Formation Of APAP-protein Adducts Research Articles

Lack of mitochondrial Cyp2E1 drives acetaminophen-induced ER stress-mediated apoptosis in mouse and human kidneys: Inhibition by 4-methylpyrazole but not N-acetylcysteine

Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury.

The mitochondrial calcium uniporter mediates mitochondrial Fe2+ uptake and hepatotoxicity after acetaminophen

Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.

Sappanone A ameliorates acetaminophen-induced acute liver injury in mice

Sappanone A (SA), a homoisoflavonoid compound extracted from the heartwood of Caesalpinia sappan Linn., exerts anti-inflammatory and antioxidant activities. However, the effects of SA on acetaminophen (APAP) overdose-induced acute liver injury (ALI) have not been determined yet. This study aims to explore the protective effects of SA and the potential mechanisms of action. Mice were pretreated with SA (25, 50, and 100 mg/kg) by intraperitoneal (i.p.) injection for seven days prior to APAP (300 mg/kg, i.p.) administration. At 12 h after APAP injection, serum and liver samples were collected. Primary murine hepatocytes were used to investigate the underlying mechanisms. SA pretreatment dose-dependently attenuated APAP-induced ALI, as validated by reduced serum alanine/aspartate aminotransferase levels, histopathologic lesions, and oxidative stress. Consistently, pretreatment with SA reduced the formation of APAP protein adducts in damaged livers of mice. Mechanistically, SA could facilitate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and thus promote cellular glutathione (GSH) synthesis. The hepatoprotective outcomes provided by SA were significantly abolished by treatment with ML385, a Nrf2 inhibitor. Besides, anti-inflammatory property of SA reduced inflammatory reaction in injured livers of mice. Of note, posttreatment with SA reveals significant therapeutic influences against APAP-induced ALI in mice. Collectively, our findings demonstrated that pretreated-SA ameliorated APAP-mediated ALI in mice, at least in part, by reducing the generation of APAP protein adducts via Nrf2-enhanced GSH synthesis, and by diminishing hepatic inflammation. Therefore, SA could be a potential hepatoprotective agent for treating ALI.

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

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

Dried blood microsamples: Suitable as an alternative matrix for the quantification of paracetamol-protein adducts?

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic drug worldwide. However, patients in several specific populations can have an increased exposure to toxic APAP metabolites. Therefore, APAP-protein adducts have been proposed as an alternative marker for the assessment of APAP intoxications and as an effective tool to study and steer APAP treatment in patients with an increased risk of APAP-induced liver damage. These adducts have been determined in plasma or serum as a matrix. Blood microsampling allows the determination of a variety of analytes, including protein adducts, in a drop of blood, facilitating convenient follow-up of patients in a home-sampling context, as well as repeated sampling of pediatric patients. We therefore evaluated the use of blood-based volumetric microsamples for the quantification of APAP-protein adducts. Quantitative methods for the determination of APAP-protein adducts in dried blood and dried plasma volumetric absorptive microsamples were developed and validated. Also a preliminary evaluation of pediatric patient dried blood microsamples was conducted.Method validation encompassed the evaluation of selectivity, carry over, calibration model, accuracy and precision, matrix effect, recovery and the effect of the hematocrit on the recovery, dilution integrity, and stability. All pre-set acceptance criteria were met, except for stability. Spiking of blank blood with APAP revealed a concentration-dependent ex vivo formation of APAP-protein adducts, resulting in a response for the measurand APAP-Cys, with an apparent role for the red blood cell fraction. Analysis of authentic samples, following intake of APAP at therapeutic dosing, revealed much higher APAP-Cys concentrations in dried blood vs. dried plasma samples, making interpretation of the results in the context of published intervals difficult. In addition, in contrast to what was observed during method validation, the data obtained for the patient samples showed a high and unacceptable variation.We conclude that, for a combination of reasons, dried blood is not a suitable matrix for the quantification of APAP-protein adducts via the measurement of the APAP-Cys digestion product. The collection of plasma or serum, either in the form of a liquid sample or a dried microsample for this purpose is advised.

Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes.

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.

Dual Role of Epidermal Growth Factor Receptor in Liver Injury and Regeneration after Acetaminophen Overdose in Mice.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Differential susceptibility to acetaminophen-induced liver injury in sub-strains of C57BL/6 mice: 6N versus 6J

Mouse models of acetaminophen (APAP) hepatotoxicity are considered relevant for the human pathophysiology. The C57BL/6 strain is most popular because it is the background strain of gene knock-out mice. However, conflicting results in the literature may have been caused by sub-strain mismatches, e.g. C57BL/6J and C57BL/6N. This study was initiated to determine the mechanism behind the sub-strain susceptibility to APAP toxicity. C57BL/6N and C57BL/6J mice were dosed with 200 mg/kg APAP and sacrificed at different time points. C57BL/6N mice developed significantly more liver injury as measured by plasma ALT activities and histology. Although there was no difference in glutathione depletion or cytochrome P450 activity between groups, C57BL/6N had a higher glutathione disulfide-to-glutathione ratio and more APAP protein adducts. C57BL/6N showed more mitochondrial translocation of phospho-JNK and BAX, and more release of mitochondrial intermembrane proteins apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), which caused more DNA fragmentation. The increased mitochondrial dysfunction was confirmed in vitro as C57BL/6N hepatocytes had a more precipitous drop in JC-1 fluorescence after APAP exposure. Conclusion: C57BL/6N mice are more susceptible to APAP-induced hepatotoxicity, likely due to increased formation of APAP-protein adducts and a subsequent enhancement of mitochondrial dysfunction associated with aggravated nuclear DNA fragmentation.

Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis. We found that APAP-AD were detected at 1h, peaked at approximately 2h, declined at 6h and almost full removed at 24h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury. Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Translational biomarkers of acetaminophen-induced acute liver injury.

Acetaminophen (APAP) is a commonly used analgesic drug that can cause liver injury, liver necrosis and liver failure. APAP-induced liver injury is associated with glutathione depletion, the formation of APAP protein adducts, the generation of reactive oxygen and nitrogen species and mitochondrial injury. The systems biology omics technologies (transcriptomics, proteomics and metabolomics) have been used to discover potential translational biomarkers of liver injury. The following review provides a summary of the systems biology discovery process, analytical validation of biomarkers and translation of omics biomarkers from the nonclinical to clinical setting in APAP-induced liver injury.

Agreement Between Caregiver Report and Hospital and School Records

S U N D A Y 539 High Dose Acetaminophen Fails to Promote Airway HyperReactivity Ex Vivo and Is Both Bronchoprotective and Bronchodilatory Richard Kurten, PhD, Sandra McCullough, BS, Stacie M. Jones, MD, Dean Roberts, PhD, Laura James, MD, Josh L. Kennedy, MD; University of Arkansas for Medical Sciences, Little Rock, AR, Arkansas Children’s Hospital Research Institute, Little Rock, AR, Slot 512-13, University of Arkansas for Medical Sciences, Little Rock, AR, University of Arkansas for Medical Sciences, Little Rock. RATIONALE: Epidemiologic studies demonstrate an association between acetaminophen (APAP) use and the development of asthma symptoms in children. No studies have examined potential relationship between APAP exposure andmechanisms for asthma exacerbations. Given established hepatotoxicity of APAP, we hypothesized that acute APAP exposure, APAP metabolism and APAP-protein adduct formation enhances bronchoconstrictor responses thereby causing airway hyperresponsiveness (AHR). METHODS: APAP metabolism and adduct formation in lung tissue was evaluated by immunocytochemistry after in vivo APAP exposure in mice and ex vivo exposure in human precision cut lung slices (PCLS). Airway bronchoconstriction and bronchodilation were measured in human PCLS airways by microscopy after acute exposure to APAP ex vivo. RESULTS: In mouse and human airways, exposure to APAP generated APAP protein adducts in airway epithelial cells, verifying drug metabolism. Neither mouse nor human airways were hyper-responsive to bronchoconstrictor agents after exposure to APAP: Carbachol-induced bronchoconstriction was reduced by 92% and 70%, respectively, in airways from mice treated in vivo with 200mg/kg APAP for 1 or 24 hours and 5mM APAP treatment ex vivoreduced bronchoconstriction in human PCLS airways by 42%. Treatment with APAP after bronchoconstriction with carbachol, histamine or IgE cross-linking resulted in dose-dependent bronchodilation (63-/+10% at 5mM) that was sustained for hours. APAP also led to bronchodilation of airways that were previously desensitized to bronchodilation by salmeterol. CONCLUSIONS: APAP is metabolized in airway epithelial cells both in vivo and ex vivo leading to protein-adduct formation. Acute exposure to APAP does not promote AHR, is protective against bronchoconstriction, and is mildly bronchodilatory.

Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Hsp72 overexpression protects from acetaminophen and DDC-associated liver injury but enhances protein aggregation

Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and possesses broad cytoprotective functions. However its hepatic function remains largely unknown. To study the importance of Hsp72 in the liver, we generated transgenic mice overexpressing Hsp72 under the control of a tissue-specific tetracycline-inducible system and crossed them with animals carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor (Hsp72-LAP mice). Acute liver injury was induced by a single intraperitoneal injection of acetaminophen (800 mg/kg). Long-term feeding (12 weeks) with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was used to induce Mallory-Denk-body (MDB) formation. Hsp72-LAP mice displayed doxycycline-regulated, robust Hsp72 overexpression in hepatocytes, but not in the other tissues or cell types tested. 18 hours after acetaminophen injection, significantly lower liver transaminase levels were noted in Hsp72-LAP mice compared to single transgenes (ALT: 933 vs. 1977, p < 0.05). No differences in acetaminophen metabolism were seen, but Hsp72 overexpression protected from formation of APAP protein adducts (p = 0.027). After DDC-feeding, Hsp72-LAP mice experienced a lower weight loss (p = 0.03) and lower ALT levels (650 vs. 875, p = 0.03). On the other hand, DDC-feeding resulted in a more abundant MDB formation in Hsp72-LAP mice vs. single transgenes. Conclusions: Our results suggest that Hsp72 overexpression protects from liver injury in two independent models but precipitates aggregate formation.

Increased susceptibility of natural killer T-cell-deficient mice to acetaminophen-induced liver injury

Acetaminophen (APAP) overdose causes severe, fulminant liver injury. The underlying mechanism of APAP-induced liver injury (AILI), studied by a murine model, displays similar characteristics of injury as those observed in patients. Previous studies suggest that aside from APAP-induced direct damage to hepatocytes, the hepatic innate immune system is activated and may contribute to the overall pathogenesis of AILI. The current study employed the use of two murine natural killer (NK) cells with T-cell receptor (NKT) cell knockout models (CD1d(-/-) and Jα18(-/-) ) to elucidate the specific role of NKT cells in AILI. Compared to wild-type (WT) mice, NKT cell-deficient mice were more susceptible to AILI, as indicated by higher serum alanine transaminase levels and mortality. Increased levels of cytochrome P450 2E1 (CYP2E1) protein expression and activities, which resulted in increased APAP protein adduct formation, were observed in livers of APAP-treated NKT cell-deficient mice, compared to WT mice. Compared to WT mice, starvation of NKT cell-deficient mice induced a higher increase of ketone bodies, which up-regulate CYP2E1 through protein stabilization. Our data revealed a novel role of NKT cells in regulating responses to starvation-induced metabolic stress. Elevated ketone body production in NKT cell-deficient mice resulted in increased CYP2E1-mediated APAP biotransformation and susceptibility to AILI.

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.

Pharmacokinetics of Acetaminophen-Protein Adducts in Adults with Acetaminophen Overdose and Acute Liver Failure

Acetaminophen (APAP)-induced liver toxicity occurs with formation of APAP-protein adducts. These adducts are formed by hepatic metabolism of APAP to N-acetyl-p-benzoquinone imine, which covalently binds to hepatic proteins as 3-(cystein-S-yl)-APAP adducts. Adducts are released into blood during hepatocyte lysis. We previously showed that adducts could be quantified by high-performance liquid chromatography with electrochemical detection following proteolytic hydrolysis, and that the concentration of adducts in serum of overdose patients correlated with toxicity. The following study examined the pharmacokinetic profile and clinical associations of adducts in 53 adults with acute APAP overdose resulting in acute liver failure. A population pharmacokinetic analysis using nonlinear mixed effects (statistical regression type) models was conducted; individual empiric Bayesian estimates were determined for the elimination rate constant and elimination half-life. Correlations between clinical and laboratory data were examined relative to adduct concentrations using nonparametric statistical approaches. Peak concentrations of APAP-protein adducts correlated with peak aminotransferase concentrations (r = 0.779) in adults with APAP-related acute liver failure. Adducts did not correlate with bilirubin, creatinine, and APAP concentration at admission, international normalized ratio for prothrombin time, or reported APAP dose. After N-acetylcysteine therapy, adducts exhibited first-order disappearance. The mean elimination rate constant and elimination half-life were 0.42 +/- 0.09 days(-1) and 1.72 +/- 0.34 days, respectively, and estimates from the population model were in strong agreement with these data. Adducts were detected in some patient samples 12 days post-ingestion. The persistence and specificity of APAP-protein adducts as correlates of toxicity support their use as specific biomarkers of APAP toxicity in patients with acute liver injury.

Mild Hypothermia Attenuates Liver Injury and Improves Survival in Mice With Acetaminophen Toxicity

Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice. Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5-37.5 degrees C) by external warming or were allowed to develop mild hypothermia (32.0-35.0 degrees C) after 2 hours from APAP administration. Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8-12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis. Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.

Macrophage migration inhibitory factor in drug-induced liver injury: a role in susceptibility and stress responsiveness

Idiosyncratic drug-induced hepatitis may depend upon many factors including a balance between pro- and anti-inflammatory mediator production levels. Using a guinea pig model of liver injury induced by bioactivation of the anesthetic drug, halothane, we found that toxicity was commensurate with an increase in serum macrophage migration inhibitory factor (MIF), a pro-inflammatory signal and counter-regulator of glucocorticoids, but only in susceptible animals. The pathogenic role of MIF was further investigated using a murine model in which liver injury was induced by the reactive metabolite of another drug, acetaminophen (APAP). MIF leakage from the liver into the sera preceded peak increases in toxicity following APAP administration. MIF null (−/−) mice were significantly less susceptible to this toxicity at 8 h. At 48 h following a 300 mg/kg dose, complete lethality was observed in wild-type mice, while 46% survival was noted in MIF−/− mice. The decreased hepatic injury in MIF−/− mice correlated with a reduction in mRNA levels of interferon-γ and a significant increase in heat shock protein expression, but was unrelated to the APAP-protein adduct formation in the liver. These findings support MIF as a critical pro-toxicant signal in drug-induced liver injury with potentially important and novel effects on heat shock protein responsiveness.

Protection against acetaminophen-induced liver injury and lethality by interleukin 10: Role of inducible nitric oxide synthase

Mechanistic study of idiosyncratic drug-induced hepatitis (DIH) continues to be a challenging problem because of the lack of animal models. The inability to produce this type of hepatotoxicity in animals, and its relative rarity in humans, may be linked to the production of anti-inflammatory factors that prevent drug-protein adducts from causing liver injury by immune and nonimmune mechanisms. We tested this hypothesis by using a model of acetaminophen (APAP)-induced liver injury in mice. After APAP treatment, a significant increase was observed in serum levels of interleukin (IL)-4, IL-10, and IL-13, cytokines that regulate inflammatory mediator production and cell-mediated autoimmunity. When IL-10 knockout (KO) mice were treated with APAP, most of these mice died within 24 to 48 hours from liver injury. This increased susceptibility to APAP-induced liver injury appeared to correlate with an elevated expression of liver proinflammatory cytokines, tumor necrosis factor (TNF)-α, and IL-1, as well as inducible nitric oxide synthase (iNOS). In this regard, mice lacking both IL-10 and iNOS genes were protected from APAP-induced liver injury and lethality when compared with IL-10 KO mice. All strains, including wild-type animals, generated similar amounts of liver APAP-protein adducts, indicating that the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity was not caused by an enhanced formation of APAP-protein adducts. In conclusion, these findings suggest that an important feature of the normal response to drug-induced liver injury may be the increased expression of anti-inflammatory factors such as IL-10. Certain polymorphisms of these factors may have a role in determining the susceptibility of individuals to idiosyncratic DIH. (HEPATOLOGY .)