title: Manganese induces oxidative impairment in cultured rat astrocytes.
abstract: Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Dejan
authors.initials: D
authors.lastname: Milatovic
authors.forename: Zhaobao
authors.initials: Z
authors.lastname: Yin
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Gupta
authors.forename: Marta
authors.initials: M
authors.lastname: Sidoryk
authors.forename: Jan
authors.initials: J
authors.lastname: Albrecht
authors.forename: Judy L
authors.initials: JL
authors.lastname: Aschner
authors.forename: Michael
authors.initials: M
authors.lastname: Aschner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D026962:Amino Acid Transport System ASC / Q000096:biosynthesis; D026921:Amino Acid Transport Systems, Neutral / Q000096:biosynthesis; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology*; D002478:Cells, Cultured; D005110:Extracellular Space / Q000187:drug effects; D028441:F2-Isoprostanes / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D020149:Manganese Poisoning / Q000378:metabolism* / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D015778:Minor Histocompatibility Antigens; D051336:Mitochondrial Membranes / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D026962:Amino Acid Transport System ASC; D026921:Amino Acid Transport Systems, Neutral; D028441:F2-Isoprostanes; D015778:Minor Histocompatibility Antigens; D012333:RNA, Messenger; C498849:Slc1a5 protein, rat; D013778:Tetrazolium Salts; D013844:Thiazoles; C403222:system N protein 1; D005973:Glutamine; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue