New microRNA biomarkers for drug-induced steatosis and their potential to predict the contribution of drugs to non-alcoholic fatty liver disease

Background and Aims: Drug-induced steatosis is a major reason for drug failure in clinical trials and post-marketing withdrawal; and therefore, predictive biomarkers are essential. These could be particularly relevant in non-alcoholic fatty liver disease (NAFLD), where most patients show features of the metabolic syndrome and are prescribed with combined chronic therapies, which can contribute to fatty liver. However, specific biomarkers to assess the contribution of drugs to NAFLD are lacking. We aimed to find microRNAs (miRNAs) responsive to steatotic drugs and to investigate if they could become circulating biomarkers for drug-induced steatosis. Methods: Human HepG2 cells were treated with drugs and changes in miRNA levels were measured by microarray and qRT-PCR. Drug-induced fat accumulation in HepG2 was analyzed by high-content screening and enzymatic methods. miRNA biomarkers were also analyzed in the sera of 44 biopsy-proven NAFLD patients and in 10 controls. Results: We found a set of 10 miRNAs [miR-22-5p, -3929, -24-2-5p, -663a, -29a-3p, -21 (5p and 3p), -27a-5p, -1260 and -202-3p] that were induced in human HepG2 cells and secreted to the culture medium upon incubation with model steatotic drugs (valproate, doxycycline, cyclosporin A and tamoxifen). Moreover, cell exposure to 17 common drugs for NAFLD patients showed that some of them (e.g., irbesartan, fenofibrate, and omeprazole) also induced these miRNAs and increased intracellular triglycerides, particularly in combinations. Finally, we found that most of these miRNAs (60%) were detected in human serum, and that NAFLD patients under fibrates showed both induction of these miRNAs and a more severe steatosis grade. Conclusion: Steatotic drugs induce a common set of hepatic miRNAs that could be used in drug screening during preclinical development. Moreover, most of these miRNAs are serum circulating biomarkers that could become useful in the diagnosis of iatrogenic steatosis

Desarrollo, análisis y optimización de modelos celulares hepáticos para estudios de fármaco-toxicología y terapia celular

Given the importance of the liver in the metabolism and maintenance of the homeostasis of the organism, many studies have been conducted in the area of toxicology and, more recently, in hepatic cellular therapy. However, the main drawback is the limited availability of viable and functional hepatocytes due to the scarcity of liver tissue. The purpose of this work was based on the development and characterization of hepatic cellular models to become an alternative to hepatocytes in toxicology studies and cellular therapy. To this end, three main objectives have been investigated: 1) to adopt a procedure of hepatocyte isolation from discarded organs for transplantation which determines the optimal conditions for the isolation and culture of hepatocytes, 2) to characterize the cells from the hepatoblastome HepG2, and 3) to develop a hepatogenic differentiation protocol to induce the hepatic differentiation in adipose-derived stem cells (ADSC). In particular, the hepatogenic differentiation of stem cells opens a wide range of possibilities to facilitate the establishment of an adult differentiated cellular model useful for pharmaco-toxicological studies and for hepatic cellular therapy. The use of adult stem cells may allow the establishment of an adult cellular model with properties that others cellular models, like HepG2, do not show. However, it is necessary to optimise the isolation and cryopreservation procedures, as well as the differentiation protocols from adult stem cells and try to acquire a wide knowledge of the cellular and molecular mechanisms that control the transdifferentiation to hepatocytes.Key Words: cellular transplant, esteatosis, human hepatocytes, hepatoblastomes, stem cells.El hígado juega un papel fundamental en el metabolismo de medicamentos y en el mantenimiento de la homeostasis del organismo y, por tanto los modelos celulares hepáticos desempeñan un papel clave para estudios fármaco-toxicológicos y más recientemente en el campo de la terapia celular. Sin embargo, la limitada disponibilidad de hepatocitos viables y funcionales debido a la falta de tejido hepático es la principal limitación para utilizar estos recursos celulares. El objetivo del presente trabajo se ha basado en el desarrollo y caracterización de modelos celulares hepáticos que puedan constituir una alternativa a los hepatocitos para este tipo de aplicaciones. Para ello se han abordado tres estrategias diferentes: 1) optimización del proceso de obtención de hepatocitos a partir de hígados enteros descartados para transplante, determinando las condiciones adecuadas para el aislamiento y cultivo de hepatocitos; 2) caracterización funcional de las células del hepatoblastoma HepG2 y 3) desarrollo de un protocolo para inducir la diferenciación hepatogénica de células madre mesenquimales adultas derivadas de tejido adiposo (ADSC). Para conseguir un buen aprovechamiento de hígados descartados para transplante resulta necesario optimizar los protocolos de aislamiento y criopreservación de hepatocitos. El estudio con células madre adultas se presenta como una alternativa muy válida para la obtención de hepatocitos-like viables y funcionalmente activos útiles a corto plazo en estudios de fármaco-toxicología y en un futuro para terapia celular hepática. El uso de células madre abre un gran abanico de posibilidades facilitando el establecimiento de un modelo celular diferenciado adulto con características que otros modelos celulares, como son el hepatoma humano HepG2, no presentan. No obstante, es necesario adquirir un mayor conocimiento de los mecanismos celulares y moleculares que controlan la transdiferenciación a hepatocitos.Palabras clave: trasplante celular, esteatosis, hepatocitos humanos, hepatoblastomas, células madre

Primary hepatocytes: Current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and crosstalk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process