Acetaminophen (APAP), a commonly used analgesic, is usually safe when administered at therapeutic doses. However, APAP overdose causes liver injury both in experimental animals and humans. APAP has been used extensively to develop an animal model of drug-induced liver injury. The toxicity is initiated by cytochrome P450 (CYP) metabolism into N-acetyl-p-benzoquinone imine (NAPQI), and the high reactivity of NAPQI with sulfhydryl groups results in depletion of reduced glutathione (GSH) in

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... tes, followed by covalent binding to intracellular proteins. 1-3) Therefore, hepatic GSH levels are important in protecting against APAP hepatotoxicity, and enhancement of hepatic GSH is a reasonable strategy for the treatment of APAP intoxication. N-Acetylcysteine (NAC) is clinically used as an antidote for APAP intoxication. 4) It is thought that NAC provides cysteine (CYS) as a precursor of GSH which traps NAPQI, leading to a decrease in toxicity (Fig. 1) . 5) However, NAC was shown to improve patient outcome after late administration 6) ; this could not be explained by trapping NAPQI. In fact, treatment of mice with NAC after APAP administration protected against hepatic necrosis without reducing covalent binding, suggesting that mechanisms other than GSH replenishment are involved in the hepatoprotective effects of NAC. [7] [8] [9] In contrast to NAC, GSH itself is not used as an antidote for APAP intoxication. It is believed that intraperitoneal administration of GSH is less effective in restoring hepatic GSH, probably because exogenous GSH can not be taken up by hepatocytes, whereas ethyl esters of GSH are proposed as GSH precursors that are taken up and hydrolyzed by hepatocytes into GSH (Fig. 1) . 10) On the other hand, intravenously administered GSH was found to protect against APAP hepatotoxicity. 11) It is considered that intravenous GSH rapidly degrades in the kidney, is absorbed as amino acids, and is used to synthesize GSH in the liver, which could led to hepatoprotection. These studies suggest the potential importance of restoring hepatic GSH levels in the protection against APAP hepatotoxicity by exogenous thiol compounds. The objective of this study was to clarify the role of GSH supplementation in the hepatoprotective action of NAC and other thiol compounds, which could lead to the development of a new hepatoprotective agent. We found that a relatively low dose of thiol compounds, GSH, NAC, CYS, and glutathione-monoethyl ester (GSH-EE), protected mice against APAP hepatotoxicity together with different abilities to re-An overdose of acetaminophen (APAP) causes liver injury both in experimental animals and humans. N-Acetylcysteine (NAC) is clinically used as an antidote for APAP intoxication, and it is thought to act by providing cysteine as a precursor of glutathione, which traps a reactive metabolite of APAP. Other hepatoprotective mechanisms of NAC have also been suggested. Here, we examined the effects of thiol compounds with different abilities to restore hepatic glutathione, on hepatotoxicity of APAP and furosemide in mice. Overnight-fasted male CD-1 mice were given APAP or furosemide intraperitoneally. NAC, cysteine, glutathione, or glutathione-monoethyl ester was administered concomitantly with APAP or furosemide. All thiol compounds used in this study effectively protected mice against APAP-induced liver injury. Only glutathione-monoethyl ester completely prevented APAP-induced early hepatic glutathione depletion. Cysteine also significantly restored hepatic glutathione levels. NAC partially restored glutathione levels. Exogenous glutathione had no effect on hepatic glutathione loss. NAC and glutathione highly stimulated the hepatic expression of cytokines, particularly interleukin-6, which might be involved in the alleviation of APAP hepatotoxicity. Furosemide-induced liver injury, which does not accompany hepatic glutathione depletion, was also attenuated by NAC and exogenous glutathione, supporting their protective mechanisms other than replenishment of glutathione. In conclusion, exogenous thiols could alleviate druginduced liver injury. NAC and glutathione might exert their effects, at least partially, via mechanisms that are independent of increasing hepatic glutathione, but probably act through cytokine-mediated and anti-inflammatory mechanisms.