yet fully investigated. Chemometric analysis revealed a clear separation of samples from the different maturation time points. Metabolite concentration evolutions could be followed and revealed strong and various metabolic alterations. The strong metabolite evolution emphasizes the brain modeling complexity during maturation, possibly reflecting physiological processes of brain tissue development. The small observed intra- and inter-experimental variabilities show the robustness of the combination of 1 H-HR-MAS NMR and 3D brain aggregates, making it useful to investigate mechanisms of toxicity that will ultimately contribute to improve predictive neurotoxicology. Graphical Abstract ᅟ.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

Neurotoxic effects of compounds can be tested in vitro using cell systems. One example is aggregating rat brain cell cultures. For the extrapolation of in vitro data to the in vivo situation, it is important to take the biokinetics of the test compound into account. In addition, the exposure in vivo is often for a longer period of time; therefore, it is crucial to incorporate this into in vitro assays as well. In this study, aggregating rat brain cell cultures were exposed to chlorpromazine (CPZ) and diazepam (DZP) for 12-days with repeated exposure. Samples were taken from the stocks, test media, cell culture media and cells at specific time points on the first and last exposure day. These samples were analysed by HPLC-UV. The amount of CPZ in the medium decreased over time, whereas the amount in the cells showed an increase. Accumulation of CPZ in the cells was seen over the 12-day repeated exposure. The amount of DZP in the medium remained stable over time and only up to 2% of DZP added was found in the cells. Different biokinetic behaviour was found for CPZ and DZP. Possible explanations are differences in uptake into the cells or efflux out of the cells. The decrease of CPZ in the medium versus the stable amount of DZP results in differences in exposure concentrations over time, which should be taken into account when interpreting in vitro effect data., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24 h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Within the Predict-IV FP7 project a strategy for measurement of in vitro biokinetics was developed, requiring the characterization of the cellular model used, especially regarding biotransformation, which frequently depends on cytochrome P450 (CYP) activity. The extrahepatic in situ CYP-mediated metabolism is especially relevant in target organ toxicity. In this study, the constitutive mRNA levels and protein localization of different CYP isoforms were investigated in 3D aggregating brain cell cultures. CYP1A1, CYP2B1/B2, CYP2D2/4, CYP2E1 and CYP3A were expressed; CYP1A1 and 2B1 represented almost 80% of the total mRNA content. Double-immunolabeling revealed their presence in astrocytes, in neurons, and to a minor extent in oligodendrocytes, confirming the cell-specific localization of CYPs in the brain. These results together with the recently reported formation of an amiodarone metabolite following repeated exposure suggest that this cell culture system possesses some metabolic potential, most likely contributing to its high performance in neurotoxicological studies and support the use of this model in studying brain neurotoxicity involving mechanisms of toxication/detoxication., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neurotoxic effects of the environmentally abundant mycotoxin Ochratoxin A (OTA) were studied in histotypic 3D rat brain cell cultures, comprising all brain cell types. Cultures were exposed to nanomolar OTA concentrations and samples were collected 48h after a single exposure, or after 10 days of repeated administration. OTA-induced changes in gene- and protein expression, as well as alterations in cell morphology were assessed. Forty-eight-hour OTA exposure resulted in a disruption of the neuronal cytoskeleton and reduced expression of several oligodendrocyte-specific markers indicative of demyelination. Astrocyte disturbances were revealed by a decrease in two astrocytic proteins involved in regulation of inflammatory responses, metallothioneins I and II. Repeated OTA administration induced a neuroinflammatory response, as visualized by an increase of isolectin B4 labelled cells, increased expression of pro-inflammatory cytokines, and detection of macrophagic ED1/CD68 positive cells, as well as an upregulation of neurodegenerative M1 microglial phenotype markers. Partial recovery from OTA-induced deleterious effects on oligodendrocytes and astrocytes was achieved by co-treatment with sonic hedgehog (SHH). In addition, metallothionein I and II co-treatment partially restored OTA-induced effects on oligodendrocytes after 48h, and modulated microglial reactivity after 10 days. These results suggest that OTA-exposure affects Shh-signalling, which in turn may influence both oligodendrocytes and astrocytes. Furthermore, the primarily astrocytic proteins MTI/MTII may affect microglial activation. Thus the neuroinflammatory response appears to be downstream of OTA-induced effects on demyelination, axonal instabilities and astrocytes disturbances. In conclusion, repeated OTA-exposure induced a secondary neuroinflammatory response characterized by neurodegenerative M1 microglial activation and pro-inflammatory response that could exacerbate the neurodegenerative process., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

As part of the ACuteTox project aimed at the development of non-animal testing strategies for predicting human acute oral toxicity, aggregating brain cell cultures (AGGR) were examined for their capability to detect organ-specific toxicity. Previous multicenter evaluations of in vitro cytotoxicity showed that some 20% of the tested chemicals exhibited significantly lower in vitro toxicity as expected from in vivo toxicity data. This was supposed to be due to toxicity at supracellular (organ or system) levels. To examine the capability of AGGR to alert for potential organ-specific toxicants, concentration-response studies were carried out in AGGR for 86 chemicals, taking as endpoints the mRNA expression levels of four selected genes. The lowest observed effect concentration (LOEC) determined for each chemical was compared with the IC20 reported for the 3T3/NRU cytotoxicity assay. A LOEC lower than IC20 by at least a factor of 5 was taken to alert for organ-specific toxicity. The results showed that the frequency of alerts increased with the level of toxicity observed in AGGR. Among the chemicals identified as alert were many compounds known for their organ-specific toxicity. These findings suggest that AGGR are suitable for the detection of organ-specific toxicity and that they could, in conjunction with the 3T3/NRU cytotoxicity assay, improve the predictive capacity of in vitro toxicity testing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: In vitro aggregating brain cell cultures containing all types of brain cells have been shown to be useful for neurotoxicological investigations. The cultures are used for the detection of nervous system-specific effects of compounds by measuring multiple endpoints, including changes in enzyme activities. Concentration-dependent neurotoxicity is determined at several time points., Methods: A Markov model was set up to describe the dynamics of brain cell populations exposed to potentially neurotoxic compounds. Brain cells were assumed to be either in a healthy or stressed state, with only stressed cells being susceptible to cell death. Cells may have switched between these states or died with concentration-dependent transition rates. Since cell numbers were not directly measurable, intracellular lactate dehydrogenase (LDH) activity was used as a surrogate. Assuming that changes in cell numbers are proportional to changes in intracellular LDH activity, stochastic enzyme activity models were derived. Maximum likelihood and least squares regression techniques were applied for estimation of the transition rates. Likelihood ratio tests were performed to test hypotheses about the transition rates. Simulation studies were used to investigate the performance of the transition rate estimators and to analyze the error rates of the likelihood ratio tests. The stochastic time-concentration activity model was applied to intracellular LDH activity measurements after 7 and 14 days of continuous exposure to propofol. The model describes transitions from healthy to stressed cells and from stressed cells to death., Results: The model predicted that propofol would affect stressed cells more than healthy cells. Increasing propofol concentration from 10 to 100 μM reduced the mean waiting time for transition to the stressed state by 50%, from 14 to 7 days, whereas the mean duration to cellular death reduced more dramatically from 2.7 days to 6.5 hours., Conclusion: The proposed stochastic modeling approach can be used to discriminate between different biological hypotheses regarding the effect of a compound on the transition rates. The effects of different compounds on the transition rate estimates can be quantitatively compared. Data can be extrapolated at late measurement time points to investigate whether costs and time-consuming long-term experiments could possibly be eliminated.

Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three-dimensional cell culture system provides optimal cell-to-cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT-PCR to measure the mRNA expression of cytokines (TNF-α, IL-1β, IL-6, IL-1ra, TGF-β, IL-15, IFN-γ), chemokines (ccl5, cxcl1, cxcl2), and i-NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases).

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Serum-free aggregating brain cell cultures are free-floating three-dimensional primary cell cultures able to reconstitute spontaneously a histotypic brain architecture to reproduce critical steps of brain development and to reach a high level of structural and functional maturity. This culture system offers, therefore, a unique model for neurotoxicity testing both during the development and at advanced cellular differentiation, and the high number of aggregates available combined with the excellent reproducibility of the cultures facilitates routine test procedures. This chapter presents a detailed description of the preparation, maintenance, and use of these cultures for neurotoxicity studies and a comparison of the developmental characteristics between cultures derived from the telencephalon and cultures derived from the whole brain. For culture preparation, mechanically dissociated embryonic brain tissue is used. The initial cell suspension, composed of neural stem cells, neural progenitor cells, immature postmitotic neurons, glioblasts, and microglial cells, is kept in a serum-free, chemically defined medium under continuous gyratory agitation. Spherical aggregates form spontaneously and are maintained in suspension culture for several weeks. Within the aggregates, the cells rearrange and mature, reproducing critical morphogenic events, such as migration, proliferation, differentiation, synaptogenesis, and myelination. For experimentation, replicate cultures are prepared by the randomization of aggregates from several original flasks. The high yield and reproducibility of the cultures enable multiparametric endpoint analyses, including "omics" approaches.

To study inflammatory reactions occurring in relation to demyelination, aggregating rat brain cell cultures were subjected to three different demyelinating insults, i.e., (i) lysophosphatidylcholine (LPC), (ii) interferon-gamma combined with lipopolysaccharide (IFN-gamma+LPS), and (iii) anti-MOG antibodies plus complement (alpha-MOG+C). Demyelination was assessed by measuring the expression of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), and the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). The accompanying inflammatory reactions were examined by the quantification of microglia-specific staining, by immunostaining for glial fibrillary acidic protein (GFAP), and by measuring the mRNA expression of a panel of inflammation-related genes. It was found that all three demyelinating insults decreased the expression of MBP and MOG, and induced microglial reactivity. LPC and alpha-MOG+C, but not IFN-gamma+LPS, decreased CNP activity; they also caused the appearance of macrophagic microglia, and increased GFAP staining indicating astrogliosis. LPC affected also the integrity of neurons and astrocytes. LPC and IFN-gamma+LPS upregulated the expression of the inflammation-related genes IL-6, TNF-alpha, Ccl5, Cxcl1, and iNOS, although to different degrees. Other inflammatory markers were upregulated by only one of the three insults, e.g., Cxcl2 by LPC; IL-1beta and IL-15 by IFN-gamma+LPS; and IFN-gamma by alpha-MOG+C. These findings indicate that each of the three demyelinating insults caused distinct patterns of demyelination and inflammatory reactivity, and that of the demyelinating agents tested only LPC exhibited general toxicity., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

Only a small percentage of neurodegenerative diseases like Alzheimer's disease and Parkinson's disease is directly related to familial forms. The etiology of the most abundant, sporadic forms seems to involve both genetic and environmental factors. Environmental compounds are now extensively studied for their possible contribution to neurodegeneration. Chemicals were found which were able to reproduce symptoms of known neurodegenerative diseases, others may either predispose to the onset of neurodegeneration, or exacerbate distinct pathogenic processes of these diseases. In any case, in vitro studies performed with models presenting various degrees of complexity have shown that many environmental compounds have the potential to cause neurodegeneration, through a variety of pathways similar to those described in neurodegenerative diseases. Since the population is exposed to a huge number of potentially neurotoxic compounds, there is an important need for rapid and efficient procedures for hazard evaluation. Xenobiotics elicit a cascade of reactions that, most of the time, involve numerous interactions between the different brain cell types. A reliable in vitro model for the detection of environmental toxins potentially at risk for neurodegenerative diseases should therefore allow maximal cell-cell interactions and multiparametric endpoints determination. The combined use of in vitro models and new analytical approaches using "omics" technologies should help to map toxicity pathways, and advance our understanding of the possible role of xenobiotics in the etiology of neurodegenerative diseases.

Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 microM in single treatment and of 1 microM and 2 microM in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 microM of THC or JWH 015, whereas the expression of TNF-alpha remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.

Brain inflammatory response is triggered by the activation of microglial cells and astrocytes in response to various types of CNS injury, including neurotoxic insults. Its outcome is determined by cellular interactions, inflammatory mediators, as well as trophic and/or cytotoxic signals, and depends on many additional factors such as the intensity and duration of the insult, the extent of both the primary neuronal damage and glial reactivity and the developmental stage of the brain. Depending on particular circumstances, the brain inflammatory response can promote neuroprotection, regeneration or neurodegeneration. Glial reactivity, regarded as the central phenomenon of brain inflammation, has also been used as an early marker of neurotoxicity. To study the mechanisms underlying the glial reactivity, serum-free aggregating brain cell cultures were used as an in vitro model to test the effects of conventional neurotoxicants such as organophosphate pesticides, heavy metals, excitotoxins and mycotoxins. This approach was found to be relevant and justified by the complex cell-cell interactions involved in the brain inflammatory response, the variability of the glial reactions and the multitude of mediators involved. All these variables need to be considered for the elucidation of the specific cellular and molecular reactions and their consequences caused by a given chemical insult.

The incidence of neurodegenerative disease like Parkinson's disease and Alzheimer's disease (AD) increases dramatically with age; only a small percentage is directly related to familial forms. The etiology of the most abundant, sporadic forms is complex and multifactorial, involving both genetic and environmental factors. Several environmental pollutants have been associated with neurodegenerative disorders. The present article focuses on results obtained in experimental neurotoxicology studies that indicate a potential pathogenic role of lead and mercury in the development of neurodegenerative diseases. Both heavy metals have been shown to interfere with a multitude of intracellular targets, thereby contributing to several pathogenic processes typical of neurodegenerative disorders, including mitochondrial dysfunction, oxidative stress, deregulation of protein turnover, and brain inflammation. Exposure to heavy metals early in development can precondition the brain for developing a neurodegenerative disease later in life. Alternatively, heavy metals can exert their adverse effects through acute neurotoxicity or through slow accumulation during prolonged periods of life. The pro-oxidant effects of heavy metals can exacerbate the age-related increase in oxidative stress that is related to the decline of the antioxidant defense systems. Brain inflammatory reactions also generate oxidative stress. Chronic inflammation can contribute to the formation of the senile plaques that are typical for AD. In accord with this view, nonsteroidal anti-inflammatory drugs and antioxidants suppress early pathogenic processes leading to Alzheimer's disease, thus decreasing the risk of developing the disease. The effects of lead and mercury were also tested in aggregating brain-cell cultures of fetal rat telencephalon, a three-dimensional brain-cell culture system. The continuous application for 10 to 50 days of non-cytotoxic concentrations of heavy metals resulted in their accumulation in brain cells and the occurrence of delayed toxic effects. When applied at non-toxic concentrations, methylmercury, the most common environmental form of mercury, becomes neurotoxic under pro-oxidant conditions. Furthermore, lead and mercury induce glial cell reactivity, a hallmark of brain inflammation. Both mercury and lead increase the expression of the amyloid precursor protein; mercury also stimulates the formation of insoluble beta-amyloid, which plays a crucial role in the pathogenesis of AD and causes oxidative stress and neurotoxicity in vitro. Taken together, a considerable body of evidence suggests that the heavy metals lead and mercury contribute to the etiology of neurodegenerative diseases and emphasizes the importance of taking preventive measures in this regard.

Ochratoxin A (OTA), a mycotoxin and widespread food contaminant, is known for its patent nephrotoxicity and potential neurotoxicity. Previous observations in vitro showed that in the CNS, glial cells were particularly sensitive to OTA. In the search for the molecular mechanisms underlying OTA neurotoxicity, we investigated the relationship between OTA toxicity and glial reactivity, in serum-free aggregating brain cell cultures. Using quantitative reverse transcriptase-polymerase chain reaction to analyze changes in gene expression, we found that in astrocytes, non cytotoxic concentrations of OTA down-regulated glial fibrillary acidic protein, while it up-regulated vimentin and the peroxisome proliferator-activated receptor-gamma expression. OTA also up-regulated the inducible nitric oxide synthase and the heme oxygenase-1. These OTA-induced alterations in gene expression were more pronounced in cultures at an advanced stage of maturation. The natural peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-delta(12,14) prostaglandin J2, and the cyclic AMP analog, bromo cyclic AMP, significantly attenuated the strong induction of peroxisome proliferator-activated receptor-gamma and inducible nitric oxide synthase, while they partially reversed the inhibitory effect of OTA on glial fibrillary acidic protein. The present results show that OTA affects the cytoskeletal integrity of astrocytes as well as the expression of genes pertaining to the brain inflammatory response system, and suggest that a relationship exists between the inflammatory events and the cytoskeletal changes induced by OTA. Furthermore, these results suggest that, by inducing an atypical glial reactivity, OTA may severely affect the neuroprotective capacity of glial cells.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to investigate the influence of glial cells on the neurotoxicity of two organophosphorus pesticides (OPs), chlorpyrifos and parathion. Mixed-cell aggregate cultures were treated continuously for 10 days between DIV 5 and 15. Parathion induced astrogliosis at concentration at which MAP-2 immunostaining, found here to be more sensitive than neuron-specific enzyme activities, was not affected. In contrast, chlorpyrifos induced a comparatively weak gliotic reaction, and only at concentrations at which neurons were already affected. After similar treatments, increased neurotoxicity of parathion and chlorpyrifos was found in aggregate cultures deprived of glial cells. These results suggest that glial cells provide neuroprotection against OPs toxicity. To address the question of the difference in toxicity between parathion and chlorpyrifos, the toxic effects of their leaving groups, p-nitrophenol and trichloropyridinol, were studied in mixed-cell aggregates. General cytotoxicity was more pronounced for trichloropyridinol and both compounds had similar toxic effects on neuron-specific enzyme activities. In contrast, trichloropyridinol induced a much stronger decrease in glutamine synthetase activity, the enzymatic marker of astrocytes. Trichloropyridinol may exert a toxic effect on astrocytes, compromising their neuroprotective function, thus exacerbating the neurotoxicity of chlorpyrifos. This is in line with the suggestion that glial cells may contribute to OPs neurotoxicity, and with the view that OPs may exert their neurotoxic effects through different mechanisms.

Phosphoinositides, synthesized from myo-inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H(+)/myo-inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense-core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo-inositol uptake. The symporter is then endocytosed in a dynamin-dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity-dependent regulation of myo-inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.

The abnormal vascular system of brain cancers inappropriately expresses membrane proteins, including proteolytic enzymes, ultimately resulting in blood extravasation. The production of inflammatory mediators, such as cytokines and nitric oxide, and tumor hypoxia have been implicated in these effects. We have previously shown that the activity of aminopeptidase A is increased in the abnormal vascular system of human and rat brain tumors. To study the mechanisms regulating the activities of peptidases in cerebral vasculature in brain tumors, we have developed a three-dimensional model of differentiated rat brain cells in aggregate cultures in which rat brain microvessels were incorporated. The secretion of interleukin-6 (IL-6) in the culture medium of aggregates was used as an indicator of inflammatory activation. Addition to these aggregates of C6 glioma cell medium (C6-CM) conditioned under hypoxic or normoxic conditions or serum mimicked tumor-dependent hypoxia or conditions of dysfunction of brain tumor vasculature. Hypoxic and normoxic C6-CM, but not serum, regulated peptidase activity in aggregates, and in particular it increased the activity of aminopeptidase A determined using histoenzymography. Serum, but not C6-CM, increased IL-6 production, but did not increase aminopeptidase A activity in aggregates. Thus soluble glioma-derived factors, but not serum-derived factors, induce dysfunctions of cerebral vasculature by directly regulating the activity of peptidases, not involving inflammatory activation. Tumor hypoxia is not necessary to modulate peptidase activity.

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

Despite a wealth of data on the neurotoxic effects of lead at the cellular and molecular levels, the reasons for its development-dependent neurotoxicity are still unclear. Here, the maturation-dependent effects of lead acetate were analyzed in immature and differentiated brain cells cultured in aggregates. Markers of general cytotoxicity as well as cell-type-specific markers of glial and neuronal cells showed that immature brain cells were more sensitive to lead than the differentiated counterparts, demonstrating that the development-dependent neurotoxicity of lead can be reproduced in aggregating brain cell cultures. After 10 days of treatment, astrocytes were found to be more affected by lead acetate than neurons in immature cultures, and microglial cells were strongly activated. Eleven days after cessation of the treatment, lead acetate caused a partial loss of astrocytes and an intense reactivity of the remaining ones. Furthermore, microglial cells expressed a macrophagic phenotype, and the loss of activity of neuron-specific enzymes was aggravated. In differentiated cultures, no reactive gliosis was found. It is hypothetized that the intense glial reactions (microgliosis and astrogliosis) observed in immature cultures contribute to the development-dependent neurotoxicity of lead., (Copyright 2002 Wiley-Liss, Inc.)

The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.

Aggregating brain cell cultures of fetal rat telencephalon can be grown in a chemically defined medium for extended periods of time. After a phase of intense mitotic activity, these three-dimensional cell cultures undergo extensive morphological differentiation, including synaptogenesis and myelination. To study the developmental toxicity of organophosphorus compounds (OP), aggregating brain cell cultures were treated with parathion. Protein content and cell type-specific enzyme activities were not affected up to a concentration of 10(5) M. Gliosis, characterized by an increased staining for glial fibrillary acidic protein (GFAP), was observed in immature and in differentiated cells. In contrast, uridine incorporation and myelin basic protein (MBP) immunoreactivity revealed strong differences in sensitivity between these two developmental stages. These results are in agreement with the view that in vivo the development-dependent toxicity is not only due to changes in hepatic detoxification, but also to age-related modifications in the susceptibility of the different populations of brain cells. Furthermore, they underline the usefulness of histotypic culture systems with a high developmental potential, such as aggregating brain cell cultures, and stress the importance of applying a large range of criteria for testing the developmental toxicity of potential neurotoxicants.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model. As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages. The results showed that FB1 did not cause cell loss and it had no effects on neurons. However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28). The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells. However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG. The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days. In summary, FB1 selectively affected glial cells. In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.

It is well known that exposure to low doses of lead causes long-lasting neurobehavioural deficits, but the cellular changes underlying these behavioural changes remain to be elucidated. A protective role of glial cells on neurons through lead sequestration by astrocytes has been proposed. The possible modulation of lead neurotoxicity by neuron-glia interactions was examined in three-dimensional cultures of foetal rat telencephalon. Mixed-brain cell cultures or cultures enriched in either neurons or glial cells were treated for 10 days with lead acetate (10(-6) m), a concentration below the limit of cytotoxicity. Intracellular lead content and cell type-specific enzyme activities were determined. It was found that in enriched cultures neurons stored more lead than glial cells, and each cell type alone stored more lead than in co-culture. Moreover, glial cells but not neurons were more affected by lead in enriched culture than in co-culture. These results show that neuron-glia interactions attenuate the cellular lead uptake and the glial susceptibility to lead, but they do not support the idea of a protective role of astrocytes.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.