- Email: [email protected]
sqstm1 in a drug-induced model of hepatotoxicity: a novel role beyond autophagy
POSTER PRESENTATIONS Conclusions: We showed that macrophage activation markers sMR and sCD163 are elevated in patients with liver injury following acetaminophen overdose and associated with the degree of liver injury. However, only sCD163 showed an association with prognosis in ALI patients. FRI-438 Characterisation of the immune response in patients with drug induced liver injury C. Bergamo1, G. Germani1, D. Bizzaro1, C. Frasson2,3, G. Basso2, A. Ferrarese1, A. Zanetto1, S. Shalaby1, I. Bortoluzzi1, M. Senzolo1, M. Gambato1, F.P. Russo1, P. Burra1. 1Department of Surgery, Oncology and Gastroenterology, Padova University Hospital, Multivisceral Transplant Unit; 2Departement of Woman and Child Health, University of Padova, Haemato-Oncology Laboratory; 3Fondazione Città della Speranza, Istituto di Ricerca Pediatrica, Padova, Italy E-mail: [email protected] Background and Aims: Drug Induced Liver Injury (DILI) represents the leading cause of acute liver failure and liver transplantation in Western countries, however pathogenesis, features and outcomes of the disease are not still well defined. The aim of the study was to assess the clinical presentation, outcome and characterisation of the immune pattern in patients with DILI. Methods: Ppatients admitted at Multivisceral Transplant Unit of Padova University Hospital for suspected DILI, from 2013 to October 2016, were included. Demographic and clinic characteristics and transplant free survival were evaluated in all patients. In a subgroup of patients mononuclear cells from peripheral blood were isolated by a density gradient centrifugation with Ficoll. Isolated cells were analyzed by flow cytometry for the following populations: monocytes (immature, early and mature), B lymphocytes, T helper and T cytotoxic lymphocytes. Data were compared with healthy controls. Results: 31 DILI patients were evaluated, 55% women with a median age of 46.7 years (range 33–70). The most frequent class of drugs involved in DILI was antimicrobials (42%). Transplant free survival rate at 1 year was 73%, with 16% patients who underwent liver transplantation. The cytofluorimetric analysis, performed in 7 DILI patients and in 4 healthy controls, showed a significant decrease of B lymphocytes in DILI patients compared to healthy controls (2.4% ± 0.9 vs. 7.9 ± 0.5%, p = 0.01). Patients with DILI due to antimicrobials showed a significant decrease of B lymphocytes (0.6 ± 0.4% vs. 7.9 ± 0.4%; p = 0.004) and of T Helper (8.3 ± 7.7% vs. 20.9 ± 12.2%; p = 0.05) compared to patients with DILI due to other drugs. Male patients showed a higher percentage of immature and early monocytes with pro-inflammatory properties (12.9 ± 3.3% vs. 2.7 ± 2.7%, p = 0.007 and 9.7 ± 1.6% vs. 3.0 ± 2.9%, p = 0.03 respectively) and a higher percentage of memory T cells (CD7-CD4+) (22.5 ± 2.8% vs. 3.8 ± 1.7%, p < 0.001) compared with female patients. No differences were detected, in terms of immune response, according different pattern of liver injury. Conclusions: The most frequent class of agents involved in DILI was antimicrobials, which seem to induce a marked immunodeficient status, especially towards B lymphocytes, suggesting a possible cytotoxic T cell-mediated liver damage. Despite the incidence of DILI was higher in women, male patients seems to have a more enhanced pro-inflammatory status. FRI-439 Drug-induced effects on mtDNA homeostasis in HepaRG cells and modulation by steatosis D. Le Guillou1, S. Bucher1, D. Hoët2, G. Labbe2, B. Fromenty1. 1INSERM U991, Université de Rennes 1, Rennes; 2SANOFI, Investigative Toxicology, Alfortville, France E-mail: [email protected] Background and Aims: Although a major mechanism of druginduced liver injury (DILI) is mitochondrial dysfunction, little is known regarding the deleterious effects of drugs on mitochondrial
DNA (mtDNA), which encodes 13 polypeptides of the respiratory chain including ND1 and COX2. However, some studies suggest that mitochondrial accumulation of drugs might favor the disturbance of mtDNA maintenance. Moreover, there is no information concerning the impact of steatosis on drug-induced impairment of mtDNA homeostasis. We have recently shown that HepaRG cells incubated with some fatty acids could be a valuable model to study druginduced hepatotoxicity in the context of NAFLD (Michaut et al., Toxicol Appl Pharmacol 2016). Nine drugs known (or suspected) to accumulate within mitochondria thanks to the membrane potential (i.e. OXPHOS uncouplers) were selected: amiodarone, atorvastatin, lovastatin, imipramine, perhexiline, troglitazone, ritonavir, terbinafine and carbamazepine. Methods: Steatotic and non-steatotic HepaRG cells were treated for 14 days with these drugs and their effects on mtDNA replication, transcription and translation were investigated. Notably, we performed preliminary investigations to determine the IC50 for cytotoxicity (ATP levels) for all drugs and to ascertain that our cell model could detect zalcitabine-induced mtDNA depletion and linezolidinduced inhibition of mtDNA translation. Results: By using drug concentrations that did not induce major cytotoxicity, we found that most of the selected drugs enhanced mtDNA levels in non-steatotic HepaRG cells while this effect was less evident in steatotic cells. These effects were in general paralleled by variations of the mRNA expression of ND1, COX2, COX4 and PGC1a/b. For ritonavir, these effects were also associated with a significant increase in TFAM and NRF1 mRNA expression. Interestingly, we found that ritonavir significantly reduced the protein expression of ND1 and COX2 but not of COX4 (suggesting a specific alteration of mtDNA translation) and strongly decreased complex I activity. However, these latter effects were less pronounced in steatotic HepaRG cells, which present lower CYP3A4 activity. Conclusions: Drugs that uncouple OXPHOS consistently trigger some mitochondrial biogenesis, which is occasionally dampened by steatosis. Ritonavir, which impairs mtDNA translation, induces a more robust mitochondrial biogenesis that was insufficient to alleviate mitochondrial dysfunction. FRI-440 p62/sqstm1 in a drug-induced model of hepatotoxicity: a novel role beyond autophagy F. Alegre1,2, M. Polo1,2, A. Marti-Rodrigo1, A.B. Moragrega1, J.V. Esplugues1,2,3, A. Blas-Garcia1,2,3, N. Apostolova1,3. 1Farmacologia, Facultad de Medicina, Universidad de Valencia; 2FISABIO; 3CIBERehd, Valencia, Spain E-mail: [email protected] Background and Aims: SQSTM1/p62 is a stress-induced scaffold protein involved in multiple cellular processes such as autophagy, inflammation and redox homeostasis. It has been associated with several liver diseases, including NASH and cancer. We have previously described a complex effect of the antiretroviral drug Efavirenz (EFV) on hepatic cells, manifested in mitochondrial dysfunction and ER stress and paralleled by enhanced autophagy. We aimed to evaluate the role of p62 in this complex model of drug-induced liver injury. Methods: Hep3B cells, primary human hepatocytes and cells with reduced mitochondrial respiratory function (Hep3B rho0) were exposed to clinically relevant concentrations (10, 25 and 50 μM) of EFV (4–48 h). Key experiments were carried out in Hep3B cells transiently transfected with p62 or CHOP (an ER stress-inducible transcription factor) siRNA. Results: Despite activation of autophagic flux, protein expression of p62 was increased in EFV-treated hepatic cells, due to enhanced p62 mRNA transcription. This increase was substantially less marked in rho° Hep3B cells. Of note, EFV produced an increase of p62 content in both mitochondrial and cytosolic extracts, but only in the cytosolic fraction when cells were exposed to pharmacologically induced ER stress (Thapsigargin). The opposite was detected with Rotenone (ETC
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POSTER PRESENTATIONS complex I inhibitor), which produced an increase in p62 only in the mitochondrial fraction. p62 levels were mediated through the transcription factor CHOP, while other well-known regulators (NFkB and Nrf2) are not involved. Western blot and RT-PCR analysis revealed that EFV enhanced expression of the NLRP3 inflammasome. Silencing of the p62 gene enhanced EFV-induced mRNA expression of the NLRP3 inflammasome and rendered hepatocytes more susceptible to EFV-induced mitochondrial damage, made evident by enhanced mitochondrial superoxide production; however, p62 silencing had no effect on LC3 expression, a key marker of autophagy. Conclusions: CHOP-induced p62 plays an autophagy-independent and protective (alleviating mitochondrial oxidative stress and exerting an anti-inflammatory action) role in an in vitro model of drug-induced hepatotoxicity characterized by mitochondrial dysfunction and ER-stress. These findings are of potential clinical relevance as they may help to understand the off-target hepatic effects of pharmacological agents. FRI-441 The S-ademetionine role in the prevention of liver injury in patients with acute leukemias in the polychemotherapy dynamics I. Skrypnyk1, G. Maslova1. 1Internal Medicine, Ukrainian Medical Stomatological Academy, Poltava, Ukraine E-mail: [email protected] Background and Aims: The polychemotherapy (PCT) is associated with a high risk of hepatotoxic reactions, that cause a review of the treatment tactics of patients with acute leukemia (AL), reduction the dose of cytotoxic drugs, increasing the interval between cycles, which reduces the effectiveness of a specific treatment. The aim – to assess the effectiveness of S-ademetionine (S-AME) in the prevention of medication liver injury in patients with AL in PCT dynamics. Methods: The study involved 82 patients with AL (61–acute myeloid leukemia (AML), 21–acute lymphoblastic leukemia (ALL)), mean age 17–72 years, men 48 (58.5%), 34 women (41.5%). The study included patients with newly diagnosed AL, ECOG I-II. The activity of alanine aminotransferase (ALT), aspartate (AST) aminotransferase, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGT) was determined before chemotherapy and after two induction of remission cycles according to the protocols of ALL and AML treatment. Depending on the assigned treatment, the patients were divided into 2 groups: I (n = 42) – PCT + S-ademetionine (S-AME) 800 mg/day; II (n = 40) – control – PCT. Results: Before chemotherapy in AL pts of group I the ALT activity was 65.11 ± 15.08 U/L, AST – 53.1 ± 12.03 U/L, alkaline phosphatase – 232.2 ± 22.21 U/L, GGT – 65.2 ± 19.26 U/L, LDH – 815.4 ± 91.07 U/L, in the group II ALT activity was 61.9 ± 16.18 U/L, AST– 59.3 ± 14.13 U/L, alkaline phosphatase – 262.1 ± 23.01 U/L, GGT– 71.04 ± 21.76 U/L, LDH– 804.5 ± 81.27 U/L. After 2 courses of chemotherapy in pts of group I, who received the S-AME, ALT activity decreased in 2.2 times (29.9 ± 13.3 U/L), AST – in 2.3 times (22.7 ± 12.3 U/L), ALP – in 1.2 times (187.1 ± 17.6 U/L), GGT – in 1.3 times (49.5 ± 11.7 U/L), LDH – in 1.7 times (421.9 ± 51.1 U/L) ( p < 0.05), reaching the value of the norm. The hepatotoxicity was observed in 5 (11.9%) pts, which was associated with low efficiency of chemotherapy. In group II pts didn’t get S-AME and the development of hepatotoxicity was observed in 17 (42.5%) pts. In general, the ALT activity in this group was 58.08 ± 16.14 U/L, AST – 41.2 ± 15.15 U/L, ALP – 244.9 ± 23.15 U/L, GGT – 53.5 ± 17.1 U/L, LDH – 641.3 ± 51.82 U/L. Hepatotoxic reactions in the comparison groups were characterized by the development of cytolytic and cholestatic syndromes of moderate activity. Conclusions: Thus, in order to prevent the development of hepatotoxic reactions in the polychemotherapy dynamics in pts with AL S-AMe 800 mg/day can be recommended.
FRI-442 Reprogramming of human umbilical cord-derived mesenchymal stromal cells into hepatocyte-like cells by over-expression of hepatocyte nuclear factor 1-alpha K. Buyl1, R.M. Rodrigues1, V. Rogiers1, J. De Kock1, T. Vanhaecke1 and In Vitro Toxicology & Dermato-Cosmetology. 1Department of In Vitro Toxicology & Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium E-mail: [email protected] Background and Aims: Stem cell technology holds great promise for the generation of human liver-based in vitro models to screen out hepatotoxic drug candidates. Recent research showed that human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) could represent a valuable cell population to generate human hepatocyte-like cells as they already express several key liver-specific transcription factors as well as hepatic progenitor markers. However, they still lack the hepatocyte nuclear factors 1-alpha (HNF1a) and 4alpha (HNF4a), indispensable for their reprogramming towards hepatocyte-like cells. Therefore, in this study, we aimed to investigate the influence of HNF1a lentiviral over-expression on the phenotype of hUC-MSCs. Methods: hUC-MSCs were transduced with HNF1a-encoding lentiviral vector using a multiplicity of infection of 1. Further, HNF1atransduced hUC-MSCs were cultured in hepatogenic differentiation medium during 21 days. Undifferentiated and differentiated hUCMSCs were used as control conditions. Whole genome microarray analysis was performed using Affymetrix microarray technology. Analysis of the transcriptomics results was carried out by using the Partek Genomics Suite and the Affymetrix Transcriptome Analysis Console. Gene expression was compared to that of human primary hepatocytes (HuHep). Statistical analysis was performed using twoway ANOVA followed by a bonferroni post hoc test (p < 0.05). Results: We found that the expression of the nuclear receptor retinoid X receptor (RXR) gamma and the nuclear transcription factor HNF4a, in HNF1a-transduced hUC-MSCs, were significantly upregulated compared to the control conditions. This expression was even higher than found in HuHep. The same was observed for the uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A family, important phase II biotransformation enzymes. Further, a significant upregulation was observed compared to the control conditions for the serum proteins alpha-foetoprotein (AFP), alpha1-antitrypsin (A1-AT), the phase I biotransformation enzymes cytochrome P450 (CYP) 1A2 and CYP2A6 and the drug transporter multidrug resistance protein (MDR) 1. Conclusions: We believe that this strategy might therefore be suitable to generate hepatocyte-like cells necessary for the development of functional human liver-based in vitro models for pharmacotoxicological purposes. However, additional experiments, including liver functionality assays are required to further characterize these hUC-MSCHNF1a-derived hepatocyte-like cells. FRI-443 Free cholesterol accumulation in liver sinusoidal endothelial cells exacerbates acetaminophen hepatotoxicity in mice, T. Teratani1, K. Tomita2, T. Suzuki1, H. Furuhashi2, R. Irie3, S. Hida4, Y. Okada2, C. Kurihara2, H. Ebinuma1, N. Nakamoto1, H. Saito5, T. Hibi1, S. Miura2, R. Hokari2, T. Kanai1. 1Department of Internal Medicine, Keio University School of Medicine, Tokyo; 2Department of Internal Medicine, National Defense Medical College, Saitama; 3Department of Pathology, National Center for Child Health and Development, Tokyo; 4Department of Molecular Oncology, Institute for Biomedical Sciences, Shinshu University Graduate School of Medicine, Matsumoto; 5Graduate School of Pharmeceutical Sciences, Keio University Faculty of Pharmacy, Tokyo, Japan E-mail: [email protected] Background and Aims: Acute acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the USA. As
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DNA (mtDNA), which encodes 13 polypeptides of the respiratory chain including ND1 and COX2. However, some studies suggest that mitochondrial accumulation of drugs might favor the disturbance of mtDNA maintenance. Moreover, there is no information concerning the impact of steatosis on drug-induced impairment of mtDNA homeostasis. We have recently shown that HepaRG cells incubated with some fatty acids could be a valuable model to study druginduced hepatotoxicity in the context of NAFLD (Michaut et al., Toxicol Appl Pharmacol 2016). Nine drugs known (or suspected) to accumulate within mitochondria thanks to the membrane potential (i.e. OXPHOS uncouplers) were selected: amiodarone, atorvastatin, lovastatin, imipramine, perhexiline, troglitazone, ritonavir, terbinafine and carbamazepine. Methods: Steatotic and non-steatotic HepaRG cells were treated for 14 days with these drugs and their effects on mtDNA replication, transcription and translation were investigated. Notably, we performed preliminary investigations to determine the IC50 for cytotoxicity (ATP levels) for all drugs and to ascertain that our cell model could detect zalcitabine-induced mtDNA depletion and linezolidinduced inhibition of mtDNA translation. Results: By using drug concentrations that did not induce major cytotoxicity, we found that most of the selected drugs enhanced mtDNA levels in non-steatotic HepaRG cells while this effect was less evident in steatotic cells. These effects were in general paralleled by variations of the mRNA expression of ND1, COX2, COX4 and PGC1a/b. For ritonavir, these effects were also associated with a significant increase in TFAM and NRF1 mRNA expression. Interestingly, we found that ritonavir significantly reduced the protein expression of ND1 and COX2 but not of COX4 (suggesting a specific alteration of mtDNA translation) and strongly decreased complex I activity. However, these latter effects were less pronounced in steatotic HepaRG cells, which present lower CYP3A4 activity. Conclusions: Drugs that uncouple OXPHOS consistently trigger some mitochondrial biogenesis, which is occasionally dampened by steatosis. Ritonavir, which impairs mtDNA translation, induces a more robust mitochondrial biogenesis that was insufficient to alleviate mitochondrial dysfunction. FRI-440 p62/sqstm1 in a drug-induced model of hepatotoxicity: a novel role beyond autophagy F. Alegre1,2, M. Polo1,2, A. Marti-Rodrigo1, A.B. Moragrega1, J.V. Esplugues1,2,3, A. Blas-Garcia1,2,3, N. Apostolova1,3. 1Farmacologia, Facultad de Medicina, Universidad de Valencia; 2FISABIO; 3CIBERehd, Valencia, Spain E-mail: [email protected] Background and Aims: SQSTM1/p62 is a stress-induced scaffold protein involved in multiple cellular processes such as autophagy, inflammation and redox homeostasis. It has been associated with several liver diseases, including NASH and cancer. We have previously described a complex effect of the antiretroviral drug Efavirenz (EFV) on hepatic cells, manifested in mitochondrial dysfunction and ER stress and paralleled by enhanced autophagy. We aimed to evaluate the role of p62 in this complex model of drug-induced liver injury. Methods: Hep3B cells, primary human hepatocytes and cells with reduced mitochondrial respiratory function (Hep3B rho0) were exposed to clinically relevant concentrations (10, 25 and 50 μM) of EFV (4–48 h). Key experiments were carried out in Hep3B cells transiently transfected with p62 or CHOP (an ER stress-inducible transcription factor) siRNA. Results: Despite activation of autophagic flux, protein expression of p62 was increased in EFV-treated hepatic cells, due to enhanced p62 mRNA transcription. This increase was substantially less marked in rho° Hep3B cells. Of note, EFV produced an increase of p62 content in both mitochondrial and cytosolic extracts, but only in the cytosolic fraction when cells were exposed to pharmacologically induced ER stress (Thapsigargin). The opposite was detected with Rotenone (ETC
Journal of Hepatology 2017 vol. 66 | S333–S542
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POSTER PRESENTATIONS complex I inhibitor), which produced an increase in p62 only in the mitochondrial fraction. p62 levels were mediated through the transcription factor CHOP, while other well-known regulators (NFkB and Nrf2) are not involved. Western blot and RT-PCR analysis revealed that EFV enhanced expression of the NLRP3 inflammasome. Silencing of the p62 gene enhanced EFV-induced mRNA expression of the NLRP3 inflammasome and rendered hepatocytes more susceptible to EFV-induced mitochondrial damage, made evident by enhanced mitochondrial superoxide production; however, p62 silencing had no effect on LC3 expression, a key marker of autophagy. Conclusions: CHOP-induced p62 plays an autophagy-independent and protective (alleviating mitochondrial oxidative stress and exerting an anti-inflammatory action) role in an in vitro model of drug-induced hepatotoxicity characterized by mitochondrial dysfunction and ER-stress. These findings are of potential clinical relevance as they may help to understand the off-target hepatic effects of pharmacological agents. FRI-441 The S-ademetionine role in the prevention of liver injury in patients with acute leukemias in the polychemotherapy dynamics I. Skrypnyk1, G. Maslova1. 1Internal Medicine, Ukrainian Medical Stomatological Academy, Poltava, Ukraine E-mail: [email protected] Background and Aims: The polychemotherapy (PCT) is associated with a high risk of hepatotoxic reactions, that cause a review of the treatment tactics of patients with acute leukemia (AL), reduction the dose of cytotoxic drugs, increasing the interval between cycles, which reduces the effectiveness of a specific treatment. The aim – to assess the effectiveness of S-ademetionine (S-AME) in the prevention of medication liver injury in patients with AL in PCT dynamics. Methods: The study involved 82 patients with AL (61–acute myeloid leukemia (AML), 21–acute lymphoblastic leukemia (ALL)), mean age 17–72 years, men 48 (58.5%), 34 women (41.5%). The study included patients with newly diagnosed AL, ECOG I-II. The activity of alanine aminotransferase (ALT), aspartate (AST) aminotransferase, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGT) was determined before chemotherapy and after two induction of remission cycles according to the protocols of ALL and AML treatment. Depending on the assigned treatment, the patients were divided into 2 groups: I (n = 42) – PCT + S-ademetionine (S-AME) 800 mg/day; II (n = 40) – control – PCT. Results: Before chemotherapy in AL pts of group I the ALT activity was 65.11 ± 15.08 U/L, AST – 53.1 ± 12.03 U/L, alkaline phosphatase – 232.2 ± 22.21 U/L, GGT – 65.2 ± 19.26 U/L, LDH – 815.4 ± 91.07 U/L, in the group II ALT activity was 61.9 ± 16.18 U/L, AST– 59.3 ± 14.13 U/L, alkaline phosphatase – 262.1 ± 23.01 U/L, GGT– 71.04 ± 21.76 U/L, LDH– 804.5 ± 81.27 U/L. After 2 courses of chemotherapy in pts of group I, who received the S-AME, ALT activity decreased in 2.2 times (29.9 ± 13.3 U/L), AST – in 2.3 times (22.7 ± 12.3 U/L), ALP – in 1.2 times (187.1 ± 17.6 U/L), GGT – in 1.3 times (49.5 ± 11.7 U/L), LDH – in 1.7 times (421.9 ± 51.1 U/L) ( p < 0.05), reaching the value of the norm. The hepatotoxicity was observed in 5 (11.9%) pts, which was associated with low efficiency of chemotherapy. In group II pts didn’t get S-AME and the development of hepatotoxicity was observed in 17 (42.5%) pts. In general, the ALT activity in this group was 58.08 ± 16.14 U/L, AST – 41.2 ± 15.15 U/L, ALP – 244.9 ± 23.15 U/L, GGT – 53.5 ± 17.1 U/L, LDH – 641.3 ± 51.82 U/L. Hepatotoxic reactions in the comparison groups were characterized by the development of cytolytic and cholestatic syndromes of moderate activity. Conclusions: Thus, in order to prevent the development of hepatotoxic reactions in the polychemotherapy dynamics in pts with AL S-AMe 800 mg/day can be recommended.
FRI-442 Reprogramming of human umbilical cord-derived mesenchymal stromal cells into hepatocyte-like cells by over-expression of hepatocyte nuclear factor 1-alpha K. Buyl1, R.M. Rodrigues1, V. Rogiers1, J. De Kock1, T. Vanhaecke1 and In Vitro Toxicology & Dermato-Cosmetology. 1Department of In Vitro Toxicology & Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium E-mail: [email protected] Background and Aims: Stem cell technology holds great promise for the generation of human liver-based in vitro models to screen out hepatotoxic drug candidates. Recent research showed that human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) could represent a valuable cell population to generate human hepatocyte-like cells as they already express several key liver-specific transcription factors as well as hepatic progenitor markers. However, they still lack the hepatocyte nuclear factors 1-alpha (HNF1a) and 4alpha (HNF4a), indispensable for their reprogramming towards hepatocyte-like cells. Therefore, in this study, we aimed to investigate the influence of HNF1a lentiviral over-expression on the phenotype of hUC-MSCs. Methods: hUC-MSCs were transduced with HNF1a-encoding lentiviral vector using a multiplicity of infection of 1. Further, HNF1atransduced hUC-MSCs were cultured in hepatogenic differentiation medium during 21 days. Undifferentiated and differentiated hUCMSCs were used as control conditions. Whole genome microarray analysis was performed using Affymetrix microarray technology. Analysis of the transcriptomics results was carried out by using the Partek Genomics Suite and the Affymetrix Transcriptome Analysis Console. Gene expression was compared to that of human primary hepatocytes (HuHep). Statistical analysis was performed using twoway ANOVA followed by a bonferroni post hoc test (p < 0.05). Results: We found that the expression of the nuclear receptor retinoid X receptor (RXR) gamma and the nuclear transcription factor HNF4a, in HNF1a-transduced hUC-MSCs, were significantly upregulated compared to the control conditions. This expression was even higher than found in HuHep. The same was observed for the uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A family, important phase II biotransformation enzymes. Further, a significant upregulation was observed compared to the control conditions for the serum proteins alpha-foetoprotein (AFP), alpha1-antitrypsin (A1-AT), the phase I biotransformation enzymes cytochrome P450 (CYP) 1A2 and CYP2A6 and the drug transporter multidrug resistance protein (MDR) 1. Conclusions: We believe that this strategy might therefore be suitable to generate hepatocyte-like cells necessary for the development of functional human liver-based in vitro models for pharmacotoxicological purposes. However, additional experiments, including liver functionality assays are required to further characterize these hUC-MSCHNF1a-derived hepatocyte-like cells. FRI-443 Free cholesterol accumulation in liver sinusoidal endothelial cells exacerbates acetaminophen hepatotoxicity in mice, T. Teratani1, K. Tomita2, T. Suzuki1, H. Furuhashi2, R. Irie3, S. Hida4, Y. Okada2, C. Kurihara2, H. Ebinuma1, N. Nakamoto1, H. Saito5, T. Hibi1, S. Miura2, R. Hokari2, T. Kanai1. 1Department of Internal Medicine, Keio University School of Medicine, Tokyo; 2Department of Internal Medicine, National Defense Medical College, Saitama; 3Department of Pathology, National Center for Child Health and Development, Tokyo; 4Department of Molecular Oncology, Institute for Biomedical Sciences, Shinshu University Graduate School of Medicine, Matsumoto; 5Graduate School of Pharmeceutical Sciences, Keio University Faculty of Pharmacy, Tokyo, Japan E-mail: [email protected] Background and Aims: Acute acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the USA. As
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