Ferroptosis and Acetaminophen Hepatotoxicity – Are We Going Down Another Rabbit Hole?

Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in the US. The mechanisms of APAP-induced liver injury have been under extensive investigations for decades and many key events of this necrotic cell death are known today. Initially, two opposing hypotheses for cell death were proposed: Reactive metabolite and protein adducts formation versus reactive oxygen and lipid peroxidation (LPO). In the end, both mechanisms were reconciled, and it is now generally accepted that the toxicity starts with formation of reactive metabolites which, after glutathione depletion, bind to cellular proteins, especially on mitochondria. This results in a mitochondrial oxidant stress, which requires amplification through a mitogen activated protein kinase cascade leading ultimately to enough reactive oxygen and peroxynitrite formation to trigger the mitochondrial membrane permeability transition and cell death. However, the earlier rejected LPO hypothesis seems to make a come-back recently under a different name: ferroptosis. Therefore, the objective of this review was to critically evaluate the available information about intracellular signaling mechanisms of APAP-induced cell death and those of ferroptosis. Conclusion: Under pathophysiologically relevant conditions, there is no evidence for quantitatively enough LPO to cause cell death and thus APAP hepatotoxicity is not caused by ferroptosis. However, the role of mitochondria localized minor LPO remains to be further investigated.

Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating the interleukin-6 – Hsp70 axis through inter-organ crosstalk

We previously demonstrated that endogenous phosphatidic acid (PA) promotes liver regeneration after acetaminophen (APAP) hepatotoxicity in mice. Based on that, we hypothesized that exogenous PA is also beneficial. To test that, we treated mice with a toxic APAP dose at 0 h, followed by PA or vehicle at multiple timepoints. We then collected blood and liver at 6, 24, and 52 h. Post-treatment with PA protected against liver injury at 6 h, and the combination of PA and N-acetyl-cysteine (NAC) further reduced injury compared to NAC alone. Interestingly, PA had no effect on major early mechanisms of APAP toxicity, including APAP bioactivation, oxidative stress, JNK activation, and mitochondrial damage. However, transcriptomics revealed that PA activated interleukin-6 (IL-6) signaling in the liver, and IL-6 was increased in serum from PA-treated mice. Furthermore, PA did not protect against APAP in IL-6-deficient mice. Additional experiments revealed that PA induced heat shock protein 70 (Hsp70) in the liver in WT mice but not in IL-6 KO mice. Furthermore, IL-6 expression increased 18-fold in adipose tissue after PA, indicating that adipose tissue is a likely source of the increased IL-6 due to PA treatment. Surprisingly, however, exogenous PA did not alter regeneration, despite the widely accepted role of IL-6 in liver repair. These data reinforce the protective role of IL-6 and Hsp70 in APAP hepatotoxicity, provide new insight into the role of IL-6 in liver regeneration, and indicate that exogenous PA or PA derivatives may one day be a useful adjunct treatment for APAP overdose with NAC.

Post-treatment with glycyrrhizin can attenuate hepatic mitochondrial damage induced by acetaminophen in mice

Overdose of acetaminophen (APAP) is responsible for the most cases of acute liver failure worldwide. Hepatic mitochondrial damage mediated by neuronal nitric oxide synthase- (nNOS) induced liver protein tyrosine nitration plays a critical role in the pathophysiology of APAP hepatotoxicity. It has been reported that pre-treatment or co-treatment with glycyrrhizin can protect against hepatotoxicity through prevention of hepatocellular apoptosis. However, the majority of APAP-induced acute liver failure cases are people intentionally taking the drug to commit suicide. Any preventive treatment is of little value in practice. In addition, the hepatocellular damage induced by APAP is considered to be oncotic necrosis rather than apoptosis. In the present study, our aim is to investigate if glycyrrhizin can be used therapeutically and the underlying mechanisms of APAP hepatotoxicity protection. Hepatic damage was induced by 300 mg/kg APAP in balb/c mice, followed with administration of 40, 80, or 160 mg/kg glycyrrhizin 90 min later. Mice were euthanized and harvested at 6 h post-APAP. Compared with model controls, glycyrrhizin post-treatment attenuated hepatic mitochondrial and hepatocellular damages, as indicated by decreased serum glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities as well as ameliorated mitochondrial swollen, distortion, and hepatocellular necrosis. Notably, 80 mg/kg glycyrrhizin inhibited hepatic nNOS activity and its mRNA and protein expression levels by 16.9, 14.9, and 28.3%, respectively. These results were consistent with the decreased liver nitric oxide content and liver protein tyrosine nitration indicated by 3-nitrotyrosine staining. Moreover, glycyrrhizin did not affect the APAP metabolic activation, and the survival rate of ALF mice was increased by glycyrrhizin. The present study indicates that post-treatment with glycyrrhizin can dose-dependently attenuate hepatic mitochondrial damage and inhibit the up-regulation of hepatic nNOS induced by APAP. Glycyrrhizin shows promise as drug for the treatment of APAP hepatotoxicity.

Acetaminophen Test Battery (ATB): A Comprehensive Method to Study Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP) overdose is the major cause of acute liver failure (ALF) in the Western world. Extensive research is ongoing to identify the mechanisms of APAP-induced ALF. APAP-induced acute liver injury is also one of the most commonly studied drug-induced liver injury models in the field of hepatotoxicity. APAP toxicity is triphasic and includes three mechanistically interlinked but temporally distinct phases of initiation, progression, and recovery/regeneration. Despite how commonly it is studied, the methods to study APAP toxicity differ significantly, often leading to confusing and contradictory data. There are number of reviews on mechanisms of APAP toxicity, but a detailed mechanism-based comprehensive method and list of assays that covers all phases of APAP hepatotoxicity are missing. The goal of this review is to provide a standard protocol and guidelines to study APAP toxicity in mice including a test battery that can help investigators to comprehensively analyze APAP toxicity in the specific context of their hypothesis. Further, we will identify the major roadblocks and common technical problems that can significantly affect the results. This acetaminophen test battery (ATB) will be an excellent guide for scientists studying this most common and clinically relevant drug-induced liver injury and will also be helpful as a roadmap for hypothesis development to study novel mechanisms.

The Late-Stage Protective Effect of Mito-TEMPO against Acetaminophen-Induced Hepatotoxicity in Mouse and Three-Dimensional Cell Culture Models

An overdose of acetaminophen (APAP), the most common cause of acute liver injury, induces oxidative stress that subsequently causes mitochondrial impairment and hepatic necroptosis. N-acetyl-L-cysteine (NAC), the only recognized drug against APAP hepatotoxicity, is less effective the later it is administered. This study evaluated the protective effect of mitochondria-specific Mito-TEMPO (Mito-T) on APAP-induced acute liver injury in C57BL/6J male mice, and a three dimensional (3D)-cell culture model containing the human hepatoblastoma cell line HepG2. The administration of Mito-T (20 mg/kg, i.p.) 1 h after APAP (400 mg/kg, i.p.) injection markedly attenuated the APAP-induced elevated serum transaminase activity and hepatic necrosis. However, Mito-T treatment did not affect key factors in the development of APAP liver injury including the activation of c-jun N-terminal kinases (JNK), and expression of the transcription factor C/EBP homologous protein (CHOP) in the liver. However, Mito-T significantly reduced the APAP-induced increase in the hepatic oxidative stress marker, nitrotyrosine, and DNA fragmentation. Mito-T markedly attenuated cytotoxicity induced by APAP in the HepG2 3D-cell culture model. Moreover, liver regeneration after APAP hepatotoxicity was not affected by Mito-T, demonstrated by no changes in proliferating cell nuclear antigen formation. Therefore, Mito-T was hepatoprotective at the late-stage of APAP overdose in mice.

The cross-talk of NLRP3 inflammasome activation and necroptotic hepatocyte death in acetaminophen-induced mice acute liver injury

Overdose acetaminophen (APAP) can result in severe liver injury, which is responsible for nearly half of drug-induced liver injury in western countries. Previous studies have found that there existed massive hepatocellular necrosis and severe inflammatory response in APAP-induced liver injury. However, the mechanistic linkage between necroptosis and NLRP3 inflammasome pathway in APAP-induced hepatotoxicity remains poorly understood. In order to investigate the relationship between inflammation and hepatocytes death in APAP hepatotoxicity, a time-course model for APAP hepatotoxicity in C57/BL6 mice was established by intraperitoneal (i.p) injection of 300 mg/kg APAP in this study. The activity of serum enzymes and pathological changes of APAP-treated mice were evaluated, and the critical molecules in necroptosis and NF-κB-NLRP3 inflammasome signaling pathway were determined by immunoblot and immunofluorescence analysis. The results demonstrated that APAP overdose resulted in a severe liver injury. Furthermore, the expression of critical molecules in NLRP3 inflammasome and necroptosis pathways peaked at 12–24 h, and then was decreased gradually, which is consistent with the pattern of pathological injury induced by APAP. Our further investigation found that the level of IL-1β in mouse liver was closely correlated with the level of phosphorylated MLKL following exposure to APAP. Furthermore, inhibition of necroptosis with necrostatin-1 significantly suppressed the activation of NLRP3 inflammasome signaling. Taken together, our results highlighted that the cross-talk between necroptosis and NLRP3 inflammasome played a critical role for promoting APAP-induced liver injury. Inhibition of the interaction of inflammation and necroptosis by pharmaceutical methods may represent a promising therapeutic strategy for APAP-induced liver injury.

HEPATOCYTE APOPTOSIS INDUCTION BY ACETAMINOPHEN THROUGH MODULATION OF CASPASE/BAX PATHWAY IN MICE

Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro. Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities. Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p<0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C. Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity.

Gender Difference on the Effect of Omega-3 Polyunsaturated Fatty Acids on Acetaminophen-Induced Acute Liver Failure

Acetaminophen (APAP) toxicity is the leading cause of drug-induced liver failure, which is closely related to mitochondrial dysfunction and oxidative damage. Studies in clinical trials and in animal models have shown that omega-3 polyunsaturated fatty acids (n-3 PUFAs) affect the progression of various types of liver damage. Interestingly, the sex-dependent effect of n-3 PUFAs on human health has also been well documented. However, it is unknown whether supplementation of n-3 PUFAs modulates the pathogenesis of APAP-induced liver failure with sex-specificity. Our results showed that both endogenous and exogenous n-3 PUFAs significantly aggravated the APAP-induced liver injury in male mice, whereas the opposite effects were observed in females. In vivo and in vitro studies demonstrated that estrogen contributes to the gender difference in the regulation of n-3 PUFAs on APAP overdose. We found that n-3 PUFA-mediated regulation of hepatic oxidative stress response and autophagy upon APAP challenge is distinct between male and female mice. Moreover, we provided evidence that β-catenin signaling activation is responsible for the sex-dependent regulation of APAP hepatotoxicity by n-3 PUFAs. Together, these findings indicated that supplementation with n-3 PUFAs displays sex-differential effect on APAP hepatotoxicity and could have profound significance in the clinical management for drug-induced liver injury.

Management of acetaminophen toxicity, a review

Acetaminophen (APAP) is a widely used drug in our environment with few adverse effects. Because of this, several patients affected by APAP hepatotoxicity unknown that the APAP dose-intake was excessive. This damage is mainly produced via one of APAP metabolites: N-acetyl-para-benzo-quinone imine (NAPQI), which is very toxic. The drug’s ingested doses as well as the length of time from APAP ingestion to N-acetylcysteine (NAC) therapy are the most essential determining factors in both the development and severity of APAP hepatotoxicity. However, there are other factors related, including alcohol intake, herbs and medications, age and genetic factors, nutritional status, and chronic liver disease. The ingestion of a toxic dose of APAP causes different clinical manifestations that depend fundamentally on the time elapsed since the intake. The diagnosis process depends on the intake (acute single overdose of after repeated overdoses). The Rumack-Matthew nomogram is acceptable after an acute single overdose, being the “possible hepatic toxicity” point 200 μg/mL at 4 hours and 25 μg/mL at 16 hours). This normogram is no applicable in after repeated overdoses. NAC is the antidote for APAP intoxication, and could be administered orally or intravenous. Finally, a multidisciplinary approach with the support of Psychiatry, Intensive Care Unit as well as Gastroenterology and Digestive Department will be necessary, especially in the case of attempted autolysis and severe liver failure.

Shikonin Attenuates Acetaminophen-Induced Hepatotoxicity by Upregulation of Nrf2 through Akt/GSK3β Signaling

Acetaminophen (APAP) overdose-induced acute liver damage is mostly due to overwhelmingly increased oxidative stress. Nuclear factor-erythroid 2-related factor2 (Nrf2) plays an important role in alleviating APAP hepatic toxicity. Shikonin (SHK) enhances Nrf2 in multiple lines of normal cells. Nevertheless, whether SHK protects against APAP-induced liver toxicity remains undefined. This study found SHK defended APAP-induced liver toxicity, as well as reversed the levels of serum alanine/aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, and reactive oxygen species (ROS), while it enhanced the liver glutathione (GSH) level in APAP-treated mice. SHK rescued the cell viability and GSH depletion, but neutralized oxidative stress in APAP-treated human normal liver L-02 cells. Mechanically, SHK increased Nrf2 expression in the exposure of APAP at the protein level but not at the mRNA level. Inhibition of Nrf2 blocked the SHK effect in APAP-treated hepatocytes. Furthermore, SHK improved Nrf2 stability through stimulating PI3K/Akt pathway, thus inhibiting GSK-3β. In vivo studies confirmed the close correlation of liver protection of SHK against APAP and Akt/GSK-3β/Nrf2 pathway. In conclusion, this study reveals that SHK prevents APAP hepatotoxicity by upregulation of Nrf2 via PI3K/Akt/GSK-3β pathway. Therefore, SHK may be a promising candidate against APAP-induced liver injury.