APAP-induced Cell Research Articles

Drug-induced oxidative stress actively prevents caspase activation and hepatocyte apoptosis

Cell death is a fundamental process in health and disease. Emerging research shows the existence of numerous distinct cell death modalities with similar and intertwined signaling pathways, but resulting in different cellular outcomes, raising the need to understand the decision-making steps during cell death signaling. Paracetamol (Acetaminophen, APAP)-induced hepatocyte death includes several apoptotic processes but eventually is executed by oncotic necrosis without any caspase activation. Here, we studied this paradoxical form of cell death and revealed that APAP not only fails to activate caspases but also strongly impedes their activation upon classical apoptosis induction, thereby shifting apoptosis to necrosis. While APAP intoxication results in massive drop in mitochondrial respiration, low cellular ATP levels could be excluded as an underlying cause of missing apoptosome formation and caspase activation. In contrast, we identified oxidative stress as a key factor in APAP-induced caspase inhibition. Importantly, caspase inhibition and the associated switch from apoptotic to necrotic cell death was reversible through the administration of antioxidants. Thus, exemplified by APAP-induced cell death, our study stresses that cellular redox status is a critical component in the decision-making between apoptotic and necrotic cell death, as it directly affects caspase activity.

Hinokiflavone exerts dual regulation on apoptosis and pyroptosis via the SIX4/Stat3/Akt pathway to alleviate APAP-induced liver injury

Hinokiflavone (HF), classified as a flavonoid, is a main bioactive compound in Platycladus orientalis and Selaginella. HF exhibits activities including anti-HIV, anti-inflammatory, antiviral, antioxidant and anti-tumor effects. The study aimed to explore the function and the mechanisms of HF on acetaminophen (APAP)-induced acute liver injury. Results indicated that HF treatment mitigated the impact of APAP on viability and restored levels of MDA, GSH and SOD on HepG2 cells. The accumulation of reactive oxygen species (ROS) mitochondrial membrane potential (MMP) in HepG2 cells stimulated by APAP were also blocked by HF. HF reduced the levels of pro-apoptotic and pro-pyroptotic proteins. Flow cytometry analysis and fluorescence staining results were consistent with western blot analysis. Following HF treatment in the APAP-induced cell model, there was observed an augmentation in the phosphorylation of Stat3 and an increase in the expression of SIX4. However, not only silenced the SIX4 protein in HepG2 cells by siRNA, but also adding the Stat3 inhibitor (Stattic), attenuated the anti-apoptotic and anti-pyroptotic effects of HF significantly. Furthermore, HF alleviated liver damage in C57BL/6 mice model. Overall, our study demonstrated that HF mitigates apoptosis and pyroptosis induced by APAP in drug-induced liver injury (DILI) through the SIX4/Akt/Stat3 pathway in vivo and in vitro. HF may have promising potential for for the treatment of DILI.

Effect of ferroptosis inhibitors in a murine model of acetaminophen-induced liver injury.

Liver injury caused by acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. The mode of APAP-induced cell death has been controversially discussed with ferroptosis emerging as a more recent hypothesis. Ferroptosis is characterized by ferrous iron-catalyzed lipid peroxidation (LPO) causing cell death, which can be prevented by the lipophilic antioxidants ferrostatin-1 and UAMC-3203. To assess the efficacy of these ferroptosis inhibitors, we used two murine models of APAP hepatotoxicity, APAP overdose alone or in combination with FeSO4 in fasted male C57BL/6J mice. APAP triggered severe liver injury in the absence of LPO measured as hepatic malondialdehyde (MDA) levels. In contrast, ferrous iron co-treatment aggravated APAP-induced liver injury and caused extensive LPO. Standard doses of ferrostatin-1 did not affect MDA levels or the injury in both models. In contrast, UAMC-3203 partially protected in both models and reduced LPO in the presence of ferrous iron. However, UAMC-3203 attenuated the translocation of phospho-JNK through downregulation of the mitochondrial anchor protein Sab resulting in reduced mitochondrial dysfunction and liver injury. Thus, APAP toxicity does not involve ferroptosis under normal conditions. The lack of effects of ferroptosis inhibitors in the pathophysiology indicates that ferroptosis signaling pathways are not relevant therapeutic targets.

Translocation of Adenosine A2B Receptor to Mitochondria Influences Cytochrome P450 2E1 Activity after Acetaminophen Overdose.

The adenosine A2B receptor (A2BAR) is a member of a family of G-protein coupled receptors (GPCRs), which has a low affinity for adenosine and is now implicated in several pathophysiological conditions. We have demonstrated the beneficial effects of A2BAR activation in enhancing recovery after acute liver injury induced by an acetaminophen (APAP) overdose. While receptor trafficking within the cell is recognized to play a role in GPCR signaling, its role in the mediation of A2BAR effects in the context of APAP-induced liver injury is not well understood. This was investigated here, where C57BL/6J mice were subjected to an APAP overdose (300 mg/kg), and the temporal course of A2BAR intracellular localization was examined. The impact of A2BAR activation or inhibition on trafficking was examined by utilizing the A2BAR agonist BAY 60-6583 or antagonist PSB 603. The modulation of A2BAR trafficking via APAP-induced cell signaling was explored by using 4-methylpyrazole (4MP), an inhibitor of Cyp2E1 and JNK activation. Our results indicate that APAP overdose induced the translocation of A2BAR to mitochondria, which was prevented via 4MP treatment. Furthermore, we demonstrated that A2BAR is localized on the mitochondrial outer membrane and interacts with progesterone receptor membrane component 1 (PGRMC1). While the activation of A2BAR enhanced mitochondrial localization, its inhibition decreased PGRMC1 mitochondria levels and blunted mitochondrial Cyp2E1 activity. Thus, our data reveal a hitherto unrecognized consequence of A2BAR trafficking to mitochondria and its interaction with PGRMC1, which regulates mitochondrial Cyp2E1 activity and modulates APAP-induced liver injury.

Role of Pyroptosis in Acetaminophen-Induced Hepatotoxicity.

Acetaminophen (APAP) is a widely used pain reliever that can cause liver injury or liver failure in response to an overdose. Understanding the mechanisms of APAP-induced cell death is critical for identifying new therapeutic targets. In this respect it was hypothesized that hepatocytes die by oncotic necrosis, apoptosis, necroptosis, ferroptosis and more recently pyroptosis. The latter cell death is characterized by caspase-dependent gasdermin cleavage into a C-terminal and an N-terminal fragment, which forms pores in the plasma membrane. The gasdermin pores can release potassium, interleukin-1β (IL-1β), IL-18, and other small molecules in a sublytic phase, which can be the main function of the pores in certain cell types such as inflammatory cells. Alternatively, the process can progress to full lysis of the cell (pyroptosis) with extensive cell contents release. This review discusses the experimental evidence for the involvement of pyroptosis in APAP hepatotoxicity as well as the arguments against pyroptosis as a relevant mechanism of APAP-induced cell death in hepatocytes. Based on the critical evaluation of the currently available literature and understanding of the pathophysiology, it can be concluded that pyroptotic cell death is unlikely to be a relevant contributor to APAP-induced liver injury.

Antioxidant and protective effects of a peptide (VTAL) derived from simulated gastrointestinal digestion of protein hydrolysates of Magallana gigas against acetaminophen-induced HepG2 cells.

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallanagigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105 ± 3.62%) was observed in 15mM APAP-induced cells when treated with 25μg/mL M.gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs.

Identification of a novel farnesoid X receptor agonist, kaempferol-7-O-rhamnoside, a compound ameliorating drug-induced liver injury based on virtual screening and in vitro validation

Farnesoid X receptor (FXR), a bile acid receptor, plays an essential role in maintaining bile acid and liver homeostasis and has been recognized as an essential target for drug-induced liver injury (DILI). This study aimed to identify potential FXR agonists by virtual screening, molecular dynamics (MD) simulation, and biological assays. First, an in-house Traditional Chinese medicine compound database was screened using a virtual approach based on molecular docking to reveal potential FXR agonists. Secondly, MD was applied to analyze the process of agonist binding. Finally, the acetaminophen (APAP)-induced L02 cells model evaluated the pharmacodynamic activity of agonists treating DILI. Virtual screening results showed that kaempferol-7-O-rhamnoside was confirmed as the FXR agonist. MD results showed that kaempferol-7-O-rhamnoside could stably bind the FXR. In addition, in vitro cell-based assay showed that kaempferol-7-O-rhamnoside could promote the expression of the FXR gene and inhibit the Cyp7a1 gene expression in APAP-induced cells, significantly reducing the activities of AST, AKP and ROS, and enhancing the expression of GSH. The current study confirmed that kaempferol-7-O-rhamnoside might improve liver function by promoting proliferation, ameliorating oxidative stress, and regulating FXR target genes as observed in vitro. Therefore, in this study, discovering the FXR agonist, kaempferol-7-O-rhamnoside, provides valuable guidance for developing novel drugs against DILI.

Creatinine accelerates APAP-induced liver damage by increasing oxidative stress through ROS/JNK signaling pathway.

Serum creatinine is an endogenous biomarker to estimate glomerular filtration rate (GFR) and is commonly used to assess renal function in clinical practice. Acetaminophen (APAP), the most available analgesic and antipyretic medication, is recommended as the drug of choice for pain control in patients with renal diseases. However, an overdose of APAP can lead to severe acute liver injury, which is also the most common cause of acute liver failure in western countries. The role of creatinine in APAP-induced liver injury is unclear and should be further explored. Herein, clinical data on patients with drug-induced liver injury revealed that the creatinine concentration between 82-442 μmol/L for female and 98–442 μmol/L for male is positively correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST). While there was no correlation between creatinine and ALT and AST when creatinine concentration is over 442 μmol/L. In addition, mice were administrated with creatinine intraperitoneally for 1 week before APAP injection to investigated the pathophysiological role of creatinine in APAP-induced acute liver injury. The results showed that creatinine administration aggravated hepatic necrosis and elevated serum lactate dehydrogenase (LDH) and ALT levels in mice upon APAP injection. The mechanism study demonstrated that creatinine could increase the production of reactive oxygen activation (ROS) and the activation of c-Jun N-terminal kinase (JNK). Furthermore, the liver injury was alleviated and the difference between APAP-treated mice and APAP combined with creatinine-treated mice was blunted after using specific ROS and JNK inhibitors. Significantly, creatinine stimulation aggravates APAP-induced cell death in HepaRG cells with the same mechanism. In summary, this study proposed that creatinine is closely related with liver function of drug-induced liver injury and exacerbates APAP-induced hepatocyte death by promoting ROS production and JNK activation, thus providing new insight into the usage of APAP in patients with kidney problems.

Antioxidant and chemoprotective peptides from simulated gastrointestinal digested (SGID) protein hydrolysate of Pyropia yezoensis against acetaminophen-induced HepG2 cells.

Excessive production of reactive oxygen and nitrogen species may result in oxidative damage to tissues and organs. Oxidative stress is a pathological mechanism that contributes to the initiation and progression of liver injury. In the present study, antioxidative peptides purified from simulated gastrointestinal-digested (SGID) protein hydrolysate of Pyropia yezoensis, showed significant antioxidant activity and also showed a protective effect against acetaminophen (N-acetyl-p-aminophenol, APAP) -induced injury in HepG2 (human liver cancer cells) cells. The antioxidant activity was increased in a dose-dependent manner. Higher cell viability (73.26 ± 0.9%) and decreasing NO levels (107.6 ± 8.9%) were observed in 15 mM APAP-induced cells when treated with the concentration of (100 μg ml−1) Pyropia peptide. Py. (pep). The sequences of the eight identified peptides present in the active fractions of the protein hydrolysate included hydrophobic and aromatic amino acids, which may have been responsible for their chemoprotective and antioxidant activities. Results indicated that the treatment with the Pyropia—peptides significantly promoted the proliferation of HepG2 cells, protecting them against APAP-mediated injury, and showed a significant antioxidant capacity. This study revealed that the Py. (pep) will be beneficial in treating drug-induced oxidative stress and liver damage conditions. Py. (pep) can also serve as a better alternative for synthetic antioxidant drugs.

Identification of gut bacterial factors linked to altered host susceptibility to acetaminophen‐induced liver injury

Acetaminophen (APAP) overdose causes acute liver injury. We previously demonstrated that differential gut microbiota modulates the host susceptibility to APAP‐induced liver injury in mice and subsequently identified 16 metabolites associated with altered susceptibility to APAP hepatotoxicity. This study aims to examine whether these metabolites modulate APAP hepatotoxicity. In primary mouse hepatocytes cultured in high‐galactose medium, phenylpropionic acid (PPA) and 3‐(4‐hydroxyphenyl)‐propionic acid (HPPA) significantly decreased APAP‐induced cell necrosis whereas 5‐aminovaleric acid (5‐AV) worsens it. In mice, PPA (in drinking water at 4 % w/v) for four weeks and HPPA (intraperitoneal injection, 50 mg/kg 2 hours prior to APAP) significantly alleviated the APAP‐induced liver injury. PPA, HPPA and 5‐AV were rarely detected, if any, in the tissue (portal vein serum or liver) of germ‐free mice, suggesting that that gut microbial production is a major determinant of host exposure to these metabolites. The biosynthetic pathways for these metabolites have been reported in bacteria. Indeed, mouse cecal bacteria readily converted L‐phenylalanine, L‐tyrosine and L‐proline to PPA, HPPA and 5‐AV, respectively. Bacterial conversion of L‐Phe to PPA is known to be mediated by the phenylalanine reductive metabolism pathway (called “Fld pathway”) or phenylalanine ammonia lyase. Examination of the deuterium‐labeled metabolites (d6‐PPA and d7‐PPA; produced via Fld pathway and phenylalanine ammonia lyase, respectively) after incubating mouse cecal bacteria with deuterium‐labeled L‐Phe (d8‐Phe) revealed that Fld pathway is the main route of PPA production from L‐Phe in mouse cecum. Interestingly, previously reported bacterial genes in Fld pathway were not detected in the mouse cecum. Also, the in vitro rate of d6‐PPA production by the cecal bacteria did not correlate with the differential levels of basal PPA levels in mouse cecum contents. The bacterial genes encoding the Fld enzymes as well as factors modulating its activities are remained to be identified. Taken together, this study presents multiple gut bacterial factors that are involved in gut microbiota’s modulation on APAP‐induced hepatotoxicity. Our next step is to study the underlying mechanisms for altered susceptibility to APAP hepatotoxicity by these metabolites.

DDAH1 Protects against Acetaminophen-Induced Liver Hepatoxicity in Mice.

In many developed countries, acetaminophen (APAP) overdose-induced acute liver injury is a significant therapeutic problem. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme for asymmetric dimethylarginine (ADMA) metabolism. Growing evidence suggests that liver dysfunction is associated with increased plasma ADMA levels and reduced hepatic DDAH1 activity/expression. The purpose of this study was to investigate the involvement of DDAH1 in APAP-mediated hepatotoxicity using Ddah1-/- and DDAH1 transgenic mice. After APAP challenge, Ddah1-/- mice developed more severe liver injury than wild type (WT) mice, which was associated with a greater induction of fibrosis, oxidative stress, inflammation, cell apoptosis and phosphorylation of JNK. In contrast, overexpression of DDAH1 attenuated APAP-induced liver injury. RNA-seq analysis showed that DDAH1 affects xenobiotic metabolism and glutathione metabolism pathways in APAP-treated livers. Furthermore, we found that DDAH1 knockdown aggravated APAP-induced cell death, oxidative stress, phosphorylation of JNK and p65, upregulation of CYP2E1 and downregulation of GSTA1 in HepG2 cells. Collectively, our data suggested that DDAH1 has a marked protective effect against APAP-induced liver oxidative stress, inflammation and injury. Strategies to increase hepatic DDAH1 expression/activity may be novel approaches for drug-induced acute liver injury therapy.

The Performance of HepG2 and HepaRG Systems through the Glass of Acetaminophen-Induced Toxicity.

Investigation of drug-induced liver injuries requires appropriate in vivo and in vitro toxicological model systems. In our study, an attempt was made to compare the hepatocarcinoma HepG2 and the stem cell-derived HepaRG cell lines both in two- and three-dimensional culture conditions to find the most suitable model. Comparison of the liver-specific characteristics of these models was performed via the extent and mechanism of acetaminophen (APAP)-induced hepatotoxicity. Investigating the detailed mechanism of APAP-induced hepatotoxicity, different specific cell death inhibitors were used: the pan-caspase inhibitor zVAD-fmk and dabrafenib significantly protected both cell lines from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and primary hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression.

G protein β5-ATM complexes drive acetaminophen-induced hepatotoxicity

Excessive ingestion of the common analgesic acetaminophen (APAP) leads to severe hepatotoxicity. Here we identify G protein β5 (Gβ5), elevated in livers from APAP overdose patients, as a critical regulator of cell death pathways and autophagic signaling in APAP-exposed liver. Liver-specific knockdown of Gβ5 in mice protected the liver from APAP-dependent fibrosis, cell loss, oxidative stress, and inflammation following either acute or chronic APAP administration. Conversely, overexpression of Gβ5 in liver was sufficient to drive hepatocyte dysfunction and loss. In hepatocytes, Gβ5 depletion ameliorated mitochondrial dysfunction, allowed for maintenance of ATP generation and mitigated APAP-induced cell death. Further, Gβ5 knockdown also reversed impacts of APAP on kinase cascades (e.g. ATM/AMPK) signaling to mammalian target of rapamycin (mTOR), a master regulator of autophagy and, as a result, interrupted autophagic flux. Though canonically relegated to nuclear DNA repair pathways, ATM also functions in the cytoplasm to control cell death and autophagy. Indeed, we now show that Gβ5 forms a direct, stable complex with the FAT domain of ATM, important for autophosphorylation-dependent kinase activation. These data provide a viable explanation for these novel, G protein-independent actions of Gβ5 in liver. Thus, Gβ5 sits at a critical nexus in multiple pathological sequelae driving APAP-dependent liver damage.

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 adduct 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 that, 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 comeback 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. 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.

Autophagy impairment as a key feature for acetaminophen-induced ototoxicity

Macroautophagy/autophagy is a highly conserved self-digestion pathway that plays an important role in cytoprotection under stress conditions. Autophagy is involved in hepatotoxicity induced by acetaminophen (APAP) in experimental animals and in humans. APAP also causes ototoxicity. However, the role of autophagy in APAP-induced auditory hair cell damage is unclear. In the present study, we investigated autophagy mechanisms during APAP-induced cell death in a mouse auditory cell line (HEI-OC1) and mouse cochlear explant culture. We found that the expression of LC3-II protein and autophagic structures was increased in APAP-treated HEI-OC1 cells; however, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence, and the activity of lysosomal enzymes decreased in APAP-treated HEI-OC1 cells. The degradation of p62 protein and the expression of lysosomal enzymes also decreased in APAP-treated mouse cochlear explants. These data indicate that APAP treatment compromises autophagic degradation and causes lysosomal dysfunction. We suggest that lysosomal dysfunction may be directly responsible for APAP-induced autophagy impairment. Treatment with antioxidant N-acetylcysteine (NAC) partially alleviated APAP-induced autophagy impairment and apoptotic cell death, suggesting the involvement of oxidative stress in APAP-induced autophagy impairment. Inhibition of autophagy by knocking down of Atg5 and Atg7 aggravated APAP-induced ER and oxidative stress and increased apoptotic cell death. This study provides a better understanding of the mechanism responsible for APAP ototoxicity, which is important for future exploration of treatment strategies for the prevention of hearing loss caused by ototoxic medications.

Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05−0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity.

Allyl Isothiocyanate Protects Acetaminophen-Induced Liver Injury via NRF2 Activation by Decreasing Spontaneous Degradation in Hepatocyte.

Acetaminophen (APAP) is one of the most frequently prescribed analgesic and anti-pyretic drugs. However, APAP-induced hepatotoxicity is a major cause of acute liver failure globally. While the therapeutic dose is safe, an overdose of APAP produces an excess of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), subsequently resulting in hepatotoxicity. Allyl isothiocyanate (AITC), a bioactive molecule in cruciferous plants, is reported to exert various biological effects, including anti-inflammatory, anti-cancer, and anti-microbial effects. Notably, AITC is known for activating nuclear factor erythroid 2-related factor 2 (NRF2), but there is limited evidence supporting the beneficial effects on hepatocytes and liver, where AITC is mainly metabolized. We applied a mouse model in the current study to investigate whether AITC protects the liver against APAP-induced injury, wherein we observed the protective effects of AITC. Furthermore, NRF2 nuclear translocation and the increase of target genes by AITC treatment were confirmed by in vitro experiments. APAP-induced cell damage was attenuated by AITC via an NRF2-dependent manner, and rapid NRF2 activation by AITC was attributed to the elevation of NRF2 stability by decreasing its spontaneous degradation. Moreover, liver tissues from our mouse experiment revealed that AITC increases the expression of heme oxygenase-1 (HO-1), an NRF2 target gene, confirming the potential of AITC as a hepatoprotective agent that induces NRF2 activation. Taken together, our results indicate the potential of AITC as a natural-product-derived NRF2 activator targeting the liver.

Mechanisms and pathophysiological significance of sterile inflammation during acetaminophen hepatotoxicity

Acetaminophen (APAP) is a widely used analgesic drug, which can cause severe liver injury after an overdose. The intracellular signaling mechanisms of APAP-induced cell death such as reactive metabolite formation, mitochondrial dysfunction and nuclear DNA fragmentation have been extensively studied. Hepatocyte necrosis releases damage-associated molecular patterns (DAMPs) which activate cytokine and chemokine formation in macrophages. These signals activate and recruit neutrophils, monocytes and other leukocytes into the liver. While this sterile inflammatory response removes necrotic cell debris and promotes tissue repair, the capability of leukocytes to also cause tissue injury makes this a controversial topic. This review summarizes the literature on the role of various DAMPs, cytokines and chemokines, and the pathophysiological function of Kupffer cells, neutrophils, monocytes and monocyte-derived macrophages, and NK and NKT cells during APAP hepatotoxicity. Careful evaluation of results and experimental designs of studies dealing with the inflammatory response after APAP toxicity provide very limited evidence for aggravation of liver injury but support of the hypothesis that these leukocytes promote tissue repair. In addition, many cytokines and chemokines modulate tissue injury by affecting the intracellular signaling events of cell death rather than toxicity of leukocytes. Reasons for the controversial results in this area are also discussed.

PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells.

Acetaminophen (APAP) is a widely used analgesic and antipyretic. It is safe at normal treatment doses; however, APAP overdose is a major cause of acute liver and kidney failure. A variety of methods to reduce the damage caused by APAP overdose have previously been evaluated. The protein-rich seaweed Pyropia yezoensis has antioxidant, antitumor and anti-inflammatory activities, and protects against cytotoxicity. However, little is known regarding the protective effects of P. yezoensis peptide against APAP-induced hepatotoxicity. The present study investigated the ability of P. yezoensis peptide (PYP1-4) to ameliorate the damage caused by APAP-induced hepatotoxicity using HepG2 as the model cell line in addition to the signaling pathways involved. Briefly, cell viability, nitric oxide, reactive oxygen species and apoptosis assays were performed in conjunction with western blot analysis and reverse transcription-quantitative PCR. First, the present study revealed the minimum toxic concentration of APAP (15 mM) and the resting concentration of PYP1-4 (0–500 ng/ml). Administration of PYP1-4 to APAP-induced cells decreased the nitric oxide and reactive oxygen species levels, and restored the levels of antioxidant-associated proteins (catalase, heme oxygenase 1, superoxide dismutase 2 and quinone oxidoreductase 1). PYP1-4 increased the translocation of nuclear factor, erythroid 2 like 2 to the nucleus and the activities of glycogen synthase kinase-3β, Akt and AMP-activated protein kinase. In addition, APAP induced apoptosis; however, PYP1-4 inhibited apoptosis by modulating the levels of pro-apoptotic markers (Bad), anti-apoptotic markers (Bcl-2 and BH3 interacting domain death agonist), caspases and poly (ADP-ribose) polymerase 1. Subsequently, the insulin-like growth factor 1 receptor signaling pathway was investigated to determine whether PYP1-4 treatment restored the levels of cell growth-associated factors during APAP-induced hepatotoxicity. PYP1-4 treatment impacted the levels of components of the insulin receptor substrate 1/PI3K/Akt and Ras/Raf/ERK signaling pathways, and promoted cell survival. Therefore, the P. yezoensis peptide PYP1-4 may be useful for preventing APAP-induced hepatotoxicity.

Emerging and established modes of cell death during acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.