Histone demethylase UTX aggravates acetaminophen overdose induced hepatotoxicity through dual mechanisms.

Acetaminophen (APAP) overdose is a major cause of acute liver failure, while the underlying mechanisms of APAP hepatotoxicity are not fully understood. Recently, emerging evidence suggests that epigenetic enzymes play roles in APAP-induced liver injury. Here, we found that Utx (ubiquitously transcribed tetratricopeptide repeat, X chromosome, also known as KDM6A), a X-linked histone demethylase which removes the di- and tri-methyl groups from histone H3K27, was markedly induced in the liver of APAP-overdosed female mice. Hepatic deletion of Utx suppressed APAP overdose-induced hepatotoxicity in female but not male mice. RNA-sequencing analysis suggested that Utx deficiency in female mice upregulated antitoxic phase II conjugating enzymes, including sulfotransferase family 2 A member 1 (Sult2a1), thus reduces the amount of toxic APAP metabolites in injured liver; while Utx deficiency also alleviated ER stress through downregulating transcription of ER stress genes including Atf4, Atf3, and Chop. Mechanistically, Utx promoted transcription of ER stress related genes in a demethylase activity-dependent manner, while repressed Sult2a1 expression through mediating H3K27ac levels independent of its demethylase activity. Moreover, overexpression of Sult2a1 in the liver of female mice rescued APAP-overdose induced liver injury. Together, our results indicated a novel UTX-Sult2a1 axis for the prevention or treatment of APAP-induced liver injury.

Decreased plasma acetaminophen glucuronide/acetaminophen concentration ratio warns the onset of acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury (AILI) is the most common cause of acute liver failure. Although the mechanisms that trigger AILI are well known, it is less understood how to halt AILI progression and facilitate liver recovery. Therefore, it is necessary to understand the pathophysiology of APAP hepatotoxicity in patients and to examine predictive/preventive markers. In a clinical study, we had a case in which aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels increased in a patient with a low ratio of APAP glucuronide concentration (AP-G)/APAP plasma concentration. Then a reverse translational study was conducted for clarifying this clinical question. The relationship between plasma AP-G/APAP concentration ratio and the levels of AST and ALT was examined by in vivo and in vitro experiments. In in vivo experiments, 10-week-old rats showed lower UGT activity, lower AP-G/APAP concentration ratios, and higher AST and ALT levels than 5-week-old rats. This suggests an inverse correlation between the AP-G/APAP concentration ratio and the AST, ALT levels in APAP-treated rats. Furthermore, as a result of the in vitro experiment, it was confirmed that the cell viability decreased when the AP-G/APAP concentration ratio in the culture medium decreased. Since the decrease in the plasma AP-G/APAP concentration ratio appears earlier than the increase of AST and ALT levels, the ratio might be a presymptomatic marker of AILI. When APAP is used for a long time, it is recommended to perform therapeutic drug monitoring of the AP-G/APAP concentration ratio, which is a predictive/preventive marker of AILI.

Isatidis Folium alleviates acetaminophen-induced liver injury in mice by enhancing the endogenous antioxidant system.

Isatidis Folium (IF) has been clinically combined with acetaminophen (APAP), but the rationality of combinational therapy is still ambiguous. In the present study, the protective effect and related mechanism of IF on APAP-induced hepatotoxicity were evaluated. Hepatic histopathology and blood biochemistry investigations clearly demonstrated that IF could restore APAP-induced hepatotoxicity. Liver distribution study indicated that the hepatoprotective effect of IF on APAP is attributed to the reduction of N-acetyl-p-benzoquinone imine (NAPQI) in liver, which is a known hepatotoxic metabolite of APAP. Further study suggested the reduction is not via decreasing the generation of NAPQI through inhibiting the enzyme activities of CYP 1A2, 2E1, and 3A4 but via accelerating the transformation of NAPQI to NAPQI-GSH by promoting GSH and decreasing GSSG contents in liver. Furthermore, IF significantly enhanced the hepatic activities of GSH-associated enzymes in APAP-treated mice. In summary, IF could alleviate APAP-induced hepatotoxicity by reducing the content of NAPQI via enhancing the level of GSH and the followed generation of NAPQI-GSH which might be ascribed to the upregulation of GSH-associated enzymes.

Impact of medication on protein and amino acid metabolism in the elderly: the sulfur amino acid and paracetamol case.

The optimisation of nutritional support for the growing number of older individuals does not usually take into account medication. Paracetamol (acetaminophen; APAP) is the first intention treatment of chronic pain that is highly prevalent and persistent in the elderly. Detoxification of APAP occurs in the liver and utilises sulfate and glutathione (GSH), both of which are issued from cysteine (Cys), a conditionally indispensable amino acid. The detoxification-induced siphoning of Cys could reduce the availability of Cys for skeletal muscle. Consequently, APAP could worsen sarcopenia, an important component of the frailty syndrome leading to dependency. The present review provides the rationale for the potential pro-sarcopenic effect of APAP then recent results concerning the effect of chronic APAP treatment on muscle mass and metabolism are discussed. The principal findings are that chronic treatments with doses of APAP comparable with the maximum posology for humans can increase the requirement for sulfur amino acids (SAA), reduce Cys availability for muscle, reduce muscle protein synthesis and aggravate sarcopenia in animals. One clinical study is in favour of an enhanced SAA requirement in the older individual under chronic treatment with APAP. Few clinical studies investigated the effect of chronic treatment with APAP combined with exercise, in nutritional conditions that probably did not affect Cys and GSH homeostasis. Whether APAP can aggravate sarcopenia in older individuals with low protein intake remains to be tested. If true, nutritional strategies based on enhancing Cys supply could be of prime interest to cut down the pro-sarcopenic effect of chronic treatment with APAP.

The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation.

Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4-6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions.

Cathelicidin promotes liver repair after acetaminophen-induced liver injury in mice.

Background & Aims: Acetaminophen (APAP)-induced acute liver injury (AILI) is a leading cause of acute liver failure (ALF). N-acetylcysteine (NAC) is only effective within 24 h after APAP intoxication, raising an urgent need for alternative approaches to treat this disease. This study aimed to test whether cathelicidin (Camp), which is a protective factor in chronic liver diseases, protects mice against APAP-induced liver injury and ALF. Methods: A clinically relevant AILI model and an APAP-induced ALF model were generated in mice. Genetic and pharmacological approaches were used to interfere with the levels of cathelicidin in vivo. Results: An increase in hepatic pro-CRAMP/CRAMP (the precursor and mature forms of mouse cathelicidin) was observed in APAP-intoxicated mice. Upregulated cathelicidin was derived from liver-infiltrating neutrophils. Compared with wild-type littermates, Camp knockout had no effect on hepatic injury but dampened hepatic repair in AILI and reduced survival in APAP-induced ALF. CRAMP administration reversed impaired liver recovery observed in APAP-challenged Camp knockout mice. Delayed CRAMP, CRAMP(1-39) (the extended form of CRAMP), or LL-37 (the mature form of human cathelicidin) treatment exhibited a therapeutic benefit for AILI. Co-treatment of cathelicidin and NAC in AILI displayed a stronger hepatoprotective effect than NAC alone. A similar additive effect of CRAMP(1-39)/LL-37 and NAC was observed in APAP-induced ALF. The pro-reparative role of cathelicidin in the APAP-damaged liver was attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced neutrophil phagocytosis of necrotic cell debris in an autocrine manner. Conclusions: Cathelicidin reduces APAP-induced liver injury and ALF in mice by promoting liver recovery via facilitating inflammation resolution, suggesting a therapeutic potential for late-presenting patients with AILI with or without ALF. Impact and implications: Acetaminophen-induced acute liver injury is a leading cause of acute liver failure. The efficacy of N-acetylcysteine, the only clinically approved drug against acetaminophen-induced acute liver injury, is significantly reduced for late-presenting patients. We found that cathelicidin exhibits a great therapeutic potential in mice with acetaminophen-induced liver injury or acute liver failure, which makes up for the limitation of N-acetylcysteine therapy by specifically promoting liver repair after acetaminophen intoxication. The pro-reparative role of cathelicidin, as a key effector molecule of neutrophils, in the APAP-injured liver is attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced phagocytic function of neutrophils in an autocrine manner.

Acetaminophen-induced reduction of NIMA-related kinase 7 expression exacerbates acute liver injury.

Background & Aims: Acetaminophen (APAP)-induced acute liver injury (ALI) is a global health issue characterised by an incomplete understanding of its pathogenesis and unsatisfactory therapies. NEK7 plays critical roles in both cell cycle regulation and inflammation. In the present study, we investigated the role and mechanism of NEK7 in APAP-induced ALI. Methods: In mice with NEK7 overexpression (hydrodynamic tail vein injection of NEK7 plasmids), hepatocyte-specific NEK7 knockout (cKO), and inducible NEK7 knockout (iKO), an overdose of APAP was administered to induce ALI. Liver injury was determined by an analysis of serum liver enzymes, pathological changes, inflammatory cytokines, and metabonomic profiles. In vitro, hepatocyte damage was evaluated by an analysis of cell viability, the reactive oxygen species levels, and mitochondrial function in different cell lines. Hepatocyte proliferation and the cell cycle status were determined by Ki-67 staining, EdU staining, and the cyclin levels. Results: NEK7 was markedly downregulated in APAP-induced injured liver and damaged hepatocytes. NEK7 overexpression in the liver significantly alleviated APAP-induced liver injury, as shown by the restored liver function, reduced pathological injury, and decreased inflammation and oxidative stress, which was confirmed in a hepatocyte cell line. Moreover, both NEK7 cKO and iKO mice exhibited exacerbation of APAP-induced ALI. Finally, we determined that cyclin B1-mediated cell cycle progression could mediate the protective effect of NEK7 against APAP-induced ALI. Conclusions: Reduced NEK7 contributes to APAP-induced ALI, possibly by dysregulating cyclins and disturbing cell cycle progression. Lay summary: Acetaminophen-induced acute liver injury is one of the major global health issues, owing to its high incidence, potential severity, and limited therapeutic options. Our current understanding of its pathogenesis is incomplete. Herein, we have shown that reduced NEK7 (a protein with a key role in the cell cycle) exacerbates acetaminophen-induced acute liver injury. Hence, NEK7 could be a possible therapeutic target for the prevention or treatment of this condition.

Determining site occupancy of acetaminophen covalent binding to target proteins in vitro.

Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.

Efficacy of broccoli and glucoraphanin in COVID-19: From hypothesis to proof-of-concept with three experimental clinical cases.

COVID-19 is described in a clinical case involving a patient who proposed the hypothesis that Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-interacting nutrients may help to prevent severe COVID-19 symptoms. Capsules of broccoli seeds containing glucoraphanin were being taken before the onset of SARS-CoV-2 infection and were continued daily for over a month after the first COVID-19 symptoms. They were found to reduce many of the symptoms rapidly and for a duration of 6-12 h by repeated dosing. When the patient was stable but still suffering from cough and nasal obstruction when not taking the broccoli capsules, a double-blind induced cough challenge confirmed the speed of onset of the capsules (less than 10 min). A second clinical case with lower broccoli doses carried out during the cytokine storm confirmed the clinical benefits already observed. A third clinical case showed similar effects at the onset of symptoms. In the first clinical trial, we used a dose of under 600 µmol per day of glucoraphanin. However, such a high dose may induce pharmacologic effects that require careful examination before the performance of any study. It is likely that the fast onset of action is mediated through the TRPA1 channel. These experimental clinical cases represent a proof-of-concept confirming the hypothesis that Nrf2-interacting nutrients are effective in COVID-19. However, this cannot be used in practice before the availability of further safety data, and confirmation is necessary through proper trials on efficacy and safety.

Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

SIRT6 as a key event linking P53 and NRF2 counteracts APAP-induced hepatotoxicity through inhibiting oxidative stress and promoting hepatocyte proliferation.

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively. Sirt6 knockdown in AML12 cells aggravated APAP-induced hepatocyte death and oxidative stress, inhibited cell viability and proliferation, and downregulated CCNA1, CCND1 and CKD4 protein levels. Sirt6 knockdown significantly prevented APAP-induced NRF2 activation, reduced the transcriptional activities of GSTµ and NQO1 and the mRNA levels of Nrf2, Ho-1, Gstα and Gstµ. Furthermore, SIRT6 showed potential protein interaction with NRF2 as evidenced by co-immunoprecipitation (Co-IP) assay. Additionally, the protective effect of P53 against APAP-induced hepatocytes injury was Sirt6-dependent. The Sirt6 mRNA was significantly down-regulated in P53 -/- mice. P53 activated the transcriptional activity of SIRT6 and exerted interaction with SIRT6. Our results demonstrate that SIRT6 protects against APAP hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation, and provide new insights in the function of SIRT6 as a crucial docking molecule linking P53 and NRF2.

Transcriptomic analysis across liver diseases reveals disease-modulating activation of constitutive androstane receptor in cholestasis.

BACKGROUND & AIMS: Liver diseases are caused by many factors, such as genetics, nutrition, and viruses. Therefore, it is important to delineate transcriptomic changes that occur in various liver diseases. METHODS: We performed high-throughput sequencing of mouse livers with diverse types of injuries, including cholestasis, diet-induced steatosis, and partial hepatectomy. Comparative analysis of liver transcriptome from mice and human samples of viral infections (HBV and HCV), alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH), and biliary atresia revealed distinct and overlapping gene profiles associated with liver diseases. We hypothesised that discrete molecular signatures could be utilised to assess therapeutic outcomes. We focused on cholestasis to test and validate the hypothesis using pharmacological approaches. RESULTS: Here, we report significant overlap in the expression of inflammatory and proliferation-related genes across liver diseases. However, cholestatic livers were unique and displayed robust induction of genes involved in drug metabolism. Consistently, we found that constitutive androstane receptor (CAR) activation is crucial for the induction of the drug metabolic gene programme in cholestasis. When challenged, cholestatic mice were protected against zoxazolamine-induced paralysis and acetaminophen-induced hepatotoxicity. These protective effects were diminished upon inhibition of CAR activity. Further, drug metabolic genes were also induced in the livers from a subset of biliary atresia patients, but not in HBV and HCV infections, AH, or NASH. We also found a higher expression of CYP2B6, a CAR target, in the livers of biliary atresia patients, underscoring the clinical importance of our findings. CONCLUSIONS: Comparative transcriptome analysis of different liver disorders revealed specific induction of phase I and II metabolic genes in cholestasis. Our results demonstrate that CAR activation may lead to variations in drug metabolism and clinical outcomes in biliary atresia. LAY SUMMARY: Transcriptomic analysis of diverse liver diseases revealed alterations in common and distinct pathways. Specifically, in cholestasis, we found that detoxification genes and their activity are increased. Thus, cholestatic patients may have an unintended consequence on drug metabolism and not only have a beneficial effect against liver toxicity, but also may require adjustments to their therapeutic dosage.

LC-MS analyses of N-acetyl-p-benzoquinone imine-adducts of glutathione, cysteine, N-acetylcysteine, and albumin in a plasma sample: A case study from a patient with a rare acetaminophen-induced acute swelling rash.

Acetaminophen (Paracetamol, APAP) has been widely used for many decades as an analgesic and antipyretic agent but APAP overdose often causes acute adverse reactions, particularly liver damage. The metabolically oxidized form of APAP, N-acetyl-p-benzoquinone imine (NAPQI), is chemically reactive and binds covalently to proteins. Therefore, NAPQI is believed to be the key metabolite that causes hepatotoxicity, especially under conditions of glutathione depletion. Other APAP-induced adverse reactions, such as skin damage, are rare and remain poorly studied. Here, we report a case study of a male patient who presented with an acute swelling skin rash (without hepatotoxicity) caused by therapeutic doses of APAP. Plasma samples were collected at 17 hr after dosing (during the manifestation of symptoms) and at one month (after recovery) and were subjected to LC-MS analysis of NAPQI-adducts. A significant concentration of NAPQI-cysteine adduct (33 pmol/mL) was found together with low concentrations of NAPQI-N-acetylcysteine adduct (2.0 pmol/mL) and NAPQI-glutathione adduct (0.13 pmol/mL). However, the NAPQI-albumin adduct was below the detection limit (below 0.001% modification on albumin) despite a previous report of high concentrations of NAPQI-albumin adduct following acute liver injury. Therefore, the observed APAP-induced skin damage may have had a different cause from APAP-induced liver injury.

Safety and Efficacy of the SNAP 12-hour Acetylcysteine Regimen for the Treatment of Paracetamol Overdose.

BACKGROUND: Acetylcysteine (NAC) is effective at preventing liver injury after paracetamol overdose. The Scottish and Newcastle Anti-emetic Pre-treatment for Paracetamol Poisoning (SNAP) Study demonstrated that a 12 h NAC regimen was associated with fewer adverse drug reactions compared with the standard 21 h regimen. Here, we describe the clinical effectiveness of the SNAP NAC regimen. METHODS: The SNAP regimen, consisting of intravenous NAC 100 mg/kg over 2 h then 200 mg/kg over 10 h, was introduced to treat all paracetamol overdose patients at the Royal Infirmary of Edinburgh, the Royal Victoria Infirmary, Newcastle and St Thomas' Hospital, London. Patient data were prospectively and systematically collected before and after the change in treatment (total patients N = 3340, 21 h N = 1488, SNAP N = 1852). Health record linkage was used to determine patient outcome after hospital discharge. FINDINGS: There was no difference in liver injury or liver synthetic dysfunction between regimens. Hepatotoxicity (peak ALT > 1000 U/L) occurred in 64 (4.3%) and 67 (3.6%) patients, respectively, in the 21 h and SNAP groups (absolute difference - 0.7%, 95% CI - 2.1 to 0.6). Multivariable logistic regression did not identify treatment regimen as an outcome-associated factor. No patients were readmitted to hospital with, or died from, liver failure within 30 days of discharge. Anti-histamine treatment (for NAC anaphylactoid drug reactions) was prescribed for 163 (11.0%) patients with the 21 h regimen and 37 (2.0%) patients with the SNAP regimen (absolute difference 9.0% (95% CI 7.3 to 10.7)). INTERPRETATION: In clinical use the SNAP regimen has similar efficacy as standard therapy for preventing liver injury and produces fewer adverse reactions.

Curcumin protects rat liver from streptozotocin-induced diabetic pathophysiology by counteracting reactive oxygen species and inhibiting the activation of p53 and MAPKs mediated stress response pathways.

Curcumin (CUR) is a highly pleiotropic molecule and possesses anti-inflammatory, hypoglycemic, antioxidative, wound-healing and antimicrobial activities. The present study was carried out to investigate whether CUR plays any beneficial role in streptozotocin (STZ) induced hepatic pathophysiology in diabetic rats. STZ exposure increased hepatic damage associated serum markers (ALT, ALP and LDH) as well as NO production in the liver tissue. Moreover, the same exposure enhanced ROS generation and lipid peroxidation; reduced GSH levels and antioxidant enzyme activities. Hyperglycemia induced hepatic pathophysiology also activated stress response pathways (involving phosphorylation of p38, ERK1/2 MAPKs and p53) and reduced mitochondrial membrane potential which in turn led to cellular apoptosis as evidenced from increased hepatic DNA fragmentation as well as FACS analysis. However, treatment with CUR effectively counteracts diabetes-induced, oxidative stress mediated hepatic damage and could act as a therapeutic in lessening liver dysfunction in diabetic subjects.

Fas receptor-deficient lpr mice are protected against acetaminophen hepatotoxicity due to higher glutathione synthesis and enhanced detoxification of oxidant stress.

UNLABELLED: Acetaminophen (APAP) overdose is a classical model of hepatocellular necrosis; however, the involvement of the Fas receptor in the pathophysiology remains controversial. Fas receptor-deficient (lpr) and C57BL/6 mice were treated with APAP to compare the mechanisms of hepatotoxicity. Lpr mice were partially protected against APAP hepatotoxicity as indicated by reduced plasma ALT and GDH levels and liver necrosis. Hepatic Cyp2e1 protein, adduct formation and hepatic glutathione (GSH) depletion were similar, demonstrating equivalent reactive metabolite generation. There was no difference in cytokine formation or hepatic neutrophil recruitment. Interestingly, hepatic GSH recovered faster in lpr mice than in wild type animals resulting in enhanced detoxification of reactive oxygen species. Driving the increased GSH levels, mRNA induction and protein expression of glutamate-cysteine ligase (gclc) were higher in lpr mice. Inducible nitric oxide synthase (iNOS) mRNA and protein levels at 6h were significantly lower in lpr mice, which correlated with reduced nitrotyrosine staining. Heat shock protein 70 (Hsp70) mRNA levels were substantially higher in lpr mice after APAP. CONCLUSION: Our data suggest that the faster recovery of hepatic GSH levels during oxidant stress and peroxynitrite formation, reduced iNOS expression and enhanced induction of Hsp70 attenuated the susceptibility to APAP-induced cell death in lpr mice.

Metabolomics evaluation of the effects of green tea extract on acetaminophen-induced hepatotoxicity in mice.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification.

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage.

An Update on Drug-induced Liver Injury.

Idiosyncratic drug-induced liver injury (DILI) is an important cause of morbidity and mortality following drugs taken in therapeutic doses. Hepatotoxicity is a leading cause of attrition in drug development, or withdrawal or restricted use after marketing. No age is exempt although adults and the elderly are at increased risk. DILI spans the entire spectrum ranging from asymptomatic elevation in transaminases to severe disease such as acute hepatitis leading to acute liver failure. The liver specific Roussel Uclaf Causality Assessment Method is the most validated and extensively used for determining the likelihood that an implicated drug caused DILI. Asymptomatic elevation in liver tests must be differentiated from adaptation. Drugs producing DILI have a signature pattern although no single pattern is characteristic. Antimicrobial and central nervous system agents including antiepileptic drugs are the leading causes of DILI worldwide. In the absence of a diagnostic test or a biomarker, the diagnosis rests on the evidence of absence of competing causes such as acute viral hepatitis, autoimmune hepatitis and others. Recent studies show that antituberculosis drugs given for active or latent disease are still a major cause of drug-induced liver injury in India and the West respectively. Presence of jaundice signifies a severe disease and entails a worse outcome. The pathogenesis is unclear and is due to a mix of host, drug metabolite and environmental factors. Research has evolved from incriminating candidate genes to genome wide analysis studies. Immediate cessation of the drug is key to prevent or minimize progressive damage. Treatment is largely supportive. N-acetylcysteine is the antidote for paracetamol toxicity. Carnitine has been tried in valproate injury whereas steroids and ursodeoxycholic acid may be used in DILI associated with hypersensitivity or cholestatic features respectively. This article provides an overview of the epidemiology, the patterns of hepatotoxicity, the pathogenesis and associated risk factors besides its clinical management.