Induced pluripotent stem cells for the treatment of liver diseases: challenges and perspectives from a clinical viewpoint.

The only curative treatment for severe end-stage liver disease (ESLD) is liver transplantation (LT) but it is limited by the shortage of organ donors. The increase of the incidence of liver disease has led to develop new therapeutic approaches such as liver cell transplantation. Current challenges that limit a wider application of this therapy include a limited cell source and the poor engraftment in the host liver of cryopreserved hepatocytes after thawing. Induced pluripotent stem cells (iPSCs) that can be differentiated into hepatocyte-like cells (HLCs) are being widely explored as an alternative to human hepatocytes because of their unlimited proliferation capacity and their potential ability to avoid the immune system. Their large-scale production could provide a new tool to produce enough HLCs for treating patients with metabolic diseases, acute liver failure (ALF), those with ESLD or patients not considered for organ transplantation. In this review we discuss current challenges for generating differentiated cells compatible with human application as well as in-depth safety evaluation. This analysis highlights the uncertainties and deficiencies that should be addressed before their clinical use but also points out the potential benefits that will produce a great impact in the field of hepatology.

Improved in vivo efficacy of clinical-grade cryopreserved human hepatocytes in mice with acute liver failure.

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 an unpredictable loss of attachment ability of the cells, which can be aggravated by cryopreservation and thawing. In this work, we focused on the use of a Good Manufacturing Practice (GMP) solution compared with the standard cryopreservation medium, the University of Wisconsin medium, for the purpose of improving the functional quality of cells and their ability to engraft in vivo, with the idea of establishing a biobank of cryopreserved human hepatocytes available for their clinical use. We evaluated not only cell viability but also specific hepatic function indicators of the functional performance of the cells such as attachment efficiency, ureogenic capability, phase I and II enzymes activities and the expression of specific adhesion molecules in vitro. Additionally, we also assessed and compared the in vivo efficacy of human hepatocytes cryopreserved in different media in an animal model of acute liver failure. Human hepatocytes cryopreserved in the new GMP solution offered better in vitro and in vivo functionality compared with those cryopreserved in the standard medium. Overall, the results indicate that the new tested GMP solution maintains better hepatic functions and, most importantly, shows better results in vivo, which could imply an increase in long-term efficacy when used in patients.

Advances in drug-induced cholestasis: Clinical perspectives, potential mechanisms and in vitro systems.

Despite growing research, drug-induced liver injury (DILI) remains a serious issue of increasing importance to the medical community that challenges health systems, pharmaceutical industries and drug regulatory agencies. Drug-induced cholestasis (DIC) represents a frequent manifestation of DILI in humans, which is characterised by an impaired canalicular bile flow resulting in a detrimental accumulation of bile constituents in blood and tissues. From a clinical point of view, cholestatic DILI generates a wide spectrum of presentations and can be a diagnostic challenge. The drug classes mostly associated with DIC are anti-infectious, anti-diabetic, anti-inflammatory, psychotropic and cardiovascular agents, steroids, and other miscellaneous drugs. The molecular mechanisms of DIC have been investigated since the 1980s but they remain debatable. It is recognised that altered expression and/or function of hepatobiliary membrane transporters underlies some forms of cholestasis, and this and other concomitant mechanisms are very likely in DIC. Deciphering these processes may pave the ways for diagnosis, prognosis and prevention, for which currently major gaps and caveats exist. In this review, we summarise recent advances in the field of DIC, including clinical aspects, the potential mechanisms postulated so far and the in vitro systems that can be useful to investigate and identify new cholestatic drugs.

Predicting drug-induced cholestasis: preclinical models.

INTRODUCTION: In almost 50% of patients with drug-induced liver injury (DILI), the bile flow from the liver to the duodenum is impaired, a condition known as cholestasis. However, this toxic response only appears in a small percentage of the treated patients (idiosyncrasy). Prediction of drug-induced cholestasis (DIC) is challenging and emerges as a safety issue that requires attention by professionals in clinical practice, regulatory authorities, pharmaceutical companies, and research institutions. Area covered: The current synopsis focuses on the state-of-the-art in preclinical models for cholestatic DILI prediction. These models differ in their goal, complexity, availability, and applicability, and can widely be classified in experimental animals and in vitro models. Expert opinion: Drugs are a growing cause of cholestasis, but the progress made in explaining mechanisms and differences in susceptibility is not growing at the same rate. We need reliable models able to recapitulate the features of DIC, particularly its idiosyncrasy. The homogeneity and the species-specific differences move animal models away from a fair predictability. However, in vitro human models are improving and getting closer to the real hepatocyte phenotype, and they will likely be the choice in the near future. Progress in this area will not only need reliable predictive models but also mechanistic insights.

Customised in vitro model to detect human metabolism-dependent idiosyncratic drug-induced liver injury.

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 described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify metabolic phenotypes with increased susceptibility to DILI. To this end, HepG2 cells with different expression levels of specific drug-metabolism enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, GSTM1 and UGT2B7) were exposed to nine drugs with reported hepatotoxicity. A panel of pre-lethal mechanistic parameters (mitochondrial superoxide production, mitochondrial membrane potential, ROS production, intracellular calcium concentration, apoptotic nuclei) was used. Significant differences were observed according to the level of expression and/or the combination of several drug-metabolism enzymes in the cells created ad hoc according to the enzymes implicated in drug toxicity. Additionally, the main mechanisms implicated in the toxicity of the compounds were also determined showing also differences between the different types of cells employed. This screening tool allowed to mimic the variability in drug metabolism in the population and showed a highly efficient system for predicting human DILI, identifying the metabolic phenotypes associated with increased DILI risk, and indicating the mechanisms implicated in their toxicity.

A Multi-Parametric Fluorescent Assay for the Screening and Mechanistic Study of Drug-Induced Steatosis in Liver Cells in Culture.

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.

Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing.

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.

Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing.

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. Recently developed human upcyte hepatocytes offer the advantage of combining many features of primary hepatocytes with the unlimited availability of hepatoma cells. We analyzed the phenotype of upcyte hepatocytes comparatively with HepG2 cells and adult primary human hepatocytes to characterize their functional features as a differentiated hepatic cell model. The transcriptomic analysis of liver characteristic genes confirmed that the upcyte hepatocytes expression profile comes closer to human hepatocytes than HepG2 cells. CYP activities were measurable and showed a similar response to prototypical CYP inducers than primary human hepatocytes. Upcyte hepatocytes also retained conjugating activities and key hepatic functions, e.g. albumin, urea, lipid and glycogen synthesis, at levels close to hepatocytes. We also investigated the suitability of this cell model for preclinical hepatotoxicity risk assessments using multiparametric high-content screening, as well as transcriptomics and targeted metabolomic analysis. Compounds with well-documented in vivo hepatotoxicity were screened after acute and repeated doses up to 1 week. The evaluation of complex mechanisms of cell toxicity, drug-induced steatosis and oxidative stress biomarkers demonstrated that, by combining the phenotype of primary human hepatocytes and the ease of handling of HepG2 cells, upcyte hepatocytes offer suitable properties to be potentially used for toxicological assessments during drug development.

Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis.

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 non-steatotic 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.

Metabolic activation and drug-induced liver injury: in vitro approaches for the safety risk assessment of new drugs.

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 understand better the idiosyncratic DILI and to identify potential risk factors. Metabolic bioactivation of drugs to form reactive metabolites is considered an initiation mechanism for idiosyncratic DILI. Reactive species may interact irreversibly with cell macromolecules (covalent binding, oxidative damage), and alter their structure and activity. This review focuses on proposed in vitro screening strategies to predict and reduce idiosyncratic hepatotoxicity associated with drug bioactivation. Compound incubation with metabolically competent biological systems (liver-derived cells, subcellular fractions), in combination with methods to reveal the formation of reactive intermediates (e.g., formation of adducts with liver proteins, metabolite trapping or enzyme inhibition assays), are approaches commonly used to screen the reactivity of new molecules in early drug development. Several cell-based assays have also been proposed for the safety risk assessment of bioactivable compounds. Copyright © 2015 John Wiley & Sons, Ltd.

LC-MS untargeted metabolomic analysis of drug-induced hepatotoxicity in HepG2 cells.

Hepatotoxicity is the number one cause for agencies not approving and withdrawing drugs for the market. Drug-induced human hepatotoxicity frequently goes undetected in preclinical safety evaluations using animal models. Human-derived in vitro models represent a common alternative to in vivo tests to detect toxic effects during preclinical testing. Most current in vitro toxicity assays rely on the measurement of nonspecific or low sensitive endpoints, which result in poor concordance with human liver toxicity. Therefore, making more accurate predictions of the potential hepatotoxicity of new drugs remains a challenge. Metabolomics, whose aim is to globally assess all the metabolites present in a biological sample, may represent an alternative in the search for sensitive sublethal markers of drug-induced hepatotoxicity. To this end, a comprehensive LC-MS-based untargeted metabolite profiling analysis of HepG2 cells, exposed to a set of well-described model hepatotoxins and innocuous compounds, was performed. It allowed to determine meaningful metabolic changes triggered by a toxic insult and gave a first estimation of the main toxicity-related pathways. Based on these metabolic patterns, a partial least squares-discriminant analysis model, able to discriminate between nontoxic and hepatotoxic compounds, was constructed. The approach described herein may provide an alternative for animal testing in preclinical stages of drug development and a controlled experimental approach to gain a better understanding of the underlying causes of hepatotoxicity.

High-content screening technology for studying drug-induced hepatotoxicity in cell models.

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.

High-content screening of drug-induced mitochondrial impairment in hepatic cells: effects of statins.

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 detail. Simultaneous assessment of mitochondrial mass and cell viability in cells exposed for 24 h to compounds provides preliminary information on the mitochondrial- or nonmitochondrial-related hepatotoxic potential of compounds. To fully address the mechanisms implicated in mitochondrial impairment, prelethal changes in mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial permeability transition, intracellular calcium concentration and apoptotic cell death were studied in cells incubated for 1 h with compounds. The assay correctly classified a set of well-known mitochondrial toxicants and negative controls and revealed high sensitivity for the detection of mitochondrial DILI and the establishment of different mitochondrial toxicity risks (low to high). This procedure was used for analysing the potential mitochondrial impairment of six statins to determine their clinical risk. All the tested statins produced mitochondrial impairment, although they showed different levels of toxicity (low-medium toxicity risk). The results suggest that this cell-based assay is a promising in vitro approach to predict the potential of drug candidates to induce mitochondrial-associated hepatotoxicity.

Competency of different cell models to predict human hepatotoxic drugs.

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 COVERED: This review describes the advantages and limitations of hepatic cell-based models for early safety risk assessment during drug development. These models include hepatocytes cultured as monolayer, collagen-sandwich; emerging complex 3D configuration; liver-derived cell lines; stem cell-derived hepatocytes. EXPERT OPINION: In vitro toxicity assays performed in hepatocytes or hepatoma cell lines can potentially provide rapid and cost-effective early feedback to identify toxic candidates for compound prioritization. However, their capacity to predict hepatotoxicity depends critically on cells' functional performance. In an attempt to improve and prolong functional properties of cultured cells, different strategies to recreate the in vivo hepatocyte environment have been explored. 3D cultures, co-cultures of hepatocytes with other cell types and microfluidic devices seem highly promising for toxicological studies. Moreover, hepatocytes derived from human pluripotent stem cells are emerging cell-based systems that may provide a stable source of hepatocytes to reliably screen metabolism and toxicity of candidate compounds.

Estado actual y perspectivas futuras del trasplante de hepatocitos / Current status and future perspectives of hepatocyte transplantation

Existe un gran número de enfermedades hepáticas para las cuales el único tratamiento efectivo es el trasplante hepático. La disparidad entre el número de potenciales beneficiarios y de órganos disponibles motiva la búsqueda de nuevas alternativas de tratamiento, entre las que se encuentra el trasplante celular hepático (TCH). Esta terapia representa una alternativa de tratamiento en estos pacientes, sin embargo, la falta de unanimidad de criterios respecto a las indicaciones y técnica, los diferentes protocolos de criopreservación así como la distinta metodología para valorar la respuesta a esta terapia pone de manifiesto la necesidad de una conferencia de consenso que unifique criterios, planteando posibles estrategias futuras que mejoren la técnica y optimicen los resultados. Nuestro objetivo es realizar una revisión y puesta al día del estado actual del TCH, enfatizando las futuras líneas de investigación que tratan de solucionar los problemas y mejorar los resultados de esta terapia

The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results. Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment

A simple transcriptomic signature able to predict drug-induced hepatic steatosis.

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.

Neonatal livers: a source for the isolation of good-performing hepatocytes for cell transplantation.

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 hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (ß1-integrin, ß-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug-metabolizing capability (cytochrome P450 and UDP-glucuronosyltransferase enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM(+) staining), which could also participate in regeneration of liver parenchyma after transplantation. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human hepatocyte transplantation applicability.

[Current status and future perspectives of hepatocyte transplantation]. / Estado actual y perspectivas futuras del trasplante de hepatocitos.

The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment.

Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering.

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-to-batch functional variability, which seriously complicates their use in routine testing. Therefore, several liver-derived cell models have been developed for drug metabolism and hepatotoxicity screening to circumvent these drawbacks. Hepatoma cell lines offer important advantages, availability, an unlimited life span and a stable phenotype, thus rendering them suitable models for such studies. However, currently available human hepatoma cell lines are not a good alternative to cultured hepatocytes as they show very limited expression for most drug-metabolising enzymes. Other approaches have been developed to generate immortalised hepatic cells with metabolic competence (use of plasmids encoding immortalising genes to transform human hepatocytes, cell lines obtained from transgenic animals, hepatocytomes or hydrid cells). Recombinant models heterologously expressing cytochrome P450 enzymes in hepatoma cells have also been generated, and are widely used in drug metabolism and toxicity evaluations. In recent years, new approaches to up-regulate the expression of drug-biotransformation enzymes in human cell lines (i.e., transfection with the expression vectors encoding key hepatic transcription factors) have also been investigated. This paper reviews the features of liver-derived cell lines, their suitability for drug metabolism and hepatotoxicity studies, and the state-of-the-art strategies pursued to generate metabolically competent hepatic cell lines.

Multiparametric evaluation of the cytoprotective effect of the Mangifera indica†L. stem bark extract and mangiferin in HepG2 cells.

OBJECTIVE: 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. METHOD: Nuclear morphology, cell viability, intracellular calcium concentration and reactive oxygen species (ROS) production were measured by using a high-content screening multiparametric assay. KEY FINDINGS: MSBE and mangiferin produced no toxicity below 500 mg/ml doses. A marked recovery in cell viability, which was reduced by the toxicants, was observed in cells pre-exposed to MSBE or mangiferin at 5-100 mg/ml doses. We also explored the possible interaction of both products over P-glycoprotein (P-gp). MSBE and mangiferin above 100 mg/ml inhibited the activity of P-gp in HepG2 cells. CONCLUSIONS: MSBE and mangiferin showed cytoprotective effects of against oxidative damage and mitochondrial toxicity induced by xenobiotics to human hepatic cells but it seemed that other constituents of the extract could contribute to MSBE protective properties. In addition, the drug efflux should be taken into account because of the inhibition of the P-gp function observed in those cells exposed to both natural products.