Basic Investigation Of The Genotoxicity And Cellular Toxicology By Environmental Phenyl Glycidyl Ether (PGE) And Polybrominated Diphenyl Ethers (PBDEs) Organic Pollutants

This research investigates the potential gene and cellular toxicology of two key environmental organic pollutants, phenyl glycidyl ether (PGE) and polybrominated diphenyl ethers (PBDEs). PGE is a material used ubiquitously wild world in the synthesis of plastics; PBDEs are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate i.e. in human biota. Humans are therefore exposed continuously to these agents; the risks of this continual exposure being unknown.In the initial research, a HPLC-MS/MS method was developed to firstly separate oligonucleotides of differing sequences and the developed method then used to investigate PGE adduct formation in specific oligonucleotides. HPLC was used to indicate PGE adduct formation and to separate the isomeric adducted oligo-products. On-line MS/MS analysis was then used to identify exactly the site of adduct formation at base resolution. The location of the adduct differed for the different oligonucleotides studied, possibly due to secondary structure formation which is known to affect adduct formation.Next, an investigation into the possible genotoxic effects of PBDEs was undertaken using a pair of cell lines (H4IIE, rat hepatoma cells and H295R, human adrenocortical carcinoma cells), using two versions of the Comet assay to assess single strand breaks and 'total-repairable' DNA damage. However, after exhaustive study, there was no evidence of any of the PBDE's exhibiting significant genotoxicity. Other cellular endpoints were then studied such as cell viability, proliferation and cell cycle arrest. Studies of cell viability and proliferation indicated the PBDEs to possess a range of effects, whilst cell cycle analysis indicated the more potent of these agents have an effect on cell cycle distribution by initiating temporary cell cycle arrest, primarily in the S phase.These studies were then followed-up by investigations of steroidogenesis and genome-wide gene expression changes by BPDEs and their derivatives. QRT-PCR analysis indicated that parent BPDEs, MeO-BDEs and OH-BDEsare able to dramatically alter steroidogenic gene expression, with the OH-moiety of the hydroxylated derivatives seeming to have a great effect. 'Microarray analysis' of genome-wide gene expression using two of the more potent OH-PBDEs (8A and 13A) indicated 13A to mediate more changes than 8A. 13A prominently altered nuclear acid metabolism genes whilst 8A altered amino acid metabolism genes. 13A changed more genes related to cell cycle and apoptosis than 8A. This is consistent with the results of the proliferation studies. Both chemicals can affect the gene expression of transcription pathways. There is no obvious concentration-dependent changes in neither 8A nor 13A induced expression upon 24 hours treatment in the present study. The Microarray data was validated, and the further PBDE concentrations tested, by real time QRT-PCR.As there was no evidence of BPDE genotoxicity apparent (Comet assay), we conclude that the biological effects of PBDEs are primarily via epigenetic routes and not via genotoxic routes. These should be further studied to more fully establish the risk of exposure and to seek possible preventative strategies.