Primary human hepatocytes-laden scaffolds for the treatment of acute liver failure.

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-hyaluronic acid (Gel-HA) scaffolds seeded with PHH for the treatment of liver failure. We first optimized the composition using Gel-HA hydrogels, looking for the mechanical properties closer to the human liver and determining HepG2 cells functionality. Gel-HA scaffolds with interconnected porosity (pore size 102 µm) were prepared and used for PHH culture and evaluation of key hepatic functions. PHH cultured in Gel-HA scaffolds exhibited increased albumin and urea secretion and metabolic capacity (CYP and UGT activity levels) compared to standard monolayer cultures. The transplant of the scaffold containing PHH led to an improvement in liver function (transaminase levels, necrosis) and ameliorated damage in a mouse model of acetaminophen (APAP)-induced liver failure. The study provided a mechanistic understanding of APAP-induced liver injury and the impact of transplantation by analyzing cytokine production and oxidative stress induction to find suitable biomarkers of cell therapy effectiveness.

Mechanistic Understanding of Idiosyncratic Drug-Induced Hepatotoxicity Using Co-Cultures of Hepatocytes and Macrophages.

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 recapitulated in current in vitro models; thus, our major goal is to develop and fully characterize a co-culture system and to evaluate its suitability for predicting iDILI. For this purpose, we used human hepatoma HepG2 cells and macrophages differentiated from a monocyte cell line (THP-1) and established the appropriate co-culture conditions for mimicking an inflammatory environment. Then, mono-cultures and co-cultures were treated with model iDILI compounds (trovafloxacin, troglitazone) and their parent non-iDILI compounds (levofloxacin, rosiglitazone), and the effects on viability and the mechanisms implicated (i.e., oxidative stress induction) were analyzed. Our results show that co-culture systems including hepatocytes (HepG2) and other cell types (THP-1-derived macrophages) help to enhance the mechanistic understanding of iDILI, providing better hepatotoxicity predictions.

In Vitro Models for Studying Chronic Drug-Induced Liver Injury.

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 animals, probably due to the well-known interspecies differences in drug metabolism, pharmacokinetics, and toxicity targets. For this reason, distinct cellular models from primary human hepatocytes or hepatoma cell lines cultured as 2D monolayers to emerging 3D culture systems or the use of multi-cellular systems have been proposed for hepatotoxicity studies. In order to mimic long-term hepatotoxicity in vitro, cell models, which maintain hepatic phenotype for a suitably long period, should be used. On the other hand, repeated-dose administration is a more relevant scenario for therapeutics, providing information not only about toxicity, but also about cumulative effects and/or delayed responses. In this review, we evaluate the existing cell models for DILI prediction focusing on chronic hepatotoxicity, highlighting how better characterization and mechanistic studies could lead to advance DILI prediction.

Long-term and mechanistic evaluation of drug-induced liver injury in Upcyte human hepatocytes.

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 hepatotoxicity. For this reason, we here evaluated the potential of using Upcytes human hepatocytes (UHH) for repeated-dose long-term exposure to drugs. The UHH were incubated with 15 toxic and non-toxic compounds for up to 21 days using a repeated-dose approach, and, in addition to conventional examination of effects on viability, the mechanisms implicated in cell toxicity were also assessed by means of high-content screening. The UHH maintained the expression and activity levels of drug-metabolizing enzymes for up to 21 days of culture and became more sensitive to the toxic compounds after extended exposures, showing inter-donor differences which would reflect variability among the population. The assay also allowed to detect the main mechanisms implicated in the toxicity of each drug as well as identifying special susceptibilities depending on the donor. UHH can be used for a long-term repeated detection of DILI at clinically relevant concentrations and also offers key mechanistic features of drug-induced hepatotoxicity. This system is therefore a promising tool in preclinical testing of human relevance that could help to reduce and/or replace animal testing for drug adverse effects.

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 lipidomic cell-based assay for studying drug-induced phospholipidosis and steatosis.

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 steatosis. Cells with and without preincubation with a mixture of free fatty acids (FFA; i.e. oleic and palmitic) were exposed to a set of well-known steatogenic and phospholipidogenic compounds. The use of FFA preloading accelerated the accumulation of phospholipids, thus leading to a better discrimination of phospholipidosis, and magnified the lipidomic alterations induced by steatogenic drugs. Phospholipidosis was characterized by increased levels of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols, while steatosis induced alterations in FA oxidation and triacylglyceride (TG) synthesis pathways (with changes in the levels of FFA, acylcarnitines, monoacylglycerides, diacylglycerides, and TG). Interestingly, palmitic and oleic acids incorporation into lipids differed. A characteristic pattern was observed in the fold of change of particular TG species in the case of steatosis (TG(54:3) > TG(52:2) > TG(50:1) > TG(48:0)). Based on the levels of those lipids containing only palmitic and/or oleic acid moieties a partial least squares-discriminant analysis model was built, which showed good discrimination among nontoxic, phospholipidogenic and steatogenic compounds. In conclusion, it has been shown that the use of FFA preincubation together with intracellular LC-MS based lipid profiling could be a useful approach to identify the potential of drug candidates to induce phospholipidosis and/or steatosis.

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.

A metabolomics cell-based approach for anticipating and investigating drug-induced liver injury.

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 a metabolomic mass spectrometry-based approach for the detection and classification of drug-induced hepatotoxicity. To this end, the metabolite profiles of human derived hepatic cells (i.e., HepG2) exposed to different well-known hepatotoxic compounds acting through different mechanisms (i.e., oxidative stress, steatosis, phospholipidosis, and controls) were compared by multivariate data analysis, thus allowing us to decipher both common and mechanism-specific altered biochemical pathways. Briefly, oxidative stress damage markers were found in the three mechanisms, mainly showing altered levels of metabolites associated with glutathione and γ-glutamyl cycle. Phospholipidosis was characterized by a decreased lysophospholipids to phospholipids ratio, suggestive of phospholipid degradation inhibition. Whereas, steatosis led to impaired fatty acids ß-oxidation and a subsequent increase in triacylglycerides synthesis. The characteristic metabolomic profiles were used to develop a predictive model aimed not only to discriminate between non-toxic and hepatotoxic drugs, but also to propose potential drug toxicity mechanism(s).

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.

Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

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 cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

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.

Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction.

BACKGROUND & AIMS: 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 is a preoperative donor liver metabolomic biosignature associated with EAD. METHODS: A comprehensive metabolomic profiling of 124 donor liver biopsies collected before transplantation was performed by mass spectrometry coupled to liquid chromatography. Donor liver grafts were classified into two groups: showing EAD and immediate graft function (IGF). Multivariate data analysis was used to search for the relationship between the metabolomic profiles present in donor livers before transplantation and their function in recipients. RESULTS: A set of liver graft dysfunction-associated biomarkers was identified. Key changes include significantly increased levels of bile acids, lysophospholipids, phospholipids, sphingomyelins and histidine metabolism products, all suggestive of disrupted lipid homeostasis and altered histidine pathway. Based on these biomarkers, a predictive EAD model was built and further evaluated by assessing 24 independent donor livers, yielding 91% sensitivity and 82% specificity. The model was also successfully challenged by evaluating donor livers showing primary non-function (n=4). CONCLUSIONS: A metabolomic biosignature that accurately differentiates donor livers, which later showed EAD or IGF, has been deciphered. The remarkable metabolomic differences between donor livers before transplant can relate to their different quality. The proposed metabolomic approach may become a clinical tool for donor liver quality assessment and for anticipating graft function before transplant.

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.

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.

HepG2 cells simultaneously expressing five P450 enzymes for the screening of hepatotoxicity: identification of bioactivable drugs and the potential mechanism of toxicity involved.

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 CYP3A4) at levels comparable to those of human hepatocytes. The potential utility of this new cell model for the in vitro screening of bioactivable drugs was assessed using a high-content screening assay that we recently developed to simultaneously measure multiple parameters indicative of cell injury. To this end, ADV-HepG2 and HepG2 cells, cultured in 96-well plates, were exposed for 24 h to a wide range of concentrations of 12 bioactivable and 3 non-bioactivable compounds. The cell viability and parameters associated with nuclear morphology, mitochondrial function, intracellular calcium concentration, and oxidative stress indicative of prelethal cytotoxicity and representative of different mechanisms of toxicity were evaluated. Bioactivable compounds showed lower IC(50) values in ADV-HepG2 cells than in HepG2 cells. Moreover, significant differences in the other parameters analyzed were observed between both cell models, while similar effects were observed for non-bioactivable compounds (negative controls). The changes in cell parameters detected in our assay for a given compound are in good agreement with the previously reported toxicity mechanism. Overall, our results indicate that this assay may be a suitable new in vitro approach for early screening of compounds to identify bioactivable hepatotoxins and the mechanism(s) involved in their toxicity.

Development of a multiparametric cell-based protocol to screen and classify the hepatotoxicity potential of drugs.

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 based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000µM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

Upgrading cytochrome P450 activity in HepG2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment.

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 impossible the study of the toxicity produced by bioactivable compounds. Genetically manipulated cells constitute promising tools for hepatotoxicity applications. HepG2 cells were simultaneously transfected with recombinant adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4 to confer them drug-metabolic competence. Upgraded cells (Adv-HepG2) were highly able to metabolize the toxin studied in contrast to the reduced metabolic capacity of HepG2 cells. Aflatoxin B1-induced hepatotoxicity was studied as a proof of concept in metabolically competent and non-competent HepG2 cells by using high content screening technology. Significant differences in mitochondrial membrane potential, intracellular calcium concentration, nuclear morphology and cell viability after treatment with aflatoxin B1 were observed in Adv-HepG2 when compared to HepG2 cells. Rotenone (non bioactivable) and citrate (non hepatotoxic) were analysed as negative controls. This cell model showed to be a suitable hepatic model to test hepatotoxicity of bioactivable drugs and constitutes a valuable alternative for hepatotoxicity testing.