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Updated: May 4, 2026
Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Published on: October 23, 2018
An integrated proteomic and transcriptomic approach to understanding azathioprine- induced hepatotoxicity in rat
Young-Eun Cho1, Pyong-Gon Moon, Moon-Chang Baek
1Department of Molecular Medicine, Cell and Matrix Biology Research Institute, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.
Abstract:
Azathioprine, an immunosuppressant, has gained a prominent position in the clinic for prevention of graft rejection in organ transplants, as well as dermatological autoimmune diseases. However, according to a number of research reports, hepatotoxicity, as one of the side effects, is a major obstacle in azathioprine therapy. In this study, an integrated toxicoproteomic and toxicotranscriptomic analysis was performed using rat primary hepatocytes, in order to gain insight into the in-depth pathway map related to azathioprine-induced hepatotoxicity. For proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to azathioprine at IC20 concentration for 24 h. In particular, 2D LC-MS/MS and informatics-assisted label-free strategy for proteomic analysis were applied in order to increase the number of identified proteins and to improve the confidence of the quantitation results. Among 119 differentially identified protein species, 69 were upregulated and 50 were downregulated in the azathioprine-treated group. At the mRNA level, results of transcriptomic analysis showed increased transcription of 340 genes and decreased transcription of 63 genes in the azathioprine-treated group. Based on the analysis of transcriptomic and proteomic results using the DAVID program, drug metabolism/oxidative stress enzymes, xenobiotic metabolism by cytochrome P450, fatty acid metabolism, primary bile acid biosynthesis, contraction, inflammation metabolism, and mitogen-activated protein kinase (MAPK) kinase (ERK/JNK/p38 kinase) pathways were affected in azathioprine-treated hepatotoxicity. The effects on genes and proteins related to several important pathways were confirmed by real-time PCR and immunoblot analysis, respectively. This study is the first to report on relevant pathways related to azathioprine-induced hepatotoxicity through performance of integrated transcriptomic and proteomic analyses.
Insights
Azathioprine causes liver damage by affecting drug metabolism, oxidative stress, and inflammatory pathways. This study used integrated toxicoproteomic and toxicotranscriptomic analyses to map these effects in rat hepatocytes.
Area of Science:
- Toxicology
- Molecular Biology
- Biochemistry
Background:
- Azathioprine is an immunosuppressant used in organ transplantation and autoimmune diseases.
- Hepatotoxicity is a significant side effect limiting azathioprine therapy.
- Understanding the molecular mechanisms of azathioprine-induced liver injury is crucial.
Purpose of the Study:
- To investigate the molecular pathways involved in azathioprine-induced hepatotoxicity.
- To utilize integrated toxicoproteomic and toxicotranscriptomic analyses for a comprehensive pathway map.
- To identify key affected biological processes in rat primary hepatocytes exposed to azathioprine.
Main Methods:
- Rat primary hepatocytes were exposed to azathioprine at IC20 concentration for 24 hours.
- Proteomic analysis employed 2D LC-MS/MS with a label-free strategy.
- Transcriptomic analysis assessed mRNA levels using high-throughput sequencing.
- DAVID program was used for pathway analysis.
- Real-time PCR and immunoblot analysis validated key findings.
Main Results:
- 119 differentially expressed proteins (69 upregulated, 50 downregulated) and altered transcription of 403 genes (340 increased, 63 decreased) were identified.
- Key affected pathways include drug metabolism, oxidative stress, xenobiotic metabolism (cytochrome P450), fatty acid metabolism, bile acid biosynthesis, inflammation, and MAPK signaling.
- Validation confirmed alterations in genes and proteins within these pathways.
Conclusions:
- Integrated transcriptomic and proteomic analyses provide a detailed pathway map of azathioprine-induced hepatotoxicity.
- The study highlights the involvement of metabolic, inflammatory, and signaling pathways in azathioprine liver injury.
- This research offers novel insights into the molecular basis of azathioprine's toxic effects on the liver.
