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PubMed | Gentoxicon BVBA, Biopredic International, Irccs Instituto Of Ricerche Farmacologiche Mario Negri, Scientific Institute of Public Health WIV ISP and Super Stellar Solutions
Type: Journal Article | Journal: Mutagenesis | Year: 2016

Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.


PubMed | Biopredic International, Innsbruck Medical University, Vrije Universiteit Brussel, Max Planck Institute for Molecular Genetics and 6 more.
Type: Journal Article | Journal: Archives of toxicology | Year: 2016

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.


PubMed | Biopredic International, French Institute of Health and Medical Research and University of Rennes 1
Type: | Journal: Scientific reports | Year: 2016

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.


Higuchi Y.,Central Institute for Experimental Animals | Kawai K.,Central Institute for Experimental Animals | Yamazaki H.,Showa Pharmaceutical University | Nakamura M.,Tokai University | And 3 more authors.
Xenobiotica | Year: 2014

1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology. © 2014 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted.


PubMed | Biopredic International, Nissan Chemical Industries, Showa Pharmaceutical University and Central Institute for Experimental Animals
Type: Journal Article | Journal: Hepatology research : the official journal of the Japan Society of Hepatology | Year: 2016

The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the 3D culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects.We compared 2D-cultured HepaRG cells with 3D-cultured HepaRG spheroids in the gene expression patterns and the metabolic functions. In the present study, we performed 3D culture of HepaRG cells using functional polymers (FP). To reveal the in vivo differentiation ability, we transplanted the 3D-cultured HepaRG spheroids into TK-NOG mice.A comparison between 2D and 3D cultures revealed that 3D-cultured HepaRG spheroids demonstrated reductions in bile duct marker expression, accelerated expression of cytochrome P450 3A4, and increases in the ratio of albumin-expressing hepatocytes. Furthermore, catalytic activities of cytochrome P450 3A4 were modified by omeprazole and rifampicin in the 3D-cultured HepaRG spheroids. Transplantation analysis revealed that 3D-cultured HepaRG spheroids formed hepatocyte-like colonies rather than cholangiocytes in vivo.Our results indicated that the enhancement of hepatic functions in 3D-cultured HepaRG cells was induced by selective hepatocyte differentiation and accelerated hepatocyte maturation. HepaRG spheroids reproduced the metabolic responses of human hepatocytes. Therefore, FP-dependent 3D-cultured HepaRG cells may serve as an excellent in vitro model for evaluating the hepatic metabolism and toxicity.


Novakova I.,University of Vienna | Subileau E.-A.,Biopredic International | Toegel S.,Medical University of Vienna | Gruber D.,University of Vienna | And 6 more authors.
PLoS ONE | Year: 2014

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the bloodbrain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison. © 2014 Novakova et al.


Kotani N.,University of Tokyo | Maeda K.,University of Tokyo | Debori Y.,University of Tokyo | Camus S.,Biopredic International | And 3 more authors.
Molecular Pharmaceutics | Year: 2012

HepaRG cells have the ability to differentiate into hepatocyte-like cells. Many papers have shown that these hepatocyte-like cells share several functional properties with intact human hepatocytes. However, although previous studies have indicated the partial maintenance of mRNA expression of drug transporters, their expression and function have not been quantitatively characterized. In the present study, the mRNA and protein expression levels and transport activities of hepatic uptake transporters, organic anion transporting polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptide (NTCP) in HepaRG cells were compared with those in cryopreserved human hepatocytes. The mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP in HepaRG cells were 22-38%, 2-15%, 82-113%, and 191-247% of those in human hepatocytes, respectively. The relative protein expression of these transporters was comparable with their mRNA expression. We observed saturable uptake of typical substrates of NTCP and OATPs except for cholecystokinin octapeptide (OATP1B3-selective substrate), and Na+-dependent uptake of taurocholate was confirmed. Their relative uptake clearances were well explained by their mRNA and protein expression levels. Additionally, inhibition potencies of 12 OATP1B1 inhibitors were investigated both in HepaRG cells and in OATP1B1-expressing HEK293 cells to demonstrate the usefulness of HepaRG cells for the characterization of OATP1B1-mediated drug-drug interactions. The K i values in both cell lines were comparable and showed significant correlation. These results suggest that the hepatic uptake transport function of OATP and NTCP transporters was relatively well maintained in HepaRG, although OATP1B3 function was too low to be detected. © 2012 American Chemical Society.


Ye Z.-W.,Catholic University of Leuven | Camus S.,Biopredic International | Augustijns P.,Catholic University of Leuven | Annaert P.,Catholic University of Leuven
Biopharmaceutics and Drug Disposition | Year: 2010

Hepatotoxicity has been reported as a side-effect in some patients on HIV protease inhibitors (PI). Since transporter interaction has been implicated as a mechanism underlying drugmediated hepatotoxicity and drug-drug interactions, the interaction of PI with the hepatic canalicular efflux transporter ABCC2 (MRP2; multidrug resistance associated protein-2) was studied. Interaction with ABCC2/Abcc2 was evaluated in human and rat sandwich-cultured hepatocytes using 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) as substrate. In rat hepatocytes, interaction with estradiol-17-β-D-glucuronide (E17G) efflux was also studied. In human hepatocytes, saquinavir, ritonavir and atazanavir were the most efficient inhibitors of ABCC2-mediated biliary excretion of CDF, whereas in rat hepatocytes indinavir, lopinavir and nelfinavir were the most efficient. No species-similarity was found for ABCC2/Abcc2 inhibition. In rat hepatocytes, the effects on Abcc2 were substrate-dependent as inhibition of biliary excretion of E17G was most pronounced for saquinavir (completely blocked), amprenavir (82% inhibition) and indinavir (68% inhibition). In conclusion, several HIV PI showed substantial ABCC2 inhibition, which, combined with the effects of PI on other hepatobiliary disposition mechanisms, will determine the clinical relevance of these in vitro interaction data. Copyright © 2010 John Wiley & Sons, Ltd.


Klein S.,Saarland University | Mueller D.,Saarland University | Schevchenko V.,Biopredic International | Noor F.,Saarland University
Journal of Applied Toxicology | Year: 2014

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30days without viability change and with high CYP activity. We also tested the acute (24h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology. © 2013 John Wiley & Sons, Ltd.


Patent
Biopredic International | Date: 2015-04-01

Methods and processes for cryopreservation and direct cell thawing and seeding or suspension after cryopreservation, including methods that eliminate the necessity of post-thaw wash, spin, and frequent practice of performing a cell count. Cell compositions and no-spin cell products produced using the methods are also described.

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