title: Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.
abstract: Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity.
authors.affiliation: University of Ottawa, Department of Biology, Center for Advanced Research in Environmental Genomics, Ottawa K1N 6N5, ON, Canada.
authors.forename: Stephan
authors.initials: S
authors.lastname: Robitaille
authors.affiliation: Memorial University of Newfoundland, Department of Biochemistry, St. John's, Newfoundland, Canada.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Mailloux
authors.affiliation: University of Ottawa, Department of Biology, Center for Advanced Research in Environmental Genomics, Ottawa K1N 6N5, ON, Canada. Electronic address: laurie.chan@uottawa.ca.
authors.forename: Hing Man
authors.initials: HM
authors.lastname: Chan
mesh_terms: D001254:Astrocytoma / Q000201:enzymology* / Q000473:pathology; D045744:Cell Line, Tumor; D016543:Central Nervous System Neoplasms / Q000201:enzymology* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D054477:Glutaredoxins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis*; D019803:Glutathione Disulfide; D006706:Homeostasis; D006801:Humans; D020262:Mercury Poisoning, Nervous System / Q000201:enzymology* / Q000378:metabolism; D008767:Methylmercury Compounds / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D011499:Protein Processing, Post-Translational
chemical_list: C516005:GLRX protein, human; D054477:Glutaredoxins; D008767:Methylmercury Compounds; D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Astrocytoma cells; Glutathione; Grx1; Methylmercury; Nrf2; Sglutahionylation