Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP-induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP-induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP-induced liver injury by modulation of danger signaling. APAP-induced liver injury was dependent, in part, on Toll-like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135-540 μg/g body weight or when delivered as a pre-, concurrent, or post-APAP therapeutic. Furthermore, BA abrogated APAP-induced cytokines and chemokines as well as high-mobility group box 1 release. Moreover, BA prevented APAP-induced inflammasome signaling as determined by interleukin (IL)-1β, IL-18, and caspase-1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell-type–specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre-tlr4−/−) were necessary for the protective effects of BA. Conclusion: BA protects against APAP-induced acute liver injury and reduced inflammasome activation in a TLR4-dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury. (Hepatology 2014;60:990–1002)
Acetaminophen (APAP) is a commonly used over-the-counter analgesic and antipyretic medicine used to relieve symptoms of mild inflammatory conditions. APAP overdoses are the leading cause of acute liver failure (ALF) in most industrialized countries. APAP toxicity accounts for approximately 50% of all cases of ALF in the United States and carries a 30% mortality.[1, 2] More than 2,600 hospitalizations and nearly 500 deaths are attributed to APAP in the United States annually. Current treatment for APAP-induced liver failure is limited to N-acetylcysteine, which works mainly by restoration of glutathione levels, and supportive care. For those who progress to liver failure, the only effective treatment is liver transplantation.
In APAP-induced injury, and other forms of sterile liver injury, endogenous damage-associated molecular patterns (DAMPs) are released to activate cellular pattern recognition receptors (PRRs) on both immune and parenchymal cells.[4-11] Release of DAMPs and activation of PRRs have numerous effects, including activation of inflammasome signaling, release of cytokines and chemokines, and cellular injury. Several studies have highlighted the role of different PRRs and inflammasome activation on APAP-induced liver injury. Additional mechanisms of APAP-induced injury have focused on mitochondrial damage and oxidant injury, which contribute to cell necrosis.[12-14]
Benzyl alcohol (BA) is an organic compound and can be produced naturally by plants and exists in many essential oils. It is commonly used as a solvent as well as a precursor to a variety of esters in industries. In the health care field, BA was used as a local anesthetic and is quite frequently added to intravenous medication solutions as a preservative because of its bacteriostatic and antipruritic properties. BA can be oxidized to benzoic acid, conjugated with glycine in the liver, and excreted as hippuric acid. Initial observations found that APAP-induced liver injury was prevented when using a delivery vehicle of a commercial formulation of bacteriostatic saline containing BA. The aim of these investigations was to test the hypothesis that BA protects against APAP-induced liver injury by prevention of mitochondrial injury and/or modulation of danger signaling.
The findings noted in this article highlight the roles of direct hepatocellular toxicity, as well as innate immune-signaling pathways, most notably TLR9, RAGE, Nalp3, and caspase-1, in mediating APAP-induced liver injury. APAP liver toxicity was also associated with release of HMGB1, which was reduced in transgenic (Tg) mice lacking TLR9 and RAGE receptors. Mice with hepatocyte-specific KO of HMGB1 were also protected against liver injury, suggesting a critical role for HMGB1 in this model. Furthermore, BA was found to be a molecule that limits APAP-induced liver injury. The mechanisms of protection of BA include direct effects on hepatocyte injury, as well as through the PRR, TLR4, specifically on myeloid cells.
A small percentage of APAP is metabolized by cytochrome p450s (CYPs) to the potentially toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI). This can cause mitochondrial injury, leading to production of ROS and stress signaling.[14, 32] The results noted in this article are consistent with these findings, demonstrating mitochondrial injury and oxidant stress. BA had an effect in vivo as well as a direct effect on hepatocytes in vitro. Possible mechanisms underlying these effects include direct influence on APAP metabolism by CYPs. BA can be a product of toluene metabolism by the same CYPs that metabolize APAP to NAPQI. It is possible that there is an inhibitory effect of BA on CYP function. Other potential mechanisms may include other signaling pathways that influence mitochondrial health.
A component of APAP-induced liver failure may be secondary to sterile injury from the innate immune response through DAMPs released from stressed or dying cells to amplify liver damage.[4, 10] DAMPs activate a group of PRRs, including TLRs, nucleotide-binding oligomerization domain (NOD)-like receptors, and RAGE. Specifically, in models of APAP-induced liver injury, Imaeda et al. found that hepatocyte DNA was a trigger of the innate immune response through TLR9 and Nalp3 signaling. This led to the activation of caspase-1 to result in cleavage of proinflammatory cytokines to active IL-1β and IL-18. Inhibition of either TLR9 signaling or Nalp3 activity or genetic depletion markedly attenuated liver injury and improved survival. Findings in this study were consistent with these previous reports. Thus, the products released from injured cells from the toxic affects of APAP can then potentially activate innate immune signaling and inflammation, which may serve to potentially exacerbate or ameliorate injury. The findings in this study and others that show increased plasma levels of mtDNA and HMGB1 suggest that these DAMPS released from injured hepatocytes may then be released to activate immune signaling.
Among the DAMPs in liver injury, HMGB1 has been shown to play a vital role, including in APAP-treated mice. HMGB1 release itself or as a complex with DNA can activate TLR2, 4, and 9. We and others have shown that HMGB1 neutralization or inhibition were protective in HIR, APAP-induced liver injury, or hemorrhagic shock and trauma.[5, 7, 9, 19, 21] In this current study, HMGB1 release was greatly attenuated by BA treatment, and HC-HMGB1 KO mice demonstrated remarkably reduced liver damage after APAP treatment. This suggests that HMGB1 signaling from hepatocytes amplifies liver injury in this model. Moreover, genetic absence of RAGE, which is a receptor of HMGB1, also reduced liver injury.
TLR4 is one of the most important sensors in innate immunity, for both pathogen-associated molecular patterns and DAMPs. Previous studies demonstrate that depletion of TLR4 protected against sterile and nonsterile liver injury. Specifically, investigators have demonstrated, in models of APAP toxicity, that mice-deficient TLR4 signaling were protected,[35, 36] whereas data presented in this article found that TLR4 signaling did not seem to be involved in APAP-induced liver injury. The differences may be attributed to use of KO mice used in this study, whereas previous studies utilized mice with mutated TLR4 receptors. More intriguing, the data presented in this article suggest that TLR4 serves as an essential receptor to mediate the protective effects of BA to limit liver injury and inflammasome activation. Clearly, the role and interplay of danger-signaling pathways and PRRs is complex. Furthermore, the cell-specific KO data in TLR4 implies a complex interplay of different cells in mediating APAP-induced liver injury. In warm HIR injury, the specific absence of TLR4 on hepatocytes and myeloid cells showed reduced liver injury, whereas DC-specific TLR4 KO exacerbated damage. The specifics of injury and the interplay of different cells and signaling pathways are seemingly highly specific to the individual insults that result in liver injury. The influence of BA through TLR4 signaling may be predominantly to prevent amplification of injury that occurs secondary to release of DAMPs from hepatocytes. The influence of BA on TLR4 signaling may negatively regulate other activated innate immune-signaling pathways.
BA has been used as a local anesthetic, and use of BA (5%) is approved by the U.S. Food and Drug Administration in treatment of head lice. High doses of BA have toxicity, including respiratory failure, vasodilation, hypotension, convulsions, and paralysis. Toxicity from BA is a potential concern, and these studies demonstrated a toxic dose in the APAP model in the dose escalation study, suggesting a therapeutic window. BA is a true neutral, water-soluble molecule that readily partitions into lipid bilayers to increase fluidity. BA can inhibit mitochondrial electron transport at several points along the respiratory chain, which may be relevant to the suggested findings in this study of protection against APAP-induced mitochondrial injury.
In summary, our study suggested BA to be a promising reagent for APAP-induced acute hepatic toxicity prevention and treatment. The protective mechanism was, at least in part, mediated by myeloid cell signaling by TLR4 as well as through direct effects on hepatocytes.