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Title: Molecular-genetics studies of organophosphate induced neurodegeneration in differentiating mammalian cell lines and neural progenitor stem cells
Author: Alyamani, Najiah
ISNI:       0000 0004 7661 0913
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2018
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Organophosphorus (OP) pesticides are widely used despite evidence they cause neurotoxicity after exposure. The primary target for OPs is acetylcholinesterase, but there is evidence they can inhibit other cellular proteins including cytoskeletal and axon growth associated proteins, which are implicated in nervous system development. Furthermore, little is known about the ability of OPs to cause genotoxicity. The objectives of this study were to evaluate the effects of selected OPs on neurite outgrowth, expression of cytoskeletal proteins and associated gene expression levels, and to investigate histone deacetylation (HDAC) activity in three types of differentiating cell models. The initial findings indicated that cell viability was unaffected by exposure to 1, 3 and 10 µM CPF, CPO and PSP in N2a and C6 cells. A high content assay was sensitive enough to rapidly detect and quantify morphological changes, including inhibition of neurite number and length. Western blot and ELISA analysis in N2a and C6 cells revealed reduced levels of the selected cytoskeletal and associated regulatory proteins (MAP-2, Tau, βIII-tubulin, GAP43, NFH and GFAP) following the treatment with at least one concentration of CPF, CPO and PSP, which could be linked to the inhibition of neurite outgrowth. Using quantitative RT-PCR analysis on the total RNA of the genes MAP-2, TUBB3, MAPT, NEFH, GAP-43, and GFAP showed a good correlation between the altered protein expression and regulation of gene levels for most markers, which suggests these OPs can cause genotoxic effects. Increased levels of HDAC activity were observed for all OPs in rodent cell lines, suggesting that epigenetic effects may be at least partly involved in some gene expression changes. RT-PCR analysis of TUBB3, NEFH, and GFAP was also carried out in ReNcell CX cells, a co-culture of neuronal and glial cells, and showed down-regulation of gene levels for at least one concentration of all the OPs, as well as increasing the level of HDAC activity in a similar pattern to the results for N2a and C6 cells. Taken together, the data in this thesis suggest a novel action of OPs altering HDAC activity, which can be correlated to some of the observed changes in gene and protein levels of selected cytoskeletal and associated regulatory proteins that can be linked to the observed disruption of neurite outgrowth and neural development. Further work is needed to identify other molecular targets invoved in these phenomena, particularly when there is no correlation with HDAC activity changes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available