ToxSci Advance Access published online on April 27, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm095
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Manganese induces oxidative impairment in cultured rat astrocytes
* Department of Pediatrics, and the Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Pharmacology, and the Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Murray State University, Breathitt Veterinary Center, Toxicology Department, Hopkinsville, KY 42241 USA
Department of Neurotoxicology, Medical Research Centre, Polish Academy of Sciences, 02-792 Warsaw, Poland
Please send request for reprints to: Michael Aschner, Ph.D., Department of Pediatrics, B-3307 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2495, Phone: 615-322-8024, FAX: 615-322-6541, E-mail: Michael.Aschner{at}vanderbilt.edu
Received February 21, 2007; revision received April 16, 2007; accepted April 18, 2007
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Abstract |
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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 (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 F2-isoprostanes (F2-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 F2-IsoPs levels at 2 h following exposure to Mn (100 µM, 500 µM or 1 mM). Consistent with this observation, Mn induced a concentration-dependant reduction in ATP and the inner mitochondrial membrane potential (
m), measured by the HPLC method and the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE), respectively. Moreover, 30 min of pretreatment with Mn (100 µM, 500 µM or 1 mM) inhibited the net uptake of glutamine (3H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 µM and 500 µM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction and alterations in glutamine/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.
Key Words: Astrocyte; Manganese; F2-isoprostanes; mitochondria; ATP; m; glutamine.
Email: Dejan Milatovic – dejan.milatovic{at}vanderbilt.edu; Zhaobao Yin – Zhaobao.yin{at}vanderbilt.edu; Ramesh C. Gupta – ramesh.gupta{at}murraystate.edu; Marta Sidoryk – marthas{at}cmdik.pan.pl; Jan Albrecht – jalb{at}cmdik.pan.pl; Judy L. Aschner – judy.aschner{at}vanderbilt.edu
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