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Title: Cellular response to DNA damage after exposure to organophosphates in vitro
Author: Remington, Seth Eli
ISNI:       0000 0004 2710 7094
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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This study aimed to investigate the potential of dichlorvos (DCV), chlorpyrifos (CPF) and sarin (GB) to induce DNA damage in vitro. DNA damage was measured by the alkaline comet assay in the neuroblastoma SHSY-5Y and lymphoblastoid TK6 cells and corroborated using phosphorylation of H2AX. Cytotoxicity was measured using the MTT assay in TK6 cells. The activation of DNA damage signalling pathways was investigated using western blotting. TK6 cells were used to investigate p53 levels and cleaved PARP-1 after exposure to OPs. Cell cycle effects were investigated in A549 cells by measuring the phosphorylation of the retinoblastoma and cdc2. Whether a slowing of the cell cycle allowed for initiation of repair mechanisms was investigated in cells lacking the DNA repair pathways base excision repair (BER) and non homologous end joining (NHEJ) after DCV. DCV induced DNA damage, the genotoxic potential was also corroborated by increased expression of γH2AX. DCV was cytotoxic at high concentrations. DCV results in increased p53 levels but no apparent phosphorylation on Ser15, which is known to be phosphorylated when exposed to other genotoxins. A G2/M block was found after exposure to dichlorvos as well as the possible initiation of DNA repair mechanisms NHEJ and BER. CPF and CPF oxon induced DNA damage in TK6 cells with a decrease in cell viability. Western blot analysis did not show a p53 response to the DNA damage with CPF. A small p53 effect at 24hours was seen with chlorpyrifos oxon but p53 may have been induced at an earlier time point. It is possible that the DNA damage observed was due to both direct DNA damage and other cellular effects, as the cells undergo apoptosis or necrosis and subsequent DNA degradation. Sarin caused low levels of DNA damage after 1 hour, which was partially repaired at 24 hours. This resulted in low level cytotoxicity and a rapid but transient increase in p53 levels and a G2/M cell cycle arrest.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available