Indocyanine green clearance varies as a function of N-acetylcysteine treatment in a murine model of acetaminophen toxicity

Standard assays to assess acetaminophen (APAP) toxicity in animal models include determination of ALT (alanine aminotransferase) levels and examination of histopathology of liver sections. However, these assays do not reflect the functional capacity of the injured liver. To examine a functional marker of liver injury, the pharmacokinetics of indocyanine green (ICG) were examined in mice treated with APAP, saline, or APAP followed by N-acetylcysteine (NAC) treatment.Male B6C3F1 mice were administered APAP (200 mg/kg IP) or saline. Two additional groups of mice received APAP followed by NAC at 1 or 4 h after APAP. At 24 h, mice were injected with ICG (10 mg/kg IV) and serial blood samples (0, 2, 10, 30, 50 and 75 min) were obtained for determination of serum ICG concentrations and ALT. Mouse livers were removed for measurement of APAP protein adducts and examination of histopathology. Toxicity (ALT values and histology) was significantly increased above saline treated mice in the APAP and APAP/NAC 4 h mice. Mice treated with APAP/NAC 1 h had complete protection from toxicity. APAP protein adducts were increased in all APAP treated groups and were highest in the APAP/NAC 1 h group. Pharmacokinetic analysis of ICG demonstrated that the total body clearance (Cl T) of ICG was significantly decreased and the mean residence time (MRT) was significantly increased in the APAP mice compared to the saline mice. Mice treated with NAC at 1 h had Cl T and MRT values similar to those of saline treated mice. Conversely, mice that received NAC at 4 h had a similar ICG pharmacokinetic profile to that of the APAP only mice. Prompt treatment with NAC prevented loss of functional activity while late treatment with NAC offered no improvement in ICG clearance at 24 h. ICG clearance in mice with APAP toxicity can be utilized in future studies testing the effects of novel treatments for APAP toxicity

Protein tyrosine phosphatase 1B modulates GSK3b/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAPinduced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B-/- hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)b/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B-/- cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B-/- mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAPinduced liver failure. © 2013 Macmillan Publishers Limited. All rights reserved.We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigaciones Biomédicasen Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Spain) (to AMV); SAF2012-38048 (MINECO, Spain) (to JM-P); PI09/0185 and Centro de Investigacion Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD, ISCIII, Spain) (to JM); AGL2010-17579 (MINECO, Spain) (to LG); and SAF2010-17822 (MINECO, Spain) (to AC).Peer Reviewe

GC-MS Determination of Amphetamine and Methamphetamine in Human Urine for 12 Hours Following Oral Administration of Dextro-Methamphetamine: Lack of Evidence Supporting the Established Forensic Guidelines for Methamphetamine Confirmation

Ten human volunteers, naive to amphetamines and divided into two groups of five each, were given an oral dose of 30 mg/70 kg D-methamphetamine in one of two different paradigms: the initial dose at 0930 h or the initial dose at 2130 h. One week later, each subject was crossed over with regard to time but given the same dose. A total of 214 urine specimens were collected either prior to dosing or at each micturition for a 12-h period post dose. Specimens were analyzed on a blind basis for methamphetamine and one of its metabolites, amphetamine, by gas chromatography-mass spectrometry (GC-MS) using coinjection of extracted sample and pentafluoropropionic anhydride and selected-ion monitoring. Approximately 20% of the D-methamphetamine was recovered unchanged from the urine specimens, and 2% was recovered as amphetamine. The mean urine methamphetamine concentration in both groups reached a maximum within 4-6 h and declined thereafter. A residual amount of methamphetamine was found in some predose specimens at the crossover evaluation, reflecting that methamphetamine may be detected in urine for up to 7 days. The amphetamine concentration reached a plateau by 4-6 h. This observation coupled with the finding that all subjects excreted approximately 2% of the methamphetamine dose as amphetamine suggested a saturable process for its biotransformation. Concentrations of both methamphetamine and amphetamine tended to be higher, but were not significantly different, for night administration. Methamphetamine concentrations were consistently greater than the 500-ng/mL cutoff in most post-dosing specimens, whereas amphetamine concentrations generally did not achieve the 200-ng/mL cutoff specified by the Substance Abuse and Mental Health Services Administration (SAMHSA) guidelines for GC-MS confirmation of methamphetamine. Some specimens containing methamphetamine had no amphetamine metabolite. The current guidelines would have resulted in 90.2% of the specimens containing methamphetamine being ruled negative by confirmation following either night or day administration, whereas one subject following the initial day administration and another following night crossover administration would have been judged positive at most time intervals. These findings suggest that the current SAMHSA guidelines select for individual metabolic variations and that GC-MS confirmation of methamphetamine will result in most occasional users being ruled negative following an oral dose of methamphetamine while some will be ruled positive

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 postingestion. 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

Essential role of Protein Tyrosine Phosphatase 1B in the modulation of insulin signaling by acetaminophen in hepatocytes

Resumen del póster presentado a la Conferencia del Centro Nacional de Investigaciones Cardiovasculares (CNIC) titulada: "Energy homeostasis and metabolic disease", celebrada en Madrid del 7 al 8 de noviembre de 2014.[Objectives]: The metabolic adverse effects of drugs develop slowly and are often missed in safety protocols, toxicity or clinical assays. In this regard, many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. Acetaminophen (APAP) has been used for over 40 years as an analgesic in the treatment of acute and chronic pain. We have investigated the cross-talk between signaling pathways activated by APAP and insulin signaling in hepatocytes with or without expression of the protein tyrosine phosphatase IB (PTPIB) and in wild-type and PTPIB-deficient mice chronically treated with APAP. [Methods]: Human primary hepatocytes, Huh7 hepatoma cells with silenced PTPIB, mouse hepatocytes from wild-type and PTPIB-deficient mice and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTPIB in the effects of APAP on glucose homeostasis and hepatic insulin signaling. Energy expenditure and islet alpha/beta cell ratio were also evaluated. [Results]: In APAP-treated human and mouse hepatocytes at concentrations that did not induce death, phosphorylation of c-jun (NH2) terminal kinase (JNK) and PTPlB expression and activity were increased. APAP pretreatment inhibited the early steps of insulin signaling and decreased Akt phosphorylation. Rosiglitazone prevented APAP effects in insulin signaling by decreasing JNK phosphorylation and PTPIB levels. Likewise, PTPIB deficiency in human or mouse hepatocytes protected against APAP-mediated impairment hepatic insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment with a protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTPIB-1- mice. [Conclusions]: our results have demonstrated a negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTPIB. Moreover, our in vivo data suggest taht chronic use o fAPAP may be associated with insulin resistance in the liver.Peer Reviewe

Targeted metabolomic profiling indicates structure-based perturbations in serum phospholipids in children with acetaminophen overdose

Phospholipids are an important class of lipids that act as building blocks of biological cell membranes and participate in a variety of vital cellular functions including cell signaling. Previous studies have reported alterations in phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) metabolism in acetaminophen (APAP)-treated animals or cell cultures. However, little is known about phospholipid perturbations in humans with APAP toxicity. In the current study, targeted metabolomic analysis of 180 different metabolites including 14 lysoPCs and 73 PCs was performed in serum samples from children and adolescents hospitalized for APAP overdose. Metabolite profiles in the overdose group were compared to those of healthy controls and hospitalized children receiving low dose APAP for treatment of pain or fever (therapeutic group). PCs and lysoPCs with very long chain fatty acids (VLCFAs) were significantly decreased in the overdose group, while those with comparatively shorter chain lengths were increased in the overdose group compared to the therapeutic and control groups. All ether linked PCs were decreased in the overdose group compared to the controls. LysoPC-C26:1 was highly reduced in the overdose group and could discriminate between the overdose and control groups with 100% sensitivity and specificity. The PCs and lysoPCs with VLCFAs showed significant associations with changes in clinical indicators of drug metabolism (APAP protein adducts) and liver injury (alanine aminotransferase, or ALT). Thus, a structure-dependent reduction in PCs and lysoPCs was observed in the APAP-overdose group, which may suggest a structure-activity relationship in inhibition of enzymes involved in phospholipid metabolism in APAP toxicity. Keywords: Metabolomics, Phospholipids, Acetaminophen, Hepatotoxicity, Dru

Essential role of protein-tyrosine phosphatase 1B in the modulation of insulin signaling by acetaminophen in hepatocytes

Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B(-/-) mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver.This work was supported by Grants SAF2012-33283, BFU2011-25420, and AGL2010-17579 from the Ministerio de Economía y Competitividad, Comunidad de Madrid Grant S2010/BMD-2423, European Foundation for the Study of Diabetes and Amylin Paul Langerhans Grant, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, and Grant PI09/0185 from Instituto de Salud Carlos III.Peer Reviewe

Correlation analysis of peak biomarker versus time to treatment with N-acetylcysteine (NAC)^*.

<p>*Log transformation of peak measurement of parameter.</p><p>Correlation analysis of peak biomarker versus time to treatment with N-acetylcysteine (NAC)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131010#t003fn001" target="_blank">*</a>}.</p