Evaluation of the neurotoxicity of pentachlorophenol and its active metabolites on SH-SY5Y neuroblastoma cells

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dc.contributor.advisor Steenkamp, Vanessa
dc.contributor.coadvisor Stander, Barend Andre
dc.contributor.postgraduate Fraser, Desiree Linda
dc.date.accessioned 2018-12-05T08:06:26Z
dc.date.available 2018-12-05T08:06:26Z
dc.date.created 2009/06/18
dc.date.issued 2018
dc.description Dissertation (MSc)--University of Pretoria, 2018.
dc.description.abstract Pentachlorophenol (PCP) is an organochloride pesticide that is ubiquitous within the environment due to its chemical stability. It is classified as a persistent organic pollutant, and has been predominantly used in the wood preservation industry. Workers and populations living close to PCP usage and production are exposed to it via inhalation and dermal absorption, and ingestion of contaminated food and water. It is lipophilic and is able to accumulate within various bodily systems, including the brain. Adverse effects of PCP have been reported to varying degrees in the immune, hepatic, and endocrine systems. Although neurological symptoms have been associated with PCP exposure, knowledge of mechanisms of neurotoxicity is limited. Elucidation of molecular mechanisms at a cellular level within neuronal cells is required to contribute toward the current gap in the knowledge of PCP neurotoxicity. The aim of the study was to evaluate the effects of PCP and its active metabolites, tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in human neuroblastoma SH-SY5Y cells. Effects on cell proliferation were assessed using the sulforhodamine B (SRB) assay. Flow cytometric analysis was employed to investigate effects on cell cycle using propidium iodide (PI), mode of cell death using Annexin V-FITC and PI, reactive oxygen species (ROS) using dichlorofluorescein, and mitochondrial membrane potential (Δᴪm) using JC-1 fluorescence. Caspase-3 activity was assessed with Ac- DEVD-AMC, and glutathione (GSH) with monochlorobimane fluorescence. Effects on acetylcholinesterase (AChE) were assessed in vitro using the Ellman esterase assay, as well as in silico via molecular docking and molecular dynamics simulation. The IC50 concentrations of PCP, TCBQ and TCHQ were 80.0, 35.4, and 63.7 μM, respectively. Cell cycle disruptions were revealed in the form of a G1 block and a G2/M block as a result of PCP and TCHQ exposure, respectively, while TCBQ resulted in a prolonged S phase traverse. The predominant mode of cell death of PCP was necrosis, while TCBQ induced apoptosis. Exposure to TCHQ resulted in one of two fates, being either predominantly apoptotic or necrotic cell death. Decreased Δᴪm was an early event for all compounds, however, differed in their involvement of inducing ROS. Oxidative stress was an evident mechanism of PCP and TCBQ toxicity, as increased ROS was accompanied by lowered GSH, while reductive stress leading to subsequent oxidative stress was indicated by increased ROS and GSH for TCHQ. All compounds yielded increased caspase-3 activity. The fate of TCHQ exposed cells was postulated as a switch from apoptosis to necrosis due to overwhelming ROS insult on apoptotic machinery, surpassing a threshold for apoptosis capability. Inhibition of AChE was observed by only TCHQ in vitro, the Ellman IC50 of which was 79.7 μM. In silico assessment supported a hypothesis of TCHQ inhibition of AChE, with TCHQ-acetate bound ligands binding AChE receptors with binding energies corresponding to the Ellman IC50. Binding stability was confirmed by molecular dynamics. Pentachlorophenol and its active metabolites exhibited different mechanisms of toxicity toward neuronal cells, leading to different modes of cell death. A new hypothesis for the molecular mechanism of TCHQ AChE inhibition was developed, and sets a platform for further investigation.
dc.description.availability Unrestricted
dc.description.degree MSc
dc.description.department Physiology
dc.identifier.citation Fraser, DL 2018, Evaluation of the neurotoxicity of pentachlorophenol and its active metabolites on SH-SY5Y neuroblastoma cells, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/68006>
dc.identifier.other S2018
dc.identifier.uri http://hdl.handle.net/2263/68006
dc.language.iso en
dc.publisher University of Pretoria
dc.rights © 2018 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.
dc.subject Unrestricted
dc.subject UCTD
dc.title Evaluation of the neurotoxicity of pentachlorophenol and its active metabolites on SH-SY5Y neuroblastoma cells
dc.type Dissertation


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