Mercury Induces Cell Cytotoxicity and Oxidative Stress and Increases β‐Amyloid Secretion and Tau Phosphorylation in SHSY5Y Neuroblastoma Cells
TL;DR: It is demonstrated that exposure of cells to 50 μg/L HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels, and results indicate that mercury may play a role in pathophysiological mechanisms of AD.
Abstract: Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, beta-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 microg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p<0.001). Preincubation of cells for 30 min with 1 microM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 microg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p<0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of beta-amyloid peptide (Abeta) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Abeta 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Abeta 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p<0.001). Preincubation of cells with melatonin resulted in an attenuation of Abeta 1-40 and Abeta 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p<0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.
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TL;DR: The three modern "faces" of mercury are the authors' perceptions of risk from the exposure of billions of people to methyl mercury in fish, mercury vapor from amalgam tooth fillings, and ethyl mercury in the form of thimerosal added as an antiseptic to widely used vaccines.
Abstract: The three modern "faces" of mercury are our perceptions of risk from the exposure of billions of people to methyl mercury in fish, mercury vapor from amalgam tooth fillings, and ethyl mercury in the form of thimerosal added as an antiseptic to widely used vaccines. In this article I review human exposure to and the toxicology of each of these three species of mercury. Mechanisms of action are discussed where possible. Key gaps in our current knowledge are identified from the points of view both of risk assessment and of mechanisms of action.
975 citations
Cites background from "Mercury Induces Cell Cytotoxicity a..."
...Other studies have indicated that mercury can cause hyperphosphorylation of the tau protein (114)....
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...Mercury is well known to cause biochemical changes in cells is consistent with oxidative stress (114)....
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TL;DR: D diagnosis of mercury toxicity can be challenging but can be obtained with reasonable reliability and effective therapies for clinical toxicity have been described.
Abstract: Mercury is a toxic heavy metal which is widely dispersed in nature. Most human exposure results from fish consumption or dental amalgam. Mercury occurs in several chemical forms, with complex pharmacokinetics. Mercury is capable of inducing a wide range of clinical presentations. Diagnosis of mercury toxicity can be challenging but can be obtained with reasonable reliability. Effective therapies for clinical toxicity have been described.
791 citations
568 citations
TL;DR: Data is discussed about the role of environmental factors in the development of idiopathic AD and PD, and their mechanisms of action, including epigenetic mechanisms by maternal nutrient supplementation and exposure to heavy metals and pesticides.
Abstract: Neurodegenerative diseases including Alzheimer (AD) and Parkinson (PD) have attracted attention in last decades due to their high incidence worldwide. The etiology of these diseases is still unclear; however the role of the environment as a putative risk factor has gained importance. More worryingly is the evidence that pre- and post-natal exposures to environmental factors predispose to the onset of neurodegenerative diseases in later life. Neurotoxic metals such as lead, mercury, aluminum, cadmium and arsenic, as well as some pesticides and metal-based nanoparticles have been involved in AD due to their ability to increase beta-amyloid (Aβ) peptide and the phosphorylation of Tau protein (P-Tau), causing senile/amyloid plaques and neurofibrillary tangles (NFTs) characteristic of AD. The exposure to lead, manganese, solvents and some pesticides has been related to hallmarks of PD such as mitochondrial dysfunction, alterations in metal homeostasis and aggregation of proteins such as α-synuclein (α-syn), which is a key constituent of Lewy bodies (LB), a crucial factor in PD pathogenesis. Common mechanisms of environmental pollutants to increase Aβ, P-Tau, α-syn and neuronal death have been reported, including the oxidative stress mainly involved in the increase of Aβ and α-syn, and the reduced activity/protein levels of Aβ degrading enzyme (IDE)s such as neprilysin or insulin IDE. In addition, epigenetic mechanisms by maternal nutrient supplementation and exposure to heavy metals and pesticides have been proposed to lead phenotypic diversity and susceptibility to neurodegenerative diseases. This review discusses data from epidemiological and experimental studies about the role of environmental factors in the development of idiopathic AD and PD, and their mechanisms of action.
474 citations
TL;DR: It is proposed that metal ion chelators, antioxidants, antiinflammatories and amyloid-lowering drugs that target the reduction of H2O2 and/or Aβ generation may be efficacious in decreasing neurotoxicity.
283 citations
Cites background from "Mercury Induces Cell Cytotoxicity a..."
...localization of cytoplasmic metal ions appear to occur concurrently with increases in cytoplasmic RNA oxidation (Nunomura et al., 2000; Nunomura et al., 2001; Olivieri et al., 2000)....
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...in cellular glutathione (Olivieri et al., 2000) and oxidative stress induced by micromolar concentrations of Aß (Olivieri et al....
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References
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TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
225,085 citations
"Mercury Induces Cell Cytotoxicity a..." refers methods in this paper
...Cell lysate protein concentrations were normalized using the protein assay of Bradford (1976)....
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...Protein concentration was assessed by protein analysis by the method of Bradford (1976), and all samples were diluted to equal protein concentrations....
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...…pellets were resuspended in a 150 mM TrisHCl buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 2 mM EDTA, and Complete Mini protease inhibitors (Roche Biochemicals) and normalized to equal protein concentrations with the protein assay of Bradford (1976)....
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TL;DR: It is shown that agents which prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3, and it is demonstrated that PKB is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC).
Abstract: Glycogen synthase kinase-3 (GSK3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin, modulation of the transcription factors AP-1 and CREB, the specification of cell fate in Drosophila and dorsoventral patterning in Xenopus embryos. GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). Here we show, however, that agents which prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and we demonstrate that it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3 (refs 10, 14), the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase.
5,158 citations
TL;DR: Data support the involvement of ApoE ϵ4 in the pathogenesis of late-onset familial and sporadic AD and suggest it may operate as a susceptibility gene (risk factor) for the clinical expression of AD.
Abstract: Apolipoprotein E, type epsilon 4 allele (APOE epsilon 4), is associated with late-onset familial Alzheimer's disease (AD). There is high avidity and specific binding of amyloid beta-peptide with the protein ApoE. To test the hypothesis that late-onset familial AD may represent the clustering of sporadic AD in families large enough to be studied, we extended the analyses of APOE alleles to several series of sporadic AD patients. APOE epsilon 4 is significantly associated with a series of probable sporadic AD patients (0.36 +/- 0.042, AD, versus 0.16 +/- 0.027, controls [allele frequency estimate +/- standard error], p = 0.00031). Spouse controls did not differ from CEPH grandparent controls from the Centre d'Etude du Polymorphisme Humain (CEPH) or from literature controls. A large combined series of autopsy-documented sporadic AD patients also demonstrated highly significant association with the APOE epsilon 4 allele (0.40 +/- 0.026, p < or = 0.00001). These data support the involvement of ApoE epsilon 4 in the pathogenesis of late-onset familial and sporadic AD. ApoE isoforms may play an important role in the metabolism of beta-peptide, and APOE epsilon 4 may operate as a susceptibility gene (risk factor) for the clinical expression of AD.
3,551 citations
"Mercury Induces Cell Cytotoxicity a..." refers background in this paper
...These findings have been extended to sporadic late-onset AD (Saunders et al., 1993)....
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TL;DR: A role for PKB in PI(3)K-mediated signal transduction is suggested and a constructed Gag–PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity.
Abstract: A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.
2,137 citations
"Mercury Induces Cell Cytotoxicity a..." refers background in this paper
...Phosphatidylinositol 3-hydroxykinase has further been shown to regulate glycogen synthase kinase-3 negatively via activation of protein kinase B and its pathway (Burgering and Coffer, 1995; Cross et al., 1995)....
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TL;DR: Evidence is presented that beta-amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes and generate free radical peptides.
Abstract: beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.
1,111 citations
Additional excerpts
...231 production (Hensley et al., 1994; Behl, 1997; Pappolla et al., 1997)....
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