Hepatic supersulfides attenuate acetaminophen-induced liver injury via enhanced detoxification and anti-inflammatory mechanisms.

Acetaminophen (APAP) is a widely used antipyretic and analgesic agent; however, overdose can lead to hepatotoxicity and, in severe cases, acute liver failure. Development of therapeutics that mitigate APAP-induced liver injury is essential to prevent progression to hepatic failure. Upon overdose, APAP is metabolized in the liver to the highly reactive electrophile N-acetyl-p-benzoquinone imine (NAPQI), which induces hepatocellular damage. Glutathione (GSH), a key intracellular nucleophile, exists partially in a modified form as glutathione hydropersulfide (GSSH), which exhibits enhanced nucleophilicity and functions as a supersulfide. While detoxification of NAPQI via GSH conjugation is well established, the role of GSSH in NAPQI detoxification has remained unknown. In this study, we investigated the protective role of hepatic supersulfides against APAP-induced liver injury using a murine model. Utilizing a newly developed tandem mass spectrometry technique, we demonstrated that supersulfides form conjugates with NAPQI, which are subsequently excreted in the urine. Moreover, administration of supersulfide donors, such as N-acetylcysteine (NAC) tetrasulfide and thioglucose tetrasulfide, elevated hepatic supersulfide levels and significantly attenuated APAP-induced liver injury. Notably, the protective effects of these donors surpassed those of conventional NAC treatment. Our findings suggest that the hepatoprotective effects of supersulfide donors involve not only enhanced detoxification of NAPQI, thereby reducing hepatocellular damage, but also suppression of inflammation. These results highlight the therapeutic potential of targeting hepatic supersulfides in the treatment of APAP overdose.