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Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides
Published on: September 16, 2020
Phosphoproteomic analyses of L-02 liver cells exposed to trichloroethylene
Xiaohu Ren1,2, Jie Li1, Bo Xia1,2
1a School of Public Health and Tropical Medicine, Southern Medical University , Tonghe District , Guangzhou , P.R. China .
Abstract:
Trichloroethylene (TCE) is an environmental and occupational toxicant that has been shown to cause serious hepatotoxicity. However, the mechanisms underlying the hepatotoxicity of TCE remain unclear. Previously, we identified several apoptosis-related proteins in TCE-induced hepatic cytotoxicity. This study is aimed to analyze the changes in phosphoproteins in L-02 liver cells exposed to TCE using iTRAQ labeling, IMAC enrichment and LC-MS/MS. We identified 1878 phosphorylation sites in 107 proteins and found that 20 sites in 16 phosphoproteins were differentially phosphorylated in L-02 cells after TCE treatment. Among these phosphoproteins, 20% were protein localization and formation processes-related proteins, 38% were metabolism-related proteins and 42% were cellular process-related proteins, including transcriptional regulation and biogenesis. Moreover, two phosphoproteins, 4E-BP1 (37T) and MCM2 (139S), were validated as TCE-induced alteration of phosphorylation at specific sites by Western-blot analysis. Taken together, our study demonstrated that TCE exposure changed the levels of multiple phosphoproteins in L-02 liver cells, and the functional analysis suggested that these differentially expressed phosphoproteins might be involved in TCE-induced hepatic cytotoxicity.
Insights
Trichloroethylene (TCE) exposure alters liver cell phosphoproteins, potentially driving liver damage. This study identifies specific protein changes linked to TCE-induced hepatotoxicity, offering new insights into its mechanisms.
Area of Science:
- Biochemistry
- Toxicology
- Proteomics
Background:
- Trichloroethylene (TCE) is a known environmental and occupational toxicant causing significant liver damage (hepatotoxicity).
- The precise molecular mechanisms behind TCE-induced hepatotoxicity are not fully understood.
- Previous research implicated apoptosis-related proteins in TCE-induced liver cell injury.
Purpose of the Study:
- To investigate alterations in phosphoproteins within L-02 liver cells upon exposure to Trichloroethylene (TCE).
- To identify specific phosphorylation changes and their potential roles in TCE-induced hepatic cytotoxicity.
Main Methods:
- Utilized iTRAQ labeling, Immobilized Metal Affinity Chromatography (IMAC) enrichment, and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to analyze phosphoproteomes.
- Quantified and identified differentially phosphorylated proteins and sites in TCE-treated L-02 cells.
- Validated specific phosphoprotein changes (4E-BP1 and MCM2) using Western-blot analysis.
Main Results:
- Identified 1878 phosphorylation sites across 107 proteins.
- Discovered 20 differentially phosphorylated sites in 16 phosphoproteins following TCE treatment.
- Found that affected phosphoproteins are involved in protein localization, metabolism, and cellular processes like transcriptional regulation and biogenesis.
- Confirmed TCE-induced phosphorylation alterations in 4E-BP1 (at 37T) and MCM2 (at 139S).
Conclusions:
- Trichloroethylene (TCE) exposure significantly alters phosphoprotein levels in liver cells.
- The identified differentially expressed phosphoproteins are potentially key players in the mechanisms of TCE-induced hepatic cytotoxicity.
- This phosphoproteomic analysis provides novel insights into the molecular pathways affected by TCE exposure.
