Abstract
Pyrrolizidine alkaloids (PAs) are one of the most significant groups of hepatotoxic phytotoxins. It is well-studied that metabolic activation of PAs generates reactive pyrrolic metabolites that rapidly bind to cellular proteins to form pyrrole–protein adducts leading to hepatotoxicity. Pyrrole–protein adducts all contain an identical core pyrrole moiety regardless of structures of the different PAs; however, the proteins forming pyrrole–protein adducts are largely unknown. The present study revealed that ATP synthase subunit beta (ATP5B), a critical subunit of mitochondrial ATP synthase, was a protein bound to the reactive pyrrolic metabolites forming pyrrole–ATP5B adduct. Using both anti-ATP5B antibody and our prepared anti-pyrrole–protein antibody, pyrrole–ATP5B adduct was identified in the liver of rats, hepatic sinusoidal endothelial cells, and HepaRG hepatocytes treated with retrorsine, a well-studied representative hepatotoxic PA. HepaRG cells were then used to further explore the consequence of pyrrole–ATP5B adduct formation. After treatment with retrorsine, significant amounts of pyrrole–ATP5B adduct were formed in HepaRG cells, resulting in remarkably reduced ATP synthase activity and intracellular ATP level. Subsequently, mitochondrial membrane potential and respiration were reduced, leading to mitochondria-mediated apoptotic cell death. Moreover, pre-treatment of HepaRG cells with a mitochondrial membrane permeability transition pore inhibitor significantly reduced retrorsine-induced toxicity, further revealing that mitochondrial dysfunction caused by pyrrole–ATP5B adduct formation significantly contributed to PA intoxication. Our findings for the first time identified ATP5B as a protein covalently bound to the reactive pyrrolic metabolites of PAs to form pyrrole–ATP5B adduct, which impairs mitochondrial function and significantly contributes to PA-induced hepatotoxicity.
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Acknowledgements
We sincerely thank Drs. Christiane Guguen-Guillouzo and Philippe Gripon from U522 INSERM and Dr. Christian Trepo from U271 INSERM for providing HepaRG cells.
Funding
The present study was supported by Research Grant Council of Hong Kong (GRF Grants nos. 14111816 and 14110714) and CUHK Direct Grant (4054302).
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All authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. This article is not an official U.S. Food and Drug Administration guidance or policy statement. No official support or endorsement by U.S Food and Drug Administration is intended or should be inferred.
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Lu, Y., Ma, J., Song, Z. et al. The role of formation of pyrrole–ATP synthase subunit beta adduct in pyrrolizidine alkaloid-induced hepatotoxicity. Arch Toxicol 92, 3403–3414 (2018). https://doi.org/10.1007/s00204-018-2309-6
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DOI: https://doi.org/10.1007/s00204-018-2309-6