Methylmercury neurotoxicity and interactions with selenium
Sonja Gray Campbell
Thesis submitted to the
Faculty of Graduate and Postdoctoral Studies, University of Ottawa
In partial fulfilment of the requirements for a M.Sc. degree in Biology with a specialisation
in Chemical and Environmental Toxicology
Department of Biology
Faculty of Science
University of Ottawa
Sonja Campbell, Ottawa, Canada, 2015
Methylmercury (MeHg) is a ubiquitous contaminant and potent neurotoxicant with
no completely effective therapy, although selenium antagonises MeHg toxicity. Furthermore,
nanoparticles are promising as a novel drug delivery system. We researched the potential of
selenium nanoparticles (SeNPs) in antagonising MeHg neurotoxicity compared to
selenomethionine (SeMet) using primary astrocyte cell cultures and examining outcomes
related to oxidative stress. We found that SeNPs were more toxic than SeMet. Increasing
SeNPs significantly decreased MeHg cellular uptake and MeHg significantly decreased
uptake of SeNPs at the highest concentration. Finally, SeNPs alone produced significantly
higher reactive oxidative species and altered the ratio of reduced-to-oxidised glutathione, but
MeHg, SeMet, and co-exposures did not. There were no significant effects on glutathione
peroxidase or reductase activity. This suggests that SeNPs are more toxic than MeHg in
cerebellar astrocytes and that they may not be suitable as a therapy at the doses and
formulation used in this research.
Le mthylmercure (MeHg) est un polluant omniprsent et une substance
neurotoxique, mais il ny a pas une thrapie compltement effective. La recherche a montr
que slnium a un effet antagonistique contre la toxicit de MeHg. De plus, les
nanoparticules ont le potentiel damliorer les proprits de mdicaments. Nous avons
recherch le potentiel de nanoparticules de slnium (NPSe) dantagoniser les effets
neurotoxicologique du MeHg en comparant avec slnomthionine (SeMet). Nous avons
utilis un modle de culture cellulaire avec des astrocytes crbelleux et tudi les effets de
stress oxydant. Nous avons trouv que les NPSe taient plus toxiques que SeMet. Aussi, il y
avait une relation inverse significative entre lexposition des NPSe et labsorption cellulaire
du MeHg, mais pas avec SeMet, et le MeHg a diminu labsorption cellulaire des NPSe
leur concentration maximum, mais pas SeMet. Enfin, les NPSe ont augment la production
des drivs ractifs de loxygne et diminu la proportion du glutathion rduit au glutathion
oxyd. Lactivit de la glutathion peroxydase et la glutathion rductase na pas chang entre
les doses diffrentes. Ces rsultats suggrent que les NPSe sont plus toxiques que le MeHg
pour les astrocytes crbelleux et quils ne sont pas appropris comme une thrapie aux
doses et formulations utilisant dans ces expriences.
List of Tables
1.1: Review of experimental results of MeHg co-exposure with selenium examining parameters of MeHg neurotoxicity. 2.1: Preparation of the MeHg, SeNP, and SeMet spiked media for the co-exposure experiments. 2.2: The MeHg, SeNP, and SeMet exposure regime for the co-exposure experiments.
List of Figures
1.1: Possible mechanisms of action for MeHg-induced oxidative stress. 2.1: Schematic of the co-exposure dosing regime. 3.1: Dose-response relationship of MeHg on primary cerebellar astrocytes using an MTT assay. 3.2: Dose-response relationship of MeHg on primary cerebellar astrocytes using a Trypan blue exclusion assay. 3.3: Dose-response relationship of selenium nanoparticles and seleno-L-methionine on primary cerebellar astrocytes using an MTT assay. 3.4: Cellular total mercury in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr. 3.5: Cellular total selenium in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr. 3.6: Cellular ROS in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr. 3.7: The ratio of reduced to oxidised glutathione (2GSH/GSSG) in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr. 3.8: Glutathione reductase activity in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr. 3.9: Glutathione peroxidase activity in primary cerebellar astrocytes exposed to MeHg and selenium nanoparticles or seleno-L-methionine for 24 hr.
Astrocytes: A type of glial cell, they are support cells in the brain that help maintain proper neuronal function and homeostasis. Antagonist: A chemical that interferes with the biochemical mechanism of another, leading to a reduction in its effects. Antioxidant: A chemical that neutralises free radicals and other cellular oxidising agents, reducing cellular oxidation. Blood-Brain Barrier: a selective membrane that allows passage of only small lipophilic compounds that can diffuse across membranes and ions and metabolic products via specific active transporter proteins. Cerebellum: A part of the central nervous system, it is classified as part of the metencephalon and is responsible for motor control. Chronic exposure: Exposure to a compound over a long time; doses may be high or low. Dose: The amount of a chemical that is taken up and absorbed by an organism. Exposure: The concentration of a chemical that an organism is exposed in the ambient environment, including air, water, or soil. Mechanism of action: The biochemical and molecular interactions of a xenobiotic with an organism by which it elicits its effects. Nanoparticle: Aggregates of a compound between 11000 nm in at least one dimension, although more stringent definitions require 1100 nm in at least one dimension. Neurotoxicant: A xenobiotic that has an adverse effect on the nervous system. Oxidative stress: An imbalance of the cellular redox system that results in high levels of reactive oxygen species that cause cell damage and can lead to cell death. Primary cell culture: Cells cultured in an artificial support medium that have originated directly from live tissue. Reactive oxidative species: highly reactive oxidative compounds produced as a bi-product of mitochondrial respiration.
Sublethal: Doses lower than the threshold to cause death in a significant proportion of the population exposed. Toxicant: A chemical with toxic properties that originates from an anthropogenic source. Toxicodynamics: The properties of the interactions of a toxic xenobiotic with an organism, such as its mechanism of action that elicits toxicity. Toxicokinetics: The properties of the interactions of an organism with a toxic xenobiotic, including its absorption, distribution, metabolism, and excretion. Xenobiotic: A compound found in a system, including the environment or within an organism, which has not been produced by that system.
ASM Astrocyte specific media BSA Bovine serum albumin DMSO Dimethyl sulfoxide DTT dithiothreitine EDTA tetrasodium ethylenediaminetetraacedic acid ETC Electron transport chain FBS Fetal bovine serum GABA Gamma aminobutyric acid GPx Glutathione peroxidase GR Glutathione reductase GSH Glutathione GSSG Glutathione disulphide HBSS Hanks balanced salt solution HPLC High performance liquid chromatography ICP-MS Inductively coupled plasma mass spectrometry LDH Lactose dehydrogenase LOEL Lowest observed effects level MDMA 3,4-methylenedioxy-methamphetamine MeHg Methylmercury MeHgCl Methylmercury chloride MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mtCK mitochondrial creatine kinase MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(tetrazolium dye) NAC N-acetyl cysteine NADPH Nicotinamide adenine dinucleotide phosphate NPSH Non-protein sylfhydryl PBS Phosphate buffered saline PSP Polysaccharide-protein complex Se Selenium SeMet Selenomethionine SeNP Selenium nanoparticle TBARS thiobarbituric acid reactive substances TrxR thioredoxin reductase UNEP United Nations Environmental Programme US EPA United States Environmental Protection Agency
This work is the product of collaboration of many people. The objectives of this
project were developed with the help of my supervisor, Dr. Laurie Hing Man Chan.
Furthermore, he provided guidance into the methodology, including: MeHg dosing
concentrations and the dosing regime of the co-exposures; the rationale to include the cell
uptake experiments; advice on data analysis and presentation; and critiques on the final
thesis. My graduate committee members, Dr. Michael Jonz, Dr. Bill Willmore, and Dr.
Alexandre Poulain provided valuable guidance throughout the course of my thesis. Dr. Ryan
Mailloux provided the suggestion to include the 2GSH/GSSG experiment in the experiment
in place of a less sensitive method. Also, he measured the 2GSH/GSSG samples and
completed the data processing. Finally, Dr. Ryan Mailloux provided invaluable advice on the
enzyme assays that allowed us to optimise the experiments effectively. Dr. Emmanuel
Yumvehoze provided advice on the cell uptake methodology, in particular helping to
optimise the selenium uptake experiment, which initially had issues with instrument
sensitivity. As well, he conducted the measurements and initial data processing of the resu