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Profiling biochemical changes associated with retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells

Manning, Alicia (2015) Profiling biochemical changes associated with retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells. Honours thesis, Murdoch University.

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SH-SY5Y neuroblastoma cells have been widely used in neuroscience research to model neurotoxic processes. It is unclear how well, in their undifferentiated, immortalised state, the cells model the original tissue type. SH-SY5Y cells can be differentiated, with compounds such as retinoic acid (RA), to become more ‘tissue-like’, and potentially better reflect the biochemistry of a mature neuronal cell. Studies that have investigated the RA-induced differentiation process have focused on upstream changes associated with a small number of genes or proteins. This study proposed to use a novel approach in untargeted gas chromatography-mass spectrometry (GC-MS) metabolomic analysis to investigate the downstream biochemical changes in the metabolome, which are closer to the phenotype associated with an RA-differentiated cell. Metabolomics was utilised to profile a wide range of biochemical changes in SH-SY5Y cells exposed to RA and how these processes were affected by exposure to malaoxon, a toxic metabolite of the pesticide malathion, to assess whether differentiated cells are more appropriate in future toxicology research.

Cultured human SH-SY5Y neuroblastoma cells were exposed to 10 μM RA for 120 hours and 1 μM malaoxon for 24 hours. The samples were quenched, harvested, extracted and derivatised before metabolomic analysis was carried out using untargeted GC-MS. The metabolites that contributed most to the variance were identified using principal component analysis. This indicated that RA-differentiated cells, and undifferentiated and differentiated cells exposed to malaoxon, were grouped in relation to metabolite profile. The important metabolites were subsequently analysed to determine changes due to RA and malaoxon exposure, with the aim to produce a profile of RA-differentiation cells and of the differential toxicity between undifferentiated and differentiated cells.

Changes were profiled in a range of metabolites including amino acids, carbohydrates, fatty acids and unknowns, indicating that exposure to RA caused a biochemical response in relation to neurite outgrowth, survival and apoptosis, decreased energy demand for cell proliferation and increased synthesis and activation of proteins involved in cell signalling. Data suggest that exposure to malaoxon induced a glutamate-mediated excitotoxic response causing oxidative stress, mitochondrial dysfunction, energy depletion and damage to the cell membrane. In the differentiated state, induction of survival and apoptotic responses may have facilitated a neuroprotective response that decreased the toxic effects of malaoxon.

This preliminary study has demonstrated that GC-MS-based metabolomic analysis can be used to detect changes in metabolite profile after RA and malaoxon exposure, illustrating the efficacy of this technique. The data indicates that there were a range of biochemical changes associated with an RA differentiated phenotype and that using such cells to study the effects of neurotoxins should be done with caution due to the potential neuroprotective response. The outcomes of this study have implications for research using RA-differentiated SH-SY5Y cells, with the decision to use differentiated cells resting on the particular neurotoxic model being investigated.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Maker, Garth, Trengove, Robert and Mullaney, Ian
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