Éditeur(s) : HAL CCSD
Résumé : International audience
Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid
Pharmaceutics

hal-01477210
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477210
DOI : 10.3390/pharmaceutics9010003
PUBMED : 28036031

Éditeur(s) : HAL CCSD MDPI
Résumé : International audience
Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug-drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug-drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.
ISSN: 1422-0067

hal-01516024
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01516024
DOI : 10.3390/ijms18040764
PUBMED : 28375174

Éditeur(s) : HAL CCSD American Society for Biochemistry and Molecular Biology
Résumé : International audience
2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxy-amino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H-NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation, by UGTs, to form, respectively, N2-(β-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gl) and N2-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-glucuronide conjugate, O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gl). AαC-HN2-O-Gl is a biologically reactive metabolite that bound to calf thymus DNA (pH 5.0 or pH 7.0) to form the N-(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) adduct at 20 to 50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (Vmax/Km) at forming AαC-N2-Gl (0.67 μL mg protein-1 min-1), UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μL mg protein-1 min-1), whereas, UGT1A1 was most efficient at forming AαC-HON2-Gl (5.0 μL mg protein-1 min-1). Human hepatocytes produced AαC-N2-Gl and AαC-HN2-O-Gl in abundant quantities, but AαC-HON2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC.
Journal of Biological Chemistry

hal-00688369
https://hal.archives-ouvertes.fr/hal-00688369
DOI : 10.1074/jbc.M111.320093
PUBMED : 22393056

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
'Alarmins' are a group of proteins or molecules that are released from cells during cellular demise to alert the host immune system. Two of them, Interleukin-33 (IL-33) and high-mobility group box-1 (HMGB1), share many similarities of cellular localization, functions and involvement in various inflammatory pathologies including hepatitis. The expressions of IL-33 and HMGB1, and their receptors ST2 and receptor for advanced glycation end products (RAGE), are substantially up-regulated during acute and chronic hepatitis. Recent data evidence a possible protective role of IL-33/ST2 axis during liver injury. A contrast in expression of IL-33 and HMGB1 alarmins were associated with type of hepatocellular death mediated by immune cells or hepato-toxic agents. The massive release of active form of IL-33 from hepatocytes may affect the recruitment and activation of its ST2-positive target immune cells in the liver to confer its alarmin functions. This review highlights the emerging roles of alarmin proteins in various liver pathologies, by focusing on classical HMGB1 and a newly discovered alarmin, the IL-33.
ISSN: 1478-3223

hal-00874209
https://hal.archives-ouvertes.fr/hal-00874209
DOI : 10.1111/j.1478-3231.2012.02802.x
PUBMED : 22530772

Éditeur(s) : HAL CCSD Springer Verlag (Germany) Springer Verlag
Résumé : Dihydroceramide ?4-desaturase 1 (DES1) catalyzes the last step of the de novo ceramide biosynthesis, which consists of the introduction of a trans ?4-double bond in the carbon chain of the dihydroceramide. It was previously observed that myristic acid binds DES1 through N-myristoylation. This N-terminal modification significantly increased the activity of the recombinant DES1 in COS-7 cells and targeted part of the enzyme initially present in the endoplasmic reticulum to the mitochondrial outer membrane, leading to an increase in ceramide levels. Since these results were obtained in a recombinant COS-7 cell model with high expression of rat DES1, the purpose of the present study was to investigate if the native DES1 enzyme was really upregulated by its N-myristoylation in cultured rat hepatocytes. We first showed that DES1 was the main dihydroceramide desaturase isoform expressed in rat hepatocytes. In this model, the wild-type myristoylable recombinant form of rat DES1 was found in both the endoplasmic reticulum and the mitochondria whereas the mutated non-myristoylable recombinant form (N-terminal glycine replaced by an alanine) was almost exclusively localized in the endoplasmic reticulum, which evidenced the importance of the myristoylation. Then, we showed that compared to other fatty acids, myristic acid was the only one to increase native DES1 activity, in both total cell lysates and mitochondrial fractions. The myristic acid-associated increase in DES1 activity was not linked to elevated mRNA or protein expression but more likely to its N-terminal myristoylation. Finally, the myristic acid-associated increase in DES1 activity slightly enhanced the number of apoptotic cells.
ISSN: 0024-4201

hal-00808026
https://hal-agrocampus-ouest.archives-ouvertes.fr/hal-00808026
DOI : 10.1007/s11745-011-3638-x

Éditeur(s) : HAL CCSD American Society for Investigative Pathology
Résumé : International audience
Recent studies suggest that psoriasis may be more severe in patients with nonalcoholic fatty liver disease, particularly in those with the inflammatory stage of steatohepatitis [nonalcoholic steatohepatitis (NASH)]. Herein, we investigated the impact of diet-induced steatohepatitis on the severity of imiquimod-induced psoriasiform dermatitis. Mice fed with a high-fat diet developed steatohepatitis reminiscent of human NASH with ballooning hepatocytes and significant liver fibrosis. Mice with steatohepatitis also displayed moderate cutaneous inflammation characterized by erythema, dermalinfiltrates of CD45(+) leukocytes, and a local production of IL-17A. Moreover, steatohepatitis was associated with an epidermal activation of caspase-1 and cutaneous overexpression of IL-1 beta. Imiquimod-induced psoriasiform dermatitis was exacerbated in mice with steatohepatitis as compared to animals fed with a standard diet. Scale formation and acanthosis were aggravated, in correlation with increased IL-17A and IL-22 expression in inflamed skins. Finally, intradermal injection of IL-17A in standard diet-fed mice recapitulated the cutaneous pathology of mice with steatohepatitis. The results show that high-fat diet induced steatohepatitis aggravates the inflammation in psoriasiform dermatitis, via the cutaneous production of IL-17A. In agreement with clinical data, this description of a novel extrahepatic manifestation of NASH should sensitize dermatologists to the screening and the management of fatty liver in psoriatic patients.
ISSN: 0002-9440

hal-01390977
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390977
DOI : 10.1016/j.ajpath.2016.05.012
PUBMED : 27423696

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their systemic effects through impairing hepatic transport of endogenous compound or drug substrates of these transporters.
ISSN: 1932-6203

Droits : http://creativecommons.org/licenses/by/
hal-01138535
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01138535
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01138535/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01138535/file/Regulation%20of%20human%20hepatic%20drug.pdf
DOI : 10.1371/journal.pone.0121232

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Hepatocellular carcinoma (HCC) is a particularly severe disease characterized by a high rate of recurrence and death even after surgical resection. Molecular characterization of HCC helps refine prognosis and may facilitate the development of improved therapy. Phosphatidylinositol 4-kinases have recently been identified as cellular factors associated with cancer. Also, phosphatidylinositol 4-kinase type IIIα (PI4KA) is necessary for the propagation of the hepatitis C virus, a major etiological factor for HCC. METHODS: Reverse transcription, quantitative real-time PCR was used to assay PI4KA mRNA. The expression levels were investigated both in a collection of molecularly and clinically characterized hepatic tissues from 344 patients with diverse liver diseases and in human hepatocyte cell lines whose proliferative and differentiation status was controlled by specific culture conditions. Analytical microarray data for 60 HCC and six normal liver tissue samples were exploited to study correlations between PI4KA mRNA levels and cell proliferation markers in vivo. Postoperative disease-specific survival and time to recurrence in a set of 214 patients with HCC were studied by univariate and multivariate analyses. RESULTS: PI4KA mRNA was more abundant in HCC than normal healthy tissues. This upregulation correlated significantly with both poor differentiation and the active proliferation rate in HCC. These associations were confirmed with in vitro models. Moreover, patients with HCC who had been treated by surgical resection and had higher PI4KA mRNA concentrations in their tumor tissue exhibited a higher risk of tumor recurrence (median time: 20 months versus 49 months, P = 0.0012) and shorter disease-specific survival (first quartile time: 16 months versus 48 months, P = 0.0004). Finally, the abundance of PI4KA mRNA proved to be an independent prognostic marker of survival for cases of HCC (hazard ratio = 2.36, P = 0.0064). CONCLUSIONS: PI4KA mRNA could be used as a new molecular marker to improve established prognostic models for HCC. These findings also indicate possible new lines of research for the development of innovative therapeutic approaches targeting PI4KA.
ISSN: 1471-2407

inserm-00934294
http://www.hal.inserm.fr/inserm-00934294
http://www.hal.inserm.fr/inserm-00934294/document
http://www.hal.inserm.fr/inserm-00934294/file/1471-2407-14-7.pdf
DOI : 10.1186/1471-2407-14-7

Éditeur(s) : HAL CCSD PeerJ
Résumé : International audience
Background: Heterocyclic aromatic amines (HAA) are environmental and food contaminants that are potentially carcinogenic for humans. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is one of the most abundant HAA formed in cooked meat. MeIQx is metabolized by cytochrome P450 1A2 in the human liver into detoxificated and bioactivated products. Once bioactivated, MeIQx metabolites can lead to DNA adduct formation responsible for further genome instability.Methods: Using a computational approach, we developed a numerical model for MeIQx metabolism in the liver that predicts the MeIQx biotransformation into detoxification or bioactivation pathways according to the concentration of MeIQx.Results: Our results demonstrate that 1) the detoxification pathway predominates, 2) the ratio between detoxification and bioactivation pathways is not linear and shows a maximum at 10µM of MeIQx in hepatocyte cell models, 3) CYP1A2 is a key enzyme in the system that regulates the balance between bioactivation and detoxification. Our analysis suggests that such a ratio could be considered as an indicator of MeIQx genotoxicity at a low concentration of MeIQx.Conclusions: Our model permits the investigation of the balance between bioactivation (i.e. DNA adduct formation pathway through the prediction of potential genotoxic compounds) and detoxification of MeIQx in order to predict the behaviour of this environmental contaminant in the human liver. It highlights the importance of complex regulations of enzyme competitions that should be taken into account in any further multi-organ models.
ISSN: 2167-8359

hal-01575579
https://hal.inria.fr/hal-01575579
PUBMED : 28879062
DOI : 10.7717/peerj.3703

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
Hepatocellular carcinoma treatment by arterial infusion of cis-diamminedichloroplatinum-II (cisplatin) exhibits certain therapeutic efficacy. However, optimizations are required and the mechanisms underlying cisplatin proapoptotic effect remain unclear. The mitogen-activated protein kinase (MAPK) pathway plays a key role in cell response to cisplatin and the functional specificity of the isoform MAPK/ERK kinase 1 and 2 (MEK1/2) and ERK1/2 could influence this response. The individual contribution of each kinase on cisplatin-induced death was thus analyzed after a transient or stable specific inhibition by RNA interference in the human hepatocellular carcinoma cells Huh-7 or in knockout mice. We demonstrated here that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas MEK1 and MEK2 acted in a redundant manner. Indeed, at clinically relevant concentrations of cisplatin, ERK1 silencing alone was sufficient to protect cells from cisplatin-induced death both in vitro, in Huh-7 cells and ERK1(-/-) hepatocytes, and in vivo, in ERK1-deficient mice. Moreover, we showed that ERK1 activity correlated with the induction level of the proapoptotic BH3-only protein Noxa, a critical mediator of cisplatin toxicity. On the contrary, ERK2 inhibition upregulated ERK1 activity, favored Noxa induction and sensitized hepatocarcinoma cells to cisplatin. Our results point to a crucial role of ERK1 in cisplatin-induced proapoptotic signal and lead us to propose that ERK2-specific targeting could improve the efficacy of cisplatin therapy by increasing ERK1 prodeath functions.
ISSN: 0143-3334

inserm-00814096
http://www.hal.inserm.fr/inserm-00814096
DOI : 10.1093/carcin/bgs317
PUBMED : 23042098