Regenerative cell therapy for the treatment of hyperbilirubinemic Gunn rats with fresh and frozen human induced pluripotent stem cells-derived hepatic stem cells.

Pluripotent stem cells have been investigated as a renewable source of therapeutic hepatic cells, in order to overcome the lack of transplantable donor hepatocytes. Whereas different studies were able to correct hepatic defects in animal models, they focused on the most mature phenotype of hepatocyte-like cells (HLCs) derived from pluripotent stem cells and needed freshly prepared cells, which limits clinical applications of HLCs. Here, we report the production of hepatic stem cells (pHSCs) from human-induced pluripotent stem cells (hiPSCs) in xeno-free, feeder-free, and chemically defined conditions using as extracellular matrix a recombinant laminin instead of Matrigel, an undefined animal-derived matrix. Freshly prepared and frozen pHSCs were transplanted via splenic injection in Gunn rats, the animal model for Crigler-Najjar syndrome. Following cell transplantation and daily immunosuppression treatment, bilirubinemia was significantly decreased (around 30% decrease, P < .05) and remained stable throughout the 6-month study. The transplanted pHSCs underwent maturation in vivo to restore the deficient metabolic hepatic function (bilirubin glucuronidation by UGT1A1). In conclusion, we demonstrate for the first time the differentiation of hiPSCs into pHSCs that (a) are produced using a differentiation protocol compatible with Good Manufacturing Practices, (b) can be frozen, and (c) are sufficient to demonstrate in vivo therapeutic efficacy to significantly lower hyperbilirubinemia in a model of inherited liver disease, despite their immature phenotype. Thus, our approach provides major advances toward future clinical applications and would facilitate cell therapy manufacturing from human pluripotent stem cells.

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection.

BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells. RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis. CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.

Using high-content screening technology for studying drug-induced hepatotoxicity in preclinical studies.

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 permits molecular, cellular and tissue-based toxicity assessments. HCS allows automated image acquisition and analysis, and provides information on multiple properties of individual cells loaded simultaneously with fluorescent dyes, which is used for drug safety evaluations. Areas covered: Herein, the authors review the principles of HCS technology and some of the most widely used HCS assays for studying drug-induced hepatotoxicity in preclinical studies in general and in the pharmaceutical industry in particular. Expert opinion: The widespread acceptation of HCS by pharmaceutical companies and academic researchers highlights the potential usefulness of this technology as a prioritization tool in drug development. The improvement of different key points such as fluorescent probes or bioinformatics tools will consolidate HCS in drug discovery.

Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases?

The worldwide shortage of donor livers for organ and hepatocyte transplantation has prompted the search for alternative therapies for intractable liver diseases. Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for acute liver failure, end-stage and congenital liver diseases, or for those patients who are not considered eligible for organ transplantation. In recent years, research to identify alternative and reliable cell sources for transplantation that can be derived by reproducible methods has been encouraged. Human pluripotent stem cells (PSCs), which comprise both embryonic and induced PSCs, may offer many advantages as an alternative to hepatocytes for liver cell therapy. Their capacity for expansion, hepatic differentiation and self-renewal make them a promising source of unlimited numbers of hepatocyte-like cells for treating and repairing damaged livers. Immunogenicity and tumorigenicity of human PSCs remain the bottleneck for successful clinical application. However, recent advances made to develop disease-corrected hepatocyte-like cells from patients' human-induced PSCs by gene editing have opened up many potential gateways for the autologous treatment of hereditary liver diseases, which may likely reduce the risk of rejection and the need for lifelong immunosuppression. Well-defined methods to reduce the expression of oncogenic genes in induced PSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells. On top of this, such new strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages.

Hepatocyte transplantation program: Lessons learned and future strategies.

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.

Transplantation of hESC-derived hepatocytes protects mice from liver injury.

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.

Culture and Functional Characterization of Human Hepatoma HepG2 Cells.

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.

General Cytotoxicity Assessment by Means of the MTT Assay.

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 goals. As such, four main classes of assays are used to monitor the response of cultured cells after treatment with potential toxicants. These methods measure viability, cell membrane integrity, cell proliferation, and metabolic activity. In this chapter, we focus on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction colorimetric assay to evaluate detrimental intracellular effects on metabolic activity. This assay is well-characterized, simple to use and remains popular in several laboratories worldwide.

Human neonatal hepatocyte transplantation induces long-term rescue of unconjugated hyperbilirubinemia in the Gunn rat.

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.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

In vitro/in vivo screening of oxidative homeostasis and damage to DNA, protein, and lipids using UPLC/MS-MS.

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 chromatography coupled to mass spectrometry (UPLC-MS/MS), was developed and validated for the quantification of 16 oxidative stress (OS) biomarkers. Some of these markers were unstable; thus, an easy sample treatment procedure, including fractionation and derivatization, was set up. The method was validated according to Food and Drug Administration (FDA) guidelines, and it provided good results in terms of intra- and inter-day precision (≤17.2 and 16 %, respectively), accuracy (relative error measurement between -16.6 and 19.8 %), and linearity (R (2) > 0.994). The approach was applied to determine the oxidative insult provoked to cultured rat hepatocytes by cumene hydroperoxide and to analyze the liver and serum samples from patients diagnosed with nonalcoholic steatohepatitis. In both studies, significant differences were found if compared to the corresponding control groups; interestingly, ophthalmic acid was shown as an OS biomarker in both models for the first time. A key advantage of the novel approach in comparison with former multi-method approaches is that now a single method is applied to assess the 16 OS biomarkers. Its comprehensive capacity to profile oxidative homeostasis and damage in both in vitro and clinical samples has been illustrated, which indicates that the proposed approach is a good choice to evaluate whether OS is involved in physiological signals, diseases, or toxic events and to what extent.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

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 dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

miR-133a regulates vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), a key protein in the vitamin K cycle.

Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silico studies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3'UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.

Clinical outcome of hepatocyte transplantation in four pediatric patients with inherited metabolic diseases.

Hepatocyte transplantation (HT) has become an effective therapy for patients with metabolic inborn errors. We report the clinical outcome of four children with metabolic inborn errors that underwent HT, describing the cell infusion protocol and the metabolic outcome of transplanted patients. Cryopreserved hepatocytes were used as this allows scheduling of treatments. Functional competence (viability, cell attachment, major cytochrome P450 and UDP-glucuronosyltransferase 1A1 activities, and urea synthesis) and microbiological safety of cell batches were assessed prior to clinical use. Four pediatric patients with liver metabolic diseases [ornithine transcarbamylase (OTC) deficiency, Crigler-Najjar (CNI) syndrome, glycogen storage disease Ia (GSD-Ia), and tyrosinemia type I (TYR-I)] underwent HT. Indication for HT was based on severity of disease, deterioration of quality of life, and benefits for the patients, with the ultimate goal to improve their clinical status whenever liver transplantation (LT) was not indicated or to bridge LT. Cells were infused into the portal vein while monitoring portal flow. The protocol included antibiotic prophylaxis and immunosuppressant therapy. After HT, analytical data on the disease were obtained. The OTC-deficient patient showed a sustained decrease in plasma ammonia levels and increased urea production after HT. Further cell infusions could not be administered given a fatal nosocomial fungus sepsis 2 weeks after the last HT. The CNI and GSD-Ia patients improved their clinical status after HT. They displayed reduced serum bilirubin levels (by ca. 50%) and absence of hypoglycaemic episodes, respectively. In both cases, the HT contributed to stabilize their clinical status as LT was not indicated. In the infant with TYR-I, HT stabilized temporarily the biochemical parameters, resulting in the amelioration of his clinical status while diagnosis of the disease was unequivocally confirmed by full gene sequencing. In this patient, HT served as a bridge therapy to LT.

Evaluation of cytochrome P450 activities in human hepatocytes in vitro.

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 assessment. This procedure allows a drastic reduction of the number of cells required in the assay and sample analysis time and increases throughput and reproducibility. Major applications of the probe cocktail strategy are P450 phenotyping of hepatocytes and induction studies.

Mechanism-based selection of compounds for the development of innovative in vitro approaches to hepatotoxicity studies in the LIINTOP project.

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 vitro models with the aim of further improving their performance and thus taking them to a pre-normative research stage. Regarding the specific area of the liver, a first basic approach was the optimisation of in vitro hepatic models and the development and optimisation of in vitro approaches for toxicity screening. New advanced technologies have been proposed and developed in order to determine cellular and molecular targets as endpoints of drug exposure. A key issue in the development and optimisation of in vitro hepatotoxicity screening methods was the selection of structurally diverse suitable hepatotoxic reference model compounds to be tested. To this end, a number of solid selection criteria were defined (drugs preferably than chemical agents, well-documented hepatotoxicity in man and well-defined mechanism/s of hepatotoxicity, commercially available no volatile compounds with unequivocal CAS number and chemical structure), the strategy followed, including all resources consulted, is described and the selected compounds are extensively illustrated.

The use of hepatocytes to investigate drug toxicity.

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 key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. Cultured hepatocytes are increasingly used by the pharmaceutical industry for the screening of hepatotoxic potential of new molecules. Hepatocytes in culture retain hepatic key functions and constitute a valuable tool to identify chemically induced cellular damage. Their use has notably contributed to the understanding of mechanisms responsible for hepatotoxicity (disruption of cellular energy status, alteration of Ca(2+) homeostasis, inhibition of transport systems, metabolic activation, oxidative stress, covalent binding, etc.). Assessment of current cytotoxicity and hepatic-specific biochemical effects is limited by the inability to measure a wide spectrum of potential mechanistic changes involved in the drug-induced toxic injury. A convenient selection of endpoints allows a multiparametric evaluation of drug toxicity. In this regard, cytomic, proteomic, toxicogenomic and metabonomic approaches help to define patterns of hepatotoxicity for early identification of potential adverse effects of the drug to the liver.

Cytochrome p450 and steatosis.

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 balloon-cell degeneration. Increased fat deposition in the liver is generally believed to be the result of an imbalance between fatty acids (FA) inflow/oxidation, and triglyceride synthesis and excretion. Fat accumulation is not necessarily a pathological condition, but has been suggested to be the setting for more severe liver diseases, including nonalcoholic steatohepatitis (NASH) or cirrhosis. Since steatosis is notably present in the Western world, there is increased interest to know its potential consequences for the liver function. However, the information available to date about the impact of steatosis on the human liver metabolism is very scarce. Specifically, the impaired metabolism of a number of drugs has been associated with fatty liver. In relation to this, changes in some cytochrome P450 (CYP) enzymes have been found in livers of patients with steatosis, in vivo models of steatosis in experimental animals and in vitro models of fat-overloaded cells. These findings suggest an association between increased lipid deposition and impaired CYP enzymes. This paper presents an overview of the impact of steatosis in the liver's drug-metabolizing capability. Moreover, the possible molecular mechanisms involved in the transcriptional regulation of the CYP expression in fatty liver are discussed.