Competency of different cell models to predict human hepatotoxic drugs.

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.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.In vitro toxicity assays performed in hepatocytes or hepatoma cell …

A newin vitroapproach for the simultaneous determination of phase I and phase II enzymatic activities of human hepatocyte preparations

Primary hepatocytes are still the best qualified in vitro system to anticipate drug metabolism in man. Recent advances in hepatocytes cryopreservation have notably increased their use not only for drug metabolism studies, but also for other applications such as cell transplantation. Evaluation of the drug-metabolizing competence of each hepatocytes preparation is needed. To date, the metabolic characterization of hepatocytes preparations relies on the assessment of phase I activities and the role of phase II enzymes receives little attention. A novel approach for the rapid assessment of the metabolic functionality of hepatocytes has been developed. A five-probe cocktail was used to simultan…

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo.

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. How…

An update on metabolism studies using human hepatocytes in primary culture

Background: 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. Objectives: As drug metabolism is the major source of pharmacokinetic variability in human beings, the main areas of current drug metabolism research in human hepatocytes are reviewed. Methods: To speed up the selection of drug candidates, the evaluation of metabolic stability, metabolite profiling and identification, and drug–drug interaction potential are key issues in drug development. Results/conclus…

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.

Evaluation of drug-metabolizing and functional competence of human hepatocytes incubated under hypothermia in different media for clinical infusion.

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 for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in Suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, b…

Assessment of the cytotoxic potential of an aqueous-ethanolic extract from Thalassia testudinum angiosperm marine grown in the Caribbean Sea

Abstract Objectives Reported antioxidant, anti-inflammatory and neuroprotective properties for one aqueous-ethanolic extract from Thalassia testudinum which grows in the Caribbean Sea compelled us to explore about extract cytotoxic effects. Methods Cell viability was assayed on tumour (HepG2, PC12, Caco-2 and 4T1) and non-tumour (VERO, 3T3, CHO, MCDK and BHK2) cell lines. The extract effects upon primary cultures of rat and human hepatocytes and human lymphocytes were assayed. Key findings The extract exhibited cytotoxicity against cancer cells compared to normal cells, and the IC50 values were 102 μg/ml for HepG2, 135 μg/ml for PC12, 165 μg/ml for Caco-2 and 129 μg/ml for 4T1 cells after 4…

Functional assessment of the quality of human hepatocyte preparations for cell transplantation.

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 functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepat…

Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

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 (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 mic…

Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell vi…

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

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 required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocyt…

Development of multiparametric cell-based protocol to assess and classify hepatotoxicity potential of drugs

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes.

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 evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrationsor = 200 microg/mL, whereas onl…

Mammalian cell metabolomics: Experimental design and sample preparation

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 design, sampling, sample processing, metabolite analysis, and data processing. Given their influence on metabolite content and biological interpretation of data, a good experimental design and the appropriate choice of a sample processing method are prerequisites for success in any metabolomic study. The use of mammalian cells in the metabolomics f…

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 reducti…

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 b…

A specific microassay for evaluating hepatic LDH activity in co-cultures of hepatocytes with other cells.

This study describes the development of a simple, rapid and reproducible microassay for determining the intracellular LDH activity of rat hepatocytes present in a co-culture system with other cells. The procedure involves treatment of cellular homogenates with an anti-LDH antiserum that specifically inhibits the LDH activity of rat hepatocytes. The assay is performed in 96-well plates and LDH activity can be measured directly in the same wells using a colorimetric method. The difference in LDH activity values measured before and after antiserum incubation reflects the LDH content of the hepatocytes in the sample. The advantages of this method are the small number of cells required, a reduct…

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 hepa…

Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.

Abstract The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25–3 mM FFA induced dose-dependent ac…

Inhibition of human P450 enzymes by natural extracts used in traditional medicine

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, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts,…

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…

Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes.

Abstract Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 μg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 μg/mL). The effect of the extract (50–400 μg/mL) on several P450 isozymes…

Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Evaluation of the effect of NCEs on human CYP450 enzyme activities is a key issue in pharmaceutical development as it may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. A liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for the fast and routine analysis of major human CYP450s enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in primary hepatocyte cell cultures. The high sensitivity and selectivity of mass …

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 …

A human hepatocellular in vitro model to investigate steatosis

The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (Cl 8: 1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incub…

Cell Lines: A Tool for In Vitro Drug Metabolism Studies

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 batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given thei…

Modulation of biotransformation and elimination systems by BM-21, an aqueous ethanolic extract from Thalassia testudinum, and thalassiolin B on human hepatocytes

Abstract 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 48 h to sub-cytotoxic concentrations of BM-21 or TB. Effects on P450 isoforms revealed significant reductions of CYP1A2, 3A4 and 2D6 activities (up to 56%, 66% and 44% inhibition, respectively) after exposition to BM-21, no changes on CYP2A6 and 2C9 activities. TB produced a concentration-dependent reduction of all P450 activities. In addition, a decrease in total UGT and UGT2…

Constitutive and inducible expression of CYP enzymes in immortal hepatocytes derived from SV40 transgenic mice

1. The expression of liver-specific transcription factors and cytochrome P450 (CYP) enzymes have been studied in three new hepatocyte-like cell lines derived from SV Delta 202 transgenic mice: AMH-Delta 202 (adult mouse hepatocytes), TAMH-Delta 202 (tumour-derived adult mouse hepatocytes) and NMH-Delta 202 (newborn mouse hepatocytes). 2. mRNA levels of liver-enriched transcription factors such as D-element binding protein (DBP), liver-enriched transcription activating protein (LAP) and the hepatic nuclear factors (HNF) 1, 2 and 3 in all Delta 202 transgenic hepatocyte lines were similar to those in the wild-type liver and in primary mouse hepatocytes. 3. Analysis of basal CYP activities and…

Strategies to In Vitro Assessment of Major Human CYP Enzyme Activities by Using Liquid Chromatography Tandem Mass Spectrometry

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologi…