APAP Metabolism Research Articles

Application of Interleukin-22 Mediates Protection in Experimental Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation invivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

Quantitative Analysis of Acetaminophen and its Primary Metabolites in Small Plasma Volumes by Liquid Chromatography-Tandem Mass Spectrometry

A fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous determination of acetaminophen (APAP) and its glucuronide and sulfate metabolites (APAP-GLU and APAP-SUL) in small plasma volumes. This method included a simple step of sample preparation and a chromatographic separation on an LC-MS-MS system equipped with an electrospray ionization source and a tandem triple quadrupole mass spectrometer in multiple reaction monitoring mode. The analytes and internal standard, APAP deuterated analog, were separated on a C18 column (3.0 µm, 2.1 × 100 mm), using aqueous 1% formic acid and methanol (80:20, v/v) as the mobile phase. The LC-MS-MS method was validated for accuracy, precision, linearity, extraction efficiency, process efficiency and matrix effect. Calibration curves were obtained by fortifying drug-free plasma and ranges of linearity were set between 0.25-20 mg/L. The mean correlation coefficients, r², were >0.99 for APAP and its metabolites. The inter-day and intra-day precision values were less than 11.75 and 13.03%, respectively, at the lower limit of quantification concentration. The usability of the method was demonstrated by studying APAP metabolism in C57BL/6J wild-type and obese ob/ob female mice, in which only small plasma volumes were available. The results showed that APAP glucuronidation was enhanced in obese mice, suggesting that changes in APAP metabolism could modify its toxicity in obesity and related fatty liver disease.

Reply:

We appreciate the comments of Drs. Craig and Simpson and Drs. Mullins and Schwartz, as well as the challenge they raise to make Acetaminophen-Induced Liver Damage (MALD) more believable, comprehensible, and usable. Acetaminophen (APAP) toxicity is indeed a complex process involving multiple steps. Although the underlying mathematics is more involved than the statistical regression models with which physicians are more familiar, MALD could easily be reduced to a web-based or hand-held format for bedside use. The mathematical model accurately fits patient data from the University of Utah. If the modeling holds up in a prospective cohort, we will certainly convert MALD to a more user-friendly format. The complexity of APAP metabolism and toxicity required us to consider the underlying mechanisms that contribute to end-stage organ damage. The relative values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and international normalized ratio (INR) are key to understanding the course of injury and, of course, are central to the model. Because of differences in decay rates, AST and ALT provide complementary information about liver damage and both should always be measured in patients with acute liver injury. Different models have different purposes. The Rumack-Matthews nomogram aids in determining the need for early N-acetylcysteine (N-Ac) treatment by relating plasma APAP level to time of overdose, as best estimated from patient history. MALD provides complementary information about the need for eventual transplant by estimating, from measured patient laboratory values, the size of overdose and elapsed time since overdose. We agree with Drs. Craig and Simpson that sequential organ failure assessment (SOFA) is a highly predictive tool, but SOFA relies on existing organ dysfunction, and its value is best realized in the context of time from overdose. Progressive organ failure is a reliable marker of poor outcome, but perhaps too late to help the patient. Our goal is early prognostication, not confirmation of events that have already occurred. We all face the same dilemma: We wish to transplant only those patients who will die or suffer irreparable organ damage without a transplant, and we want to give ourselves maximum time to procure a suitable organ when necessary. By using only data obtained on admission, MALD serves as the earliest warning of need for transplant. Figure 3 shows how closely the model predicts actual enzyme curves after admission, indicating that data collected after treatment with N-AC provides little, if any, additional information regarding timing and size of overdose. Drs. Mullins and Schwartz raise a number of interesting points, but have misinterpreted some of our statements. First, we state that AST is approximately twice ALT at peak, not at baseline, or in patients without acute liver injury. Second, we never discourage anyone from using N-Ac after 24 hours. Survival depends on physiological response to injury even after N-Ac, as modeled by MALD. Third, the fraction of APAP converted to N-acetyl-p-benzoquinone-imide is one of the patient-specific parameters that has no effect on the predictions of outcome because it merely rescales the lethal dose. We disagree on the issue of arbitrary parameters—values were carefully chosen and referenced (where references existed). We also did not state that MALD was superior to King's College Criteria (KCC). We said it compared favorably and should be tested prospectively. We regret that data limitations prevented us from using the complete KCC score—this deficit will be corrected in a prospective study that compares as many methods as possible. Like many royalists, Mullins and Schwartz vow fealty to a king who they themselves have nearly decapitated (e.g., by reducing the INR criterion in KCC by 70%). We have no doubt that MALD requires testing and refinement, but we do not understand the objection to further research on a new, promising approach to a problem. The spirit of science lies with further investigation, rather than with loud huzzahs for the past. Christopher H. Remien*, Frederick R. Adler* , Terry D. Box , Lindsey Waddoups , Norman L. Sussman§, * Department of Mathematics, University of Utah, Salt Lake City, UT, Department of Biology, University of Utah, Salt Lake City, UT, Department of Gastroenterology, University of Utah, Salt Lake City, UT, § Department of Surgery, Baylor College of Medicine, Houston, TX.

Vα14iNKT cell deficiency prevents acetaminophen-induced acute liver failure by enhancing hepatic glutathione and altering APAP metabolism

Acetaminophen (APAP) overdose is widely regarded as a major cause of acute liver failure in the United States. Intentional or accidental overdose of APAP in man or rodent elicits direct hepatocellular injury that is accompanied by hepatic depletion of the antioxidant, glutathione (GSH). In recent years, the innate immune response has also been shown to promote the development of APAP hepatotoxicity via indirect liver damage. In the present study, we demonstrate that Jα18−/− mice, which are selectively deficient in the innate immune T cell, Vα14iNKT cells, were resistant to APAP hepatotoxicity relative to WT mice as reflected by biochemical and histological liver injury markers. In parallel, improvement in the biochemical and histological parameters of liver injury in Jα18−/− mice was associated with a significant increase in hepatic levels of GSH, which detoxified APAP metabolites to attenuate hepatic oxidative stress, liver injury and necrosis. Notably, the protective effect of hepatic GSH during Vα14iNKT cells deficiency was demonstrated by its depletion in Jα18−/− mice using dl-buthionine-[S,R]-sulfoximine which exacerbated hepatic oxidative and nitrosative stress as well as liver necrosis and caused mice mortality. Extraordinarily, APAP metabolism in Jα18−/− mice was altered in favor of hepatic GSH conjugates and decreased glucuronide conjugates. In summary, we reveal a novel finding establishing a unique association between hepatic innate immunity and GSH levels in altering APAP metabolism to suppress liver injury and necrosis during Vα14iNKT cells deficiency in Jα18−/− mice.

Acetaminophen protein adduct formation following low-dose acetaminophen exposure: comparison of immediate-release vs extended-release formulations

Acetaminophen (APAP) protein adducts are a biomarker of APAP metabolism, reflecting oxidation of APAP and generation of the reactive metabolite N-acetyl-p-benzoquinone imine. High levels of adducts correspond to liver toxicity in patients with APAP-related acute liver failure. Adduct formation following low-dose exposure to APAP has not been well studied. APAP protein adducts were measured in blood samples collected from fasted individuals who participated in a crossover study of APAP (80 mg/kg) comparing extended release (ER) and immediate release (IR) formulations. Adducts were quantified in all postdose blood samples using a validated high-performance liquid chromatography electrochemical detection (HPLC-EC) assay. Comparison of pharmacokinetic parameters for adducts did not reveal significant differences between ER and IR formulations, with one exception. Formation rates for adducts were faster for IR than the ER formulation (0.420 ± 0.157 vs. 0.203 ± 0.080 1/h), respectively. Maximum plasma concentrations (Cmax) of adducts for IR and ER were 0.108 (±0.020) and 0.100 (±0.028) nmol/ml serum, respectively, and were two orders of magnitude lower than adduct levels previously reported in adults with acute liver failure secondary to APAP. APAP protein adducts are rapidly formed following nontoxic ingestion of APAP at levels significantly lower than those associated with acute liver failure.

Deficiency of Interleukin-15 Enhances Susceptibility to Acetaminophen-Induced Liver Injury in Mice

Hepatocytes have a direct necrotic role in acetaminophen (APAP)-induced liver injury (AILI), prolonged secondary inflammatory response through innate immune cells and cytokines also significantly contributes to APAP hepatotoxicity. Interleukin 15 (IL-15), a multifunction cytokine, regulates the adaptive immune system and influences development and function of innate immune cells. To better understand the role of IL-15 in liver injury, we treated wild-type (WT) and IL-15-knockout (Il15−/−) mice with a hepatotoxic dose of APAP to induce AILI and evaluated animal survival, liver damage, APAP metabolism in livers and the inflammatory response. Production of pro-inflammatory cytokines/chemokines was greater in Il15−/− than WT mice. Subanalysis of hepatic infiltrated monocytes revealed greater neutrophil influx, along with greater hepatic induction of inducible nitric oxide synthase (iNOS), in Il15−/− than WT mice. In addition, the level of hepatic hemeoxygenase 1 (HO-1) was partially suppressed in Il15−/− mice, but not in WT mice. Interestingly, elimination of Kupffer cells and neutrophils did not alter the vulnerability to excess APAP in Il15−/− mice. However, injection of galactosamine, a hepatic transcription inhibitor, significantly reduced the increased APAP sensitivity in Il15−/− mice but had minor effect on WT mice. We demonstrated that deficiency of IL-15 increased mouse susceptibility to AILI. Moreover, Kupffer cell might affect APAP hepatotoxicity through IL-15.

Acetaminophen-induced liver injury in rats and mice: Comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity.

AMAP, the alleged non-toxic isomer of acetaminophen, is toxic in rat and human liver

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

Evaluation of pharmacokinetic differences of acetaminophen in pseudo germ‐free rats

To evaluate the metabolic interaction between host and gut microflora on drug metabolism, pseudo germ-free rats were prepared with an antibiotics cocktail to change their gut conditions. The usefulness of the pseudo germ-free model was evaluated for observing the DMPK of acetaminophen (APAP). Pseudo germ-free rats were prepared by orally administering antibiotic cocktails consisting of bacitracin, streptomycin and neomycin, and then APAP was orally administered to control and pseudo germ-free rats. The plasma concentration of APAP and its six metabolites were quantified using a validated LC-MS/MS method. A non-compartment model estimated the pharmacokinetic parameters of APAP and its metabolites, and the ratios of the area under curve (AUC; AUC(metabolite) /AUC(APAP) ) were also observed to evaluate the change of APAP metabolism. The AUCs of APAP and APAP-Glth (glutathione) were higher and the AUC(APAP-Sul) /AUC(APAP) (metabolic efficiency of sulfate conjugation) was lower in pseudo germ-free rats than those in the control rats. The decrease in metabolic efficiency of sulphate conjugation could result from the reduction of the sulphate supply, causing an increase of the AUC of APAP and APAP-Glth. The activities of gut microflora can affect the state of hepatic sulphate for drug conjugation, indirectly leading to characteristic APAP metabolism. These results indicate that gut microflora may play an important role in the pharmacokinetics and metabolism of APAP. Thus, the metabolic interaction between host and gut microflora should be considered upon drug administration and pseudo germ-free rats prepared in the present study can be competent for investigating the metabolic interaction between host and gut microflora on drug metabolism.

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug-drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C(18) column and quantitated with a 16 min run in a triple-quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC-MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats.

Korean red ginseng prevents APAP‐induced hepatotoxicity and mortality through metabolic regulation: The role of ginsenoside Rg3, a protopanaxadiol

Inappropriate use of acetaminophen (APAP) can lead to significant morbidity and mortality secondary to hepatic necrosis. We determined the beneficial effect and molecular mechanism of Korean red ginseng (KRG) on the APAP‐mediated hepatotoxicity and identified a major component of KRG for protection. Pretreatment with high doses of KRG for 1 week significantly reduced mortality at the LD50 of APAP. APAP increased AST and ALT activities, which was abrogated by low doses of KRG for 5 weeks. KRG also delayed APAP metabolism in plasma through CYP2E1 inhibition. These protective effects of KRG were consistent with the results from histopathological examinations. KRG induced antioxidant/phase 2 gene battery in rat liver and H4IIE. Exposure of KRG increased the nuclear Nrf2 and C/EBPβ, which are important transcriptional factors for those genes including GSTA2. These effects were downstream of multiple cellular signaling, including PI3K, JNK or PKA. Rg3 but not Rb1, Rc and Rg1 significantly increased those gene expressions. Rg3 resulted in the transcriptional activation of GSTA2 through Nrf2 and C/EBP downstream of the cellular signaling. These results demonstrate that KRG is efficacious in protection from APAP‐induced hepatotoxicity and mortality and that Rg3 is a major component of KRG for the hepatoprotection, implying that Rg3 should be considered to be a potential hepatoprotective agent.

Role of Galectin-3 in Acetaminophen-Induced Hepatotoxicity and Inflammatory Mediator Production

Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and inflammatory mediator production. In the present studies, we analyzed the role of Gal-3 in liver inflammation and injury induced by acetaminophen (APAP). Treatment of wild-type (WT) mice with APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was associated with increased hepatic expression of Gal-3 messenger RNA and protein. Immunohistochemical analysis showed that Gal-3 was predominantly expressed by mononuclear cells infiltrating into necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-3-deficient mice. This was most pronounced at 48-72 h post-APAP and correlated with decreases in APAP-induced expression of 24p3, a marker of inflammation and oxidative stress. These effects were not due to alterations in APAP metabolism or hepatic glutathione levels. The proinflammatory proteins, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and MIP-3α, as well as the Gal-3 receptor (CD98), were upregulated in livers of WT mice after APAP intoxication. Loss of Gal-3 resulted in a significant reduction in expression of iNOS, MMP-9, MIP-3α, and CD98, with no effects on IL-1β. Whereas APAP-induced increases in MIP-2 were augmented at 6 h in Gal-3(-/-) mice when compared with WT mice, at 48 and 72 h, they were suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also upregulated after APAP, a response dependent on Gal-3. Moreover, exaggerated APAP hepatotoxicity in mice lacking TNFR1 was associated with increased Gal-3 expression. These data demonstrate that Gal-3 is important in promoting inflammation and injury in the liver following APAP intoxication.

The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation

Acetaminophen (APAP) overdose is the predominant cause of acute liver failure in the United States. Toxicity begins with a reactive metabolite that binds to proteins. In rodents, this leads to mitochondrial dysfunction and nuclear DNA fragmentation, resulting in necrotic cell death. While APAP metabolism is similar in humans, the later events resulting in toxicity have not been investigated in patients. In this study, levels of biomarkers of mitochondrial damage (glutamate dehydrogenase [GDH] and mitochondrial DNA [mtDNA]) and nuclear DNA fragments were measured in plasma from APAP-overdose patients. Overdose patients with no or minimal hepatic injury who had normal liver function tests (LTs) (referred to herein as the normal LT group) and healthy volunteers served as controls. Peak GDH activity and mtDNA concentration were increased in plasma from patients with abnormal LT. Peak nuclear DNA fragmentation in the abnormal LT cohort was also increased over that of controls. Parallel studies in mice revealed that these plasma biomarkers correlated well with tissue injury. Caspase-3 activity and cleaved caspase-3 were not detectable in plasma from overdose patients or mice, but were elevated after TNF-induced apoptosis, indicating that APAP overdose does not cause apoptosis. Thus, our results suggest that mitochondrial damage and nuclear DNA fragmentation are likely to be critical events in APAP hepatotoxicity in humans, resulting in necrotic cell death.

Polyinosinic-polycytidylic acid suppresses acetaminophen-induced hepatotoxicity independent of type I interferons and toll-like receptor 3

Viral infections are often linked to altered drug metabolism in patients; however, the underlying molecular mechanisms remain unclear. Here we describe a mechanism by which activation of antiviral responses by the synthetic double-stranded RNA ligand, polyinosinic-polycytidylic acid (polyI:C), leads to decreased acetaminophen (APAP) metabolism and hepatotoxicity. PolyI:C administration down-regulates expression of retinoic X receptor-α (RXRα) as well as its heterodimeric partner pregnane X receptor (PXR) in mice. This down-regulation results in suppression of downstream cytochrome P450 enzymes involved in conversion of APAP to its toxic metabolite. Although the effects of polyI:C on drug metabolism are often attributed to interferon production, we report that polyI:C can decrease APAP metabolism in the absence of the type I interferon receptor. Furthermore, we demonstrate that polyI:C can attenuate APAP metabolism through both its membrane-bound receptor, Toll-like receptor 3 (TLR3), as well as cytoplasmic receptors. This is the first study to illustrate that in vivo administration of polyI:C affects drug metabolism independent of type I interferon production or in the absence of TLR3 through crosstalk between nuclear receptors and antiviral responses.

Protective effects of goldenseal (Hydrastis canadensis L.) on acetaminophen-induced hepatotoxicity through inhibition of CYP2E1 in rats

Background:Goldenseal (Hydrastis canadensis L.) inhibits various cytochrome P450 (CYP) isoforms such as CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A in vitro. High doses of acetaminophen (APAP) generate the highly reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI), catalyzed mainly by CYP2E1. The aim of this study was to investigate the hepatoprotective effects of orally administrated goldenseal against APAP-induced acute liver failure (ALF) via inhibition of CYP2E1.Materials and Methods:Male Wistar rats were treated orally with goldenseal (300 and 1000 mg/kg) 2, 18, and 26 h before and 6 h after oral APAP (400 mg/kg) administration. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities as well as serum APAP concentration were evaluated.Results:Goldenseal extract inhibited CYP1A2, CYP2D6, CYP2E1, and CYP3A activity, and the inhibitory effect on CYP2E1 was the strongest (IC50 4.32 μg/mL). Treatment with goldenseal (300 mg/kg) significantly attenuated the APAP-induced increase in serum AST and ALT, and the hepatoprotective effect of goldenseal was stronger than that of silymarin (200 mg/kg). Moreover, serum APAP concentration was increased by goldenseal treatment, presumably as a result of the inhibitory effect of goldenseal on the metabolism of APAP to NAPQI.Conclusion:These results suggest that goldenseal ameliorates APAP-induced ALF and that this protection can likely be attributed to the inhibition of CYP2E1 activity, which generates the highly reactive intermediate of APAP.

Diet Restriction Inhibits Apoptosis and HMGB1 Oxidation and Promotes Inflammatory Cell Recruitment during Acetaminophen Hepatotoxicity

Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology.

Lipopolysaccharide binding protein inhibitory peptide protects against acetaminophen-induced hepatotoxicity

Acetaminophen (APAP)-induced liver injury remains the main cause of acute liver failure in the United States. Our previous work demonstrated that LPS binding protein (LBP) knockout mice are protected from APAP-induced hepatotoxicity. LBP is known to bind avidly to LPS, facilitating cellular activation. In this study, we sought to specifically inhibit the interaction between LBP and LPS to define the role of this interaction in APAP-induced liver injury. The peptide LBPK95A was able to inhibit LBP-mediated LPS activation of RAW 267.4 cells in a dose-dependent manner in vitro. In vivo, C57Bl/6 mice were treated with either LBPK95A or vehicle control concurrently with the administration of APAP (350 mg/kg). Mice treated with LBPK95A had significantly lower serum aspartate aminotransferase and alanine aminotransferase levels. Morphometric analysis of the liver tissue showed significantly less liver injury in mice treated with LBPK95A. To assess whether the LBPK95A altered glutathione depletion and APAP metabolism, we measured total glutathione levels in the liver after APAP. We found no difference in the glutathione levels and APAP-adduct formation between LBPK95A vs. vehicle control both at baseline and after APAP. In conclusion, our results support the hypothesis that LBP-induced liver injury after APAP is due to its ability to mediate activation by endogenous LPS. Our results suggest that blocking LBP-LPS interactions is a potential therapeutic avenue for the treatment of APAP-induced liver injury.

Role of caspase-1 and interleukin-1β in acetaminophen-induced hepatic inflammation and liver injury

Acetaminophen (APAP) overdose can result in serious liver injury and potentially death. Toxicity is dependent on metabolism of APAP to a reactive metabolite initiating a cascade of intracellular events resulting in hepatocellular necrosis. This early injury triggers a sterile inflammatory response with formation of cytokines and innate immune cell infiltration in the liver. Recently, IL-1β signaling has been implicated in the potentiation of APAP-induced liver injury. To test if IL-1β formation through caspase-1 is critical for the pathophysiology, C57Bl/6 mice were treated with the pan-caspase inhibitor Z-VD-fmk to block the inflammasome-mediated maturation of IL-1β during APAP overdose (300 mg/kg APAP). This intervention did not affect IL-1β gene transcription but prevented the increase in IL-1β plasma levels. However, APAP-induced liver injury and neutrophil infiltration were not affected. Similarly, liver injury and the hepatic neutrophilic inflammation were not attenuated in IL-1-receptor-1 deficient mice compared to wild-type animals. To evaluate the potential of IL-1β to increase injury, mice were given pharmacological doses of IL-1β after APAP overdose. Despite increased systemic activation of neutrophils and recruitment into the liver, there was no alteration in injury. We conclude that endogenous IL-1β formation after APAP overdose is insufficient to activate and recruit neutrophils into the liver or cause liver injury. Even high pharmacological doses of IL-1β, which induce hepatic neutrophil accumulation and activation, do not enhance APAP-induced liver injury. Thus, IL-1 signaling is irrelevant for APAP hepatotoxicity. The inflammatory cascade is a less important therapeutic target than intracellular signaling pathways to attenuate APAP-induced liver injury.

Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice

Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.

Rifampicin-Activated Human Pregnane X Receptor and CYP3A4 Induction Enhance Acetaminophen-Induced Toxicity

Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.