Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various invivo and invitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable invitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na+-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasi

Effect of the antioxidant anthocyanins on the functions of chicken testicular cells

International audienc

Monoclonal antibodies against human translation termination factor eRF3 and their utilization for sub-cellular localization of eRF3.

International audienceEukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes the stop codon and promotes nascent peptide chain release, while eRF3 facilitates this peptide release in a GTP-dependent manner. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay. Despite extensive investigation, the complete understanding of eRF3 function have been hampered by the lack of specific anti-eRF3 monoclonal antibodies (Mabs). The purpose of the study was production of recombinant eRF3a/GSPT1, development of anti-eRF3a/GSPT1 Mabs and their utilization for eRF3a/GSPT1 sub-cellular localization. Plasmid encoding C-terminal part of human GSPT1/eRF3a was constructed. Purified protein, which was predominantly present in the inclusion bodies, was used for the development of Mabs. Characterization of the regions recognized by Mabs using GSPT1/eRF3a mutants and its visualization in the 3D space suggested that Mabs recognize different epitopes. Consistent with its function in translational termination, immunostaining of the cells with developed Mabs revealed that the endogenous GSPT1/eRF3a localized in endoplasmic reticulum. Taking into account the important role of eRF3 for the fundamental research one can suggests that developed Mabs have great prospective to be used as a research reagent in a wide range of applications

Direct cooperation between estrogen receptors and astrocyte-specific factors elicits high activity of the zebrafish brain aromatase gene

conférence invitée présentée par A. MenuetInternational audienc

Distribution of aromatase mRNA and protein in the brain and pituitary of female rainbow trout: Comparison with estrogen receptor alpha.

International audienceRecent data indicate that estrogens locally produced in the brain by aromatization of androgens could be important for neurogenesis and brain repair. In this respect, fish are interesting because of the extremely high aromatase activity of their brain. In this study, the rainbow trout brain aromatase was cloned and riboprobes were used to map the distribution of cells expressing the corresponding mRNAs. A very strong hybridization signal was detected in the pituitary and in cells bordering the ventricles in the telencephalon and ventral diencephalon, with the highest expression in the preoptic area and hypothalamus. A weaker signal was detected in the ependymal layer bordering the torus semicircularis and optic tectum. This localization was fully confirmed by immunohistochemistry using antibodies against a teleost aromatase. In addition, this antibody showed that aromatase expression in fact corresponds to radial glial cells because immunoreactive cells had long cytoplasmic processes extending toward the pial surface. Because brain aromatase was shown to be upregulated by estradiol in fish, the distribution of aromatase mRNAs was compared with that of rainbow trout estrogen receptor alpha (rtERalpha) on adjacent sections. Although the highest aromatase expression was found in regions expressing rtERalpha, no obvious coexpression was found, as rtERalpha was never observed in radial cells. However, reverse transcriptase-polymerase chain reaction experiments performed on brain cell cultures enriched in glial cells suggest that a weak expression of rtERalpha in glial cells cannot be excluded. The possible role of the high brain aromatase content in fish could be related to the continuous growth of their central nervous system during adulthood

A practical multi-step synthesis of ethyl N-functionalized β-amino benzimidazole acrylate derivatives as promising cytotoxic agents

International audienceA series of 16 new ethyl [Formula: see text]-amino benzimidazole acrylate derivatives 12(a-p) with a (2E)-s-cis/trans conformation and bearing two points of diversity was designed and synthesized by using a multi-step strategy (reductive amination, deprotection in acidic media and transamination) in moderate to good yields from ethyl 3-dimethylamino-2-(1H-benzimidazol-2-yl)acrylate (5) and monosubstituted N-Boc diamines (7a,7b) as starting building blocks. Products 12 were evaluated for their in vitro cytotoxic potential against six selected human cell lines (Huh7-D12, Caco2, MDA-MB231, HCT116, PC3 and NCI-H727). Compounds 12a, 12e and 12l exhibited selective and micromolar antitumor activities against Huh7-D12 and Caco2 cell lines

Hypoxia differentially modulates the genomic stability of clinical-grade ADSCs and BM-MSCs in long-term culture

International audienceLong-term cultures under hypoxic conditions have been demonstrated to maintain the phenotype of mesenchymal stromal/stem cells (MSCs) and to prevent the emergence of senescence. According to several studies, hypoxia has frequently been reported to drive genomic instability in cancer cells and in MSCs by hindering the DNA damage response and DNA repair. Thus, we evaluated the occurrence of DNA damage and repair events during the ex vivo expansion of clinical-grade adipose-derived stromal cells (ADSCs) and bone marrow (BM)-derived MSCs cultured with platelet lysate under 21% (normoxia) or 1% (hypoxia) O2 conditions. Hypoxia did not impair cell survival after DNA damage, regardless of MSC origin. However, ADSCs, unlike BM-MSCs, displayed altered γH2AX signaling and increased ubiquitylated γH2AX levels under hypoxic conditions, indicating an impaired resolution of DNA damage-induced foci. Moreover, hypoxia specifically promoted BM-MSC DNA integrity, with increased Ku80, TP53BP1, BRCA1 and RAD51 expression levels and more efficient non-homologous end joining and homologous recombination repair. We further observed that hypoxia favored mtDNA stability and maintenance of differentiation potential after genotoxic stress. We conclude that long-term cultures under 1% O2 were more suitable for BM-MSCs as suggested by improved genomic stability compared with ADSCs. This article is protected by copyright. All rights reserved

Simple elaboration of drug-SPION nanocapsules (hybridosomes®) by solvent shifting: effect of the drug molecular structure and concentration

International audienceDrug nanocapsules coated with iron oxide nanoparticles (SPION) were elaborated by the simultaneous nanoprecipitation of the drug and the nanoparticles, through solvent shifting. We examined four drugs: sorafenib, sorafenib tosylate, α-tocopherol and paclitaxel, to cover the cases of molecular solids, ionic solids, and molecular liquids. We first investigated the formation of the drug core in the final mixture of solvents at different concentrations. A Surfactant Free Micro-Emulsion domain (SFME, thermodynamically stable) was observed at low drug concentration and an Ouzo domain (metastable) at high drug concentration, except for the case of paclitaxel which crystallizes at high concentration without forming an Ouzo domain. When co-nanoprecipitated with the molecular drugs in the Ouzo domain (sorafenib or α-tocopherol), the SPION limited the coalescence of the drug particles to less than 100 nm, forming capsules with a drug encapsulation efficiency of ca 80 %. In contrast, larger capsules were formed from the SFME or when using the ionic form (sorafenib tosylate). Finally, the sorafenib-SPION capsules exhibit a similar chemotherapeutic effect as the free drug on the hepatocellular carcinoma in vitro

Impact des principales fusariotoxines, seules ou en association, sur les fonctions testiculaires du poulet de chair

Mycotoxins are secondary metabolites produced by molds. The presence of these contaminants in animal feed isa major problem because mycotoxins have various adverse effects on health, affecting in particular certainfunctions such as reproduction. The objective of our approach is to evaluate the impact of the main fusariotoxins,in vivo and in vitro, on the testicular functions of broiler chickens. For the in vivo test, 5 groups of 12 broilers(Ross PM3) received the control or experimentally contaminated diet from hatch to slaughter (35 days of age).The diets were contaminated in: deoxynivalenol (DON, 5 mg / kg), fumonisins (FBs, 20 mg / kg), zearalenone(ZEA, 0.5 mg / kg) in single contamination or containing the 3 mycotoxins. The mycotoxins required for theexperiments were obtained by culturing toxigenic fungal strains.Analysis of the testis growth and histological examination has shown that the proliferative activity of the testis isincreased in animals exposed to FBs compared to the control group. On the other hand, no difference was foundin markers of cell death (caspase 3 activity and TUNEL immunostaining), of inflammation (IL6, IL1β andIFNɣ), or in oxidative stress (antioxidant capacity and catalase activity). In vitro experiments using purifiedmycotoxins diluted in a solvent (DMSO) were tested on chicken testicular cell cultures after 96 h of exposure.The results confirmed the in vivo data showed no effect at low dose. However, high doses (FB1: 100 nM, ZEA:30 μM, DON 1 μM) have an impact on cell viability.In conclusion, in vitro, only high doses can lead to testicular lesions but in vivo the maximum concentrationsacceptable did not induce any significant effect on the testes of 35 days-old chicken.Les mycotoxines sont des métabolites secondaires produits par certaines moisissures. La présence de cescontaminants dans les aliments destinés aux animaux est un problème majeur, car les mycotoxines peuvent avoir des effets néfastes variés sur leur santé, en impactant notamment certaines fonctions telle que la reproduction. L’objectif de notre démarche est d’évaluer l’impact des principales fusariotoxines, in vivo et in vitro sur les fonctions testiculaires du poulet de chair. Pour l’essai in vivo, 5 groupes de 12 poulets de chair (Ross PM3) ont reçu de la naissance à l’abattage (35 jours d’âge) les aliments suivants : un aliment témoin sain ou un des 4 aliments expérimentalement contaminés avec du déoxynivalénol (DON, 5mg/kg), des fumonisines (FBs, 20 mg/kg), de la zéaralénone (ZEA, 0,5 mg/kg) en contamination unique ou contenant les 3 mycotoxines. Les mycotoxines nécessaires aux expérimentations ont été obtenues par culture de souches fongiques toxinogènes. L’analyse de la croissance et l’examen histologique du testicule ont montré que l’activité proliférative du testicule, est augmentée chez les animaux exposés aux FBs, en comparaison du groupe témoin. Par contre, aucune différence sur les marqueurs de mortalité cellulaire (activité caspase 3 et immunomarquage TUNEL), d’inflammation (IL6, IL1β et IFNɣ), ou de stress oxydatif (capacité antioxydante et activité de la catalase) n’a été révélée entre les lots. Des mycotoxines purifiées et diluées dans un solvant (DMSO) ont été testés sur des cultures testiculaires de poulets après 96h d’exposition. Les résultats ont confirmé les données in vivo montrant aucun effet à faible dose et un effet négatif des doses élevées (FB1: 100 nM ; ZEA : 30 μM, DON 1μM). En conclusion, in vitro, seules des fortes doses peuvent conduire à des lésions testiculaires mais in vivo les doses maximales autorisées n’induisent pas d’effet négatif notable sur les testicules de poulets élevés jusqu’à 35j

Identification of small molecule regulators of the nuclear receptor HNF4alpha based on naphthofuran scaffolds.

1 : Régulation Spatio-Temporelle de la Transcription (SPARTE) : R. Le Guével, F. Oger, G. Salbert - RMN et Intéractions Lipides Protéines (RMN-ILP) : A. Bondon - R. Le Guével, F. Oger : These two authors contributed equally to this study.International audienceNuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4alpha (NR2A1) is a recently 'deorphanized' nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4alpha is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4alpha in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4alpha in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4alpha was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4alpha-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4alpha activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans