Evaluation of the Importance of Astrocytes When Screening for Acute Toxicity in Neuronal Cell Systems
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Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC50 values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.
KeywordsIn vitro Human Neurons NT2.N NT2.A Co-culture
The authors are grateful to the Humane Research Trust for financial support for this study.
- Clemedson C, Kolman A, Forsby A (2007) The integrated acute systemic toxicity project (ACuteTox) for the optimisation and validation of alternative in vitro tests. ATLA-Altern Lab Anim 35:33–38Google Scholar
- Dringen R (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 64:9–671Google Scholar
- Eyer CL, Rio C, Smith JR (2000) Trimethyltin reduces ATP levels and MTT reduction in the LRM55 rat astrocytoma cell line. Vitro Mol Toxicol 13:263–268Google Scholar
- Garcia de Arriba S, Wegner F, Gruener K, Verdaguer E, Pallas M, Camins A, Wagner A, Wohlfahrt K, Allgaier C (2006) Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons. Neurochem Int 49:466–474CrossRefPubMedGoogle Scholar
- Gegg ME, Beltran B, Salas-Pino S, Bolanos JP, Clark JB, Moncada S, Heales SJ (2003) Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurones: implications for neuroprotection/neurodegeneration? J Neurochem 86:228–237CrossRefPubMedGoogle Scholar
- Kaariainen TM, Piltonen M, Ossola B, Kekki H, Lehtonen S, Nenonen T, Lecklin A, Raasmaja A, Mannisto PT (2008) Lack of robust protective effect of quercetin in two types of 6-hydroxydopamine-induced parkinsonian models in rats and dopaminergic cell cultures. Brain Res 1203:149–159CrossRefPubMedGoogle Scholar
- Larson EM, Doughman DJ, Gregerson DS, Obritsch WF (1997) A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Invest Ophth Vis Sci 38:1929–1933Google Scholar
- Page B, Page M, Noel C (1993) A new fluorometric assay for cytotoxicity measurements in vitro. Int J Oncol 3:473–476Google Scholar
- Pleasure SJ, Page C, Lee VMY (1992) Pure, postmitotic, polarized human neurons derived from Ntera-2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci 2:1802–1815Google Scholar
- Wullner U, Seyfried J, Groscurth P, Beinroth S, Winter S, Gleichmann M, Heneka M, Loschmann PA, Schulz JB, Weller M, Klockgether T (1999) Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function. Brain Res 826:53–62CrossRefPubMedGoogle Scholar
- Xu LJ, Lee JE, Giffard RG (1999) Overexpression of bcl-2, bcl-x(L) or hsp70 in murine cortical astrocytes reduces injury of co-cultured neurons. Neurosci Lett 19:3–197Google Scholar