Effects of Chlorpyrifos-Based Pesticide Formulation on the Mouse (Mus musculus) Neuronal Cell Line (N2a): Neurotoxicity and Inflammatory Response

Background: The widespread use of pesticides has raised concerns about their potential 
impacts on non-target species and environmental health. Chlorpyrifos, a commonly used 
organophosphate insecticide, has been linked to neurotoxic effects, but its impact on 
neuronal cells at the cellular level needs further investigation. Methods: This study 
evaluated the neurotoxicity and inflammatory response of a chlorpyrifos-based pesticide 
formulation using the mouse (Mus musculus) neuronal cell line (N2a). Characterization of 
the pesticide formulation included gas chromatography-mass spectrometry (GC-MS) and 
thermal analyses (thermogravimetric analysis (TGA) and differential scanning calorimetry 
(DSC)). Results:Neurotoxicity assays, including the [3(4,5-dimethyl)-2 bromide-5 diphenyl 
tetrazolium] (MTT) assay and lactate dehydrogenase (LDH) release assay, showed IC50 
values of 5.23 µg/mL and 3.78 µg/mL, respectively. The median IC50 of 4.51 µg/mL was 
used in inflammatory response assays, revealing increased production of pro-inflammatory 
cytokines (IL-6 and TNF-α) and elevated nitric oxide (NO) levels. Additionally, the 
expression of inflammatory markers, such as COX-2 and iNOS, was upregulated. 
Conclusion: This study is the first to report in vitro neurotoxicity and inflammatory 
responses induced by chlorpyrifos in the N2a cell line, highlighting the importance of in vitro 
assays for assessing the toxic effects of pesticide formulations on neuronal health and the 
environment.