The importance of timing. : The influence of circadian rhythms on organophosphate-induced neurotoxicity in SH-SY5Y cells.

This project aims to investigate whether the neurotoxic effects of the model compound chlorpyrifos vary over the circadian rhythm. The circadian rhythm is a 24-hour cycle that regulates the expression of many genes and is crucial to many cellular and physiological mechanisms. Chlorpyrifos affects nerve cells by inhibiting acetylcholinesterase in the postsynaptic nerve, leading to acetylcholine accumulation in the synaptic cleft, thus allowing continuous stimulation of nerve impulses, even fatal to humans in high doses. To address this aim, time-dependent effects of chlorpyrifos exposure on acetylcholinesterase activity in undifferentiated SH-SY5Y cells exhibiting a synchronised circadian rhythm were compared to unsynchronised cells. In this project, the cells synchronized to the circadian rhythm were later exposed to chlorpyrifos for 24 hours, with a group of cells being exposed every four hours. After 24 hours of exposure, the acetylcholinesterase assay was performed to measure acetylcholinesterase activity, and cells were lysed for qPCR to analyse clock gene expression. In the acetylcholinesterase assay, chlorpyrifos-exposed, controls, and the background controls followed similar rhythmicity. In the acetylcholinesterase test, chlorpyrifos-exposed, controls, and background controls followed a similar rhythm. This may indicate that other external factors may have affected the cells. Furthermore, the samples treated with chlorpyrifos, and the control showed no oscillation in the gene expression of clock genes, while the background control appeared to oscillate. The oscillation of gene expression of circadian rhythm genes shows how synchronized the cells are to the circadian rhythm. In conclusion, the samples treated with chlorpyrifos were affected by chlorpyrifos, so they became desynchronized. Furthermore, why the control samples have non-oscillation can be explained by the cells becoming less synchronized after 48 hours from entrained, as well as by the desynchronization that occurred during the medium change. Future research should conduct larger experiments where it is ensured that the cells was not affected by external factors. Despite these difficulties, this is a new and important area of research in chronotoxicology, as this research can be further applied to in vitro experimental designs, i.e., how circadian rhythms affect an experiment. Additionally, advances in this field may be important in how we deal with toxins in the future.