Laia Tolosa - Academia.edu (original) (raw)

Papers by Laia Tolosa

Biomaterials advances, Apr 1, 2022

Current protocols in toxicology, May 1, 2017

Human hepatic cells have been used for drug safety risk evaluations throughout early development ... more Human hepatic cells have been used for drug safety risk evaluations throughout early development phases. They provide rapid, cost‐effective early feedback to identify drug candidates with potential hepatotoxicity. This unit presents a cell‐based assay to evaluate the risk of liver damage associated with steatogenic drugs. Detailed protocols for cell exposure to test compounds and for the assessment of steatosis‐related cell parameters (intracellular lipid content, reactive oxygen species production, mitochondrial impairment, and cell death) are provided. A few representative results that illustrate the utility of this procedure for the screening of drug‐induced steatosis are shown. © 2017 by John Wiley & Sons, Inc.

Springer eBooks, 2013

Mesenchymal stem cells (MSCs) are multipotent cells, able to differentiate into elements of the m... more Mesenchymal stem cells (MSCs) are multipotent cells, able to differentiate into elements of the mesodermal lineage. Bone marrow and adipose tissue represent the main sources for MSCs isolation. In the last decade, several studies have reported the plasticity of MSCs toward a hepatocyte-like phenotype. Both types of cells have been cultured under similar pro-hepatogenic conditions by sequential exposure to cytokines, growth factors and hormones reflecting their temporal expression during in vivo hepatogenesis. The different capacities of hepatic differentiation of the two subsets in vitro have been investigated.

Archives of Toxicology, Mar 19, 2015

High-content screening is the application of automated microscopy and image analysis to both cell... more High-content screening is the application of automated microscopy and image analysis to both cell biology and drug discovery. Over the last decade, this technique has emerged as a useful technology that allows the simultaneous measurement of different parameters at a single-cell level. Hepatotoxicity is a compelling reason for drug nonapprovals and withdrawals. It is recognized that the safety of a compound cannot be based on a single in vitro assay, and existing methods are not predictive of drug-induced toxicity. However, different HCS assays have been recently demonstrated as being powerful for identifying different mechanisms implicated in drug-induced toxicity with high sensitivity and specificity. These assays integrate the data obtained from different cell function indicators and can be easily incorporated into basic screening processes for the safety evaluation and selection of drug candidates; thus, they contribute greatly to lessen the likelihood of drug failure. Exploring the use of cellular imaging technology in drug-induced liver injury by reviewing the different tests proposed provides evidence that this technology has a strong impact on drug discovery.

Revista de toxicología, 2017

Food and Chemical Toxicology, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Xenotransplantation, Jul 25, 2019

Brief communication 2 3 Title: Regenerative cell therapy for the treatment of hyperbilirubinemic ... more Brief communication 2 3 Title: Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and 4 frozen human iPSCs-derived hepatic stem cells 5 6

Expert Opinion on Drug Metabolism & Toxicology, Sep 27, 2016

Introduction: Drug attrition rates due to hepatotoxicity are an important safety issue considered... more Introduction: Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an 'artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno-and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

Neuroscience, Feb 1, 2009

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective degeneration and death of motoneurons in the spinal cord, brainstem and motor cortex which causes progressive muscle weakness and paralysis. Although the molecular mechanisms causing the disease remain unknown, excitotoxicity and loss of trophic support have been proposed as causes of degeneration. The present study was designed to elucidate the mechanisms of motoneuron death induced by serum deprivation and the potential neuroprotective effects of vascular endothelial growth factor (VEGF) in dissociated and organotypic rat spinal cord cultures. Serum withdrawal induced apoptotic cell death in dissociated spinal cord cultures, which was prevented in the presence of VEGF. In organotypic spinal cord cultures, low serum-induced motoneuron death was mediated by the stress-related kinase p38 mitogen-activated protein kinase (p38MAPK), as it was reversed by the p38MAPK inhibitor SB203580. In these cultures, exposure to VEGF blocked p38MAPK phosphorylation and prevented the demise of motoneurons. These effects of VEGF were mediated through the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway, as they were blocked in the presence of the PI3-K inhibitor LY294002. In addition, serum deprivation induced down-regulation of the anti-apoptotic protein Bcl-2 and this effect was prevented both by SB203580 and by VEGF via the PI3-K/Akt pathway. Therefore, Bcl-2 could also play an important role in the neuroprotection induced by VEGF in spinal cord cultures. Together, these findings indicate that VEGF prevents motoneuron death induced by serum deprivation blocking the activity of p38MAPK via the PI3-K/Akt signaling pathway.

Toxicology and Applied Pharmacology, Jul 1, 2016

Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. T... more Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and nonsteatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis.

Neuroscience, Sep 1, 2009

Nasal contribution to exhaled nitric oxide at rest and during breathholding in humans. Am J Respi... more Nasal contribution to exhaled nitric oxide at rest and during breathholding in humans. Am J Respir Crit Care Med 153: 829-836, 1996. 4) Dear JW, Scadding GK, Foreman JC: Reduction by NG-nitro-L-arginine methyl ester (L-NAME) of antigen-induced nasal airway plasma extravasation in

Revista de Toxicología, 2014

Drug-induced liver injury is a significant leading cause of liver disease and post-market attriti... more Drug-induced liver injury is a significant leading cause of liver disease and post-market attrition of approved drugs. Several hepatic cell-based models have been used for early safety risk assessment during drug development. Their capacity to predict hepatotoxicity depends on cells' functional performance. Cultured hepatocytes have contributed to increase knowledge of the metabolic patterns and mechanisms involved in drug toxicity. A major limitation of monolayer hepatocytes is that they undergo rapid loss of hepatic functionality over time, particularly drug metabolising capability. The sandwich culture model promotes polarised cell surface and stabilises hepatocyte functionality, particularly transport systems, better than monolayer cultures. As 3D spatial organisation and complex heterotypic cell interactions are essential for the functional homeostasis of the liver, hepatocyte models (3D cultures, co-cultures with NPCs and microfluidic systems) that mimic cell-cell, cellmatrix interactions and nutrient flow characteristic of the liver microenvironment have been shown to improve the metabolic competency of hepatocytes and have been proposed for better in vitro predictions of drug hepatotoxicity. In addition to hepatocytes, other cell-based models have been proposed for hepatotoxicity studies. Hepatoma cell lines are metabolically poor compared to hepatocytes, but offer key advantages, such as unlimited life span, reproducibility, high availability and easy handling, which make them useful for screening purposes. Alternatively, hepatic cell lines engineered for stable or transient expression of key drug-metabolising enzymes have also been used. Finally, stem cell-derived hepatocytes are emerging in vitro systems that would provide a stable source of hepatocytes from individuals with highly valuable particular polymorphic characteristics for preclinical drug metabolism and toxicity prediction of new drugs.

Antioxidants, Dec 30, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Methods in molecular biology, Nov 3, 2014

Hepatoma cell lines are frequently used as in vitro alternatives to primary human hepatocytes. Ce... more Hepatoma cell lines are frequently used as in vitro alternatives to primary human hepatocytes. Cell lines are characterized by their unlimited life span, stable phenotype, high availability, and easy handling. However, their major limitation is the lower expression of some metabolic activities compared with hepatocytes. HepG2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies. HepG2 cells are nontumorigenic cells with high proliferation rates and an epithelial-like morphology that perform many differentiated hepatic functions. In this chapter, freezing, thawing, and subculturing procedures for HepG2 cells are described. We further provide protocols for evaluating lipid accumulation, glycogen storage, urea synthesis, and phase I and phase II drug metabolizing activities in HepG2 cells.

Stem Cell Research & Therapy, Apr 18, 2023

Background High-throughput pharmaco-toxicological testing frequently relies on the use of establi... more Background High-throughput pharmaco-toxicological testing frequently relies on the use of established liverderived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable. Methods We have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions. Results Clonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks. Conclusion Our strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

Gels, Oct 20, 2022

Archives of Toxicology, Feb 14, 2022

Drug-induced liver injury (DILI) is one of the most common and serious adverse drug reactions and... more Drug-induced liver injury (DILI) is one of the most common and serious adverse drug reactions and a major cause of drug development failure and withdrawal. Although different molecular mechanisms are implicated in DILI, enhanced ROS levels have been described as a major mechanism. Human-derived cell models are increasingly used in preclinical safety assessment because they provide quick and relatively inexpensive information in early stages of drug development. We have analyzed and compared the phenotype and functionality of two liver cell models (Upcyte human hepatocytes and HepaRG cells) to demonstrate their suitability for long-term hepatotoxicity assessments and mechanistic studies. The transcriptomic and functional analysis revealed the maintenance of phase I and phase II enzymes, and antioxidant enzymes along time in culture, although the differences found between both test systems underlie the differential sensitivity to hepatotoxins. The evaluation of several mechanisms of cell toxicity, including oxidative stress, by high-content screening, demonstrated that, by combining the stable phenotype of liver cells and repeated-dose exposure regimes to 12 test compounds at clinically relevant concentrations, both Upcyte hepatocytes and HepaRG offer suitable properties to be used in routine screening assays for toxicological assessments during drug preclinical testing.

Cellular Signalling, Nov 1, 2005

Wasting of skeletal muscle (cachexia) is associated with a variety of chronic or inflammatory dis... more Wasting of skeletal muscle (cachexia) is associated with a variety of chronic or inflammatory disorders and has long been recognized as a poor prognostic sign. It is currently accepted that the cytokine tumor necrosis factor alpha (TNF-alpha; cachectin) plays a key role in the development of this condition. TNF-alpha-induced apoptotic cell death represents a potential mechanism by which muscle wasting can occur. Evidence has accumulated that the cytokine interferon gamma (IFN-gamma) may act as a modulator of TNF-alpha signalling. Thus, the present study was designed to elucidate if TNF-alpha can directly induce apoptosis in differentiated myotubes, to assess the potential anti-apoptotic properties of IFN-gamma and to get insight into the signalling pathways implicated in the modulatory effects of IFN-gamma. Myoblasts of the murine cell line C2C12 were allowed to differentiate in a low serum containing media and myogenesis assessed by muscle specific protein expression. Non-proliferating, polynucleated, fully differentiated myotubes were obtained after seven days in differentiation media. Exposure of C2C12 myotubes to TNF-alpha for 48 h induced apoptosis characterized by enhanced caspase-3 activity, which resulted in poly(ADP-ribose) polymerase (PARP) cleavage and increased histone-associated-DNA fragmentation. These effects were fully reverted in the presence of IFN-gamma. This cytokine induced down-regulation of the subtype 2 of TNF-alpha receptors (TNF-R2), enhanced TNF-alpha-induced NF-kappaB translocation to the nucleus and binding to DNA and increased the immunoreactivity of the protein c-IAP1, a member of the inhibitor of apoptosis (IAP) gene family whose synthesis is stimulated by NF-kappaB at the transcriptional level. Together, these results demonstrate that TNF-alpha directly induces apoptosis in differentiated myotubes and suggest that the cytokine IFN-gamma, might represent a new immunoadjuvant therapeutic tool for managing cachexia.

Molecular and Cellular Neuroscience, 2011

Besides glutamate excitotoxicity, the neuroinflammatory response is emerging as a relevant contri... more Besides glutamate excitotoxicity, the neuroinflammatory response is emerging as a relevant contributor to motoneuron loss in amyotrophic lateral sclerosis (ALS). In this regard, high levels of circulating proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) have been shown both in human patients and in animal models of ALS. The aim of this work was to study the effects of TNF-α on glutamateinduced excitotoxicity in spinal cord motoneurons. In rat spinal cord organotypic cultures chronic glutamate excitotoxicity, induced by the glutamate-uptake inhibitor threohydroxyaspartate (THA), resulted in motoneuron loss that was associated with a neuroinflammatory response. In the presence of TNF-α, THAinduced excitotoxic motoneuron death was potentiated. Co-exposure to TNF-α and THA also resulted in down-regulation of the astroglial glutamate transporter 1 (GLT-1) and in increased extracellular glutamate levels, which were prevented by nuclear factor-kappaB (NF-κB) inhibition. Furthermore, TNF-α and THA also cooperated in the induction of oxidative stress in a mechanism involving the NF-κB signalling pathway as well. The inhibition of this pathway abrogated the exacerbation of glutamate-mediated motoneuron death induced by TNF-α. These data link two important pathogenic mechanisms, excitotoxicity and neuroinflammation, suggested to play a role in ALS and, to our knowledge, this is the first time that TNF-α-induced NF-κB activation has been reported to potentiate glutamate excitotoxicity on motononeurons.