Evaluation of the mechanisms of neurotoxcity using metabolomic strategies

Student thesis: Doctoral Thesis

Abstract

There are many neurotoxic substances that affect humans. Cobalt and mephedroneare examples of neurotoxic compounds with little known about their mode of action.Cobalt neurotoxicity was investigated in human astrocytoma and neuroblastoma cellsusing proliferation assays coupled with LC-MS based metabolomics and transcriptomics techniques. Cells were treated with a range of cobalt concentrations between 0 and 200 μM. The metabolism of mephedrone at 100 μM was first studied in primary rat hepatocytes and then the neurotoxicity of mephedrone at 100 μM was assessed in human neuroblastoma and astrocytoma cells using proliferation assays coupled with LC-MS based metabolomics.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayr evealed that cobalt was cytotoxic and decreased cell metabolism and its effect was dose and time dependent in both cell lines. Metabolomic analysis revealed several altered metabolites particularly those related to DNA deamination and methylation pathways. One of the increased metabolites was uracil which can be generated from DNA deamination or from fragmentation of RNA. To investigate the origin of uracil genomic DNA was isolated and analysed by LC-MS. Interestingly, the source of uracil, which is uridine was increased significantly in the DNA of both cell lines.Additionally, the results of the qRT-PCR showed an increase in the production of five genes Mlh1, Sirt2, MeCP2, UNG and TDG in both cell lines. These genes are related to DNA strand breakage, hypoxia, methylation and base excision repair.Cultured primary hepatocytes were able to metabolise mephedrone and produce many of the metabolites produced by previous research on freshly isolated hepatocytes. However, metabolic capacity was impaired in the cultured hepatocytes with lower quantities of metabolites being produced in comparison with freshly isolated hepatocytes. MTT and neutral red (NR) assays results showed no negative effect on cells metabolism or growth. There was an increase in cell metabolism at 100 μM of mephedrone. This concentration of mephedrone was used in the metabolomic experiments to further investigate metabolomics changes associated with this concentration. The metabolomic analysis of neuroblastoma revealed no strong pattern of effect of mephedrone on the cells. However, the analysis of the metabolome of astrocytoma revealed a marked effect after treatment with mephedrone. The predominant change was on lipids particularly ether lipids that play an important role in controlling membrane fluidity.Overall, metabolomic analysis was helpful in revealing the changes that both cobalt and mephedrone could induce in human neuronal derived cell lines. These findings could help in understanding the effect of these cytotoxic compounds on human brain.
Date of Award1 Apr 2018
LanguageEnglish
Awarding Institution
  • University Of Strathclyde
Supervisor (Supervisor)David Watson (Supervisor)

Cite this