Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells |
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Authors: | Sohee Phark So-Young Park Seonyoung ChoiZhi Zheng Eunkyung ChoMin Lee Ji-youn LimJong Bok Seo Nam Hee WonWoon-Won Jung Donggeun Sul |
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Institution: | a Graduate School of Medicine, Korea University, Seoul, 136-705, Republic of Koreab Pharmacognosy Lab., College of Pharmacy, Dankook Univeristy, Cheonan, 330-714, Republic of Koreac Metabolome Analysis Team, Korea Basic Science Institute, Seoul, 136-713, Republic of Koread Department of Pathology, College of Medicine, Korea University, Seoul, 136-705, Republic of Koreae Department of Clinical Pathology, College of Health Science, Korea University, Seoul, 136-703, Republic of Koreaf Environmental Toxico-Genomic and Proteomic Center, College of Medicine, Korea University, Seoul, 136-705, Republic of Korea |
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Abstract: | Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF. |
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Keywords: | 2 3 4 7 8-PCDF 2 3 4 7 8-pentachlorodibenzofuran MTT 3-(4 5-Dimethyl thiazol-2-yl)-2 5-diphenyltetrazolium bromide LC-ESI-MS/MS liquid chromatography electrospray ionization-tandem mass spectrometry TOF2 MS time-of-flight 2 mass spectrometry PCDFs polychlorinated dibenzofurans NMA normal point melting agarose LMA low point melting agarose IEF isoelectric focusing SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis 2-DE two dimensional gel electrophoresis PNPO pyridoxine-5&prime -phosphate oxidase UDP-GlcDH UDP-glucose 6-dehydrogenase PAI-1 plasminogen activator inhibitor I precursor PAI-3 plasminogen activator inhibitor-3 precursor PA28α proteasome activator complex subunit 1 14-3-3 σ isoform 1 of 14-3-3 protein sigma PPIase A peptidyl-prolyl cis-trans isomerase A 14-3-3 υ 14-3-3 protein gamma DJ-1 protein DJ-1 NDK A nucleoside diphosphate kinase A |
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