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Title: Exploring oxidative stress pathways in epilepsy
Author: Al-Mosawi, R. H.
ISNI:       0000 0004 7970 3699
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Background: Oxidative stress is now believed to play a role in the evolution of epilepsy and antioxidants have been proposed as putative antiepileptic agents. The experimental evidence suggested that nuclear factor, erythroid 2 like 2 (Nrf2) could be a promising therapeutic target for epilepsy. The main aims of this study were to investigate the effects of common AEDs on oxidative stress biomarkers in a human SH-SY5Y cell-line, and to explore the association of common variants in the (NFE2L2) gene with clinical characteristics of epilepsy. Methods: SH-SY5Y human cells were exposed to H2O2 (0-1000µM), LPS (0-100 µg/ml), and pilocarpine (0-100 µM) for 1, 4, and 24 hours. Cells were exposed to carbamazepine (CBZ; 0-100µM)), valproic acid (VPA; 0-1000µM) levetiracetam (LEV; 0-300µM), and lamotrigine (LTG; 0-100µM) for 1, 4, and 24hrs, with or without 100µM H2O2 for further 4hrs. AED effects were assessed against basal oxidative stress, determined by malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and reduced/oxidised glutathione (GSH/GSSG) ratio. The expression of Nrf2, haemoxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1) was determined using real time PCR and western blot. The genotyping of the five SNPs in NFE2L2 gene was carried out using TaqMan® genotyping assays. Results: MDA level was increased in a time and concentration-dependent manner up to maximum of 3-fold, following exposure to H2O2, LPS, and pilocarpine. However, SOD activity, GSH/GSSG ratio decreased by 5-fold, 15-fold, respectively, after exposure to H2O2, LPS, and pilocarpine. MDA level was elevated in a concentration-dependent-manner, up to a maximum of 13-fold, 5.5-fold, 15-fold and 9.5-fold, following exposure to CBZ, VPA, LEV and LTG, respectively. In contrast, SOD activity was reduced, again in a concentration-dependent manner, by a factor of up to 2, 2.5, 1.5 and 1.3 after incubation with CBZ, VPA, LEV and LTG, respectively. The GSH/GSSG ratio was also reduced after exposure to CBZ (up to 5.8-fold), VPA (up to 4-fold) LEV (up to 5-fold), and LTG (up to 3-fold). Further exposure to 100µM H2O2 amplified AEDs effects on MDA concentration, SOD activity and GSH/GSSG ratio by up to 3-fold. Expression of the Nrf2 gene was increased by 4-fold following exposure to both 100µM CBZ and 1000µM VPA, while expression of HO-1 was increased by 2.7-fold and 3-fold, respectively. NQO-1 gene expression was increased after incubation with 100µM LTG and 1000µM VPA by 2.3-fold and 2.9-fold, respectively. Additional exposure to 100µM H2O2 augmented AED effects on Nrf2, HO-1 and NQO-1 gene expression by up to 5-fold. Relative expression of Nrf2, HO-1 and NQO-1 proteins was increased by 1.3-fold, 1.2-fold and 1.2-fold after exposure to CBZ, VPA and LTG, respectively. There was no effect of LEV. Small additional increases (up to 10%) in protein expression were seen after further 24 hours incubation with 100µM H2O2. Association was observed between NFE2L2 genotype, rs2886161, and the remission on any AED. Nonetheless, a multivariate predictive model did not show any significant contribution of NFE2L2 SNP genotypes, demographic and clinical predictors and the response to AED treatment. Conclusions: These findings imply that several existing AEDs have pro-oxidant rather than antioxidant effects, albeit at relatively high concentrations. This may limit their antiseizure activity and/or contribute to their adverse effect profile. There was no evidence from this pharmacogenomics investigation that common variants in the NFE2L2 gene have any influence on the presentation of epilepsy or on its prognosis.
Supervisor: Pirmohamed, Munir ; Marson, Anthony ; Sills, Graeme Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral