ToxSci Advance Access published online on August 13, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm210
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Proteomic Analysis of Rat Striatal Synaptosomes During Acrylamide Intoxication At a Low Dose-rate
1 Center for Environmental and Human Toxicology, University of Florida, Building 471, Mowry Rd., Gainesville, FL 32611-0885 2 Protein Chemistry Core, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32611-0885 3 Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, 111 E. 210th st., Bronx, NY 10467
Corresponding author: Richard M. LoPachin, Ph.D., Montefiore Medical Center, Moses Research Tower – 7, 111 E. 210th St., Bronx, NY 10467, (718) 920-5054 (telephone), (718) 515-4903 (fax), lopachin{at}aecom.yu.edu (e-mail)
Received June 6, 2007; revision received July 30, 2007; accepted August 1, 2007
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Abstract |
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We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical presynaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins. Synaptosomes were prepared from striatum of ACR-intoxicated rats (21 mg/kg/d x 7, 14 or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light (12C9 - control) or heavy (13C9 - ACR) ICAT reagent. Results show that ACR intoxication caused a progressive reduction in the ICAT labeling of many nerve terminal proteins. A label-free mass spectrometric approach (multi-dimensional protein identification) was used to show that the observed reductions in ICAT incorporation were not due to general changes in protein abundance and that ACR formed adducts with cysteine residues on peptides which also exhibited reduced ICAT incorporation. The decrease in labeling was temporally correlated to the development of neurological toxicity and confirmed previous findings that cysteine adducts of ACR accumulate as a function of exposure. The accumulation of adduct is consistent with the cumulative neurotoxicity induced by ACR and suggests that cysteine adduct formation is a necessary neuropathogenic step. Furthermore, our analyses identified specific proteins (e.g., v-ATPase, dopamine transporter, N-ethylmaleimide sensitive factor) that were progressively and significantly adducted by ACR and might, therefore, be neurotoxicologically relevant targets.
Key Words: isotope-coded affinity tag; toxic neuropathy; neurotoxicity; adduct formation; nerve terminal; proteomic analysis.
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