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Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes
Published on: April 12, 2015
Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation
Verónica O Berríos1, Nawal M Boukli2, Jose W Rodriguez3
1Department of Biochemistry, Universidad Central del Caribe, Ave. Laurel #100, Santa Juanita, P.O. Box 60327, Bayamón, PR, 00960-6032, USA.
Abstract:
Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyridostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 μM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 µM pyridostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.
Insights
Organophosphate (OP) exposure can harm developing brains. While OP toxicity primarily stems from acetylcholinesterase (AChE) inhibition, this study found OPs may affect neural progenitor cell development through other mechanisms.
Area of Science:
- Neuroscience
- Toxicology
- Developmental Biology
Background:
- Organophosphates (OPs) are a significant human health concern, particularly for brain development.
- OP toxicity is mainly attributed to acetylcholinesterase (AChE) inhibition, but alternative mechanisms are suspected.
- Understanding OP effects on neural progenitor cells is crucial for assessing developmental risks.
Purpose of the Study:
- To investigate the impact of chronic acetylcholinesterase (AChE) inhibition by OPs on rat neural progenitor cell differentiation.
- To determine if AChE inhibition is the sole mechanism behind OP-induced neurotoxicity in developing neural cells.
- To explore potential alternative mechanisms of OP toxicity affecting neural development.
Main Methods:
- Primary rat neural progenitor cells were cultured and exposed to paraoxon (an OP) or pyridostigmine (a reversible AChE inhibitor).
- Cell viability was assessed using the MTT assay.
- Cell differentiation and phenotype distribution (neurons, glial cells) were analyzed via flow cytometry.
- Neurite outgrowth was evaluated to assess developmental patterns.
Main Results:
- Paraoxon reduced cell viability at concentrations >50 μM, but this effect was not dose-dependent.
- Pyridostigmine did not affect cell viability, even after prolonged exposure, indicating AChE inhibition alone did not cause toxicity.
- While protein expression patterns changed, the proportions of differentiated neurons and glial cells remained unaltered.
- Both paraoxon and pyridostigmine treatments led to decreased neurite outgrowth, suggesting impacts on developmental patterns beyond differentiation.
Conclusions:
- Acetylcholinesterase (AChE) inhibition by pyridostigmine did not impact neural progenitor cell viability or differentiation.
- Organophosphate (OP) paraoxon's toxicity was not solely due to AChE inhibition.
- OPs may affect neural progenitor cell development, specifically neurite outgrowth, through mechanisms independent of AChE inhibition.
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