Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.

Acetaminophen (APAP) overdose results in the production of reactive oxygen species (ROS), hepatocyte necrosis, and cell death, and leads to acute liver failure. Interleukin-17 (IL-17), a pro-inflammatory cytokine, plays a key role in the recruitment of neutrophils into sites of inflammation and subsequent damage after liver ischemia-reperfusion injury. In this study, we employed IL-17 knockout (KO) mice to investigate the role of IL-17 in APAP-induced hepatotoxicity. IL-17 wide type (WT) and IL-17 KO mice received an intraperitoneal injection of APAP (300 mg/kg). After 16 h of treatment, the hepatic injury, inflammatory mediators, immune cell infiltration, and western blotting were examined and analyzed. The serum alanine transferase (ALT) enzyme levels and hepatic myeloperoxidase (MPO) activity were significantly elevated 16 h after APAP treatment in the WT mice. IL-17 deficiency significantly attenuates APAP-induced liver injury, MPO activity, pro-inflammatory cytokines (tumor necrosis factor-α, IL-6 and interferon-γ) levels and inflammatory cell (neutrophils, macrophage) infiltration in the liver. Moreover, phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased at 16 h after APAP treatment in the IL-17 KO mice compared with the IL-17 WT mice. Our data suggests that IL-17 plays a pivotal role in APAP-induced hepatotoxicity through modulation of inflammatory response, and perhaps in part through the ERK signaling pathway. Blockade of IL-17 could be a potential therapeutic target for APAP-induced hepatotoxicity.

Acetaminophen (paracetamol, APAP) poisoning is a prominent global cause of drug-induced liver injury. While N-acetylcysteine (NAC) is an effective antidote, it has therapeutic limitations in massive overdose or delayed presentation. The objective is to comprehensively review the literature on fomepizole as a potential adjunct antidote for acetaminophen toxicity.

Primary human hepatocytes (hHEP), human HepaRG and HepG2 cell lines are the most used human liver-based in vitro models for hepatotoxicity testing, including screening of drug-induced liver injury (DILI)-inducing compounds. hHEP are the reference hepatic in vitro system, but their availability is limited and the cells available for toxicology studies are often of poor quality. Hepatic cell lines on the other hand are highly proliferative and represent an inexhaustible hepatic cell source. However, these hepatoma-derived cells do not represent the population diversity and display reduced hepatic metabolism. Alternatively, stem cell-derived hepatic cells, which can be produced in high numbers and can differentiate into multiple cell lineages, are also being evaluated as a cell source for in vitro hepatotoxicity studies. Human skin-derived precursors (hSKP) are post-natal stem cells that, after conversion towards hepatic cells (hSKP-HPC), respond to hepatotoxic compounds in a comparable way as hHEP. In the current study, four different human hepatic cell systems (hSKP-HPC, hHEP, HepaRG and HepG2) are evaluated for their capacity to predict hepatic toxicity. Their hepatotoxic response to acetaminophen (APAP) exposure is compared to data obtained from patients suffering from APAP-induced acute liver failure (ALF). The results indicate that hHEP, HepaRG and hSKP-HPC identify comparable APAP-induced hepatotoxic functions and that HepG2 cells show the slightest hepatotoxic response. Pathway analyses further points out that HepaRG cells show the highest predicted activation of the functional genes related to 'damage of liver', followed by hSKP-HPC and hHEP cells that generated similar results. HepG2 did not show any activation of this function.

Acetaminophen (APAP) poisoning is the most common cause of drug-induced acute liver injury (ALI). Our results showed that toll-like receptor 5 (TLR5) was abundantly expressed in hepatocytes and dramatically downregulated in the toxic mouse livers. Hence, we herein investigated the role of TLR5 signaling after APAP overdose. Mice were intraperitoneally (i.p.) injected with APAP to induce ALI, and then injected with flagellin at one hour after APAP administration. Flagellin attenuated APAP-induced ALI based on decreased histopathologic lesions, serum biochemical, oxidative stress, and inflammation. Furthermore, the protective effects of flagellin were abolished by TH1020 (a TLR5 antagonist) treatment. These results suggest that flagellin exerted protective effects on ALI via TLR5 activation. Mechanistically, flagellin injection promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus in hepatocytes. Consistent with the in vivo results, flagellin increased the activation of Nrf2 in hepatocytes, resulting in decreased APAP toxicity. ML385, a selective inhibitor of Nrf2, abolished the flagellin-mediated hepatoprotective effects in damaged livers and hepatocytes. Additionally, the flagellin-induced Nrf2 translocation was dependent upon the activation of TLR5-JNK/p38 pathways. These findings suggest that TLR5 signaling-induced Nrf2 activation, at least partially, contributed to the protection against APAP-induced ALI by flagellin treatment.

Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in many countries. In the present study, we developed stable mouse models of acute drug-induced hepatic injury (DILI) and acute drug-induced hepatic failure (DILF) by sub-lethal and lethal APAP injection respectively. The differences in hepatic transcriptome profiling between these two models were compared by RNA sequencing, which were validated by qPCR, western-blot and ELISA. In results, serum IL-6, TNF-a and IL-10 levels are higher in DILF than in DILI. The upregulated genes in DILF compared with DILI were mostly enriched in the areas of "cellular development process", "cell division", "multicellular organism development," etc. The downregulated genes in DILF compared with DILI were mostly enriched in the areas of "cellular response to chemical stimulus", "cellular response to stress", "cell activation," etc. Sub-lethal doses of APAP increased Myc, Bag3 and Btc expression in mouse liver, but lethal doses of APAP did not, which suggested that these three genes might play important roles in adaptive protection reactions in DILI. The serum Btc level might be a potential biomarker of drug induced liver injury with good prognosis. Our data can help us better understand the mechanisms of hepatotoxicity that influence prognosis and seek novel prognostic indicators of DILI.

Formation of acetaminophen (APAP) protein adducts are a critical feature of APAP hepatotoxicity, and circulating protein adducts have recently been utilized in bioassays for identification of APAP overdose in humans. Despite their clinical significance, mechanisms of adduct release into the circulation are not well understood. Extracellular vesicles (EVs) are discrete membrane bound vesicles, which package cellular cargo and function in extracellular transport. Clarification of their role in transport of APAP adducts is relevant since adduct packaging within these vesicles could shield them from detection by antibody based methods, resulting in under-estimating adduct levels. Hence, this study evaluated EV release after APAP overdose in primary mouse hepatocytes and human HepaRG cells in vitro, in mice and APAP overdose patients in vivo and examined their role in transport of APAP-protein adducts. EVs were characterized by size and protein composition and the levels of APAP-protein adducts were measured. Significant elevations in circulating EV numbers were observed 6 h after APAP overdose in vivo and by 4 h in primary mouse hepatocytes in culture. EVs were also elevated in media from HepaRG cells by 24 h after APAP exposure, an effect recapitulated in APAP overdose patients, where EV numbers were elevated compared to healthy controls. Although APAP-protein adducts were elevated in circulation and media parallel to the increased exosome release, no detectable adducts were observed within EVs. This suggests that although APAP overdose enhances EV release from hepatocytes in mice and humans, it is not a significant mechanism of release of APAP protein adducts into circulation.

Overdose use of acetaminophen (APAP) often occurs a severe liver injury, and its liver injury is lethal in some cases. Macrophage migration inhibitory factor (MIF) is expressed in a variety of cells and has multifunctional roles. However, the role of MIF in APAP-induced liver injury has not been fully investigated. In this study, we investigated whether treatment with (S,R)-3-(4-hydroxyphenil)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), a MIF inhibitor, protected mice from acute APAP-induced liver injury. Acute liver injury was induced by injection of APAP (300 mg/kg body weight). Mice were treated with a single injection of ISO-1(15 mg/kg body weight) 1 h (h) before APAP administration. Histological, biochemical and molecular analyses were performed in liver of mice 12 h after APAP administration. ISO-1 remarkably improved the histological findings of APAP-induced liver injury in mice. The increases in serum levels of alanine aminotransferase (ALT), and macrophage inflammatory protein-2 (MIP-2) by APAP were inhibited by ISO-1. In addition, ISO-1 reduced the increased number of the myeloperoxidase-staining cells and that of TUNEL-positive staining cells in the liver of mice with APAP-induced liver injury. Up-regulation of hepatic receptor interacting protein kinase (RIPK)3 and heat shock protein70 by APAP was suppressed in the liver of mice given ISO-1. These results provide the additional evidence that inhibition of MIF activity may be clinically effective for treatment of acute APAP-induced liver injury.

Acetaminophen (APAP) overdosage results in hepatotoxicity, but the underlying molecular mechanisms are still not completely understood. In the current study, we focused on mitochondrial-specific oxidative liver injury induced by APAP exposure. Owning to genetic polymorphisms in the CYP2E1 gene or varying inducibility by xenobiotics, the CYP2E1 mRNA level and protein activity vary extensively among individuals. As CYP2E1 is a known ROS generating enzyme, we chose HepG2 to minimize CYP2E1-induced ROS formation, which will help us better understand the APAP induced mitochondrial-specific hepatotoxicity in a subpopulation with low CYP2E1 activity. HepG2 cells were exposed to a low and toxic dose (0.5 and 10mM) of APAP and analyzed at four time points for genome-wide gene expression. Mitochondria were isolated and electron spin resonance spectroscopy was performed to measure the formation of mitochondrial ROS. The yield of ATP was measured to confirm the impact of the toxic dose of APAP on cellular energy production. Our results indicate that 10mM APAP significantly influences the expression of mitochondrial protein-encoding genes in association with an increase in mitochondrial ROS formation. Additionally, 10mM APAP affects the expression of genes encoding the subunits of electron transport chain (ETC) complexes, which may alter normal mitochondrial functions by disrupting the assembly, stability, and structural integrity of ETC complexes, leading to a measurable depletion of ATP, and cell death. The expression of mitochondrium-specific antioxidant enzyme, SOD2, is reduced which may limit the ROS scavenging ability and cause imbalance of the mitochondrial ROS homeostasis. Overall, transcriptome analysis reveals the molecular processes involved in the observed APAP-induced increase of mitochondrial ROS formation and the associated APAP-induced oxidative stress.

Resveratrol (RES) has been shown to possess many pharmacological activities including protective effect against liver damage induced by hepatotoxins. In the present study, the hepato-protective effect of RES against acetaminophen (APAP)-induced liver injury in mice and the involved mechanisms was investigated. This study clearly demonstrated that administration of RES three days before APAP treatment significantly alleviated APAP-induced hepatotoxicity, as evidenced by morphological, histopathological, and biochemical assessments such as GSH content and serum ALT/AST activity. Treatment with RES resulted in significant inhibition of CYP2E1, CYP3A11, and CYP1A2 activities, and then caused significant inhibition of the bioactivation of APAP into toxic metabolite NAPQI. Pretreatment with RES significantly reduced APAP-induced JNK activation to protect against mitochondrial injury. Additionally, RES treatment significantly induced SIRT1 and then negatively regulated p53 signaling to induce cell proliferation-associated proteins including cyclin D1, CDK4, and PCNA to promote hepatocyte proliferation. This study demonstrated that RES prevents APAP-induced hepatotoxicity by inhibition of CYP-mediated APAP bioactivation and regulation of SIRT1, p53, cyclin D1 and PCNA to facilitate liver regeneration following APAP-induced liver injury.

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.

The zebrafish embryo (ZFE) is a promising alternative, non-rodent model in toxicology, which has an advantage over the traditionally used models as it contains complete biological complexity and provides a medium to high-throughput setting. Here, we assess how the ZFE compares to the traditionally used models for liver toxicity testing, i.e., in vivo mouse and rat liver, in vitro mouse and rat hepatocytes, and primary human hepatocytes. For this comparison, we analyzed gene expression changes induced by three model compounds for cholestasis, steatosis, and necrosis. The three compounds, cyclosporine A, amiodarone, and acetaminophen, were chosen because of their relevance to human toxicity and these compounds displayed hepatotoxic-specific changes in the mouse in vivo data. Compound induced expression changes in the ZFE model shared similarity with both in vivo and in vitro. Comparison on single gene level revealed the presence of model specific changes and no clear concordance across models. However, concordance was identified on the pathway level. Specifically, the pathway "regulation of metabolism - bile acids regulation of glucose and lipid metabolism via FXR" was affected across all models and compounds. In conclusion, our study with three hepatotoxic model compounds shows that the ZFE model is at least as comparable to traditional models in identifying hepatotoxic activity and has the potential for use as a pre-screen to determine the hepatotoxic potential of compounds.

Diallyl sulfide (DAS) has been shown to prevent xenobiotic (e.g. ethanol, acetaminophen) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. Therefore, the goal of the current study was to investigate the structural analogs of DAS for their improved toxicity profiles and their effectiveness in reducing xenobiotic-induced toxicity and HIV-1 replication. Previously, we identified commercially available analogs that possessed CYP2E1 inhibitory capacity greater than or equal to that of DAS. In this study, we evaluated the toxicity and efficacy of these analogs using hepatocytes, monocytes, and astrocytes where CYP2E1 plays an important role in xenobiotic-mediated toxicity. Our results showed that thiophene, allyl methyl sulfide, diallyl ether, and 2-prop-2-enoxyacetamide are significantly less cytotoxic than DAS in these cells. Moreover, these analogs reduced ethanol- and acetaminophen-induced toxicity in hepatocytes and HIV-1 replication in monocytes more effectively than DAS. Overall, our findings are significant in terms of using these DAS analogs as a tool in vitro and in vivo, especially to examine chronic xenobiotic-induced toxicity and disease pathogenesis that occurs through the CYP2E1 pathway.

We describe a strategy using an in vitro metabolomics assay with tubular rat NRK-52E cells to investigate the Modes of Action (MoAs) of nephrotoxic compounds. Chemicals were selected according to their MoAs based on literature information: acetaminophen, 4-aminophenol and S-(trichlorovinyl-)L-cysteine (TCVC), (covalent protein binding); gentamycin, vancomycin, polymycin B and CdCl2 (lysosomal overload) and tenofovir and cidofovir (mitochondrial DNA-interaction). After treatment and harvesting of the cells, intracellular endogenous metabolites were quantified relative to vehicle control. Metabolite patterns were evaluated in a purely data-driven pattern generation process excluding published information. This strategy confirmed the assignment of the chemicals to the respective MoA except for TCVC and CdCl2. Finally, TCVC was defined as unidentified and CdCl2 was reclassified to the MoA "covalent protein binding". Hierarchical cluster analysis of 58 distinct metabolites from the patterns enabled a clear visual separation of chemicals in each MoA. The assay reproducibility was very good and metabolic responses were consistent. These results support the use of metabolome analysis in NRK-52E cells as a suitable tool for understanding and investigating the MoA of nephrotoxicants. This assay could enable the early identification of nephrotoxic compounds and finally reduce animal testing.

The toxic industrial chemical (TIC1) phosgene remains an important chemical intermediate in many industrial processes. Inhalation of phosgene can cause an acute lung injury (ALI) which, in severe cases may result in death. There are currently no effective pharmacological therapies or evidence-based treatment guidelines for managing exposed individuals. N-acetylcysteine (NAC) is a commercially available drug licensed in the UK and elsewhere for the treatment of paracetamol (acetaminophen) overdose. It has a number of mechanisms of action which may provide therapeutic benefit for the treatment of phosgene-induced ALI. It has previously been shown to provide therapeutic efficacy against the lung damaging effects of sulfur mustard vapour exposure, when given by the inhaled route, in the pig (Jugg et al., 2013). Our research objective was to determine whether inhaled NAC might also be therapeutic for other chemicals, in this case, phosgene. This study has demonstrated that multiple nebulised doses, administered from 30 min after exposure of terminally anaesthetised pigs to phosgene, is not an effective therapy when administered at the times and doses employed in this study. There remains no pharmacological treatment for phosgene-induced lung injury.

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain- and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (FcγR)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

Acetaminophen (APAP) overdose can induce acute liver injury (ALI) with significant morbidity and mortality. Propacetamol is an APAP prodrug, which is clinically bioequivalent to APAP. Kaempferol, a dietary flavonoid, has antioxidant, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the protective effect of kaempferol on propacetamol-induced ALI and its underlying mechanism in mice. Kaempferol pretreatment (125 mg/kg) before propacetamol injection significantly decreased propacetamol-induced serum ALT and AST activities, and DNA fragmentation. Kaempferol administration also reduced propacetamol-induced oxidative stress by inhibiting thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine (3-NT) formation partly through downregulation of cytochrome P450 2E1 (CYP2E1) expression, upregulation of UDP glucuronosyltransferase family 1 member A1 (UGT1A1) expression, restoration of the activities of antioxidant enzymes including SOD, GPx and catalase toward normal, recovery of propacetamol-suppressed Nrf2 and GCLC expressions, and maintenance of normal glutathione level. Furthermore, kaempferol markedly attenuated APAP-induced serum TNF-α and IL-6 productions, downregulated APAP-induced phosphorylations of JNK and ERK, and decreased early hepatic apoptosis via decreasing Bax/Bcl-2 ratio and caspase 3 activation. Furthermore, administration of N-acetylcysteine (NAC) and kaempferol significantly rescued more mice than a low dose of NAC only did when a lethal dose of propacetamol injected and therapized at a delayed time point. These data suggested that kaempferol protects the liver against propacetamol-induced injury through anti-oxidative, anti-inflammatory and anti-apoptotic activities.