Teresa Donato

• University of Valencia

Drug-induced cholestasis is one of the major mechanisms implicated in drug-induced hepatotoxicity that poses a serious problem in terms of patient morbidity and mortality, healthcare system expenses and efficacy of newly developed drugs. Impaired bile acid homeostasis due to transporter alterations, hepatocellular injury or canalicular abnormalitie...

Liver tissue engineering offers a remarkable tool for toxicological screening and an approach to disease modeling, and provides an alternative solution to liver transplantation in treating end-stage liver diseases. As the liver is a highly specialized multicellular organ, distinctive features such as diversity of cell types, extracellular matrix co...

Cell-based liver therapies based on retrieving and steadying failed metabolic function(s) for acute and chronic diseases could be a valuable substitute for liver transplants, even though they are limited by the low engraftment capability and reduced functional quality of primary human hepatocytes (PHH). In this paper we propose the use of gelatin-h...

Hepatotoxicity or drug-induced liver injury (DILI) is a major safety issue in drug development as a primary reason for drug failure in clinical trials and the main cause for post-marketing regulatory measures like drug withdrawal. Idiosyncratic DILI (iDILI) is a patient-specific, multifactorial, and multicellular process that cannot be recapitulate...

Drug-induced liver injury (DILI) is a major clinical problem in terms of patient morbidity and mortality, cost to healthcare systems and failure of the development of new drugs. The need for consistent safety strategies capable of identifying a potential toxicity risk early in the drug discovery pipeline is key. Human DILI is poorly predicted in an...

Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations , mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation cause...

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 safet...

Nonalcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in western countries. The molecular mechanisms leading to NAFLD are only partially understood, and effective therapeutic interventions are clearly needed. Therefore, preclinical research is required to improve knowledge about NAFLD physiopathology and to iden...

Drug-induced liver injury (DILI) remains a major cause of drug development failure, post-marketing warnings and restriction of use. An improved understanding of the mechanisms underlying DILI is required for better drug design and development. Enhanced reactive oxygen species (ROS) levels may cause a wide spectrum of oxidative damage, which has bee...

The development of suitable and reproducible liver cell models is fundamental for regenerative medicine, drug screening, and disease modeling. Human primary hepatocytes are the gold standard not only for hepatic cell therapy but also for preclinical toxicological screening or the study of liver disease; however, their limited availability, variabil...

Safety evaluation of thousands of chemicals that are directly added to or come in contact with food is needed. Due to the central role of the liver in intermediary and energy metabolism and in the biotransformation of foreign compounds, the hepatotoxicity assessment is essential. New approach methodologies have been proposed for the safety evaluati...

Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations, mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation causes...

Drug-induced liver injury (DILI) constitutes one of the most frequent reasons of restricted-use warnings as well as withdrawals of drugs in postmarketing and poses an important concern for the pharmaceutical industry. The current hepatic in vivo and in vitro models for DILI detection have shown clear limitations, mainly for studies of long-term hep...

Drug-induced liver injury is a major cause of drug discovery failure in clinical trials and a leading cause of liver disease. Current preclinical drug testing does not predict hepatotoxicity which highlights the importance of developing highly predictive cell-based models. The use of stem cell technology and differentiation into hepatocyte-like cel...

Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been desc...

Background: The aqueous extract of the Allophylus cominia (L) Sw (Sapindaceae) leaves has shown anti-diabetic, anti-obesity and anti-inflammatory properties. In the Caribbean region, it is typically used for the treatment of type-2 diabetes. Methods: Considering the herb–drug interaction, the aim of this study was to evaluate the potential effects...

Phospholipidosis and steatosis are two toxic effects, which course with overaccumulation of different classes of lipids in the liver. MS-based lipidomics has become a powerful tool for the comprehensive determination of lipids. LC-MS lipid profiling of HepG2 cells is proposed as an in vitro assay to study and anticipate phospholipidosis and steatos...

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 cel...

Idiosyncratic hepatotoxicity occurs unpredictably in a small group of susceptible individuals with no direct dose relationship. Although the mechanisms involved in these adverse drug reactions remain unclear, several hypotheses have been proposed to explain their idiosyncratic nature, including drug conversion into reactive metabolites (bioactivati...

Determining long-term hepatotoxicity of new drugs is essential for the pharmaceutical industry development; however, it is limited by the lack of suitable cell-based models able to maintain hepatic functions over time. In the present work we explored the suitability of a new cellular model called Human Hepatocytes Upcytes (HHU) which preserves live...

Introduction: The need for alternatives to animal experimentation and traditional testing methods has been widely discussed in recent years. This has led scientists and regulatory authorities to investigate alternative methods for toxicity testing. High-content screening (HCS) has emerged as a powerful tool in predictive toxicology since it permit...

Disruption of the vectorial bile acid transport in the liver is a key feature of cholestatic drugs, although many causal and mechanistic aspects are still unknown. The aim of the present study was to explore if cholestatic drugs can repress or induce the expression of hepatic transporters. To this end, sandwich-cultured rat hepatocytes were treated...

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 upgrad...

In preclinical stages of drug development, anticipating potential adverse drug effects such as toxicity is an important issue for both saving resources and preventing public health risks. Current in vitro cytotoxicity tests are restricted by their predictive potential and their ability to provide mechanistic information. This study aimed to develop...

The capacity of human hepatic cell-based models to predict hepatotoxicity depends on the functional performance of cells. The major limitations of human hepatocytes include the scarce availability and rapid loss of the hepatic phenotype. Hepatoma cells are readily available and easy to handle, but are metabolically poor compared with hepatocytes. R...

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 com...

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 ce...

Drug-induced liver injury (DILI) is a significant leading cause of hepatic dysfunction, drug failure during clinical trials and post-market withdrawal of approved drugs. Many cases of DILI are unexpected reactions of an idiosyncratic nature that occur in a small group of susceptible individuals. Intensive research efforts have been made to understa...

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 th...

Cytotoxicity assays were among the first in vitro bioassay methods used to predict toxicity of substances to various tissues. In vitro cytotoxicity testing provides a crucial means for safety assessment and screening, and for ranking compounds. The choice of using a particular cytotoxicity assay technology may be influenced by specific research goa...

Hepatotoxicity is the number one cause for agencies not approving and withdrawing drugs for the market. Drug-induced human hepatotoxicity frequently goes undetected in pre-clinical safety evaluations using animal models. Human-derived in vitro models represent a common alternative to in vivo tests to detect toxic effects during pre-clinical testing...

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 UDP-glucuronosyl transferase activity. Hepatocyte transplantation has been proposed as an alternative treatment for Crigler-Najjar syndrome, but is still limited by the quality, and the lo...

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...

La lesión del hígado por fármacos es una de las causas principales de enfermedad hepática y de retirada del mercado de fármacos autorizados. Son varios los modelos de células hepáticas utilizados durante el desarrollo de fármacos para la valoración temprana de su seguridad. Los estudios basados en hepatocitos cultivados han contribuido al conocimie...

Drug-induced liver injury is a significant leading cause of hepatic dysfunction, drug failures during clinical trials and post-market withdrawal of approved drugs. Hepatotoxicity may result from either the drug itself or, more frequently, the bioactivation process and the production of reactive metabolites. Evaluation of potential hepatotoxicity re...

Introduction: The liver is the most important target for drug-induced toxicity. This vulnerability results from functional liver features and its role in the metabolic elimination of most drugs. Drug-induced liver injury is a significant leading cause of acute, chronic liver disease and an important safety issue when developing new drugs. Areas c...

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing...

Currently, there is increasing interest in developing accurate methods for the quantitative analysis of bile acids (BAs) in biological samples. We have developed a sensitive, fast, and reproducible UPLC-MRM-MS method for BA profiling in serum, liver tissue, or cultured cells of different species (human, rat, and mouse). This method, validated accor...

A frequent mechanism for drug-induced liver injury (DILI) is mitochondrial impairment, and early evaluation of new drugs for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. To this end, we designed a high-content screening assay to study mitochondrial-induced hepatotoxicity in HepG2 cells in de...

Multiple analytical methods are required to comprehensively assess oxidative homeostasis and specific damage to macromolecules. Our aim was to develop a straightforward strategy for the fast assessment of global oxidative status and specific damage to DNA, proteins, and lipids. To this end, an analytical method, based on ultra-performance liquid ch...

Early allograft dysfunction (EAD) dramatically influences graft and patient outcome after orthotopic liver transplantation and its incidence is strongly determined by donor liver quality. Nevertheless, objective biomarkers, which can assess graft quality and anticipate organ function, are still lacking. This study aims to investigate whether there...

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...

One key issue in the pharmaceutical development of new compounds is knowledge on metabolism, the enzymes involved and the potential hepatotoxicity of a drug. Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, human hepatocytes show phenotypic instability and have restricted accessibility and high batch-...

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatical...

Mango (Mangifera indica L.) stem bark extract (MSBE) is a natural product with biological properties and mangiferin is the major component. This paper reported the evaluation of the protective effects of MSBE and mangiferin against the toxicity induced in HepG2 cells by tert-butyl hydroperoxide or amiodarone. Nuclear morphology, cell viability, int...

Metabolomics represents the global assessment of metabolites in a biological sample and reports the closest information to the phenotype of the biological system under study. Mammalian cell metabolomics has emerged as a promising tool with potential applications in many biotechnology and research areas. Metabolomics workflow includes experimental d...

Fluorimetric methods to assess cytochrome P450 (P450) activities that do not require metabolite separation have been developed. These methods make use of non- or low-fluorescent P450 substrates that produce highly fluorescent metabolites in aqueous solutions. The assays are based on the direct incubation of intact cells in culture with appropriate...

Use of the HepG2 cell line to assess hepatotoxicity induced by bioactivable compounds is hampered by their low cytochrome P450 expression. To overcome this limitation, we have used adenoviral transfection to develop upgraded HepG2 cells (ADV-HepG2) expressing the major P450 enzymes involved in drug metabolism (CYP1A2, CYP2D6, CYP2C9, CYP2C19, and C...

Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA p...

The liver plays a key role in fat metabolism, and excessive lipid accumulation in liver cells is characterised by a large spectrum of lesions, e.g., steatosis and phospholipidosis. Steatosis is increased lipid accumulation, mainly as triglycerides, in the liver, while phospholipidosis is a lysosomal storage disorder characterised by intracellular a...

The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, th...

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds base...

BM-21 is an extract obtained from Thalassia testudinum marine plant with pharmacological properties. The effects of BM-21 and thalassiolin B (TB), its main component, on enzyme and transport proteins involved in drug metabolism and excretion in human cultured hepatocytes were evaluated. Cells were exposed for 48h to sub-cytotoxic concentrations of...

Major hepatic cytochrome P450 activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) can be simultaneously examined in human hepatocytes by incubation with a cocktail of multiple specific probes. Cocktail strategy in combination with mass spectrometry is shown to be a robust, fast, and sensitive procedure for P450 activity...

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 impossib...

In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyz...

Alternative and/or complementary sources of cells such as hepatic progenitor cells (HPC) are under investigation for hepatic cell therapy purposes. Steatotic livers are those most commonly rejected for clinical transplantation and are also unsuitable for good quality hepatocyte isolation. Taken together these two facts, our aim was to investigate w...

Steatosis, or excessive accumulation of lipids in the liver, is a generally accepted previous step to the development of more severe conditions like nonalcoholic steatohepatitis, fibrosis, and cirrhosis. We aimed to characterize the metabolic profile that defines simple steatosis in human tissue and to identify potential disturbances in the hepatic...

Storage of human hepatocytes is essential for their use in research and liver cell transplantation. However, cryopreservation and thawing (C/T) procedures have detrimental effects on the viability and functionality compared with fresh cells. The aim of this study was to upgrade the standard C/T methodology to obtain better quality hepatocytes for c...

The 6th European Framework Programme project LIINTOP was specifically raised to optimise and provide established protocols and experimental in vitro models for testing intestinal and liver absorption, metabolism and toxicity of molecules of pharmacological interest. It has been focused on some of the most promising existing liver and intestine in v...

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kin...

The liver is the most important target for toxicity caused by drugs. This vulnerability is a consequence of the functional features of the liver and their role in the metabolic elimination of most drugs. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. The liver is very active in metaboli...

The liver is the most important target for toxicity caused by drugs. This vulnerability is a consequence of the functional features of the liver and their role in the metabolic elimination of most drugs. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. The liver is very active in metaboli...

The liver is very active in metabolizing foreign compounds and the major target for toxicity caused by drugs. Hepatotoxicity may be the result of the drug itself or, more frequently, a result of the bioactivation process and the production of reactive metabolites. Prioritization of compounds based on human hepatotoxicity potential is currently a ke...

UDP-glucuronsyltransferases (UGTs) are a family of conjugating enzymes that participate in the metabolism of many drugs. The study of potential drug-drug interactions involving UGTs has been largely hindered by the limited availability of selective functional assays for individual UGT enzymes. We propose a sensitive and reproducible procedure for t...

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are req...

Four new fluorescent derivatives of cholic acid have been synthesized; they incorporate a dansyl moiety at 3alpha-, 3beta-, 7alpha- or 7beta- positions. These cholic acid analogs are UV photoactive and also exhibit green fluorescence. In addition, they have been demonstrated to be suitable for studying the kinetics of bile acid transport by flow cy...

The term fatty liver identifies a liver in which lipids account for more than 5% of the liver's wet weight. When fat accumulates, the lipids primarily stored as triglycerides (TG) result in steatosis and provide substrates for lipid peroxidation. Accumulation of neutral lipids in hepatocytes leads to micro- and macro-vesicular steatosis and to ball...

Drugs are capable of inducing hepatic lipid accumulation. When fat accumulates, lipids are primarily stored as triglycerides which results in steatosis and provides substrates for lipid peroxidation. An in vitro multiparametric flow cytometry assay was performed in HepG2 cells by using fluorescent probes to analyze cell viability (propidium iodide,...

Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6...

One of the most common mechanisms of hepatotoxicity is drug-induced cholestasis. Hence, new approaches for screening the cholestatic potential of drug candidates are desirable. In this context, we describe herein the use of synthetic 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent conjugates of cholic acid (ChA) at positions 3alpha, 3beta, 7alpha,...

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionalit...

A fast and sensitive method was developed for the assessment of CYP induction in human hepatocytes cultured in 96-well plates. The effects on CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were examined by means of mass spectrometry and the well-known cocktail strategy. The performance of the method was tested by using prototypic inducers such...

The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-...

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue f...

Cultured human hepatocytes are the closest in vitro model to human liver and constitute a very predictive model for drug metabolism in vivo. The variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of cytochrome P450 expression in human liver. As drug metabolism is the major source of pharmacokinetic variability...

Cholylamidofluorescein (CamF) has been selected as a fluorescent bile acid scaffold to perform a full characterization of its photophysical properties. In aqueous medium, under nitrogen, the absorption spectrum of CamF was expectedly dependent on pH. Under air, the presence of CO(2) resulted in a partial protonation of the photoactive form, reducin...

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the c...

Polyphenols are a family of natural compounds with many biological properties. This review focuses on their potential interaction on the cytochrome P450 system. Effects of phenolic acids, anthocyanins, stilbenes, catechins and other flavonoids on the drug metabolising function are revised. Their daily intake and presence in herbal medicines justify...

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluate...

It is now recognized that necrosis is not the only mechanism responsible for chemically-induced cell death. It is believed that apoptosis could be the major form of cell death induced by toxicants and that necrosis is associated only with circumstances of gross cell injury. The liver is a key target organ for drug toxicity and an important effort i...

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high...