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Title: The effect of heavy metals on differentiated neuronal and glial cells
Author: Lawton, Michelle
ISNI:       0000 0001 3606 2025
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2007
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Heavy metal poisoning poses a serious health risk among populations worldwide. The symptoms presented by exposure are varied and depend upon the species of the metal, the age of the individual and the exposure dose. All heavy metals have debilitating effects on the CNS. Children are especially sensitive to the neurological effects due to the intense growth and activity of a developing nervous system and inadequately developed defences. The aims of this study were to determine the effects of sub-lethal concentrations of numerous heavy metals on neuronal and glial cell differentiation. Using established cellular models, the toxicity of zinc, lead, mercury, methylmercury and thimerosal were investigated using assays of cell viability and morphology on differentiating N2a and C6 cells. Initial research revealed thimerosal, methylmercury and cadmium to be the most toxic compounds tested, in terms of their ability to inhibit the outgrowth of neurites in both cell lines at sub-lethal concentrations. Although cadmium chloride showed similar patterns of toxicity to the mercury compounds, thimerosal and MeHgCl were chosen for further investigation at a molecular level. Methylmercury chloride a common environmental pollutant and thimerosal; a preservative found in many medicines, were chosen for further investigation, as previous work has demonstrated the health risks posed by the two organic mercury compounds but little is known about non-lethal changes that occur in the nervous system, especially with thimerosal. Both thimerosal and MeHgCl inhibited MTT reduction and neurite outgrowth after 4 and 24 hours exposure at sub-lethal concentrations (0.1 and 1 µM). The inhibition of neurite outgrowth by sub-lethal concentrations of MeHgCl and thimerosal was accompanied by cytoskeletal changes in the cells. At 4 hours in C6 cells there was no change in the levels of tyrosinated a-tubulin, whereas in N2a cells the level of tubulin tyrosination was shown to be reduced compared to the control. Both cell lines exhibited a fall in total a-tubulin, tyrosinated a-tubulin and total ß-tubulin after 24 hours of exposure to organic mercury compounds, indicating proteolysis and/or reduced synthesis of the tubulin subunit. N2a cells also showed a decrease in the levels of phosphorylation in the neurofilament heavy chain after 4 hours of exposure to thimerosal and MeHgCl, whereas after 24 hours there appeared to be proteolytic degradation, as the total neurofilament heavy chain levels were reduced compared to the untreated controls. Reduced levels of tubulin and NFH were confirmed by immunofluorescence staining of fixed cell monolayers. Western blotting analysis also indicated increased ERK activation in glial cells incubated with 0.1 and 1 µM thimerosal for 4 hours, followed by reduced activation after 24 hours exposure, whereas exposure to MeHgCl decreased the levels of ERK activation at both time points. In the neuronal cell line ERK activation was suppressed at both 4 and 24 hours and with both concentrations of the organic mercury compounds. As ERK activation plays a key role in the regulation of neurite outgrowth and NFH phosphorylation, both of which were inhibited by the addition of thimerosal and MeHgCl, the findings are consistent with a role for disrupted ERK signalling in the sub-lethal toxicity of these compounds. Both thimerosal and MeHgCl caused redistribution of SERCA and ryanodine receptors, both of which are mechanisms by which intracellular Ca2+ concentrations are maintained. As the endoplasmic reticulum (ER) houses both SERCA and ryanodine receptors, the reorganisation may indicate that organic mercury compounds cause redistribution of the ER. Such disruption may lead to sustained increases in intracellular Ca2+, causing elevated activity in Ca2+ dependant enzymes. Indeed, western blotting analysis and enzyme assays showed that calpain activity (particularly calpain 1) increased in response to sub-lethal concentrations of the organic mercury compounds. As calpains target cytoskeletal proteins, the increased activity may be at least partly responsible for reduced levels of tubulin and NFH.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available