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Drug-induced liver injury (DILI) development is commonly associated with acetaminophen (APAP) overdose, where glutathione scavenging leads to mitochondrial dysfunction and hepatocyte death. DILI is a severe disorder without effective late-stage treatment, since N-acetyl cysteine must be administered 8 h after overdose to be efficient. Ammonia homeostasis is altered during liver diseases and, during DILI, it is accompanied by decreased glycine N-methyltransferase (GNMT) expression and S-adenosylmethionine (AdoMet) levels that suggest a reduced methionine cycle. Anti-miR-873-5p treatment prevents cell death in primary hepatocytes and the appearance of necrotic areas in liver from APAP-administered mice. In our study, we demonstrate a GNMT and methionine cycle activity restoration by the anti-miR-873-5p that reduces mitochondrial dysfunction and oxidative stress. The lack of hyperammoniemia caused by the therapy results in a decreased urea cycle, enhancing the synthesis of polyamines from ornithine and AdoMet and thus impacting the observed recovery of mitochondria and hepatocyte proliferation for regeneration. In summary, anti-miR-873-5p appears to be an effective therapy against APAP-induced liver injury, where the restoration of GNMT and the methionine cycle may prevent mitochondrial dysfunction while activating hepatocyte proliferative response.
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REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) is a global strategy and regulation policy of the EU that aims to improve the protection of human health and the environment through the better and earlier identification of the intrinsic properties of chemical substances. It entered into force on 1st June 2007 (EC 1907/2006). REACH and EU policies plead for the use of robust high-throughput "omic" techniques for the in vitro investigation of the toxicity of chemicals that can provide an estimation of their hazards as well as information regarding the underlying mechanisms of toxicity. In agreement with the 3R's principles, cultured cells are nowadays widely used for this purpose, where metabolomics can provide a real-time picture of the metabolic effects caused by exposure of cells to xenobiotics, enabling the estimations about their toxicological hazards. High quality and robust metabolomics data sets are essential for precise and accurate hazard predictions. Currently, the acquisition of consistent and representative metabolomic data is hampered by experimental drawbacks that hinder reproducibility and difficult robust hazard interpretation. Using the differentiated human liver HepG2 cells as model system, and incubating with hepatotoxic (acetaminophen and valproic acid) and non-hepatotoxic compounds (citric acid), we evaluated in-depth the impact of several key experimental factors (namely, cell passage, processing day and storage time, and compound treatment) and instrumental factors (batch effect) on the outcome of an UPLC-MS metabolomic analysis data set. Results showed that processing day and storage time had a significant impact on the retrieved cell's metabolome, while the effect of cell passage was minor. Meta-analysis of results from pathway analysis showed that batch effect corrections and quality control (QC) measures are critical to enable consistent and meaningful estimations of the effects caused by compounds on cells. The quantitative analysis of the changes in metabolic pathways upon bioactive compound treatment remained consistent despite the concurrent causes of metabolomic data variation. Thus, upon appropriate data retrieval and correction and by an innovative metabolic pathway analysis, the metabolic alteration predictions remained conclusive despite the acknowledged sources of variability.
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Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Hígado/efectos de los fármacos , Metabolómica/métodos , Acetaminofén/toxicidad , Línea Celular Tumoral , Ácido Cítrico/toxicidad , Células Hep G2 , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Metaboloma/efectos de los fármacos , Metaboloma/genética , Control de Calidad , Reproducibilidad de los Resultados , Ácido Valproico/toxicidad , Xenobióticos/toxicidadRESUMEN
Extraction of meaningful biological information from the vast array of data that metabolomics analyses generate is a major challenge in the field. A variety of computational and visual tools that help to identify changes in metabolic pathways have been proposed including functional analysis and pathway analysis. Meta-analysis of metabolomic data has emerged as a powerful source of information. In this work, the applicability of the Mantel's test for the correlation of functional results from metabolic pathway analysis is shown using experimental and simulated data sets as evaluation examples. The statistical significance of the correlation coefficient can be assessed by permutation testing requiring practically no computation time. The use of the Mantel's test can assist the critical comparison of different phenotypes, studies, methods, platforms, or data preprocessing strategies, as well as help to identify inconsistencies between metabolomic study outcomes, making this algorithm attractive for data interpretation and meta-analysis on a routine basis.
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Redes y Vías Metabólicas , Metabolómica , Proyectos de InvestigaciónRESUMEN
The estimation of steatosis in a liver graft is mandatory prior to liver transplantation, as the risk of graft failure increases with the level of infiltrated fat. However, the assessment of liver steatosis before transplantation is typically based on a qualitative or semiquantitative characterization by visual inspection and palpation and histological analysis. Thus, there is an unmet need for transplantation surgeons to have access to a diagnostic tool enabling an in situ fast classification of grafts prior to extraction. In this study, we have assessed an attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic method compatible with the requirements of an operation room for the evaluation of the lipid contents in human livers. A set of 20 human liver biopsies obtained from organs intended for transplantation were analyzed by expert pathologists, ATR-FTIR spectroscopy, lipid biochemical analysis, and UPLC-ESI(+/-)TOFMS for lipidomic profiling. Comparative analysis of multisource data showed strong correlations between ATR-FTIR, clinical, and lipidomic information. Results show that ATR-FTIR captures a global picture of the lipid composition of the liver, along with information for the quantification of the triradylglycerol content in liver biopsies. Although the methodology performance needs to be further validated, results support the applicability of ATR-FTIR for the in situ determination of the grade of liver steatosis at the operation room as a fast, quantitative method, as an alternative to the qualitative and subjective pathological examination.
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Trasplante de Hígado , Quirófanos , Espectrofotometría Infrarroja/métodos , Humanos , Factores de TiempoRESUMEN
The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because VDR deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including MOGAT1, LPGAT1, AGPAT2, and DGAT1 (glycerolipid metabolism); CDS1, PCTP, and MAT1A (phospholipid metabolism); and FATP2, SLC6A12, and AQP3 (uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4-6 h) upon VDR activation by 10 nM VitD or 100 µM lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids.
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Regulación de la Expresión Génica , Hepatocitos/metabolismo , Fosfolípidos/biosíntesis , Receptores de Calcitriol/metabolismo , Triglicéridos/biosíntesis , Animales , Células Hep G2 , Humanos , Ratones , Ratones Noqueados para ApoE , Fosfolípidos/genética , Receptores de Calcitriol/genética , Triglicéridos/genéticaRESUMEN
Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.
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Ácidos y Sales Biliares/sangre , Colestasis/inducido químicamente , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Congéneres de la Testosterona/toxicidad , Adulto , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colestasis/metabolismo , Ácido Cólico/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/genética , Masculino , Receptores Androgénicos/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/genética , Esteroide 12-alfa-Hidroxilasa/genética , Esteroide 12-alfa-Hidroxilasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
SCOPE: Gut microbiota contributes to non-alcoholic fatty liver disease (NAFLD) pathogenesis by multiple mechanisms not yet completely understood. Novel differential features between germ-free mice (GFm) transplanted with protective or non-protective cecal microbiota against NAFLD are investigated. METHODS AND RESULTS: Gut microbiota composition, plasma, and fecal bile acids (BAs) and liver mRNAs are quantified in GFm recipients from four donor mice differing in NAFLD severity (control diet, high-fat diet [HFD]-responder, HFD-non-responder, and quercetin-supplemented HFD). Transplanted GFm are on control or HFD for 16-weeks. Multivariate analysis shows that GFm colonized with microbiota from HFD-non-responder and quercetin supplemented-HFD donors (protected against NAFLD) clusters together, whereas GFm colonized with microbiota from control and HFD-responder mice (non-protected against NAFLD) establishes another cluster. Protected phenotype is associated with increased gut Desulfovibrio and Oscillospira, reduced gut Bacteroides and Oribacterium, lower primary and higher secondary BAs in plasma and feces, induction of hepatic BA transporters, and repression of hepatic lipogenic and BA synthesis genes. CONCLUSION: Protective gut microbiota associates with increased specific secondary BAs, which likely inhibit lipogenic pathways and enhance bile flow in the liver. This novel cross-talk between gut and liver, via plasma BAs, that promotes protection against NAFLD may have clinical and nutritional relevance.
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Ácidos y Sales Biliares/sangre , Microbioma Gastrointestinal , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Animales , Dieta Alta en Grasa , Etanol/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
Understanding central mechanisms underlying drug-induced toxicity plays a crucial role in drug development and drug safety. However, a translation of cellular in vitro findings to an actual in vivo context remains challenging. Here, physiologically based pharmacokinetic (PBPK) modeling was used for in vivo contextualization of in vitro toxicity data (PICD) to quantitatively predict in vivo drug response over time by integrating multiple levels of biological organization. Explicitly, in vitro toxicity data at the cellular level were integrated into whole-body PBPK models at the organism level by coupling in vitro drug exposure with in vivo drug concentration-time profiles simulated in the extracellular environment within the organ. PICD was exemplarily applied on the hepatotoxicant azathioprine to quantitatively predict in vivo drug response of perturbed biological pathways and cellular processes in rats and humans. The predictive accuracy of PICD was assessed by comparing in vivo drug response predicted for rats with observed in vivo measurements. To demonstrate clinical applicability of PICD, in vivo drug responses of a critical toxicity-related pathway were predicted for eight patients following acute azathioprine overdoses. Moreover, acute liver failure after multiple dosing of azathioprine was investigated in a patient case study by use of own clinical data. Simulated pharmacokinetic profiles were therefore related to in vivo drug response predicted for genes associated with observed clinical symptoms and to clinical biomarkers measured in vivo. PICD provides a generic platform to investigate drug-induced toxicity at a patient level and thus may facilitate individualized risk assessment during drug development.
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Azatioprina/toxicidad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Modelos Teóricos , Farmacocinética , Adulto , Animales , Azatioprina/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Sobredosis de Droga/etiología , Humanos , Masculino , Ratas , Reproducibilidad de los Resultados , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad Aguda/métodosRESUMEN
MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.
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Cromatografía Liquida/métodos , Hígado/metabolismo , Metaboloma , Metabolómica/métodos , Animales , Adhesión Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión/métodos , Técnicas Citológicas , Células Hep G2/química , Células Hep G2/metabolismo , Humanos , Extracción Líquido-Líquido/métodos , Hígado/citología , Ratas , Espectrometría de Masa por Ionización de Electrospray/métodos , Flujo de TrabajoRESUMEN
This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.
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Hepatocitos/trasplante , Fallo Hepático Agudo/cirugía , Trasplante de Hígado/métodos , Errores Innatos del Metabolismo/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Separación Celular/métodos , Supervivencia Celular , Criopreservación/métodos , Difusión de Innovaciones , Selección de Donante , Femenino , Predicción , Supervivencia de Injerto , Humanos , Lactante , Recién Nacido , Fallo Hepático Agudo/diagnóstico , Fallo Hepático Agudo/metabolismo , Trasplante de Hígado/efectos adversos , Trasplante de Hígado/tendencias , Masculino , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/metabolismo , Persona de Mediana Edad , Selección de Paciente , Factores de Riesgo , Resultado del TratamientoRESUMEN
BACKGROUND: Hepatic cell therapy has become a viable alternative to liver transplantation for life-threatening liver diseases. However, the supply of human hepatocytes is limited due to the shortage of suitable donor organs required to isolate high-quality cells. Human pluripotent stem cells reflect a potential renewable source for generating functional hepatocytes. However, most differentiation protocols use undefined matrices or factors of animal origin; as such, the resulting hepatocytes are not Good Manufacturing Practice compliant. Moreover, the preclinical studies employed to assess safety and function of human embryonic stem cell (hESC)-derived hepatocytes are generally limited to immunodeficient mice. In the present study, we evaluate the generation of hepatocytes under defined conditions using a European hESC line (VAL9) which was derived under animal-free conditions. The function capacity of VAL9-derived hepatocytes was assessed by transplantation into mice with acetaminophen-induced acute liver failure, a clinically relevant model. METHODS: We developed a protocol that successfully differentiates hESCs into bipotent hepatic progenitors under defined conditions, without the use of chromatin modifiers such as dimethyl sulphoxide. These progenitors can be cryopreserved and are able to generate both committed precursors of cholangiocytes and neonate-like hepatocytes. RESULTS: Thirty days post-differentiation, hESCs expressed hepatocyte-specific markers such as asialoglycoprotein receptor and hepatic nuclear factors including HNF4α. The cells exhibited properties of mature hepatocytes such as urea secretion and UGT1A1 and cytochrome P450 activities. When transplanted into mice with acetaminophen-induced acute liver failure, a model of liver damage, the VAL9-derived hepatocytes efficiently engrafted and proliferated, repopulating up to 10 % of the liver. In these transplanted livers, we observed a significant decrease of liver transaminases and found no evidence of tumourigenicity. Thus, VAL9-derived hepatocytes were able to rescue hepatic function in acetaminophen-treated animals. CONCLUSIONS: Our study reveals an efficient protocol for differentiating VAL9 hESCs to neonatal hepatocytes which are then able to repopulate livers in vivo without tumour induction. The human hepatocytes are able to rescue liver function in mice with acetaminophen-induced acute toxicity. These results provide proof-of-concept that replacement therapies using hESC-derived hepatocytes are effective for treating liver diseases.
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Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Células Madre Embrionarias/citología , Hepatocitos/citología , Hepatocitos/trasplante , Acetaminofén/toxicidad , Animales , Sistema Biliar/citología , Diferenciación Celular , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Crigler-Najjar type 1 disease is a rare inherited metabolic disease characterized by high levels of unconjugated bilirubin due to the complete absence of hepatic uridine diphosphoglucuronate-glucuronosyltransferase activity. Hepatocyte transplantation (HT) has been proposed as an alternative treatment for Crigler-Najjar syndrome, but it is still limited by the quality and the low engraftment and repopulation ability of the cells used. Because of their attachment capability and expression of adhesion molecules as well as the higher proportion of hepatic progenitor cells, neonatal hepatocytes may have an advantage over adult cells. Adult or neonatal hepatocytes were transplanted into Gunn rats, a model for Crigler-Najjar disease. Engraftment and repopulation were studied and compared by immunofluorescence (IF). Additionally, the serum bilirubin levels, the presence of bilirubin conjugates in rat serum, and the expression of uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) in rat liver samples were also analyzed. Here we show that neonatal HT results in long-term correction in Gunn rats. In comparison with adult cells, neonatal cells showed better engraftment and repopulation capability 3 days and 6 months after transplantation, respectively. Bilirubinemia decreased in the transplanted animals during the whole experimental follow-up (6 months). Bilirubin conjugates were also present in the serum of the transplanted animals. Western blots and IF confirmed the presence and expression of UGT1A1 in the liver. This work is the first to demonstrate the advantage of using neonatal hepatocytes for the treatment of Crigler-Najjar in vivo.
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Síndrome de Crigler-Najjar/terapia , Hepatocitos/trasplante , Regeneración Hepática , Anciano , Anciano de 80 o más Años , Animales , Bilirrubina/sangre , Proliferación Celular , Femenino , Glucuronosiltransferasa/metabolismo , Humanos , Recién Nacido , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Propranolol , Ratas GunnRESUMEN
The small heterodimer partner (SHP) (NR0B2) is an atypical nuclear receptor that lacks a DNA-binding domain. It interacts with and inhibits many transcription factors, affecting key metabolic processes, including bile acid, cholesterol, fatty acid, and drug metabolism. Our aim was to determine the influence of steatotic drugs and nonalcoholic fatty liver disease (NAFLD) on SHP expression and investigate the potential mechanisms. SHP was found to be repressed by steatotic drugs (valproate, doxycycline, tetracycline, and cyclosporin A) in cultured hepatic cells and the livers of different animal models of NAFLD: iatrogenic (tetracycline-treated rats), genetic (glycine N-methyltransferase-deficient mice), and nutritional (mice fed a methionine- and choline-deficient diet). Among the different transcription factors investigated, CCAAT-enhancer-binding protein α (C/EBPα) showed the strongest dominant-repressive effect on SHP expression in HepG2 and human hepatocytes. Reporter assays revealed that the inhibitory effect of C/EBPα and steatotic drugs colocalize between -340 and -509 base pair of the SHP promoter, and mutation of a predicted C/EBPα response element at -473 base pair abolished SHP repression by both C/EBPα and drugs. Moreover, inhibition of major stress signaling pathways demonstrated that the mitogen-activated protein kinase kinase 1/2 pathway activates, while the phosphatidylinositol 3 kinase pathway represses SHP in a C/EBP-dependent manner. We conclude that SHP is downregulated by several steatotic drugs and in advanced NAFLD. These conditions can activate signals that target C/EBPα and consequently repress SHP, thus favoring the progression and severity of NAFLD.
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Hígado Graso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Ciclosporina/toxicidad , Doxiciclina/toxicidad , Hígado Graso/inducido químicamente , Humanos , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Tetraciclina/toxicidad , Tiazepinas/toxicidad , Transcripción Genética , Ácido Valproico/toxicidadRESUMEN
It is estimated that only a few marketed drugs are able to directly induce liver steatosis. However, many other drugs may exacerbate or precipitate fatty liver in the presence of other risk factors or in patients prone to non-alcoholic fatty liver disease. On the other hand, current in vitro tests for drug-induced steatosis in preclinical research are scarce and not very sensitive or reproducible. In the present study, we have investigated the effect of well-characterized steatotic drugs on the expression profile of 47 transcription factors (TFs) in human hepatoma HepG2 cells and found that these drugs are able to up- and down-regulate a substantial number of these factors. Multivariate data analysis revealed a common TF signature for steatotic drugs, which consistently and significantly repressed FOXA1, HEX and SREBP1C in cultured cells. This signature was also observed in the livers of rats and in cultured human hepatocytes. Therefore, we selected these three TFs as predictive biomarkers for iatrogenic steatosis. With these biomarkers, a logistic regression analysis yielded a predictive model, which was able to correctly classify 92 % of drugs. The developed algorithm also predicted that ibuprofen, nifedipine and irinotecan are potential steatotic drugs, whereas troglitazone is not. In summary, this is a sensitive, specific and simple RT-PCR test that can be easily implemented in preclinical drug development to predict drug-induced steatosis. Our results also indicate that steatotic drugs affect expression of both common and specific subsets of TF and lipid metabolism genes, thus generating complex transcriptomic responses that cause or contribute to steatosis in hepatocytes.
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Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/genética , Toxicogenética/métodos , Factores de Transcripción/genética , Anciano , Algoritmos , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Células Hep G2 , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Transcripción/metabolismoRESUMEN
Liver fatty acid binding protein (FABP1) prevents lipotoxicity of free fatty acids and regulates fatty acid trafficking and partition. Our objective is to investigate the transcription factors controlling the human FABP1 gene and their regulation in nonalcoholic fatty liver disease (NAFLD). Adenovirus-mediated expression of multiple transcription factors in HepG2 cells and cultured human hepatocytes demonstrated that FOXA1 and PPARα are among the most effective activators of human FABP1, whereas C/EBPα is a major dominant repressor. Moreover, FOXA1 and PPARα induced re-distribution of FABP1 protein and increased cytoplasmic expression. Reporter assays demonstrated that the major basal activity of the human FABP1 promoter locates between -96 and -229bp, where C/EBPα binds to a composite DR1-C/EBP element. Mutation of this element at -123bp diminished basal reporter activity, abolished repression by C/EBPα and reduced transactivation by HNF4α. Moreover, HNF4α gene silencing by shRNA in HepG2 cells caused a significant down-regulation of FABP1 mRNA expression. FOXA1 activated the FABP1 promoter through binding to a cluster of elements between -229 and -592bp, whereas PPARα operated through a conserved proximal element at -59bp. Finally, FABP1, FOXA1 and PPARα were concomitantly repressed in animal models of NAFLD and in human nonalcoholic fatty livers, whereas C/EBPα was induced or did not change. We conclude that human FABP1 has a complex mechanism of regulation where C/EBPα displaces HNF4α and hampers activation by FOXA1 and PPARα. Alteration of expression of these transcription factors in NAFLD leads to FABP1 gen repression and could exacerbate lipotoxicity and disease progression.
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Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hígado Graso/metabolismo , Hígado Graso/terapia , Factor Nuclear 3-alfa del Hepatocito/metabolismo , PPAR alfa/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/genética , Hígado Graso/genética , Células Hep G2 , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , PPAR alfa/genética , Unión ProteicaRESUMEN
Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid ß-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.
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Hígado Graso/genética , Hígado Graso/metabolismo , Factor Nuclear 3-alfa del Hepatocito/fisiología , Hepatocitos/metabolismo , Metabolismo de los Lípidos/genética , Adulto , Anciano , Animales , Células Cultivadas , Regulación hacia Abajo/genética , Hígado Graso/patología , Femenino , Células Hep G2 , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Hepatocitos/patología , Humanos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico , Cultivo Primario de Células , Ratas , Adulto JovenRESUMEN
In a number of adverse drug reactions leading to hepatotoxicity, drug metabolism is thought to be involved by the generation of reactive metabolites from non-toxic drugs. The use of hepatoma cell lines, such as HepG2 cell line, for the evaluation of drug-induced hepatotoxicity is hampered by their low cytochrome P450 expression which makes impossible the study of the toxicity produced by bioactivable compounds. Genetically manipulated cells constitute promising tools for hepatotoxicity applications. HepG2 cells were simultaneously transfected with recombinant adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4 to confer them drug-metabolic competence. Upgraded cells (Adv-HepG2) were highly able to metabolize the toxin studied in contrast to the reduced metabolic capacity of HepG2 cells. Aflatoxin B1-induced hepatotoxicity was studied as a proof of concept in metabolically competent and non-competent HepG2 cells by using high content screening technology. Significant differences in mitochondrial membrane potential, intracellular calcium concentration, nuclear morphology and cell viability after treatment with aflatoxin B1 were observed in Adv-HepG2 when compared to HepG2 cells. Rotenone (non bioactivable) and citrate (non hepatotoxic) were analysed as negative controls. This cell model showed to be a suitable hepatic model to test hepatotoxicity of bioactivable drugs and constitutes a valuable alternative for hepatotoxicity testing.
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Adenoviridae/genética , Aflatoxina B1/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Modelos Biológicos , Aflatoxina B1/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Ácido Cítrico/administración & dosificación , Ácido Cítrico/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A/genética , Vectores Genéticos , Células Hep G2 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Rotenona/administración & dosificación , Rotenona/metabolismo , TransfecciónRESUMEN
Currently, the only effective treatment for end-stage liver disease is liver transplantation. The number of patients on the waiting list increases considerably each year, giving rise to a wide imbalance between supply and demand for healthy livers. Knowledge of stem cells and their possible use have awakened great interest in the field of hepatology, these cells being one of the most promising short-term alternatives. Hepatic stem cell therapy consists of the implantation of healthy cells capable of performing the functions that damaged cells are unable to carry out. Recent observations indicate that several stem cells can differentiate into distinct cell lineages. Hepatic differentiation of adult stem cells from several origins has yielded highly promising results. Adipose tissue in adults contains a reservoir of stem cells that can be induced and differentiated into different types of cells, showing a high degree of plasticity. Because of its abundance and easy access, adipose tissue is a promising source of adult stem cells for hepatic stem cell therapy. The present article reviews the progress made in the differentiation of adult stem cells from adipose tissue into cells with hepatic phenotype. We also discuss the potential application of this technique as a therapy for temporary metabolic support in patients with end-stage liver failure awaiting whole organ transplantation, as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and as a treatment in patients with genetic metabolic defects in vital liver functions.
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Tejido Adiposo/citología , Fallo Hepático/cirugía , Trasplante de Células Madre , Diferenciación Celular , Predicción , Hepatocitos , Humanos , Células Madre Mesenquimatosas , FenotipoRESUMEN
The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.
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Ácidos Grasos no Esterificados/metabolismo , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácidos Grasos no Esterificados/farmacología , Hígado Graso/inducido químicamente , Hígado Graso/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Técnicas In Vitro , Rojo Neutro/metabolismo , Ácido Oléico/metabolismo , Ácido Oléico/farmacología , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologíaRESUMEN
Chromaffin cells in culture show high neuropathy target esterase (NTE) activity. It is well known that inhibition and specific modification of NTE by some organophosphorus (OPs) compounds induces a neurodegenerative neuropathy. It has been suggested that NTE is responsible for phosphatidylcholine homeostasis, although its role in neuropathy induction remains unclear. The cDNA of human NTE (4.4kbp) was inserted into an adenoviral vector. Bovine chromaffin cells cultured at 50,000 cells/well were incubated with the vector for 2h and after removing the volume of infection, cells were maintained in the incubator. After 24h, NTE activity was 6.8+/-0.5mU/10(6) cells in untreated cells and 14.8+/-1.5mU/10(6) cells, 19.3+/-2.9mU/10(6) cells, 24.8+/-0.9mU/10(6) cells and 30.9+/-1.0mU/10(6) cells in cells incubated with 2, 4, 8 and 16microl of vector, respectively. After 60min of inhibition with mipafox increased concentrations, the calculated I(50) (60min) values were 5.5, 6.2 and 6.6microM for cells infected with 0, 2 and 10microl of vector preparation. We confirm that the adenoviral vector containing the human NTE gene is active in bovine chromaffin cells in culture and that the NTE activity expressed by the vector shows the same inhibition pattern by the neuropathic OP mipafox as the NTE activity of bovine chromaffin cells and cells remained viable after the high NTE activity expression.