Evaluation of the Importance of Astrocytes When Screening for Acute Toxicity in Neuronal Cell Systems

Abstract

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 IC(50) 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.

NT2 Derived Neuronal and Astrocytic Network Signalling

“…It has also been found that NT2A cells are coupled via connexin 43 containing gap junctions [20]. In addition, in our laboratory we have used the NT2N/NT2A co-culture system, to demonstrate that astrocytes confer significantly increased neuronal resilience to chemical toxicity and that neurons and astrocytes display markedly differential sensitivities to a number of CNS toxins [22], [23]. Such findings therefore provide evidence of the distinct functionality of NT2A cells.…”

Single-Cell ELISA and Flow Cytometry as Methods for Highlighting Potential Neuronal and Astrocytic Toxicant Specificity

“…Numerous discrete biochemical effects can lead to cytoskeleton changes and the degradation of NFs is widely considered a mandatory component of neurodegeneration following treatment with many neurotoxicants, irrespective of their primary mode of action (Schmuck and Kahl 2009). Indeed, the use of flow cytometry and S-C ELISAs can be complemented by assays focussed on the cellular consequences of reactive species generation, such as GSH, mitochondrial membrane potential and ATP determination (Woehrling et al 2010), thus illuminating the roles of such species in the cell structural responses seen in the present study.…”

“…Differentiation of the NT2.D1 Cell Line to Give NT2.N, NT2.N/A or NT2.A Cultures Production of NT2.N, NT2.N/A or NT2.A cultures from the NT2.D1 cells was performed as previously described (Woehrling et al 2010). Briefly, NT2 cells were differentiated using advanced DMEM medium, supplemented with 2% (v/v) foetal bovine serum (FBS), 2 mM L-glutamine, 1% (v/v) pen/strep (10,000 U penicillin and 10 mg streptomycin/ml concentrate) and 1 9 10 -5 M all-trans retinoic acid (RA) for 4 weeks.…”

“…As mentioned previously, an isolated condition is an alien state for neuronal cells that could result in an altered response to test chemicals in vitro when compared with the in vivo situation. We have previously employed general biochemical endpoints to propose the well-defined NT2 neuronal and astrocytic (NT2.N/A) cell system as a useful in vitro model of the importance of astrocytic modulation of the neurotoxic response (Woehrling et al 2010). The NT2.N/A co-culture is produced via terminal differentiation of the pleuripotent human embryonal carcinoma (EC) NT2.D1 cell line to give a post-mitotic network of NF positive neuronal (NT2.N) cells and GFAP positive astrocytic (NT2.A) cells, with a consistent (1:2) neuronal-astrocytic ratio (Andrews 1984;Bani-Yaghoub et al 1999).…”

“…We selected eight test chemicals with different mechanisms of action from the reference compounds employed by the A-CuteTox in vitro testing project, including the neurotoxicity work package (WP7.1; Table 1); these agents ranged from relatively potent neurotoxins (acrylamide. atropine) to nonneurotoxins (rifampicin, verapamil) (Woehrling et al 2010;Kinsner-Ovaskainen et al 2009;Forsby and Blaauboer 2007). A number of approaches can be applied to discriminate between neuronal and astrocytic cell death, the most basic and time consuming being manual cell counting (Anderl et al 2009).…”