title: Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane.
abstract: 1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn(2+))-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p<0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn(2+)-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity.
authors.affiliation: Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300, PR China; Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
authors.forename: Zhenlie
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507, Japan.
authors.forename: Sahoko
authors.initials: S
authors.lastname: Ichihara
authors.affiliation: Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Oikawa
authors.affiliation: Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507, Japan.
authors.forename: Jie
authors.initials: J
authors.lastname: Chang
authors.affiliation: Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510, Japan.
authors.forename: Lingyi
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300, PR China.
authors.forename: Shijie
authors.initials: S
authors.lastname: Hu
authors.affiliation: Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300, PR China. Electronic address: huanghl@gdoh.org.
authors.forename: Hanlin
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510, Japan. Electronic address: gak@rs.tus.ac.jp.
authors.forename: Gaku
authors.initials: G
authors.lastname: Ichihara
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism*; D006842:Hydrocarbons, Brominated / Q000633:toxicity; D008297:Male; D008345:Manganese / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D011916:Rats, Inbred F344; D012997:Solvents / Q000633:toxicity*
chemical_list: D051017:Apoptosis Regulatory Proteins; D006842:Hydrocarbons, Brominated; D010750:Phosphoproteins; D012997:Solvents; D008345:Manganese; C118559:1-bromopropane
keywords: 1-Bromopropane; 14-4-3 θ; Antiapoptosis; Hippocampus; Phosphoproteomics