Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury

Gouri Shanker, Judy L. Aschner, Tore Syversen, Michael Aschner

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

Oxidative stress has been implicated in neurotoxic damage associated with various metals, including methylmercury (MeHg). Although the mechanism(s) of MeHg-induced neurotoxicity remains unclear, evidence supports a mediatory role for astrocytes, a cell type that preferentially accumulates MeHg. Using scanning confocal microscopy (LSCM), the present study was undertaken to examine the role of astrocytes as the site of reactive oxygen species (ROS). Three redox-sensitive fluorescent probes were used for ROS analysis, (a) CM-H 2DCFDA (chloromethyl derivative of dichlorodihydrofluorescein diacetate), a probe for intracellular hydrogen peroxide (H2O 2); (b) hydroethidine (HETH), a probe for superoxide anion (·O2 -), and (c) CM-H2XRos (chloromethyl derivative of dihydro X-rosamine), and a probe that is selective for mitochondrial reactive oxygen intermediates. Astrocytes were treated with 10 μM MeHg for 30 min, following which the various fluorescent probes were added; 20 min later LSCM images were collected. Astrocytes loaded with CM-H 2DCFDA and HE demonstrated a significant MeHg-induced increase in fluorescence intensity indicative of increased intracellular H2O 2 and·O2 -, respectively. Similar results were obtained with the mitotracker dye, CM-H2XRos. Additionally, exposure of astrocytes for 24 h to 100 μM buthionine-L-sulfoxane (BSO), a glutathione (GSH) synthesis inhibitor, caused a significant increase in ROS formation. Furthermore, BSO pretreatment significantly enhanced the MeHg-induced formation of·O2 -, indicating an important role for GSH in the maintenance of optimal cellular redox status. Time-course experiments performed in the simultaneous presence of CM-H2XRos and CM-H2DCFDA demonstrated that the MeHg-induced CM-H2XRos fluorescence changes preceded those of CM-H2DCFDA, suggesting that the mitochondria represent an early primary site for ROS formation. Taken together, these studies illustrate that MeHg induces the generation of astrocyte-derived ROS and support a role for astrocytic ROS in MeHg-associated neurotoxic damage.

Original languageEnglish (US)
Pages (from-to)48-57
Number of pages10
JournalMolecular Brain Research
Volume128
Issue number1
DOIs
StatePublished - Sep 10 2004
Externally publishedYes

Fingerprint

Astrocytes
Free Radicals
Reactive Oxygen Species
Fluorescent Dyes
Oxidation-Reduction
Fluorescence
Confocal Microscopy
Superoxides
Hydrogen Peroxide
Glutathione
Mitochondria
Oxidative Stress
Coloring Agents
Metals
Maintenance
Oxygen
buthionine
2',7'-dichlorodihydrofluorescein diacetate

Keywords

  • Astrocytes
  • Disorders of the nervous system
  • Glutathione
  • Laser scanning confocal microscopy
  • Methylmercury
  • Mitochondria
  • Neurotoxicity
  • Neurotoxicology
  • Reactive oxygen species
  • Redox status

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury. / Shanker, Gouri; Aschner, Judy L.; Syversen, Tore; Aschner, Michael.

In: Molecular Brain Research, Vol. 128, No. 1, 10.09.2004, p. 48-57.

Research output: Contribution to journalArticle

Shanker, Gouri ; Aschner, Judy L. ; Syversen, Tore ; Aschner, Michael. / Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury. In: Molecular Brain Research. 2004 ; Vol. 128, No. 1. pp. 48-57.
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