Drug metabolism and toxicity


Role of Parkin in Acetaminophen-induced Mitophagy and Liver Injury

Jessica A. Williams, Hong-Min Ni, Wen-Xing Ding

University of Kansas Medical Center, Kansas City, KS

Autophagy is a catabolic process that degrades proteins and damaged organelles to promote cell survival. Mitophagy is a selective form of autophagy specific for degradation of mitochondria. Acetaminophen (APAP) overdose causes liver injury by inducing necrosis following mitochondrial damage, and we previously demonstrated that pharmacological induction of autophagy by rapamycin protected against APAP-induced liver injury in mice by degrading damaged mitochondria. However, the mechanism for this mitochondria removal by autophagy is unknown. Parkin, an E3 ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkininduced mitophagy in protection against APAP-induced liver injury using wild type (WT) and Parkin knockout (KO) mice. Parkin translocated to mitochondria in WT mouse livers after APAP treatment followed by mitophagy induction. We expected Parkin KO mice to have increased liver injury after APAP-overdose compared to WT mice due to their inability to induce mitophagy in the absence of Parkin. However, Parkin KO mice were actually protected against APAP-induced liver injury compared to WT mice as demonstrated by analysis of serum enzyme activity and liver tissue histology, and electron microscopy analysis surprisingly revealed that mitophagy still occurred in Parkin KO mice after APAP treatment. In addition, APAP increased mitochondrial protein ubiquitination and p62 mitochondrial translocation in both WT and Parkin KO mice, which provided further evidence of mitophagy induction in these mice. Even though it has been shown that Parkin is required for mitophagy induction in vitro, our data suggest that Parkin may not be essential for mitophagy induction in vivo. In addition, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK) and increased hepatocyte proliferation after APAP treatment in their livers compared to WT mice. In conclusion, these data suggest that Parkin is not essential for mitophagy induction in liver. Furthermore, Parkin may promote APAP-induced liver injury by enhancing APAPinduced JNK activation and impairing the liver repair process by inhibiting hepatocyte proliferation independent of its role in mitophagy.


The following people have nothing to disclose: Jessica A. Williams, Hong-Min Ni, Wen-Xing Ding


Short-term and Long-term exposure of IL-22 show opposite effects on Acetaminophen induced liver injury

Dechun Feng1, Yan Wang1, Hua Wang1, Heng Liu2, Honglei Weng2, Xiaoni Kong3, Brittany V. Martin-Murphy4, Yongmei Li1, Steven Dooley2, Cynthia Ju4, Bin Gao1

1laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD; 2Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany; 3School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China; 4Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO

Interleukin-22 (IL-22) is a well-documented hepatoprotective cytokine that protects against hepatocellular damage in various models of liver injury. Here we demonstrated that pretreatment with IL-22 protected mice from acetaminophen (APAP) hepatotoxicity. The protective effects were dependent on STAT3 because IL-22 pretreatment did reduce APAP hepatotoxicity in liver-specific STAT3 knockout (STAT3Hep-/-) mice. However, to our surprise, IL-22 transgenic (IL-22TG) mice were more susceptible to APAP-induced liver injury than WT mice. Overexpression of IL-22 by injection of adenovirus IL-22 for 6 weeks also aggravated APAP-induced liver injury. In agreement with these findings, more APAP adducts and accelerated GSH depletion were found in IL-22 TG mice after APAP administration. Furthermore, hepatic expression of Cyp2E1 was much higher in IL-22TG mice than that in WT mice. The elevated hepatic Cyp2E1 expression and APAP-induced liver injury in IL22TG were not altered in IL-22TGSTAT3Hep-/- double mutant mice, suggesting these elevations were independent of liver STAT3. In contrast, the enhanced liver damage in IL-22TG mice was almost completely blocked in IL-22TGCyp2E1 double mutant mice. Finally, HNF1 a, a transcriptional factor that plays a key role in the control Cyp2E1 gene transcription, was markedly upregulated in the liver of IL-22TG mice. Conclusion: Short-term pretreatment with IL-22 protects mice from APAPinduced liver injury through hepatic STAT3 activation; however, long-term exposure of IL-22 exacerbates APAP hepatotoxicity by inducing Cyp2E1 and HNF-1 α expression.


The following people have nothing to disclose: Dechun Feng, Yan Wang, Hua Wang, Heng Liu, Honglei Weng, Xiaoni Kong, Brittany V. Martin-Murphy, Yongmei Li, Steven Dooley, Cynthia Ju, Bin Gao


Efavirenz induces endoplasmic stress and UPR in human hepatocytes: implication of mitochondria

Nadezda Apostolova1, Fernando Alegre2, 1, Miriam Polo2, 1, Haryes A. Funes2, 1, Ana Blas-Garcia2, 1, Juan V. Esplugues1,2

1Department of Pharmacology, Universitat de Valencia, Valencia, Spain; 2FISABIO- Hospital Universitario Dr. Peset, Valencia, Spain

Background. Both mitochondrial dysfunction and altered function of the endoplasmic reticulum (ER-stress”UPR), play a major role in hepatic pathophysiology, including drug-induced liver damage. Efavirenz (EFV), a non-nucleoside analog reverse transcriptase inhibitor, is a cornerstone of current anti-HIV1 therapy. Despite being generally safe, EFV produces hepatotoxicity in up to 10% of patients. Recent evidence has revealed that shortterm exposure (24h) of human hepatic cells to EFV triggers mitochondrial dysfunction and ER-stress with UPR activation. Aim. To analyze the implication of mitochondria in the effect of ER stress/UPR triggered by EFV. Methods. The human hepatoma line Hep3B and cells lacking functional mitochondria (Hep3B rho-zero obtained through pharmacological interruption of mtDNA replication) were exposed to clinically relevant concentrations (10 and 25μM) of EFV (24h). Results. The concentration-dependent increase in both mRNA and protein expression of GADD153/CHOP (CCAAT/enhancer binding protein) and GRP78 (Glucose-regulated protein 78) was considerably lower in rho-zero cells. Likewise, unlike WT cells, which displayed altered ER morphology and increased ER signal (fluorescence microscopy) under EFV treatment, rho-zero cells manifested no such changes. The specific interconnection between ER-stress and mitochondria was also evident when Ca2+ levels were studied. Similarly to the classic ER-stressor thapsigargin, EFV enhanced [Ca2+]c, though the effect occurred through a different mechanism not the Ca2+ transporter SERCA. Unlike thapsigargin, EFV produced a decrease in [Ca2+]m, probably due to diminished activity of the mitochondrial membrane potential-dependent Ca2+ uniporter. Interestingly, the overall increase in [Ca2+]c in WT Hep3B was not altered in rho-zero cells. Moreover, in this model, EFV has previously been shown to induce autophagic degradation of mitochondria. Western blot studies of the specific marker protein LC3 (light chain of the microtubule-associated protein) revealed that LC3-II formation triggered by EFV was reduced in cells lacking functional mitochondria. When general viability/proliferation (cell count) was assessed (by static cytometry), the cytotoxic effect of EFV was found to be less pronounced in rho-zero cells. Conclusions. Mitochondria are specifically implicated in the ERstress induced in human hepatic cells with clinically relevant concentrations of Efavirenz. These findings expand our knowledge of the mechanisms that trigger ER-stress and throw light on the mitochondria/ER interplay in drug-induced hepatic challenge, with specific relevance for patients undergoing EFV-containing therapy.


Juan V. Esplugues - Speaking and Teaching: Abbvie, MSD, AstraZeneca

The following people have nothing to disclose: Nadezda Apostolova, Fernando Alegre, Miriam Polo, Haryes A. Funes, Ana Blas-Garcia


Acetaminophen (APAP)-induced Hepatotoxicity is Characterized by Greater Susceptibility of Cells in S or G2/M and Prolonged Arrest in G0/G1 with Inability to Reenter Cell Cycle Accounts for Failure of Hepatic Regeneration in Acute Liver Failure (ALF)

Preeti Viswanathan1, Sriram Bandi2, Sanjeev Gupta2

1Pediatric Gastroenterology and Hepatoloqy, Childrens Hospital at Montefiore, Albert Einstein College of Medicine, New York, NY; 2Medicine and Pathology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY

Understanding the molecular basis of failed hepatic regeneration will provide novel pathophysiological and therapeutic mechanisms for drug-induced ALF. To define reasons for the characteristic observation of active DNA synthesis but not cell division in residual hepatocytes in liver explants after ALF, we studied the effects of APAP on HuH-7 cells, mouse hepatocytes and intact mice. C57BL/6 mice were given LD50 dose of APAP i.p to induce ALF. Liver injury was characterized by encephalopathy, liver test abnormalities, hepatic inflammation and perivenous necrosis, and mortality. Culture of HuH-7 cells or mouse hepatocytes with APAP in IC50 concentrations caused cytotoxicity as confirmed by MTT assays. Gene expression arrays from APAP-treated cells or mice showed disturbances in ATM signaling pathway and western blot of tissue and cell lysates confirmed ATM-related DNA damage responses (DDR), including pATM, pATR, pH2AX, pChek1 and pChek2 expression. This DDR in the setting of ATM dysregulation was verified by Comet assays with extensive double-strand DNA breaks. To evaluate greatest susceptibility of cell subpopulations to APAP, we analyzed HuH-7 cells by FACS, and found cells in S or G2/M were lost within 4 h, whereas cells in G0/G1 survived over long-term. This was confirmed when HuH-7 cells synchronized by hydroxyurea in late S were rapidly destroyed by APAP. By contrast, G0/G1 cells exposed to APAP stopped proliferating and failed to enter cell cycle, despite removal of APAP from culture medium. These cells in G0/G1 displayed significant DNA damage, as indicated by gene expression arrays, pH2AX staining and Comet assays. Next, to determine whether APAP-induced arrest of cell cycle could be reversed by G-CSF, which was previously found to improve outcomes in ALF, we performed further studies. Remarkably, after G-CSF treatment, HuH-7 cells exposed to APAP regained the ability to overcome G0/G1 arrest and entered the cell cycle. Similarly, mice treated with G-CSF after induction of APAP toxicity showed improved survival and superior liver regeneration, with greater Ki67 expression compared with mice receiving APAP alone. This improvement correlated with less pH2AX staining and comet formation, indicating decreased DNA damage in G-CSFtreated animals. Conclusions: Actively cycling cells in S or G2/M were highly susceptible to APAP toxicity. By contrast, G0/G1 cells survived APAP-induced DNA damage but were prevented from cycling. The inability to reenter cell cycle will help explain failure of residual hepatocytes to regenerate liver in APAP-induced ALF. This molecular process should offer further new directions for therapeutic development in ALF.


The following people have nothing to disclose: Preeti Viswanathan, Sriram Bandi, Sanjeev Gupta


Acute and Chronic Ethanol Administration Differentially Affect Hepatic Autophagy and the Nuclear content of Transcription Factor EB (TFEB)

Paul G. Thomes, Casey S. Trambly Kusum K. Kharbanda, Natalia A. Osna, Terrence M. Donohue

Internal Medicine, Liver study unit, Univ Nebraska-Omaha, Omaha, NE

Background: Liver enlargement, due to accumulation of lipids and proteins in hepatocytes is common in heavy drinkers. We have demonstrated hepatomegaly in EtOH-fed rodents, which exhibit decelerated hepatic protein catabolism. In contrast, rodents subjected to acute ethanol administration exhibit no proteopathy. Here, we compared the effects of acute and chronic EtOH feeding on autophagy, the highly- regulated radation by lysosomes of a cell's cytoplasmic components. also measured the intracellular distribution of TFEB, the transcription factor that controls autophagy and lysosome biogenesis. Methods: C57Bl/6 mice transgenic for the fusion protein GFP-LC3, an autophagosome (AV) marker protein, were gavaged with EtOH (6g/kg) or PBS 12 hr before death. Separate mice were chronically pair-fed (35 to 62 days) EtOH or control liquid diets. Livers or hepatocytes were harvested from the animals and analyzed. Results: Acute EtOH caused a 1. 8-fold elevation of AVs over PBS controls. Elevated levels of GFP, the degradation product of GFP-LC3 confirmed that acute ethanol enhanced autophagy flux. Furthermore, EtOH-gavaged mice had 2. 3-fold higher TFEB nuclear content than PBS-gavaged mice, as judged by the nuclear to cytoplasmic ratio of the protein. Mice subjected to chronic EtOH feeding exhibited hepatomegaly, associated with proteopathy and steatosis, with evidence of mild injury, as judged by elevated serum ALT/AST. AV levels in livers of EtOH-fed mice were higher but lysosome levels were 25% lower than pair fed controls, but the level of P62, another marker of lysosomal degradation was elevated, indicating that higher AVs in livers of these mice represented their accumulation due to reduced AV degradation by lysosomes. The activity of lysosomal acid lipase, which degrades hepatic lipids was lower in livers of EtOH-fed mice. In contrast to acutely-treated mice, chronically EtOH-fed mice had 2-fold lower TFEB nuclear to cytoplasmic ratio than pair-fed controls. Conclusion: Our findings indicate that acute EtOH enhanced autophagy, as judged by elevated AVs, enhanced GFP-LC3 catabolism and higher TFEB nuclear localization. Conversely, chronic EtOH-feeding disrupted autophagy, as indicated by AV accumulation, lower LAL activity, lysosomal substrate accumulation (P62, triglycerides and hepatic proteins) and lower nuclear TFEB accumulation, which slows lysosome biogenesis and autophagy. These findings partially explain previous reports of disturbances in protein and lipid catabolism, which result in their accumulation in livers of EtOH-fed rodents and of problem drinkers. Supported by Dean's Reviewed Research Grant of the UNMC.


The following people have nothing to disclose: Paul G. Thomes, Casey S. Trambly, Kusum K. Kharbanda, Natalia A. Osna, Terrence M. Donohue


Abacavir inhibits mitochondrial function and increases vulnerability to acetaminophen-induced hepatotoxicity

Ana Blas-Garcia2, 1, Victor M. Victor2, 1, Haryes A. Funes2, 1, Nadezda Apostolova1, Juan V. Esplugues1, 2

1Department of Pharmacology, Universitat de Valencia, Valencia, Spain; 2FISABIO-Hospital Universitario Dr. Peset, Valencia, Spain

Background. Liver disease is the second cause of mortality in HIV-infected patients treated with High Activity Antiretroviral Therapy (HAART) and has been related in some cases to antiretroviral drugs. Nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) are essential components of HAART and have been associated with chronic liver mitochondrial toxicity due to their interference with mitochondrial DNA (mtDNA) replication. However, as recently shown for other drugs used in HAART, mitochondrial dysfunction can be generated by mechanisms unrelated to mtDNA replication. Since acetaminophen, a wellknown hepatotoxic drug, also interferes with the mitochondria when administered in overdose, we hypothesize that its the combination with antiretroviral can exacerbate the mitotoxic effect of these drugs. Aim. To evaluate the acute effects of clinically-relevant concentrations of the most widely used NRTI, alone or in combination with acetaminophen, on mitochondrial function and cellular viability in hepatic cells. Methods. Several parameters of mitochondrial function (oxygen consumption, mitochondrial membrane potential -Δψm-, reactive oxygen species production, intracellular ATP levels) and cellular viability were assessed in non-HIV-infected Hep3B cells treated (124h) with the pyrimidine analogues Lamivudine, Zidovudine and Emtricitabine, the purine analogues Abacavir (ABC) and Didanosine (ddI), or the nucleotide analogue Tenofovir. Further experiments were performed in the presence of different concentrations of acetaminophen. Data were reported as mean+/SEM, and their statistical significance versus vehicle was analyzed by one-way ANOVA. Results. Clinical concentrations of ABC and ddI, but not of the other NRTI, produced an immediate and significant decrease in mitochondrial function, which was evident in a concentration-dependent inhibition of O2 consumption, a increased production of reactive oxygen species, and a reduction of Δψm and intracellular ATP levels. This mitochondrial dysfunction did not compromise cell survival, as the aforementioned parameters were restored to previous values after 24h treatment. However, co-administration of these drugs with acetaminophen concentrations below those considered toxic in hepatic cellular models exacerbated the deleterious effects of both treatments on mitochondrial function and cellular viability. Conclusions. The combination of ABC or ddI with low concentrations of acetaminophen significantly increases the risk of acetaminophen-mediated liver injury. Our findings are of considerable relevance given that acetaminophen is currently prescribed to some patients taking NRTI.


Juan V. Esplugues - Speaking and Teaching: Abbvie, MSD, AstraZeneca

The following people have nothing to disclose: Ana Blas-Garcia, Victor M. Victor, Haryes A. Funes, Nadezda Apostolova


Metabonomic Biomarkers for Drug-induced Liver Injury in Rats and Humans: Better than Alanine Aminotransferase?

Jia-bo Wang1, Zheng-sheng Zou1, Yan-ling Zhao1, Lu-shan Qin1, Qi Li1, Zhi-jie Ma1, Xiao-xi Du2, Xiao-he Xiao1

1 302 Military Hospital of China, Beijing, China; 2Center for Drug Reevaluation, China Food and Drug Administration, Beijing, China

Although alanine aminotransferase (ALT) is a universal adopted clinical and regulatory tool for detecting liver injury, especially drug-induced liver injury (DILI),ALT assay is not indeed a test of liver function. The identification and evaluation for novel translational biomarkers are obligatorily needed for both non-clinical and clinical assessment of DILI. In this study, the blood samples were collected from rats intoxicated by acetaminophen or a well documented hepatotoxic herb- Polygonum multiflorum (PM) and then screened for potential metabonomic biomarkers by LC-Q-TOF. Fifteen PM -intoxicated patients were also screened for biomarkers compared to rats, as well as healthy volunteers (10 cases), DILI patients caused by the other drugs (30 cases), or the other types of liver injuries, including viral (36 cases) and autoimmune (30 cases) liver diseases. The results showed the serum ALT activity did not change dramatically in the early intoxication stage of PM in rats. Totally 41 metabonomic biomarkers of PM were identified in rat serum of better sensitivity than ALT and 13 among them were identical in patients. Furthermore, 4 biomarkers, such as LysoPC(20: 4(8Z,11Z,14Z,17Z)) and PE(15: 0/22: 0), were found of strong correlations with the extent of liver injury. Through bioinformatic analysis, the metabolic pathways associated with the hepatotoxicity of PM were concentrated to phospholipids, linolenate and arachidonate metabolic process. In summary, we found that ALT is not sensitive to diagnose liver injury of PM in its early stage of intoxication, while the metabonomic biomarkers have desirable sensitivity to detect liver injury of the herb. Our results provide data on clinically potent biomarkers in clinic diagnosis for patients undergoing DILI related to PM or other herbal preparations. The PM DILI could be clearly discriminated from HBV infection caused liver failure (HBV-LF) and autoimmune hepatitis (AIH), but not DILI patients caused by the other drugs (left panel). The metabonomics biomarkers related to PM DILI could be concentrated to an integrated network including 10 primary network targets, such as linolenate and arachidonate (right panel).



The following people have nothing to disclose: Jia-bo Wang, Zheng-sheng Zou, Yan-ling Zhao, Lu-shan Qin, Qi Li, Zhi-jie Ma, Xiao-xi Du, Xiao-he Xiao


Influence of oxygen-binding cytoglobin expressed in hepatic stellate cells on acetaminophen-induced acute hepatocyte damage: In vivo and in vitro studies

Yuga Teranishi1, Tsutomu Matsubara2, Keiko Iwaisako2, Kazuki Nakatani2, Thuy T. Le1, Frank J. Gonzalez3, Kazuo Ikeda2, Norifumi Kawada1

1Hepatology Osaka City University Osaka, Japan; 2Anatomy and Regenerative Biology Osaka city university Osaka, Japan; 3Center for Cancer Research, National Cancer Bethesda, MD

Purpose: Oxygen is required for cytochrome P450-dependent drug metabolism. Cytoglobin (Cygb) is a unique globin expressed exclusively in hepatic stellate cells (HSC); its role in oxygen-dependent metabolism in neighboring hepatocytes (Hc) has remained unknown. We wanted to assess the correlation between Cygb in HSC and xenobiotic metabolism in Hc. Methods: Acute liver injury was induced in wild-type (WT) and Cygb-null mice by administrating acetaminophen (APAP, 300 mg/kg), carbon tetrachloride (CCl4, 0. 5 mg/kg), or lipopolysaccharide (5 μg/kg)/D-galactosamine (700 mg/kg) (LPS/D-GalN). Liver damage was evaluated by measuring serum levels of alanine transaminase (ALT) and liver histology. Hc and HSC were isolated from mice. APAP-induced hepatotoxicity was assessed under normoxia and hypoxia (5% O2). In co-culture studies, Hc and HSC were exposed to 30 mM APAP under hypoxic condition. Hepatotoxicity was determined by MTT assay and propidium iodide staining. Results: In APAPinduced acute liver injury, serum ALT levels were higher in WT mice than Cygb-null mice (1 3, 970 and 4, 699 U/L, respectively). Liver histology showed more severe necrosis around the central vein area in WT than Cygb-null mice, indicating that Cygb deficiency markedly attenuated APAP-induced liver damage. There were no differences in mRNA and protein levels of Cyp2e1 and total glutathione concentration in the livers. However, serum APAP metabolites were decreased by half in Cygbnull mice. Liver mRNA expression levels of pro-inflammatory cytokines remained unchanged between WT and Cygb-null. In in vitro experiments, damage of Hc was triggered by 10 and 20 mM APAP under normoxia, which was markedly alleviated under hypoxia. There was no difference in Hc cell death between HcWT and HCCygb-null in the presence of various concentrations of APAP. Co-culture studies revealed that HSCWT, but not HCCygb-null, deteriorated APAP-induced injury of Hc. Cygb deficiency also alleviated acute liver injury induced by CCI4 but not by LPS/D-GalN. Conclusions: This study demonstrates that Cygb in HSC contributes to cytochrome P450-mediated metabolism of xenobiotics in Hc, presumably in an oxygen-dependent manner. It is suggested that HSC interact with Hc in xenobiotic degradation.


The following people have nothing to disclose: Yuga Teranishi, Tsutomu Matsubara, Keiko Iwaisako, Kazuki Nakatani, Thuy T. Le, Frank J. Gonzalez, Kazuo Ikeda, Norifumi Kawada


Protein Aggregation in Parental & Recombinant Hep g2 Cells after Etoh or Proteasome Inhibitor Exposure

Paul G. Thomes, Casey S. Trambly, Natalia A. Osna, Kusum K. Kharbanda, Dahn L. Clemens, Terrence M. Donohue

Internal Medicine, Liver study unit, Univ Nebraska-Omaha, Omaha, NE

Background: Aggresomes appear as Mallory-Denk bodies in patients with alcoholic liver injury. The proteasome destroys modified proteins before they become prone to aggregation. Proteasome inhibition therefore triggers aggresome formation and these are eliminated by autophagy. Here, we compared the effect of the proteasome inhibitor, MG132, with ethanol (EtOH) on the ability of each to cause aggresome development in parental and recombinant HepG2 cells. Methods: EtOHmetabolizing VL-17A cells and non-metabolizing HepG2 cells were exposed to zero, 25, 50 or 100 mM EtOH for 24 to 72 hr or to the proteasome inhibitor MG132 (2. 5μM) for 18 hr. Aggresomes were then detected using Proteostat® aggresome detection kit (Enzo, Inc. ) and quantified by confocal microscopy and flow cytometry. Results: VL-17A cells exposed to MG132 exhibited six- and 1. 6-fold increases in the number and size, respectively, of protein aggregates over controls. 24 hr exposure of cells to 25, 50 and 100mM EtOH caused increases in aggregate number 1. 8-, 1. 7, - and 1. 7-fold respectively, with 1. 4-, 1. 3- and 1. 3-fold rises in aggregate size, respectively, over control cells. These same EtOH concentrations caused a dose-dependent decline in 20S proteasome activity. VL-17A cells exposed 72 hr to 25, 50 and 100 mM EtOH enhanced aggregate numbers (2. 7-, 2. 4- and 2. 5-fold, respectively) with similar increases in aggregate sizes over control cells. Again, proteasome activity in EtOH-exposed cells declined dosedependently. Flow cytometry revealed that exposure of VL-17A cells to 50 mM EtOH caused 1. 7 and 2. 2-fold higher fluorescent signal after 48 and 72 hr, respectively, indicative of aggresome formation. EtOH-induced aggregation was dependent on EtOH metabolism as non-metabolizing HepG2 cells exhibited aggregation after MG132, but not after 50 mM EtOH treatment. When we exposed Hep G2 and VL-17A cells to MG132 (16 hr) and then rapamycin (100 nM) four hr before harvest, aggresome content in MG132-treated cells declined significantly. Conclusion: Our findings indicate that EtOH- and MG132-induced proteasome down-regulation in VL-17A cells caused accumulation of protein aggregates. Aggresomes increased in an alcohol dose-independent but a time-dependent manner. Because an increasing degree of proteasome inhibition occurred with rising EtOH doses, which did not intensify protein aggregation, we postulate that other compensatory mechanisms prevent further aggresome accumulation. Supported by Grant 1-R01-AA16546 from the NIAAA.


The following people have nothing to disclose: Paul G. Thomes, Casey S. Trambly, Natalia A. Osna, Kusum K. Kharbanda, Dahn L. Clemens, Terrence M. Donohue


Poor Adherence to Isoniazid (INH) Monitoring Guidelines is Common in Cases of INH Hepatotoxicity and Associated with More Severe Liver Injury: Experience of The Drug Induced Liver Injury Network (DILIN)

Paul H. Hayashi1, Timothy J. Davern2, Robert J. Fontana3, Naga P Chalasani4, Andrew Stolz5, Jayant A. Talwalkar6, Victor J. Navarro7, William M. Lee8, David E. Kleiner9, Jiezhun Gu10, Jay H.Hoofnagle11

1Division of Gastroenterology & Hepatology, University of North Carolina, Chapel Hill, NC; 2Division of Gastroenterology, California Pacific Medical Center, San Francisco, CA; 3Division of Gastroenterology, University of Michigan, Ann Arbor, MI; 4Division of Gastroenterology, Indiana University School of Medicine, Indianapolis, IN; 5Division of Gastroenterology, University of Southern California, Los Angeles, CA; 6Division of Gastroenterology Mayo Clinic, Rochester, MN; 7Division of Gastroenterology, Albert Einstein Medical Center, Philadelphia, PA; 8Division of Gastroenterology, Southwestern University, Dallas, TX; 9Laboratory of Pathology, National Cancer Institute National Institutes of Health, Bethesda, MD; 10Duke Clinical Research Institute, Duke University Durham, NC; 11 Liver Disease Section, NIDDK, National Institutes of Health, Bethesda, MD

Background: Over 250, 000 Americans annually receive isoniazid (INH). In 2006, the American Thoracic Society (ATS) established guidelines for when to stop INH for hepatotoxicity. However it is unclear if these guidelines can reduce the frequency or severity of liver injury. Aim: To analyze the presenting features of patients with well-characterized INH hepatotoxicity and determine correlates of DILI severity. Methods: Patients with INH hepatotoxicity enrolled in the DILIN from Sep 2004 through April 2013 with a causality score of definite, very likely, or probable were identified. Delay in stopping INH was defined as time from meeting ATS stopping criteria (hepatitis symptoms and/or liver enzyme elevation) to INH discontinuance. Case severity was assessed by the 5-point DILIN Severity Index Score (SIS) that ranges from enzyme elevations without jaundice (SIS=1) to transplant or death (SIS=5). Several variables including age, sex, race, body mass index (BMI), presence of underlying liver disease, and delay in stopping INH were examined for associations with SIS. Results: Amongst 1091 DILIN patients, INH was the 2nd most commonly implicated agent with 69 cases, of which 60 met inclusion criteria [48% definitely, 38% highly likely and 13% probably attributable to INH]. Median age was 49 years (range 4 to 68), 70% were female, 27% Caucasian, 25% Hispanic, 18% African American and 8% Asian. 58 (97%) took INH for latent TB. Patients presented with either hepatocellular (92%) or mixed cholestatic-hepatocellular (7%) biochemical injury patterns; 72% were jaundiced. Cases were evenly distributed across the 5-point SIS scale with 13 (22%) undergoing transplant and/or dying. Median delay between reaching ATS stopping criteria and INH discontinuance was 9 days (range 0-99). Only 27 (45%) stopped INH within 7 days of meeting criteria, 15 (25%) remained on therapy for 15-28 days and 9 (15%) continued >28 days. Most delays occurred when patients and/or providers ignored symptoms. On univariate analysis, only higher BMI and delay in discontinuing INH were associated with higher SIS (p-values <0. 05). Amongst 13 fatal or transplanted cases, 4 (31%) remained on the INH for 8-21 days after meeting stopping criteria and 5 (39%) for >21 days. Summary: Isoniazid treatment for latent TB continues to be a leading cause of DILI in the US. Poor adherence to ATS guidelines for INH discontinuance is common in cases of hepatotoxicity and associated with more severe liver injury including death and need for transplant. Adherence to ATS guidelines should be assessed for community effectiveness.


Robert J. Fontana - Consulting: GlaxoSmithKline, tibotec; Grant/Research Support: Gilead, vertex, Ocera

Naga P. Chalasani - Consulting: Salix, Abbott, Merck, Lilly, Enterome, Aegerion; Grant/Research Support: Intercept, Lilly, GenFit, Gilead, Enterome, Cumberland, Galectin

Jayant A. Talwalkar - Consulting: Lumena; Grant/Research Support: Intercept, Salix, Gilead

William M. Lee - Consulting: Eli Lilly, Novartis; Grant/Research Support: Gilead, Roche, Vertex, BI, Anadys, BMS, merck; Speaking and Teaching: Merck

The following people have nothing to disclose: Paul H. Hayashi, Timothy J. Davern, Andrew Stolz, Victor J. Navarro, David E. Kleiner, Jiezhun Gu, Jay H. Hoofnagle


Synergistic cytotoxicity of Temozolomide and ABT-888 in dual-drug targeted Polymeric Nanoparticles

Jose Antonio Munoz-Gamez1, Laura Sanjuan1, 3, Rosa Quiles1, 2, Andres Barrientos4, Julian Lopez-Viota1, Josefa León 1, 2, Angel Carazo1, Jorge Casado1, Esther-José Pavón-Castillero1, Ana Belen Martίn1, 3, Angeles Ruiz-Extremera2, 5 Javier Salmeron2, 3

1Unidad de Apoyo a la Investigación, Hospital Universitario San Cecilio, Granada, Spain; 2Centro de Investigación Biomedica en Red de Enfermedades Hepticas y Digestivas, CIBERehd, Granada, Spain; 3Departamento de Medicina, Universidad de Granada, Granada, Spain; 4Unidad Clίnica de Aparato Digestivo, Hospital Universitario San Cecilio, Granada, Spain; 5Unidad de Pediatrίa, Hospital Universitario San Cecilio, Granada, Spain

Background and aims: Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide and its incidence has been increasing in recent years. Because current therapies are rarely able to achieve complete tumor ablation, it is necessary to study any new therapeutic strategy that arises. Accordingly, we propose a new and interesting strategy for HCC treatment, namely the use of poly (ADP-ribose) polymerase (PARP-1) inhibitors (ABT-888) together with temozolomide (TMZ, a DNA-damaging agent) incorporated into magnetic nanoparticles (MNPs). Method: Magnetic Fe/Fe3O4 cores were synthetized using thermal decomposition methods, and a final layer of silica was incorporated to coat the composite MNPs. The simultaneous adsorption of TMZ and ABT888 PARP-1 inhibitor was monitored by electrophoretic mobility measurements. In vitro tests were performed with HepG2, Hep3B and PLC-PRF-5 tumoral cell lines and with WRL-68 nontumoral cells. Results: The MNPs were loaded simultaneously with TMZ and different concentrations of ABT888, had a final size of 16 ± 4 nm. A high degree of stability in culture medium was achieved and 50% of both drugs had been released about 10-15 hours after their dissolution in the culture medium. Laser confocal microscopy images showed that the MNPs had entered the liver tumor cells and that both drugs were released into the cells. The DNA damage induced by TMZ triggered PARP-1 activation, but this stimulus was reduced in the presence of ABT-888 coated NPs, inducing the following effects: G2/M cell cycle arrest (67% for MNPs/TMZ/ABT-888 vs. 24% in the control group, P>0. 05), accumulation of DNA damage (P<0. 05), mitochondrial depolarization (54% for MNPs/TMZ/ABT-888 vs. 10% in the control group, P>0. 01) and apoptotic cell death (free drugs 34. 5% vs. MNP-coated drugs 53. 5%, P=0. 001). Conclusions: TMZ and ABT888 can be incorporated simultaneously into MNPs and thus released to an extended degree and gradually, over time. The nanocarriers were able to enter the tumor cells and release both drugs inside them. The apoptotic effect thus induced was greater than that produced by non-vehiculized drugs.


The following people have nothing to disclose: Jose Antonio Munoz-Gamez, Laura Sanjuan, Rosa Quiles, Andrés Barrientos, Julian Lopez-Viota, Josefa León, Angel Carazo, Jorge Casado, Esther-José Pavón-Castillero, Ana Belen Martin, Angeles Ruiz-Extremera, Javier Salmeron


Trimethoprim/Sulfamethoxazole hepatotoxicity: Analysis of 31 cases

Lafaine Grant1, Jiezhun Gu2, Saleh Alqahtani3, Don C. Rockey4, William M. Lee1

1UT Southwestern Medical Center at Dallas, Dallas, TX; 2Duke Clinical Research Institute, Durham, NC; 3The Johns Hopkins Hospital, Baltimore, MD; 4The Medical University of South Carolina, Charleston, SC

Aim: To describe the clinical features of trimethoprim/sulfamethoxazole (TMP/SMZ) drug-induced liver injury (DILI) among patients enrolled in the Drug-Induced Liver Injury Network (DILIN). Methods: 67 suspected cases of DILI due to TMP/SMZ were identified within 1, 257 patients enrolled in DILIN between 2004 and April 2013. 31 cases were adjudicated and scored as definite (> 95%), highly likely (75% - 95%) or probable (50%-74%). Results: Table 1 depicts clinical features. Patients commonly presented with immuno-allergic signs/symptoms (fever, rash). Jaundice and abnormal liver enzymes were identified soon thereafter and usually peaked early during the course of the liver injury with mean peak ALT of 685 U/L, AST 579 U/L, alkaline phosphatase 493 U/L and total bilirubin 13. 7 mg/dL occurring at days 3, 3, 18 and 16, respectively after onset. The pattern of liver injury varied from hepatocellular (11/30, 37%), cholestatic (11/30, 37%) and mixed (8/30, 27%) types. Eight patients (26%) had a history of other drug allergies; 5/30 (17%) had a positive ANA, 7/28 (25%) a positive ASMA, and 5/30 eosinophilia. Injury was typically moderate to severe and required hospitalization in 77% of cases. Resolution was slow, with most patients remaining symptomatic for more than 4 weeks. Normalization of liver tests took up to 6 months. Of the 27 patients with follow-up available, 7 (26%) still had abnormal serum enzymes or clinical, findings of liver disease beyond 6 months. There was 1 liver-related death; no patient required transplantation. Conclusion: TMP/SMZ hepatotoxicity has a distinct phenotype with a short latency and immuno-allergic features. The pattern of biochemical injury varies but is typically moderate to severe and slow in resolving. Thus, TMP/SMZ remains a common cause of DILI but is rarely fatal.

Table 1: Clinical Characteristics of TMP/SMZ Liver Injury N=31

Age (y) mean (min, max)44 (19,82)
Gender (males)17 (55%)
Race (self-reported)
White or Caucasian21(68%)
Black or African American7 (23%)
Other/Multiracial/Latino3 (10%)
Latency, mean days (min, max)
Drug to start of symptoms14 (0,56)
Drug to start DILI onset24 (6,74)
Signs and Symptoms
Itching24 (77%)
Jaundice23 (74%)
Rash20 (64%)
Fever20 (64%)
Nausea20 (64%)
Abdominal pain15 (48%)


Don C. Rockey - Consulting: Ono; Grant/Research Support: Sucampo, Sucampo, Hyperion, Actelion

William M. Lee - Consulting: Eli Lilly, Novartis; Grant/Research Support: Gilead, Roche, Vertex, BI, Anadys, BMS, merck; Speaking and Teaching: Merck

The following people have nothing to disclose: Lafaine Grant, Jiezhun Gu, Saleh Alqahtani


Immunohistochemical analysis of the carcinogenic process of cholangiocarcinoma cases epidemically developing among workers of a printing company in Japan

Yasunori Sato1, Shoji Kubo 2, Yasuni Nakanuma1

1Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Department of Hepato-Biliary-Pancreatic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan

Background: Recently, cholangiocarcinoma cases have epidemically developed among offset color proof-printing workers of a printing company in Japan. In this series, DNA damage of biliary epithelial cells due to inhalation of organic solvents including 1, 2-dichloropropane and/or dichloromethane (DCM) is supposed to be associated with the carcinogenic process. The metabolism of DCM proceeds through two pathways; a cytochrome P450 (CYP) 2E1 dependent pathway and a Thetaclass glutathione S-transferase (GST) T1-1-catalyzed pathway, where the latter has been implicated in carcinogenicity. Aim: This study was performed to examine the carcinogenic process of cholangiocarcinoma cases developing among workers of the printing company. Methods: Immunostaining of GST T1-1, CYP2E1, and gamma-H2AX was performed using formalinfixed, paraffin-embedded tissue sections of the cholangiocarcinoma cases of the printing company (n = 5). For comparison, tissue sections of cholangiocarcinoma associated with hepatolithiasis (n =16) as well as normal livers (n =10) were used. All cholangiocarcinoma cases were surgically resected, and were histologically associated with biliary intraepithelial neoplasia (BilIN). Gamma-H2AX was used as a marker of DNA double strand break. Results: The immunohistochemical expression of GST T1-1 was observed in biliary epithelial cells of normal biliary tract and hepatocytes. The expression of GST T1-1 was also observed in the foci of BilIN and invasive adenocarcinoma for all cholangiocarcinoma cases used. The immunohistochemical expression of CYP2E1 was observed in hepatocytes of normal livers, while normal biliary epithelial cells as well as BilIN and cholangiocarcinoma were typically negative. Gamma-H2AX was detected in foci of invasive adenocarcinoma in 4 of 5 cholangiocarcinoma cases of the printing company, and 3 cases further showed occasional expression of gamma-H2AX in non-neoplastic biliary epithelial cells as well as BilIN. In the cases of cholangiocarcinoma associated with hepatolithiasis, 7 of 16 cases showed the expression of gamma-H2AX in the invasive foci, whereas non-neoplastic biliary epithelial cells and BilIN were typically negative. Conclusions: These results suggest that the inhalation of organic solvents may act as a carcinogen for biliary epithelial cells by causing DNA damage through the GST T1-1-catalyzed pathway, and provide evidence that supports the causal relation between organic solvent inhalation and cholangiocarcinoma development in the patients.


The following people have nothing to disclose: Yasunori Sato, Shoji Kubo, Yasuni Nakanuma


Pharmacogenomics of Hepatic Transaminase Elevation following Initiation of Antiretroviral Therapy

Fausta A. Difah1, Daniel H. Johnson1, Paul Leger1, Eric S. Daar2, Roy M. Gulick3, Richard Haubrich4, Gregory K. Robbins5, Paul McLaren5, 6, David W. Haas1

1Vanderbilt Univ Sch of Med, Nashville, TN; 2Los Angeles Biomed Res Inst at Harbor-UCLA Medical Center, Torrance, CA; 3Weill Cornell Coll of Med, New York, NY; 4University of California San Diego, San Diego, CA; 5Harvard Univ, Boston, MA; 6Broad Institute of MIT and Havard, Cambridge, MA

Introduction: Drug induced hepatotoxicity is a commonly cited reason for withdrawal of FDA-approved drugs. Many studies have associated single nucleotide polymorphisms (SNPs) with antiretroviral therapy (ART) pharmacokinetics and/or toxicities, and ART-induced hepatotoxicity has been well described. We examined potential associations between SNPs and hepatic transaminase elevations (hereafter called hepatotoxicity) following initiation of ART in prospective clinical trials, including interactions between genetic and non-genetic factors. Methods: This retrospective cohort analysis utilized data from prospective clinical trials of the AIDS Clinical Trials Group (ACTG). The ART-naive ACTG studies A5202, A5142, A5095, and ACTG 384 were included in the analyses. Protocol-defined regimens comprised various 3- or 4-drug combinations of nucleoside analogues, non-nucleoside analogues and/or protease inhibitors. Genetic consent was obtained under ACTG protocol A5128. Genotyping utilized Illumina HumanHap 650Y or 1MDuo platforms. Unassayed SNPs were imputed. Hepatotoxicity was defined as grade 3 or higher elevations in transaminases (ALT or AST) (>5x upper limit of normal (ULN)) within the first 96 weeks of study enrollment. Subjects with baseline ALT or AST >160 units/L were censored from analyses. Logistic regression analyses were performed using the PLINK statistical software. Results: A total of 2485 subjects had genetic and clinical data available, of which 107 had incident grade 3 (n=73) or grade 4 (n=34) ALT or AST elevations on study; median time to event was 16 and 24 weeks respectively. Higher baseline AST and ALT values were associated with incident hepatotoxicity (p<0. 003). After adjusting for baseline ALT, body mass index, CD4 count, as well as sex, ACTG protocol, and top four genetic principal components, no SNP achieved genome-wide significance for association with hepatotoxicity (P<5x10-8). One of the 10 lowest P-value SNPs was rs9994893 in ARHGAP24 (Rho GTPase-activating protein 24, OR 3. 9 [2. 3 6. 7], P-value 4. 9 x 10-7). Conclusions: Among patients who initiated ART regimens in prospective clinical trials, no SNP was associated with incident hepatotoxicity at genome-wide significance. One of the lowest P-value SNPs was rs9994893 in ARHGAP24. Interestingly, in a recent Japanese genome-wide study of hepatotoxicity in childhood acute lymphoblastic leukemia or lymphoma, the lowest P value SNP was in ARHGAP24, although not rs9994893. A potential association in ARHGAP24 may be spurious but warrants further replication.


Eric S. Daar - Advisory Committees or Review Panels: Gilead; Consulting: Bristol Myers Squibb, Merck, ViiV, Janssen; Grant/Research Support: Abbott, Merck, Gilead, ViiV, Pfizer, Bristol Myers Squibb

Roy M. Gulick - Grant/Research Support: Janssen, Pfizer, ViiV

Richard Haubrich - Advisory Committees or Review Panels: Abbott, Bristol-Myers Squibb, Gilead Sciences, GSK, Merck, Tibotec and ViiV; Grant/Research Support: Abbott, GlaxoSmithKline, Merck, Pfizer and ViiV

Gregory K. Robbins - Grant/Research Support: Gilead

David W. Haas - Consulting: Merck; Grant/Research Support: Merck, Boehringer-Ingelheim, Bristol-Myers Squibb, Gilead

The following people have nothing to disclose: Fausta A. Ditah, Daniel H. Johnson, Paul Leger, Paul McLaren


An International Effort to Assess Hepatotoxicity Associated with Some Herbalife® Products

Dina Halegoua-De Marzio2, Maricruz Vega1, Joel Schifter Weber1, Guruprasad P Aithal3, Raul J. Andrade4, Fernando Bessone5, Einar Bjornsson6, Helgi K. Bjornsson6, Dominique G. Larrey7, Maribel Lizarzabal8, M. I. Lucena4, Inmaculada Medina Cáliz4 , Edgardo Mengual8, Sigurdur Olafsson6, Marie-Pierre Ripault7, Leonard B. Seeff, Jose Serrano10, Daniel Shouval11, C. Stephens4, Felix Stickel12, Victor J. Navarro1

1Einstein Medical Center, Philadelphia, PA; 2Thomas Jefferson University Hospital, Philadelphia, PA; 3The University of Nottingham, Nottingham, United Kingdom; 4Universidad de Malaga, Malaga, Spain; 5University of Rosario School of Medicine, Rosario, Argentina; 6Landspitali University Hospital, Reykjavik, Iceland; 7Saint-Eloi Hospital, Montpellier, France; 8Universitary Hospital of Maracaibo, Maracaibo, Venezuela, Bolivarian Republic of; 9Center for Drug Evaluation and Research (CDER), Silver Spring, MD; 10National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, MD; 11Hadassah University Hospital, Jerusalem, Israel; 12University of Berne,Berne, Switzerland

Background: Reports of hepatotoxicity attributed to various Dietary Supplements distributed by Herbalife® (DSH) exist. Cases of positive rechallenge suggest causation. Structured causality assessment of published and unpublished cases can support or refute the notion that some DSH have hepatotoxic potential. The Roussel Uclaf Causality Assessment Method (RUCAM), although not developed specifically for dietary supplements, has been used to assess causality in cases of suspected hepatotoxicity. Aim: To review cases of hepatotoxicity associated with DSH from the US, Europe, and South America, and assess causation with the RUCAM. Methods: 29 cases of suspected hepatotoxicity due to DSH (some published) were contributed by investigators in the US, Europe, and South America. 83 products were implicated in these cases. A standardized case report form was completed by the site investigator. Factors used in calculating the RUCAM, such as timing of onset and recovery, risk factors, exposure to other drugs, and exclusion of other causes for liver injury were ascertained. Results: Four cases occurred between1990-99, 13 between 2000-07, and 12 between 2008-12. The majority were female (22, 76%), median age 46 yrs (range 21 to 70). The products were used most commonly for weight loss and health promotion. Based on the RUCAM scale, 1 case was highly probable, 6 were probable, 9 were possible and 4 cases were considered unlikely to have liver injury due to DSH products. Four cases (13. 8%) had positive rechallenge. The remaining 9 cases (31%) had insufficient data to determine scores. For the 16 cases determined to have at least possible causal association, the median latency from ingestion to injury was 117 days (range 12 to 729). Most (15, 94%) were symptomatic at presentation. The most common symptoms were jaundice (69%), lethargy (50%), abdominal discomfort (31%), nausea (19%), and rash (19%). Median peak ALT was 1715 IU/L (range 231 to 2929), median peak alkaline phosphatase was 275. 5 IU/L (range 95 to 459), and the median peak bilirubin was 9. 6 mg/dL (range 0. 4 to 29. 0). The majority presented with hepatocellular liver injury (mean R ratio 18. 5). No patients in this series required liver transplantation; however, 1 liver-related death was reported in a patient with possible DSH hepatotoxicity. Conclusions: This analysis suggests that some DSH have hepatotoxic potential. Hepatotoxicity, typically hepatocellular, occurred more commonly in women and had a variable latency. Increased awareness is advised until this potential association between DSH or some of its ingredients and hepatotoxicity is further clarified.


Guruprasad P. Aithal - Advisory Committees or Review Panels: Aegerion Pharmaceuticals, Abbott, UK, LtD, Falk Pharma; Consulting: Biogen Idec, OTSUKA PHARMACEUTICAL EUROPE LTD, Basilea Pharmaceutica; Speaking and Teaching: Lilly

Fernando Bessone - Advisory Committees or Review Panels: Schering Plough, Gilead, Glaxo; Speaking and Teaching: Bristol Myers Squibb, Janssen, Bayer

Dominique G. Larrey - Board Membership: ROCHE, MSD, TIBOTEC/JANSSEN, ABBOTT, BOEHRINGER, BMS, GILEAD; Consulting: BAYER, SANOFI, PFIZER, SERVIER, HELSINN, MMV, BIAL, TEVA; Grant/Research Support: Roche, Boehringer, BMS, GILEAD; Independent Contractor: ABBOTT

Daniel Shouval - Advisory Committees or Review Panels: Scigen; Board Membership: Scigen; Consulting: Scigen

The following people have nothing to disclose: Dina Halegoua-De Marzio, Maricruz Vega, Joel Schifter Weber, Raul J. Andrade, Einar Bjornsson, Helgi K. Bjornsson, Maribel Lizarzabal, M. I. Lucena, Inmaculada Medina Cáliz, Edgardo Mengual, Sigurdur Olafsson, Marie-Pierre Ripault, Leonard B. Seeff, Jose Serrano, C. Stephens, Felix Stickel, Victor J. Navarro


Acute liver injury during cotreatment with levetiracetam and temozolomide

Tawfik R. Khoury Shmuel Chen, Meir Mizrahi

Gastroenterology and liver disease institute, Hadassah medical center, Jerusalem, Israel

Background: Drug-induced liver injury (DILI) accounts for approximately 10 percent of all cases of acute hepatitis. Temozolomide (TMZ) is an alkylating, anti-neoplastic agent used for the treatment of refractory anaplastic astrocytoma, glioblastoma multiforme (GBM). Levetiracetam (LEV) is an established as antiepileptic drug. When administered separately each drugs is considered to be relatively safe. however, LEV and TMZ are commonly used together in the treatment of brain malignancies. Aim: To determine the rate of liver injury due to combination therapy with TMZ and LEV. Methods: We retrospectively compared records of patients with and without the combination of TMZ and LEV in our institution (2007-2013). Data included demographics, liver injury reflected by liver enzymes and patients outcome Results: 32 patients with combination therapy (group A) were compared to 73 age/sex matched patients with monotherapy (group B). Groups were similar in underlying indication for treatment, There were 64 men and 52 women, mean age 53 ±14 vs. 51 ± 19 years (A vs. B, P=NS). Indications for treatment were: Astrocytoma 50. 4% vs. GBM 49. 6% (P=NS), body surface area was 1. 92±0. 2 vs. 1. 82±0. 2 (P=NS comparing group A vs. group B), O6-methylguanine methyltransferase (MGMT) in the brain tissue was 28% vs. 16. 4% (P=0. 2, comparing group A vs. B), no difference in daily dose of LEV 1. 71±0. 6G vs. 1. 82±0. 99G (P=NS) comparing group A vs. B, as for liver injury, the initial levels of liver enzymes were similar between group A and B ( 30 vs. 26 for ALT, 24 vs. 27 for AST, 79 vs. 72 for ALK-P, 62 vs. 68 for GGT and bilirubin levels 6. 41mmol/ml P=NS) but comparing liver enzymes during dual treatment was different with 241 VS. 26. 5 for ALT, 118 vs. 26 for AST, 164 vs. 70 for ALK-P, 228 vs. 62 for GGT and 46 vs. 8. 6 for bilirubin levels P=0. 008), on liver US 4/32 in group A vs. 3/73 had evidence of fatty liver 19% vs. 4% p=0. 01). One patient in group A died due to liver failure compared to no patient in group B. conclusion: combination therapy with TMZ and LEV can provoke liver injury and even death, based on this findings we suggest that every patient undergoing dual treatment will be screened for liver enzymes every 2 months.


The following people have nothing to disclose: Tawfik R. Khoury, Shmuel Chen, Meir Mizrahi


The analgesic flupirtine may induce severe hepatocellular liver injury in women

Ahanasia Ziagaki, Stephan Boehm, Chrisfin Felkel, Eleni Koukouliofi, Balazs Fueloep, Albrecht Boehlig, Adam Herber, Johannes Wiegand, Thomas Berg, Florian van Boemmel

Hepatology Section, University Hospital Leipzig, Leipzig, Germany

Background: Flupirtine, a central acting non-opioid analgesic used in many countries, was recently described to induce drug induced liver injury (DILI) in some patients. We have studied clinical courses of flupirtine associated DILI and compared it to DILI caused by other drugs. Methods: All patients from one German center who were hospitalized between 2010 and 2013 for DILI were retrospectively analyzed. DILI was defined by elevation of ALT>3x the upper limit of normal (ULN) and history of drug intake within the past 6 months after exclusion of viral, autoimmune and metabolic liver diseases. Age, weight, sex, levels of ALT, bilirubin, prothrombin time rates and clinical outcomes were compared between patients with DILI associated with flupirtine or with other drugs at days 0, 3, 7 and 14 after admission to hospital. Results: A total of 51 patients were identified. Four patients were excluded because of intended intoxication with high dosed paracetamol. DILI was very likely associated with flupirtine in 18 (38%) and with other drugs in 29 (62%) patients. Patients in both groups had similar age (59±13 [range, 35-80] vs. 56±17[20-84] years, p=n. s. ). Patients in the flupirtine group were mostly female (17/1 vs. 16/13, p=0. 01) and had lower body weight compared to the control group (69±12[50-90] vs. 80±16[49-124] kg; p=0. 03). The mean time between onset of symptoms and admission to hospital was 10±7[0-30] and 6±8[0-30] days (p=n. s). Mean ALT levels were similar in both groups at days 0, 3, 7 and 14 of hospitalization (32±31[3-90] vs. 39±58[3-196], 24. 5±23. 3[1. 5-72] vs. 28. 4±28 [2. 2-93], 8. 3±8. 5[1. 4-24. 6] vs. 6. 5±5[1. 2-20] and 3. 3±4[0. 8-12] vs. 3. 2±3. 7[0. 7-13. 5] x ULN, respectively; p=n. s. ). Bilirubin levels were initially higher and increased further until day 14 in the flupirtine group (15. 1±9. 4[2. 6-29] vs. 6. 7±8. 8[0. 1-33], 16. 2±8. 9[3. 4-32. 5] vs. 9. 1±9. 9[0. 4-33], 19. 1±7. 1[3. 2-27. 9] vs. 4. 2±5. 8[0. 316. 2] and 12. 4±8[0. 4-23] vs. 1. 8±2. 8[0. 3-10. 2] x ULN; p=0. 06, 0. 049, <0. 000 and 0. 007, respectively). Mean prothrombine time rates were lower in the flupirtine group between days 0 and 14 (47±21[47-87] vs. 84±29[25-124], 42±21[2081]vs. 68±15[50-97], 61 ±32[24-119] vs. 98±22[44-131] and 73±32[13-113] vs. 101 ±17[70-128]; p=<0. 000, 0. 002, 0. 004, 0. 047, respectively). In both groups 12 and 10 patients received treatment with prednisone or acetylcysteine (p=0. 033). Two patients in the flupirtin group underwent liver transplantation. No patient died from DILI. Conclusion: Flupirtine may lead to severe courses of hepatocellular liver injury and liver failure, especially in women. Monitoring ALT levels may be recommendable for patients receiving flupirtine.


Thomas Berg - Advisory Committees or Review Panels: Gilead, BMS, Roche, Tibotec, Vertex, Jannsen, Novartis, Abbott, Merck; Consulting: Gilead, BMS, Roche, Tibotec; Vertex, Janssen; Grant/Research Support: Gilead, BMS, Roche, Tibotec; Vertex, Jannssen, Schering Plough, Boehringer Ingelheim, Novartis; Speaking and Teaching: Gilead, BMS, Roche, Tibotec; Vertex, Janssen, Schering Plough, Novartis, Merck, Bayer

Florian van Boemmel - Advisory Committees or Review Panels: Roche Pharma; Board Membership: Gilead Sciences; Grant/Research Support: Gilead Sciences, Roche Pharma, BMS; Speaking and Teaching: Gilead Sciences, Roche Pharma, BMS, MSD, Janssen-Cilag, Siemens

The followinq people have nothing to disclose: Athanasia Ziagaki, Stephan Boehm, Christin Felkel, Eleni Koukoulioti, Balazs Fueloep, Albrecht Boehlig, Adam Herber, Johannes Wiegand


The role of hepatobiliary transporters in toxic hepatitis

Jorge A. López-Velázquez1, Varenka J. Barbero-Becerra1, Vicente Sánchez- Valle1, Ylse Gutiérrez-Grobe1, Norberto C. ChavezTapia1, José M. Ramίrez-Jaramillo2, Fredy Chablé-Montero2, Misael N. Uribe-Esquivel1, Nahum Méndez-Sanchéz1

1Liver Research Unit, Medica Sur Clinic & Foundation, Mexico City Mexico; 2Pathology Research Unit, Medica Sur Clinic & Foundation, Mexico City, Mexico

Background. Drug compounds have been shown to exert certain effects on pathophysiological mechanisms involving hepatobiliary transporters in toxic hepatitis (TH) leading to liver dysfunction. Localization and individualized substrate specificities have been proposed to be crucial in TH development. Aims. Evaluate the expression of MDR1, MRP2, MRP3 and BSEP and their association with drug type administered in liver biopsies of patients with TH. Methods. We analyzed data from 16 patients with clinical, biochemical and histopathological diagnosis of TH and 16 without TH (Non-TH). TH patients presented a multidrug-therapy pattern which includes principally immunosuppressive, analgesics and antibiotic drugs. Hepatobiliary transporter expression was analyzed by quantitative real-time PCR and immunohistochemistry. RUCAM score was used to associate a specific expression pattern for a particular drug. Results. Sixteen patients (7 women and 9 men) with a mean age of 43. 6 years. mRNA expression was significantly lower in TH group than in Non-TH group in MRP2 (p< 0. 03), MRP3 (p< 0. 05) and BSEP (p< 0. 0009). In protein expression MDR1 and MRP2 showed a 100% positivity expression in both groups; however in terms of intensity, MDR1 expression was higher in TH group (75%) than in Non-TH group (45%) (P<0. 01), opposite to MRP2 expression which was lower In TH group (31%) than in Non-TH group (45%) (P<0. 01). Regarding MRP3, TH group showed a 56% of positivity expression; meanwhile it was not detectable in Non-TH group. Finally, BSEP presented a lower positivity expression (50%) in comparison to Non-TH group (95%)(Figure). Conclusion. In TH patients there was a down-regulation in BSEP, an increased expression in MRP3 and apparently no change in MRP2 and MDR1 expression. One of the main pathophysiological mechanisms involves the modulation of hepatobiliary transporter expression by drugtype administered in TH. Further studies which precise and determine the molecular mechanism involved in the regulation of TH development and drug therapy in order to design therapeutic options that become applicable to improve the prognostic in these patients.



The following people have nothing to disclose: Jorge A. López-Velázquez, Varenka J. Barbero-Becerra, Vicente Sánchez- Valle, Ylse Gutiérrez-Grobe, Norberto C. Chavez-Tapia, José M. Ramírez-Jaramillo, Fredy Chablé-Montero, Misael N. Uribe-Esquivel, Nahum Méndez-Sanchéz