Abstract
Acetaminophen (APAP) overdose is a common cause of drug-induced liver injury (DILI). Ferroptosis has been recently implicated in APAP-induced liver injury (AILI). However, the functional role and underlying mechanisms of mitochondria in APAP-induced ferroptosis are unclear. In this study, the voltage-dependent anion channel (VDAC) oligomerization inhibitor VBIT-12 and ferroptosis inhibitors were injected via tail vein in APAP-injured mice. Targeted metabolomics and untargeted lipidomic analyses were utilized to explore underlying mechanisms of APAP-induced mitochondrial dysfunction and subsequent ferroptosis. As a result, APAP overdose led to characteristic changes generally observed in ferroptosis. The use of ferroptosis inhibitor ferrostatin-1 (or UAMC3203) and iron chelator deferoxamine further confirmed that ferroptosis was responsible for AILI. Mitochondrial dysfunction, which is associated with the tricarboxylic acid cycle and fatty acid β-oxidation suppression, may drive APAP-induced ferroptosis in hepatocytes. APAP overdose induced VDAC1 oligomerization in hepatocytes, and protecting mitochondria via VBIT-12 alleviated APAP-induced ferroptosis. Ceramide and cardiolipin levels were increased via UAMC3203 or VBIT-12 in APAP-induced ferroptosis in hepatocytes. Knockdown of Smpd1 and Taz expression responsible for ceramide and cardiolipin synthesis, respectively, aggravated APAP-induced mitochondrial dysfunction and ferroptosis in hepatocytes, whereas Taz overexpression protected against these processes. By immunohistochemical staining, we found that levels of 4-hydroxynonenal (4-HNE) protein adducts were increased in the liver biopsy samples of patients with DILI compared to that in those of patients with autoimmune liver disease, chronic viral hepatitis B, and non-alcoholic fatty liver disease (NAFLD). In summary, protecting mitochondria via inhibiting VDAC1 oligomerization attenuated hepatocyte ferroptosis by restoring ceramide and cardiolipin content in AILI.
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Abbreviations
- APAP :
-
Acetaminophen
- DILI :
-
Drug-induced liver injury
- 4-HNE :
-
4-Hydroxynonenal
- VDAC1 :
-
Voltage-dependent anion channel 1
- AILI :
-
APAP-induced liver injury
- NAPQI :
-
N-Acetyl-p-benzoquinone imine
- GSH :
-
Glutathione
- ROS :
-
Reactive oxygen species
- AIH :
-
Autoimmune liver disease
- CHB :
-
Chronic viral hepatitis B
- NAFLD :
-
Non-alcoholic fatty liver disease steatosis
- ALD :
-
Alcoholic liver disease
- DFO :
-
Deferoxamine
- siRNA :
-
Small interfering RNA
- ALT :
-
Alanine aminotransferase
- AST :
-
Aspartate aminotransferase
- H&E :
-
Hematoxylin and eosin
- MMP :
-
Mitochondrial membrane potential
- MDA :
-
Malondialdehyde
- OCR :
-
Oxygen consumption rate
- MOM :
-
Mitochondrial outer membrane
- BSA :
-
Bovine serum albumin
- PA :
-
Palmitic acid
- LC-MS :
-
Liquid chromatography-mass spectrometry
- H-SCORE :
-
Histological scores
- ALP :
-
Alkaline phosphatase
- RNA-seq :
-
RNA sequencing
- GSEA :
-
Gene set enrichment analysis
- Fer-1 :
-
Ferrostatin-1
- TUNEL :
-
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
- PMHs :
-
Primary mouse hepatocytes
- DCFH-DA :
-
Dichloro-dihydro-fluorescein diacetate
- TCA :
-
Tricarboxylic acid
- ETC :
-
Electron transport chain
- CL :
-
Cardiolipin
- CER :
-
Ceramide
- Taz :
-
Tafazzin
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This work was supported by the National Natural Science Foundation of China (NSFC 31771308, NSFC 81970513, and NSFC 81670524), the Shanghai Municipal Natural Science Foundation (17ZR1401800), the Innovative Research Team of High-level Local Universities in Shanghai, Shanghai Key Laboratory of Bioactive Small Molecules (ZDSYS14005), the Major Project of National Thirteenth Five Plan (2017ZX09304016), the Project of Shanghai Shenkang Hospital Development Center (16CR2009A), and the Project of Clinical Research Center, Shanghai Jiao Tong University School of Medicine (DLY201607).
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B.N performed mouse and cell experiments and implemented the data analysis. Q.X and XH.L performed clinical evaluation and collected clinical samples. XH. L, Y.J, D.X, L.M, and J.L helped collecting samples andperformed western blot analysis and quantitative real-time PCR analysis. X.Z assisted with pathology and scoring. Y.Z. helped with bioinformatics analysis. XB.L, Y. Mao, and X.Z conceived and supervised the project and analyzed the data. XB.L. and B.N. wrote the manuscript. Prof. Sun Ning helped with manuscript reviewing and editing. All authors discussed the results and revised the manuscript.
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All animal experiments were approved by the Animal Ethics Committee of Fudan University School of Basic Medical Sciences. Animal research was conducted in accordance with international guidelines. The clinical research was approved by the Ethics Committee of Shanghai Jiaotong University Renji Hospital in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.
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Highlights
1. Ferroptosis is responsible for APAP-induced hepatocyte cell death.
2. Protecting mitochondria via inhibiting VDAC1 oligomerization attenuates APAP-induced ferroptosis.
3. Taz and Smpd1, responsible for CL and CER synthesis, affect APAP-induced mitochondrial dysfunction and subsequent ferroptosis in hepatocytes.
4. Levels of 4-HNE protein adducts are increased in the liver samples of patients with DILI.
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Niu, B., Lei, X., Xu, Q. et al. Protecting mitochondria via inhibiting VDAC1 oligomerization alleviates ferroptosis in acetaminophen-induced acute liver injury. Cell Biol Toxicol 38, 505–530 (2022). https://doi.org/10.1007/s10565-021-09624-x
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DOI: https://doi.org/10.1007/s10565-021-09624-x