Detection of neurotoxicity using in vitro neuronal cell systems

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Detection of Neurotoxicity Using In Vitro Neuronal Cell Systems

Implementation of the EC White Paper, "Strategy for a future chemicals policy" (2001), is estimated to require the testing of approximately 30,000 'existing' chemicals by 2012. In vitro tests were recommended for use where possible. This study evaluated a novel in vitro testing strategy for neurotoxicity, as neurotoxic data is a requisite of the White Paper (2001). Sensitivities of undifferentiated and differentiated PC12 cells and primary CGC cultures to identify neurotoxins were compared. Cytotoxicants and neurotoxicants used in other neurotoxicity studies and covering a range of mechanisms and potencies were used. Undifferentiated PC12 cells were used to indicate basal cytotoxicity to which sensitivities of neuronal-like models were compared. Collectively, the tested toxins failed to significantly distinguish cell system responses as measured using the following endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton changes. As p53 is involved in mechanisms of neurotoxicity, including neurodegeneration and neurodisease, a p53 transfected PC12 cell line was characterised. The general functionality of the transfected cells was found to be questionable and use of the transfected p53 PC12 cell line was not recommended. All cell system responses produced IC50 values, some with at least 3x differences in cell system responses, due to differentiated cell systems being consistently less sensitive to both neurotoxicants and cytotoxicants. Comparisons between IC50 values and rat acute oral LD50 values did not give significant differences. This was hypothesised to be due to the differentiated cell systems post-mitotic state and the presence of elevated defence mechanisms. More neurospecific endpoints were recommended for inclusion in a testing strategy in order to elucidate the greater sensitivity of the neuronal-like cell systems.JRC.I.2-Validation of biomedical testing method

Nitric Oxide Stimulates PC12 Cell Proliferation via cGMP and Inhibits at Higher Concentrations Mainly via Energy Depletion

We investigated how nitric oxide (NO) regulates proliferation of pheochromocytoma PC12 cells, rat aortic endothelial cells and mouse astrocytes. In all cells a NO donor (DETA/NO) stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation in PC12 cells was prevented by an inhibitor of soluble guanylate cyclase, and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase, or in cells lacking p53. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors caused complete cytostasis of PC12 and endothelial cells. However, uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO, although they did so in the additional presence of N-acetyl-cysteine. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production and ATP level, and a specific glycolytic inhibitor (2-deoxy-D-glucose) caused complete cytostasis. Our results suggest that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration.JRC.I.2-Validation of biomedical testing method

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 diVerentiated PC12 cells and primary cerebellum granule cells (CGC) were compared to undiVerentiated 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 signiWcantly diVerent cell system responses using all endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton modiWcations. For all chemicals tested, neuronal-like cell systems were generally less sensitive than undiVerentiated PC12 cells. Acute oral rodent LD50 values correlated with cytotoxicity IC50 values for the respective chemicals tested in each cell system. This study concluded that although simple non-speciWc assays are required to distinguish basal cytotoxicity from speciWc neurotoxicity by using diVerent cell systems with diVerent states of neuronal diVerentiation, further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants.JRC.I.2-Validation of biomedical testing method

Metabolism and Neurotoxicity: the Significance of Genetically Engineered Cell Lines and New Three-Dimensional Cell Cultures.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Development of a Mechanistically-based Genetically Engineered PC12 Cell System to Detect p53-mediated cytotoxicity

The human wild type p53 gene, key for apoptosis, was introduced into the pheochromocytoma (PC12) cell line, to create a mechanistically-based in vitro test model for the detection of p53-mediated toxicity. Expression of the wt p53 gene was regulated by a system, which allowed or blocked expression p53 by absence or presence of tetracycline in the culture media. Western blot analyses confirmed an inducible and tetracycline-dependent expression of the wt p53 protein. Functionality of the p53 protein was verified by camptothecin treatment, known to induce p53-dependent apoptosis. Results showed that p53-expressing cells were significantly more sensitive to camptothecin induced cytotoxicity compared to non-expressing cells, and presented a significantly higher incidence of apoptosis. A screening study on 31 metal compounds, showed that the classified human carcinogens (NaAsO(2), CdSO(4).8H(2)O, Na(2)CrO(4).4H(2)O, MnCl(2), (NH(4))(2)PtCl(6)) significantly increased cytotoxicity in p53-expressing cells compared to non-expressing cells, suggesting that their cytotoxicity was p53-mediated. Finally, acute and subchronic treatment with methyl mercury showed no significant differences in cytotoxicity and the percentage of apoptosis or necrosis between p53-expressing and non-expressing differentiated cells, suggesting that methyl mercury cytotoxicity was p53-independent.JRC.I.2-Validation of Alternative Method

The Value of Alternative Testing for Neurotoxicity in the Context of Regulatory Needs

Detection and characterization of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalized mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.JRC.I.2-Validation of biomedical testing method