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Sample records for adult human hepatocytes

  1. Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

    PubMed

    Duret, Cedric; Moreno, Daniel; Balasiddaiah, Anangi; Roux, Solene; Briolotti, Phillipe; Raulet, Edith; Herrero, Astrid; Ramet, Helene; Biron-Andreani, Christine; Gerbal-Chaloin, Sabine; Ramos, Jeanne; Navarro, Francis; Hardwigsen, Jean; Maurel, Patrick; Aldabe, Rafael; Daujat-Chavanieu, Martine

    2015-01-01

    Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation. PMID:25622096

  2. Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

    PubMed Central

    Auth, Marcus K.H.; Boost, Kim A.; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H.; Bechstein, Wolf-Otto; Blaheta, Roman A.

    2005-01-01

    AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors. PMID:15810072

  3. Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes

    PubMed Central

    Tarlow, Branden D.; Pelz, Carl; Naugler, Willscott E.; Wakefield, Leslie; Wilson, Elizabeth M.; Finegold, Milton J.; Grompe, Markus

    2014-01-01

    Summary Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts and subsequently contribute to restoration of the hepatocyte mass. PMID:25312494

  4. Mouse liver repopulation with hepatocytes generated from human fibroblasts

    PubMed Central

    Zhu, Saiyong; Rezvani, Milad; Harbell, Jack; Mattis, Aras N.; Wolfe, Alan R.; Benet, Leslie Z.; Willenbring, Holger; Ding, Sheng

    2014-01-01

    Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1–3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy. PMID:24572354

  5. In utero Transplanted Human Hepatocytes Allows for Postnatal Engraftment of Human Hepatocytes in Pigs

    PubMed Central

    Fisher, James E; Lillegard, Joseph B; Mckenzie, Travis J; Rodysill, Brian R; Wettstein, Peter J; Nyberg, Scott L

    2012-01-01

    In utero cell transplantation (IUCT) can lead to postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCTs have involved hematopoietic stem cells. It is unknown if human hepatocytes used for IUCT in fetal pigs will lead to engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1×107 human hepatocytes in utero and were delivered by cesarean-section at term. Piglets received a second direct liver injection of 5×107 human hepatocytes 1 week postnatally. Serum was analyzed for human albumin at 2, 4, and 6 weeks post-engraftment. Piglet livers were harvested 6 weeks after transplantation and examined by immunohistochemistry, PCR and fluorescence in situ hybridization for human specific sequences. Piglets receiving IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all post-engraftment time points. Human albumin gene expression, the presence of human hepatocytes and the presence of human beta-2 microglobulin were all confirmed 6 weeks post-engraftment. IUCT in fetal pigs using human hepatocytes early in gestation allowed for engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large scale expansion of human hepatocytes in genetically-engineered pigs. PMID:23280879

  6. Metabolism of lipoproteins by human fetal hepatocytes

    SciTech Connect

    Carr, B.R.

    1987-12-01

    The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of (/sup 125/I)iodo-LDL and (/sup 125/I)iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. (/sup 125/I)Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas (/sup 125/I)iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues.

  7. Generation of functional hepatocytes from human spermatogonial stem cells

    PubMed Central

    Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

  8. Toxicogenomics directory of chemically exposed human hepatocytes.

    PubMed

    Grinberg, Marianna; Stöber, Regina M; Edlund, Karolina; Rempel, Eugen; Godoy, Patricio; Reif, Raymond; Widera, Agata; Madjar, Katrin; Schmidt-Heck, Wolfgang; Marchan, Rosemarie; Sachinidis, Agapios; Spitkovsky, Dimitry; Hescheler, Jürgen; Carmo, Helena; Arbo, Marcelo D; van de Water, Bob; Wink, Steven; Vinken, Mathieu; Rogiers, Vera; Escher, Sylvia; Hardy, Barry; Mitic, Dragana; Myatt, Glenn; Waldmann, Tanja; Mardinoglu, Adil; Damm, Georg; Seehofer, Daniel; Nüssler, Andreas; Weiss, Thomas S; Oberemm, Axel; Lampen, Alfons; Schaap, Mirjam M; Luijten, Mirjam; van Steeg, Harry; Thasler, Wolfgang E; Kleinjans, Jos C S; Stierum, Rob H; Leist, Marcel; Rahnenführer, Jörg; Hengstler, Jan G

    2014-12-01

    A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the

  9. Hypothermic storage of human hepatocytes for transplantation.

    PubMed

    Gramignoli, Roberto; Dorko, Kenneth; Tahan, Veysel; Skvorak, Kristen J; Ellis, Ewa; Jorns, Carl; Ericzon, Bo-Goran; Fox, Ira J; Strom, Stephen C

    2014-01-01

    Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since most patients require multiple cell infusions over an extended period of time, we investigated hepatic functions in cells maintained in University of Wisconsin solution at 4°C up to 72 h. Eleven different assessments of hepatic viability and function were investigated both pre- and posthypothermic storage, including plating efficiency, caspase-3/7 activity, ammonia metabolism, and drug-metabolizing capacity of isolated hepatocytes. Long-term function, basal, and induced cytochrome P450 activities were measured after exposure to prototypical inducing agents. Cells from 47 different human liver specimens were analyzed. Viability significantly decreased in cells cold stored in UW solution, while apoptosis level and plating efficiency were not significantly different from fresh cells. Luminescent and fluorescent methods assessed phases I and II activities both pre- and post-24-72 h of cold preservation. A robust induction (up to 200-fold) of phase I enzymes was observed in cultured cells. Phase II and ammonia metabolism remained stable during hypothermic storage, although the inductive effect of culture on each metabolic activity was eventually lost. Using techniques that characterize 11 measurements of hepatic viability and function from plating efficiency, to ammonia metabolism, to phases I and II drug metabolism, it was determined that while viability decreased, the remaining viable cells in cold-stored suspensions retained critical hepatic functions for up to 48 h at levels not significantly different from those observed in freshly isolated cells. PMID:23768881

  10. Thyroid hormone effect in human hepatocytes.

    PubMed

    Miler, Eliana A; Ríos de Molina, María Del Carmen; Domínguez, Gabriela; Guerra, Liliana N

    2008-01-01

    We have already demonstrated that a combined treatment of methimazole and an antioxidant mixture improved the condition of hyperthyroid patients both biochemically and clinically. Elevated thyroid hormone levels might trigger signs and symptoms of hyperthyroidism through the increase of free radicals. To study the direct effect of thyroid hormone on cellular markers of oxidative stress, we carried out in vitro assays in which 0.1-20.0 nM T3 (6.5-1300.0 ng/dl) doses were added to culture media of the human hepatocyte cell line Hep G2 for 1-24 h. T3 increased malondialdehyde (MDA) and intracellular oxidized glutathione (GSSG) levels; SOD activity was also higher with hormone treatment, whereas catalase and glutathione peroxidase activities showed no variation at different T3 doses and during all experimental times. When ascorbic acid was added to the culture, the MDA level decreased and SOD activity was increased. With higher doses of T3 (e.g. 200 nM), cell death occurred (69% of apoptotic cells). The increase in SOD activity was not enough to overcome the effect of T3 since MDA and GSSG remained high during a 24-h experiment. We showed a beneficial effect of ascorbic acid when cells were exposed to a T3 dose of 20 nM, a higher level of hormone than that achieved in hyperthyroidism. PMID:18647489

  11. Polygonal networks, "geodomes", of adult rat hepatocytes in primary culture.

    PubMed

    Mochizuki, Y; Furukawa, K; Mitaka, T; Yokoi, T; Kodama, T

    1988-01-01

    Polygonal networks, "geodomes", in cultured hepatocytes of adult rats were examined by both light and electron microscopy. On light microscopical examinations of specimens stained with Coomassie blue after the treatment with Triton X-100, the networks were detected 5 days after culture, which consisted of triangles arranged mainly in hexagonal patterns. They surrounded main cell body, looking like a headband, or were occasionally situated over nuclei, looking like a geodesic dome. Scanning electron microscopical observations after Triton treatment revealed that these structures were located underneath surface membrane. Transmission electron microscopical investigations revealed that the connecting fibers of networks consisted of microfilaments which radiated in a compact bundle from electron-dense vertices. PMID:3396075

  12. INTERINDIVIDUAL VARIATION IN THE METABOLISM OF ARSENIC IN HUMAN HEPATOCYTES

    EPA Science Inventory


    The liver is the major site for the enzymatic methylation of inorganic arsenic (iAs) in humans. Primary cultures of normal human hepatocytes isolated from tissue obtained at surgery or from donor livers have been used to study interindividual variation in the capacity of live...

  13. Mutagenicity towards Salmonella typhimurium of some known genotoxic agents, activated by isolated hepatocytes of monkey (Macaca fascicularis). Comparison with isolated human hepatocytes.

    PubMed

    Neis, J M; Roelofs, H M; van Gemert, P J; Henderson, P T

    1986-06-01

    This paper describes some striking differences between isolated human and monkey hepatocytes in their capacity to activate some known genotoxic agents into products mutagenic towards Salmonella typhimurium. Isolated monkey hepatocytes, in contrast to human hepatocytes, appeared to activate benzidine (BZ), N-acetylbenzidine (MABZ), N,N'-diacetylbenzidine (DABZ), 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) poorly. With monkey hepatocytes BZ was slightly more mutagenic than DABZ, whereas with human hepatocytes DABZ was more active than BZ. N-Nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were also found to be poorly mutagenic when activated by monkey hepatocytes, unlike the human hepatocytes. However, the polycyclic arylhydrocarbons benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthracene (7,12-DMBA) were highly active in the presence of monkey hepatocytes, unlike the human hepatocytes. A metabolic study showed that monkey liver preparations seem to possess a higher monooxygenase activity towards B[a]P than human liver preparations.

  14. Xeno-repopulation of Fah -/- Nod/Scid mice livers by human hepatocytes.

    PubMed

    Su, Baoliang; Liu, Changcheng; Xiang, Dao; Zhang, Haibin; Yuan, Siming; Wang, Minjun; Chen, Fei; Zhu, Haiying; He, Zhiying; Wang, Xin; Hu, Yiping

    2011-03-01

    Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics, infection of hepatitis viruses, and the efficacy of hepatitis vaccines. Significant levels of liver xeno-repopulation have been reported in Fah (-/-) Rag2 (-/-) Il2rg (-/-) mice. However, A new model, termed Fah (-/-) Nod/Scid mice, which combines the advantages of liver repopulation in Fah (-/-) mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding. Fah (-/-) Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities. FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah (-/-) Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes; the proportion of engrafted human hepatocytes reached 33.6%. The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis, the expression of characteristic proteins, and the biochemical analysis of liver function. These results show that Fah (-/-) Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.

  15. Induction of digitoxigenin monodigitoxoside UDP-glucuronosyltransferase activity by glucocorticoids and other inducers of cytochrome P-450p in primary monolayer cultures of adult rat hepatocytes and in human liver.

    PubMed

    Schuetz, E G; Hazelton, G A; Hall, J; Watkins, P B; Klaassen, C D; Guzelian, P S

    1986-06-25

    We have recently proposed that glucocorticoids induce cytochrome P-450p, a liver microsomal hemoprotein originally isolated from rats treated with the antiglucocorticoid pregnenolone 16 alpha-carbonitrile (PCN), through a mechanism that involves a stereospecific recognition system clearly distinguishable from the classic glucocorticoid receptor (Schuetz, E. G., Wrighton, S. A., Barwick, J. L., and Guzelian, P. S. (1984) J. Biol. Chem. 259, 1999-2012). We now report that digitoxigenin monodigitoxoside UDP-glucuronosyltransferase (DIG UDP-glucuronosyltransferase), a liver microsomal enzyme activity induced by PCN in rats, is also inducible, as is P-450p, in primary monolayer cultures of adult rat hepatocytes. DIG UDP-glucuronosyltransferase activity closely resembled reported characteristics of induction of P-450p in its time course of induction, concentration-response relationships, exclusivity of induction by steroids with glucocorticoid properties, unusual rank order of potency of glucocorticoid agonists, unusually high ED50 for induction by glucocorticoids, enhanced induction rather than inhibition by anti-glucocorticoids in the presence of glucocorticoids, and finally, induction by nonsteroidal inducers of P-450p. DIG UDP-glucuronosyltransferase activity was also readily detected in human liver microsomes and was elevated in two patients who had received inducers of P-450p. We conclude that the liver enzymes controlled by the postulated PCN recognition system include not only P-450p but also one or more UDP-glucuronosyltransferases.

  16. Type III interferons are expressed by Coxsackievirus-infected human primary hepatocytes and regulate hepatocyte permissiveness to infection

    PubMed Central

    Lind, K; Svedin, E; Utorova, R; Stone, V M; Flodström-Tullberg, M

    2014-01-01

    Hepatitis is a common and potentially fatal manifestation of severe Coxsackievirus infections, particularly in newborn children. Little is known of the immune-mediated mechanisms regulating permissiveness to liver infection. It is well established that type I interferons (IFNs) play an important role in the host innate immune response to Coxsackievirus infections. Recent studies have highlighted a role for another IFN family, the type III IFNs (also called IFN-λ), in anti-viral defence. Whether type III IFNs are produced by hepatocytes during a Coxsackievirus infection remains unknown. Moreover, whether or not type III IFNs protects hepatocytes from a Coxsackievirus infection has not been addressed. In this study, we show that primary human hepatocytes respond to a Coxsackievirus B3 (CVB3) infection by up-regulating the expression of type III IFNs. We also demonstrate that type III IFNs induce an anti-viral state in hepatocytes characterized by the up-regulated expression of IFN-stimulated genes, including IFN-stimulated gene (ISG15), 2′-5′-oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1). Furthermore, our study reveals that type III IFNs attenuate CVB3 replication both in hepatocyte cell lines and primary human hepatocytes. Our studies suggest that human hepatocytes express type III IFNs in response to a Coxsackievirus infection and highlight a novel role for type III IFNs in regulating hepatocyte permissiveness to this clinically relevant type of virus. PMID:24773058

  17. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    SciTech Connect

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  18. Effect of matrine on primary human hepatocytes in vitro.

    PubMed

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  19. Human hepatocytes and endothelial cells in organotypic membrane systems.

    PubMed

    Salerno, Simona; Campana, Carla; Morelli, Sabrina; Drioli, Enrico; De Bartolo, Loredana

    2011-12-01

    The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell-cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell-cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices. PMID:21871658

  20. 3D Cultivation Techniques for Primary Human Hepatocytes

    PubMed Central

    Bachmann, Anastasia; Moll, Matthias; Gottwald, Eric; Nies, Cordula; Zantl, Roman; Wagner, Helga; Burkhardt, Britta; Sánchez, Juan J. Martínez; Ladurner, Ruth; Thasler, Wolfgang; Damm, Georg; Nussler, Andreas K.

    2015-01-01

    One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  1. Primary monolayer culture of adult mouse hepatocytes -- a model for the study of hepatotropic viruses.

    PubMed

    Arnheiter, H

    1980-01-01

    Primary monolayer cultures of adult mouse hepatocytes isolated by collagenase perfusion of the liver in situ were exposed to 2 hepatotropic viruses, an avian influenza A virus adapted to grow in mouse liver in vivo and a herpes simplex type I virus. Influenza virus infection led to lysis ofindividual hepatocytes and total monolayer destruction within 18 to 120 hours after infection according to the virus dose used. Virus replication was evidenced by assaying hepatocyte supernates for hemagglutinin and infectivity, by immunofluorescent staining and by electron microscopy. Herpes virus infection resulted in polykaryocyte formation followed by nuclear pycnosis and cell lysis. Virus replication was assayed by titration of supernate infectivity.

  2. Identification of small molecules for human hepatocyte expansion and iPS differentiation

    PubMed Central

    Shan, Jing; Schwartz, Robert E.; Ross, Nathan T.; Logan, David J.; Thomas, David; Duncan, Stephen A.; North, Trista E.; Goessling, Wolfram; Carpenter, Anne E.; Bhatia, Sangeeta N.

    2013-01-01

    Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo and human hepatocyte sourcing has been limiting many fields of research for decades. Here, we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. One class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted differentiation of iPS-derived hepatocytes, toward a phenotype more mature than what was previously obtainable. The identification of these small molecules can help to address a major challenge impacting many facets of liver research and may lead to the development of novel therapeutics for liver diseases. PMID:23728495

  3. Cryopreservation of isolated human hepatocytes for transplantation: State of the art.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Hughes, Robin D

    2006-10-01

    Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.

  4. Cryopreservation of isolated human hepatocytes for transplantation: State of the art.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Hughes, Robin D

    2006-10-01

    Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation. PMID:16793034

  5. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    PubMed

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

  6. Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes

    SciTech Connect

    Chojkier, M.; Brenner, D.A.; Leffert, H.L.

    1989-06-05

    The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts. In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+. However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with (5-3H)proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic (8-arg)vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with (35S)methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific (32P)cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment.

  7. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    PubMed

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  8. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    SciTech Connect

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  9. Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

    PubMed

    Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

    2016-06-01

    Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

  10. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  11. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

    PubMed Central

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  12. Role of Periductal and Ductular Epithelial Cells of the Adult Rat Pancreas in Pancreatic Hepatocyte Lineage

    PubMed Central

    Rao, M. Sambasiva; Dwivedi, Rama S.; Yeldandi, Anjana V.; Subbarao, V.; Tan, Xiaodi; Usman, Mohammed I.; Thangada, Shobha; Nemali, Mohan R.; Kumar, Sujata; Scarpelli, Dante G.; Reddy, Janardan K.

    1989-01-01

    Development of pancreatic hepatocytes in adult rats maintained on copper dificient diet containing 0.6% trien (CuDT) has been reported recently. To elucidate the histogenesis of hepatocytes a sequential study was undertaken using morphologic, histochemical, immunochemical, in situ hybridization, and Northern blot analysis. Male F-344 rats weighing 80 to 90 g were fed CuDT for 8 weeks and returned to normal rat chow. Beginning from 4 weeks of copper depletion, there was a progressive loss of acinar cells and by 8 weeks more than 90% of the acinar tissue was lost. During this period, there was an increase in the number of adipocytes in the interstitium, and in the number of interstitial and ductular cells. Morphologic observations were confirmed by immunoblot and Northern blot analysis, in which the amount of pancreatic proteins and their mRNAs decreased between 5 and 8 weeks. During this period, a progressive increase in the level of albumin mRNA was observed. In situ hybridization, performed at 7 weeks of copper deficiency, showed localization of albumin mRNA over interstitial and ductular cells. Pancreatic hepatocytes were identified immediately after the rats were returned to a normal diet and gradually increased in number. The hepatocytes occupied almost 60% of the pancreatic volume by 8 weeks. During the early recovery phase, hepatocytes were identified in ductules as well as in the interstitium. Based on these studies, it is concluded that both the ductular cells and interstitial cells, which resemble oval cells of liver, are capable of transforming into pancreatic hepatocytes and these cells may be considered stem-cell equivalent. ImagesFigure 9Figure 10Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8Figure 11Figure 12Figure 13Figure 14Figure 15Figure 16 PMID:2470253

  13. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    PubMed

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  14. Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF.

    PubMed

    Mitaka, T; Kojima, T; Mizuguchi, T; Mochizuki, Y

    1996-09-01

    To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

  15. In vitro metabolism of thyroxine by rat and human hepatocytes.

    PubMed

    Richardson, Vicki M; Ferguson, Stephen S; Sey, Yusupha M; Devito, Michael J

    2014-05-01

    1. The liver metabolizes thyroxine (T(4)) through two major pathways: deiodination and conjugation. Following exposure to xenobiotics, T(4) conjugation increases through the induction of hepatic uridine diphosphate glucuronosyltransferase (UGT) in rodents; however, it is uncertain to what degree different species employ deiodination and conjugation in the metabolism of T(4). The objective of this study was to compare the metabolism of T4 in untreated and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-treated primary sandwich-cultured hepatocytes from rat (SCRH) and human (SCHH). 2. Basal metabolite concentrations were 13 times higher in the medium of SCRH compared to SCHH. Metabolite distribution in the medium of SCRH versus SCHH was as follows: T(4)G, (91.6 versus 5.3%); T4S, (3.6 versus 4.4%) and T(3) + rT(3), (4.9 versus 90.3%). PCB 153 induced T(4)G in the medium of SCRH and SCHH; however, T(4)S and T(3) + rT(3) were changed but to a much lesser degree. 3. The results indicate that baseline T(4) glucuronidation is greater in SCRH compared to SCHH. These data also suggest that glucuronidation may be a more important pathway for T(4) metabolism in rats and deiodination may be a favored pathway in humans; however, with PCB 153 treatment these data support glucuronidation as a primary route of T(4) metabolism in both rat and humans.

  16. Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt.

    PubMed

    Schulze-Bergkamen, Henning; Brenner, Dirk; Krueger, Andreas; Suess, Dorothee; Fas, Stefanie C; Frey, Christian R; Dax, Andreas; Zink, Dorothea; Büchler, Peter; Müller, Martina; Krammer, Peter H

    2004-03-01

    CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x(L). HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine(727)--but not tyrosine(705)--phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases. PMID:14999683

  17. Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

    PubMed Central

    Giri, Shibashish; Bader, Augustinus

    2014-01-01

    Background Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Methods Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. Results After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5–7 days. The remaining transfected hepatocytes persisted for 2–4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose

  18. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    SciTech Connect

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  19. COVALENT BINDING OF TRICHLOROETHYLENE TO PROTEINS IN HUMAN AND RAT HEPATOCYTES. (R826409)

    EPA Science Inventory

    The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte c...

  20. Modeling of Hepatocytes Proliferation Isolated from Proximal and Distal Zones from Human Hepatocellular Carcinoma Lesion

    PubMed Central

    Montalbano, Mauro; Curcurù, Giuseppe; Shirafkan, Ali; Vento, Renza; Rastellini, Cristiana; Cicalese, Luca

    2016-01-01

    Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatocyte function and transformation capacity. However, limited information is available in comparing the growth and proliferation of human hepatocytes obtained from different areas of the same cirrhotic liver in relation to their distance from the HCC lesion. In this study, hepatocytes from 10 patients with cirrhosis and HCC undergoing surgical resections from specimens obtained at a proximal (CP) and distal (CD) distance from the HCC lesion were isolated and placed in primary culture. CP hepatocytes (CP-Hep) were isolated between 1 to 3 cm (leaving at least 1cm margin to avoid cancer cells and/or satellite lesions), while CD hepatocytes (CD-Hep) were isolated from more than 5 cm or from the contralateral-lobe. A statistical model was built to analyze the proliferation rates of these cells and we evaluated expression of HCC markers (Glypican-3 (GPC3), αSmooth Muscle Actin (α-SMA) and PCNA). We observed a significant difference in proliferation and in-vitro growth showing that CP-Hep had a proliferation pattern and rate significantly different than CD-Hep. Based on these data, this model can provide information to predict growth of human hepatocytes in primary culture in relation to their pre-cancerous state with significant differences in the HCC markers expression. This model provides an important innovative tool for in-vitro analysis of HCC. PMID:27074018

  1. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

  2. Expression of human. alpha. sub 1 -antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes

    SciTech Connect

    Kay, M.A.; Baley, P.; Rothenberg, S.; Leland, F; Fleming, L.; Ponder, K.P.; Liu, Tajen; Finegold, M.; Darlington, G.; Pokorny, W.; Woo, S.L.C. )

    1992-01-01

    The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. The authors have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here they demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. In two animals they have transplanted hepatocytes transduced with a retroviral vector containing the human {alpha}{sub 1}-antitrypsin cDNA under transcriptional control of the cytomegalovirus promotor. Both animals had significant human {alpha}{sub 1}-antitrypsin in the serum for 1 month. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstruction with the use of promoters of cellular genes that are active in the normal liver.

  3. Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells

    PubMed Central

    Ma, Xiaocui; Tschudy-Seney, Benjamin; Roll, Garrett; Behbahan, Iman Saramipoor; Ahuja, Tijess P.; Tolstikov, Vladimir; Wang, Charles; McGee, Jeannine; Khoobyari, Shiva; Nolta, Jan A.; Willenbring, Holger

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultraperformance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies. PMID:23681950

  4. Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Lehec, Sharon C; Hughes, Robin D

    2005-12-01

    Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and alpha-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E(1), and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, alpha-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. alpha-lipoic acid preincubation (0.5-5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM alpha-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation.

  5. Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Lehec, Sharon C; Hughes, Robin D

    2005-12-01

    Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and alpha-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E(1), and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, alpha-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. alpha-lipoic acid preincubation (0.5-5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM alpha-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation. PMID:16315306

  6. The effects of immunosuppressive agents on the function of human hepatocytes in vitro.

    PubMed

    Serrano, Trinidad; Mitry, Ragai R; Terry, Claire; Lehec, Sharon C; Dhawan, Anil; Hughes, Robin D

    2006-01-01

    Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by

  7. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    PubMed

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  8. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes

    PubMed Central

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P.; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J.; Gathercole, Laura L.

    2015-01-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  9. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    PubMed

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

  10. IFNL3 genotype is associated with differential induction of IFNL3 in primary human hepatocytes

    PubMed Central

    Kurbanov, Fuat; Kim, Yonghak; Latanich, Rachel; Chaudhari, Pooja; El-Diwany, Ramy; Knabel, Matt; Kandathil, Abraham J; Cameron, Andrew; Cox, Andrea; Jang, Yoon-Young; Thomas, David L; Balagopal, Ashwin

    2016-01-01

    Background Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive. Methods IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3. Results PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07). Conclusions Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection. PMID:26109548

  11. Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice.

    PubMed

    Sakai, Yusuke; Yamanouchi, Kosho; Ohashi, Kazuo; Koike, Makiko; Utoh, Rie; Hasegawa, Hideko; Muraoka, Izumi; Suematsu, Takashi; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Kuroki, Tamotsu; Eguchi, Susumu

    2015-10-01

    Subcutaneous liver tissue engineering is an attractive and minimally invasive approach used to curative treat hepatic failure and inherited liver diseases. However, graft failure occurs frequently due to insufficient infiltration of blood vessels (neoangiogenesis), while the maintenance of hepatocyte phenotype and function requires in vivo development of the complex cellular organization of the hepatic lobule. Here we describe a subcutaneous human liver construction allowing for rapidly vascularized grafts by transplanting engineered cellular sheets consisting of human primary hepatocytes adhered onto a fibroblast layer. The engineered hepatocyte/fibroblast sheets (EHFSs) showed superior expression levels of vascularization-associated growth factors (vascular endothelial growth factor, transforming growth factor beta 1, and hepatocyte growth factor) in vitro. EHFSs developed into vascularized subcutaneous human liver tissues contained glycogen stores, synthesized coagulation factor IX, and showed significantly higher synthesis rates of liver-specific proteins (albumin and alpha 1 anti-trypsin) in vivo than tissues from hepatocyte-only sheets. The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel cell therapies for liver diseases.

  12. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

    PubMed

    Tobita, Takamasa; Guzman-Lepe, Jorge; Takeishi, Kazuki; Nakao, Toshimasa; Wang, Yang; Meng, Fanying; Deng, Chu-Xia; Collin de l'Hortet, Alexandra; Soto-Gutierrez, Alejandro

    2016-01-01

    There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes. PMID:26890260

  13. Lineage restriction and differentiation of human embryonic stem cells into hepatic progenitors and zone 1 hepatocytes.

    PubMed

    Pei, Haiyun; Yang, Yinxiang; Xi, Jiafei; Bai, Zongliang; Yue, Wen; Nan, Xue; Bai, Cixian; Wang, Yunfang; Pei, Xuetao

    2009-03-01

    Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

  14. Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes.

    PubMed

    Grossman, M; Raper, S E; Wilson, J M

    1991-11-01

    Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans. PMID:1767337

  15. The impact of solute carrier (SLC) drug uptake transporter loss in human and rat cryopreserved hepatocytes on clearance predictions.

    PubMed

    Lundquist, Patrik; Lööf, Johan; Sohlenius-Sternbeck, Anna-Karin; Floby, Eva; Johansson, Jenny; Bylund, Johan; Hoogstraate, Janet; Afzelius, Lovisa; Andersson, Tommy B

    2014-03-01

    Cryopreserved hepatocytes are often used as a convenient tool in studies of hepatic drug metabolism and disposition. In this study, the expression and activity of drug transporters in human and rat fresh and cryopreserved hepatocytes was investigated. In human cryopreserved hepatocytes, Western blot analysis indicated that protein expression of the drug uptake transporters [human Na(+)-taurocholate cotransporting polypeptide (NTCP), human organic anion transporting polypeptides (OATPs), human organic anion transporters, and human organic cation transporters (OCTs)] was considerably reduced compared with liver tissue. In rat cryopreserved cells, the same trend was observed but to a lesser extent. Several rat transporters were reduced as a result of both isolation and cryopreservation procedures. Immunofluorescence showed that a large portion of remaining human OATP1B1 and OATP1B3 transporters were internalized in human cryopreserved hepatocytes. Measuring uptake activity using known substrates of OATPs, OCTs, and NTCP showed decreased activity in cryopreserved as compared with fresh hepatocytes in both species. The reduced uptake in cryopreserved hepatocytes limited the in vitro metabolism of several AstraZeneca compounds. A retrospective analysis of clearance predictions of AstraZeneca compounds suggested systematic lower clearance predicted using metabolic stability data from human cryopreserved hepatocytes compared with human liver microsomes. This observation is consistent with a loss of drug uptake transporters in cryopreserved hepatocytes. In contrast, the predicted metabolic clearance from fresh rat hepatocytes was consistently higher than those predicted from liver microsomes, consistent with retention of uptake transporters. The uptake transporters, which are decreased in cryopreserved hepatocytes, may be rate-limiting for the metabolism of the compounds and thus be one explanation for underpredictions of in vivo metabolic clearance from cryopreserved

  16. Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism.

    PubMed

    Ballard, T Eric; Wang, Shuai; Cox, Loretta M; Moen, Mark A; Krzyzewski, Stacy; Ukairo, Okechukwu; Obach, R Scott

    2016-02-01

    Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

  17. Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPα, and PROX1 transduction.

    PubMed

    Nakamori, Daiki; Takayama, Kazuo; Nagamoto, Yasuhito; Mitani, Seiji; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-15

    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs. PMID:26679606

  18. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  19. Enhancement of hepatocyte differentiation from human embryonic stem cells by Chinese medicine Fuzhenghuayu

    PubMed Central

    Chen, Jiamei; Gao, Wei; Zhou, Ping; Ma, Xiaocui; Tschudy-Seney, Benjamin; Liu, Chenghai; Zern, Mark A; Liu, Ping; Duan, Yuyou

    2016-01-01

    Chinese medicine, Fuzhenghuayu (FZHY), appears to prevent fibrosis progression and improve liver function in humans. Here we found that FZHY enhanced hepatocyte differentiation from human embryonic stem cells (hESC). After treatment with FZHY, albumin expression was consistently increased during differentiation and maturation process, and expression of metabolizing enzymes and transporter were also increased. Importantly, expression of mesenchymal cell and cholangiocyte marker was significantly reduced by treatment with FZHY, indicating that one possible mechanism of FZHY’s role is to inhibit the formation of mesenchymal cells and cholangiocytes. Edu-labelled flow cytometric analysis showed that the percentage of the Edu positive cells was increased in the treated cells. These results indicate that the enhanced proliferation involved hepatocytes rather than another cell type. Our investigations further revealed that these enhancements by FZHY are mediated through activation of canonical Wnt and ERK pathways and inhibition of Notch pathway. Thus, FZHY not only promoted hepatocyte differentiation and maturation, but also enhanced hepatocyte proliferation. These results demonstrate that FZHY appears to represent an excellent therapeutic agent for the treatment of liver fibrosis, and that FZHY treatment can enhance our efforts to generate mature hepatocytes with proliferative capacity for cell-based therapeutics and for pharmacological and toxicological studies. PMID:26733102

  20. Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells.

    PubMed

    Allameh, Abdolamir; Esmaeli, Shahnaz; Kazemnejad, Somaieh; Soleimani, Masoud

    2009-06-01

    The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

  1. Insulin and IGFs enhance hepatocyte differentiation from human embryonic stem cells via the PI3K/AKT pathway.

    PubMed

    Magner, Nataly L; Jung, Yunjoon; Wu, Jian; Nolta, Jan A; Zern, Mark A; Zhou, Ping

    2013-10-01

    Human embryonic stem cells (hESCs) can be progressively differentiated into definitive endoderm (DE), hepatic progenitors, and hepatocytes, and thus provide an excellent model system for the mechanistic study of hepatocyte differentiation, which is currently poorly understood. Here, we found that insulin enhanced hepatocyte differentiation from hESC-derived DE. Insulin activated the PI3K/AKT pathway, but not the mitogen-activated protein kinase pathway in the DE cells, and inhibition of the PI3K/AKT pathways by inhibitors markedly inhibited hepatocyte differentiation. In addition, insulin-like growth factor 1 (IGF1) and IGF2 also activated the PI3K/AKT pathway in DE cells and their expression was robustly upregulated during hepatocyte differentiation from DE. Furthermore, inhibition of IGF receptor 1 (IGF1R) by a small molecule inhibitor PPP or knockdown of the IGF1R by shRNA attenuated hepatocyte differentiation. Moreover, simultaneous knockdown of the IGF1R and the insulin receptor with shRNAs markedly reduced the activation of AKT and substantially impaired hepatocyte differentiation. The PI3K pathway specifically enhanced the expression of HNF1 and HNF4 to regulate hepatocyte differentiation from DE. Although inhibition of the PI3K pathway was previously shown to be required for the induction of DE from hESCs, our study revealed a positive role of the PI3K pathway in hepatocyte differentiation after the DE stage, and has advanced our understanding of hepatocyte cell fate determination.

  2. Bile Acid-Induced Necrosis in Primary Human Hepatocytes and in Patients with Obstructive Cholestasis

    PubMed Central

    Woolbright, Benjamin L.; Dorko, Kenneth; Antoine, Daniel J.; Clarke, Joanna I.; Gholami, Parviz; Li, Feng; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Fan, Fang; Jenkins, Rosalind E.; Park, B. Kevin; Hagenbuch, Bruno; Olyaee, Mojtaba; Jaeschke, Hartmut

    2015-01-01

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. PMID:25636263

  3. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    PubMed

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

  4. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    PubMed

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming. PMID:27053247

  5. APPARENT SEXUAL DIFFERENCES IN METABOLISM OF INORGANIC ARSENIC IN HUMAN HEPATOCYTES

    EPA Science Inventory

    APPARENT SEXUAL DIFFERENCES IN METABOLISM OF INORGANIC ARSENIC IN HUMAN HEPATOCYTES. M Styblo1, G A Hamilton1, E L LeCluyse1 and D J Thomas2. 1University of North Carolina, Chapel Hill, NC, USA; 2US EPA, ORD, NHEERL, Research Triangle Park, NC, USA.
    The liver is considered a m...

  6. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  7. Pluripotent Stem Cell-Derived Hepatocyte-Like Cells

    PubMed Central

    Schwartz, R. E.; Fleming, H.E.; Bhatia, S. N.

    2014-01-01

    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and function. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for pharmacology and toxicology drug screening. iPS cells can be differentiated in a stepwise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iPS-derived hepatocyte-like cells resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iPS-derived hepatocyte-like cells express fetal markers such as alpha fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (0.1%) of many detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iPS-derived hepatocyte-like cells and adult hepatocytes have limited the use of stem cells as a renewable source of functional adult human hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte

  8. Comparative metabolism of geranyl nitrile and citronellyl nitrile in mouse, rat, and human hepatocytes.

    PubMed

    Kemper, Raymond A; Nabb, Diane L; Gannon, Shawn A; Snow, Timothy A; Api, Anne Marie

    2006-06-01

    Geranyl nitrile (GN) and citronellyl nitrile (CN) are fragrance components used in consumer and personal care products. Differences in the clastogenicity of these two terpenes are postulated to result from differential biotransformation, presumably involving the conjugated nitrile moiety. The metabolic clearance and biotransformation of GN and CN were compared in primary hepatocytes from mice, rats, and humans. For determination of intrinsic clearance, GN and CN were incubated with hepatocytes in sealed vials, and the headspace was sampled periodically by solid-phase microextraction and analyzed by gas chromatography/mass spectrometry. For metabolite identification, GN and CN were incubated with hepatocytes from each species for 60 min, and reaction mixtures were extracted and analyzed by mass spectroscopy. Both GN and CN were rapidly metabolized in hepatocytes from all species (T1/2, 0.7-11.6 min). Within a species, intrinsic clearance was similar for both compounds and increased in the order human < rat < mouse. Major common pathways for biotransformation of GN and CN involved 1) epoxidation of the 6-alkenyl moiety followed by conjugation with glutathione, 2) hydroxylation of the terminal methyl group(s) followed by direct conjugation with glucuronic acid in rodents or further oxidation to the corresponding acid in human cells, and 3) hydroxylation of the allylic C5 position. No evidence for either phase I or phase II metabolism of the conjugated nitrile moiety was obtained. Thus, the presumed metabolic basis for differences in genotoxicity remains elusive.

  9. The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

    PubMed Central

    Koj, A; Gauldie, J; Regoeczi, E; Sauder, D N; Sweeney, G D

    1984-01-01

    Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines. Images Fig. 3. PMID:6083778

  10. Human hepatocyte isolation and relationship of cell viability to early graft function.

    PubMed

    Mitry, Ragai R; Hughes, Robin D; Aw, Marion M; Terry, Claire; Mieli-Vergani, Giorgina; Girlanda, Raffaele; Muiesan, Paolo; Rela, Mohamed; Heaton, Nigel D; Dhawan, Anil

    2003-01-01

    Hepatocyte transplantation is emerging as an additional modality of treatment for patients with acute liver failure or liver-based metabolic disorders. The procedure requires isolation of high-quality hepatocytes from unused donor livers. Hepatocytes were isolated from 20 donor livers (11 right lobes, 3 left lateral segments, 6 whole livers) using a collagenase perfusion technique. Cell viability (median 56%, range 13-95%) and yield (median 1.4 x 10(9) cells, range 2.0 x 10(6)-1.8 x 10(10) cells) varied according to the tissue available. Fatty livers rejected for transplantation gave lower cell viability (median 45%, range 25-59%). There was a significant correlation between age of donor (median 21 years, range 7-66 years) and viability of isolated hepatocytes in vitro (r = -0.683, p = 0.001). The 13 segments of livers were from reduced/split grafts used for clinical transplantation in 9 children and 4 adults. There was no significant correlation between in vitro cell viability and clinical parameters including intensive care stay, serum aspartate aminotransferase,and international normalized ratio (in the first 7 days), and allograft rejection or other early posttransplant complications, in patients transplanted with the corresponding tissue. PMID:12693666

  11. Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells

    NASA Astrophysics Data System (ADS)

    Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

    2016-08-01

    Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

  12. Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

    PubMed

    Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

    2016-08-01

    Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases. PMID:27379400

  13. Human neonatal hepatocyte transplantation induces long-term rescue of unconjugated hyperbilirubinemia in the Gunn rat.

    PubMed

    Tolosa, Laia; López, Silvia; Pareja, Eugenia; Donato, María Teresa; Myara, Anne; Nguyen, Tuan Huy; Castell, José Vicente; Gómez-Lechón, María José

    2015-06-01

    Crigler-Najjar type 1 disease is a rare inherited metabolic disease characterized by high levels of unconjugated bilirubin due to the complete absence of hepatic uridine diphosphoglucuronate-glucuronosyltransferase activity. Hepatocyte transplantation (HT) has been proposed as an alternative treatment for Crigler-Najjar syndrome, but it is still limited by the quality and the low engraftment and repopulation ability of the cells used. Because of their attachment capability and expression of adhesion molecules as well as the higher proportion of hepatic progenitor cells, neonatal hepatocytes may have an advantage over adult cells. Adult or neonatal hepatocytes were transplanted into Gunn rats, a model for Crigler-Najjar disease. Engraftment and repopulation were studied and compared by immunofluorescence (IF). Additionally, the serum bilirubin levels, the presence of bilirubin conjugates in rat serum, and the expression of uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) in rat liver samples were also analyzed. Here we show that neonatal HT results in long-term correction in Gunn rats. In comparison with adult cells, neonatal cells showed better engraftment and repopulation capability 3 days and 6 months after transplantation, respectively. Bilirubinemia decreased in the transplanted animals during the whole experimental follow-up (6 months). Bilirubin conjugates were also present in the serum of the transplanted animals. Western blots and IF confirmed the presence and expression of UGT1A1 in the liver. This work is the first to demonstrate the advantage of using neonatal hepatocytes for the treatment of Crigler-Najjar in vivo. PMID:25821167

  14. Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

    PubMed

    Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L; Berrueta, Lisbeth; Salmen, Siham

    2016-05-01

    Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection.

  15. CD56+ T Cells Inhibit Hepatitis C Virus Replication in Human Hepatocytes

    PubMed Central

    Ye, Li; Wang, Xu; Wang, Shihong; Wang, Yanjian; Song, Li; Hou, Wei; Zhou, Lin; Li, He; Ho, Wenzhe

    2009-01-01

    CD56+ T cells are abundant in liver and play an important role in defense against viral infections. However, the role of CD56+ T cells in control of HCV infection remains to be determined. We investigated the noncytolytic anti-HCV activity of primary CD56+ T cells in human hepatocytes. When HCV JFH-1-infected hepatocytes were co-cultured with CD56+ T cells or incubated in media conditioned with CD56+ T cell culture supernatants (SN), HCV infectivity and replication were significantly inhibited. The antibodies to interferon (IFN)-γ or IFN-γ receptor could largely block CD56+ T cell-mediated anti-HCV activity. Investigation of mechanism(s) responsible for CD56+ T cell-mediated noncytolytic anti-HCV activity showed that CD56+ T SN activated the multiple elements of janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and enhanced the expression of IFN regulatory factors (IRFs) 1, 3, 7, 8 and 9, resulting in the induction of endogenous IFN-α/β expression in hepatocytes. Moreover, CD56+ T SN treatment inhibited the expression of HCV-supportive miRNA-122 and enhanced the levels of anti-HCV miRNA-196a in human hepatocytes. Conclusion: These findings provide direct in vitro evidence at cellular and molecular levels that CD56+ T cells may have an essential role in innate immune cell-mediated defense against HCV infection. PMID:19085952

  16. Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.

    PubMed

    Vildhede, Anna; Wiśniewski, Jacek R; Norén, Agneta; Karlgren, Maria; Artursson, Per

    2015-08-01

    Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

  17. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  18. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer

    PubMed Central

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C.; Dandri, Maura

    2016-01-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  19. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

    PubMed

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C; Dandri, Maura; Pollok, Joerg-Matthias

    2016-05-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  20. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

    PubMed

    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  1. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  2. Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites.

    PubMed

    Wang, Wendy WeiWei; Khetani, Salman R; Krzyzewski, Stacy; Duignan, David B; Obach, R Scott

    2010-10-01

    Metabolism is one of the important determinants of the overall disposition of drugs, and the profile of metabolites can have an impact on efficacy and safety. Predicting which drug metabolites will be quantitatively predominant in humans has become increasingly important in the research and development of new drugs. In this study, a novel micropatterned hepatocyte coculture system was evaluated for its ability to generate human in vivo metabolites. Twenty-seven compounds of diverse chemical structure and subject to a range of drug biotransformation reactions were assessed for metabolite profiles in the micropatterned coculture system using pooled cryopreserved human hepatocytes. The ability of this system to generate metabolites that are >10% of dose in excreta or >10% of total drug-related material in circulation was assessed and compared to previously reported data obtained in human hepatocyte suspensions, liver S-9 fraction, and liver microsomes. The micropatterned coculture system was incubated for up to 7 days without a change in medium, which offered an ability to generate metabolites for slowly metabolized compounds. The micropatterned coculture system generated 82% of the excretory metabolites that exceed 10% of dose and 75% of the circulating metabolites that exceed 10% of total circulating drug-related material, exceeds the performance of hepatocyte suspension incubations and other in vitro systems. Phase 1 and phase 2 metabolites were generated, as well as metabolites that arise via two or more sequential reactions. These results suggest that this in vitro system offers the highest performance among in vitro metabolism systems to predict major human in vivo metabolites.

  3. Small-Molecule-Driven Hepatocyte Differentiation of Human Pluripotent Stem Cells

    PubMed Central

    Siller, Richard; Greenhough, Sebastian; Naumovska, Elena; Sullivan, Gareth J.

    2015-01-01

    Summary The differentiation of pluripotent stem cells to hepatocytes is well established, yet current methods suffer from several drawbacks. These include a lack of definition and reproducibility, which in part stems from continued reliance on recombinant growth factors. This has remained a stumbling block for the translation of the technology into industry and the clinic for reasons associated with cost and quality. We have devised a growth-factor-free protocol that relies on small molecules to differentiate human pluripotent stem cells toward a hepatic phenotype. The procedure can efficiently direct both human embryonic stem cells and induced pluripotent stem cells to hepatocyte-like cells. The final population of cells demonstrates marker expression at the transcriptional and protein levels, as well as key hepatic functions such as serum protein production, glycogen storage, and cytochrome P450 activity. PMID:25937370

  4. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

  5. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

    PubMed Central

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  6. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  7. Flame Retardant BDE-47 Effectively Activates Nuclear Receptor CAR in Human Primary Hepatocytes

    PubMed Central

    Sueyoshi, Tatsuya

    2014-01-01

    Polybrominated diphenyl ether BDE-47 (2,2′,4,4′-tetrabromodiphenyl ether) is a thyroid hormone disruptor in mice; hepatic induction of various metabolic enzymes and transporters has been suggested as the mechanism for this disruption. Utilizing Car −/− and Pxr −/− mice as well as human primary hepatocytes, here we have demonstrated that BDE-47 activated both mouse and human nuclear receptor constitutive activated/androstane receptor (CAR). In mouse livers, CAR, not PXR, was responsible for Cyp2b10 mRNA induction by BDE-47. In human primary hepatocytes, BDE-47 was able to induce translocation of YFP-tagged human CAR from the cytoplasm to the nucleus andCYP2B6 and CYP3A4 mRNAs expressions. BDE-47 activated human CAR in a manner akin to the human CAR ligand CITCO (6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) in luciferase-reporter assays using Huh-7 cells. In contrast, mouse CAR was not potently activated by BDE-47 in the same reporter assays. Furthermore, human pregnane X receptor (PXR) was effectively activated by BDE-47 while mouse PXR was weakly activated in luciferase-reporter assays. Our results indicate that BDE-47 induces CYP genes through activation of human CAR in addition to the previously identified pathway through human PXR. PMID:24218150

  8. DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes

    PubMed Central

    Nauwelaers, Gwendoline; Bessette, Erin E.; Gu, Dan; Tang, Yijin; Rageul, Julie; Fessard, Valérie; Yuan, Jian-Min; Yu, Mimi C.; Langouët, Sophie; Turesky, Robert J.

    2011-01-01

    DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 107 DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation. PMID:21456541

  9. Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line.

    PubMed

    Green, Charlotte J; Johnson, Deborah; Amin, Harsh D; Sivathondan, Pamela; Silva, Michael A; Wang, Lai Mun; Stevanato, Lara; McNeil, Catriona A; Miljan, Erik A; Sinden, John D; Morten, Karl J; Hodson, Leanne

    2015-09-15

    The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile(148)Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.

  10. Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line

    PubMed Central

    Green, Charlotte J.; Johnson, Deborah; Amin, Harsh D.; Sivathondan, Pamela; Silva, Michael A.; Wang, Lai Mun; Stevanato, Lara; McNeil, Catriona A.; Miljan, Erik A.; Sinden, John D.; Morten, Karl J.

    2015-01-01

    The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile148Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored. PMID:26126685

  11. Comparison of human hepatocytes isolated from livers accepted or discarded for orthotopic transplantation.

    PubMed

    Groothuis, G M; Sandker, G W; Pruim, J; Weert, B; Slooff, M J; Meijer, D K

    1995-12-01

    The aim of this study was to compare human hepatocytes isolated from livers accepted and from livers discarded for transplantation with respect to viability and drug transport function. In addition, the influence of age of the donor and preservation time of the liver on cell viability was determined. Cell viability was assessed by trypan blue exclusion, MTT reduction, morphological integrity and ATP content, and drug transport function by uptake and excretion of taurocholic acid. Hepatocytes could be isolated successfully from livers accepted as well as from livers discarded for transplantation, with a median yield of 5.0 x 10(6) cells/g (range 0.1 to 42.4) and 0.7 x 10(6) cells/g (range 0.0 to 22.7), respectively (not significantly different). These cells were not significantly different with respect to viability and transport rate of taurocholate. Neither the age of the donor nor the duration of liver preservation (6-43 hr in University of Wisconsin solution) significantly influenced cell yield and viability. It is concluded that because of this overlap in cell viability, hepatocytes isolated both from accepted and from discarded livers can in principle be used to investigate drug transport functions in the human liver. PMID:20650173

  12. Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-culture with Plated Primary Hepatocytes and HepaRG.

    PubMed

    Bonn, Britta; Svanberg, Petter; Janefeldt, Annika; Hultman, Ia; Grime, Ken

    2016-04-01

    A key requirement in drug discovery is to accurately define intrinsic clearance (CL(int)) values of less than 1 µl/min/10(6) hepatocytes, which requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHHs), and the HµREL human hepatocyte/stromal cell co-cultures for determination of low CL(int) values. The investigation demonstrated that the systems were capable of providing statistically significant CL(int) estimations down to 0.2 µl/min/10(6) cells. The HµREL assay provided a higher level of reproducibility, with repeat significant CL(int) values being defined in a minimum of triplicate consecutive assays for six of seven of the low CL(int) compounds compared with four of seven for PHHs and two of seven for HepaRG. The assays were also compared with a suspension assay using drugs with higher CL(int) values and diverse enzymology. The CL(int) values from the PHH and HµREL assays were similar to those defined by a hepatocyte suspension assay, indicating that they can be used interchangeably alongside a standard assay. Finally, data from these two assays could also predict in vivo hepatic metabolic CL(int) to within 3-fold for greater than 70% of the compounds tested, with average fold errors (AFE) of 1.6 and 2.3, respectively, whereas the HepaRG data were predictive to within 3-fold for only 50% of compounds (AFE 2.9). In summary, all systems have utility for low CL(int) determination, but the HµREL co-culture appears slightly superior regarding overall assay performance. PMID:26851239

  13. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    PubMed

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  14. Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes.

    PubMed

    De Gottardi, Andrea; Vinciguerra, Manlio; Sgroi, Antonino; Moukil, Moulay; Ravier-Dall'Antonia, Florence; Pazienza, Valerio; Pugnale, Paolo; Foti, Michelangelo; Hadengue, Antoine

    2007-08-01

    Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH

  15. Priming of hepatocytes enhances in vivo liver transduction with lentiviral vectors in adult mice.

    PubMed

    Pichard, Virginie; Boni, Sébastien; Baron, William; Nguyen, Tuan Huy; Ferry, Nicolas

    2012-02-01

    Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.

  16. Comparison of cryopreserved HepaRG cells with cryopreserved human hepatocytes for prediction of clearance for 26 drugs.

    PubMed

    Zanelli, Ugo; Caradonna, Nicola Pasquale; Hallifax, David; Turlizzi, Elisa; Houston, J Brian

    2012-01-01

    Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL(int)) in the recently developed cryopreserved HepaRG cell system with CL(int) in human cryopreserved pooled hepatocytes and with CL(int) in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL(int) in HepaRG cells and human hepatocytes, which was linear throughout the range of CL(int) (1-2000 ml · min(-1) · kg(-1)) and not dependent on particular cytochrome P450 involvement. Prediction of CL(int) in HepaRG cells was on average within 2-fold of in vivo CL(int) (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance-limited CL(int) in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool. PMID:21998403

  17. Cross-species transmission of gibbon and orangutan hepatitis B virus to uPA/SCID mice with human hepatocytes.

    PubMed

    Sa-Nguanmoo, Pattaratida; Tanaka, Yasuhito; Ratanakorn, Parntep; Sugiyama, Masaya; Murakami, Shuko; Payungporn, Sunchai; Sommanustweechai, Angkana; Mizokami, Masashi; Poovorawan, Yong

    2011-06-01

    To investigate the potential of cross-species transmission of non-human primate HBV to humans, severe combined immunodeficiency mice transgenic for urokinase-type plasminogen activator, in which the mouse liver has been engrafted with human hepatocytes, were inoculated with non-human primate HBV. HBV-DNA positive serum samples from a gibbon or orangutan were inoculated into 6 chimeric mice. HBV-DNA, hepatitis B surface antigen (HBsAg), and HB core-related antigen in sera and HBV cccDNA in liver were detectable in 2 of 3 mice each from the gibbon and orangutan. Likewise, applying immunofluorescence HBV core protein was only found in human hepatocytes expressing human albumin. The HBV sequences from mouse sera were identical to those from orangutan and gibbon sera determined prior to inoculation. In conclusion, human hepatocytes have been infected with gibbon/orangutan HBV.

  18. The farnesoid X receptor induces fetuin-B gene expression in human hepatocytes

    PubMed Central

    Murakami, Takeshi; Walczak, Robert; Caron, Sandrine; Duhem, Christian; Vidal, Vincent; Darteil, Raphaël; Staels, Bart

    2007-01-01

    FXR (farnesoid X receptor), a nuclear receptor activated by BAs (bile acids), is a key factor in the regulation of BA, lipid and carbohydrate metabolism. The recent development of synthetic FXR agonists and knockout mouse models has accelerated the discovery of FXR target genes. In the present study, we identify human fetuin-B as a novel FXR target gene. Treatment with FXR agonists increased fetuin-B expression in human primary hepatocytes and in the human hepatoma HepG2 cell line. In contrast, fetuin-B expression was not responsive to FXR agonist treatment in murine primary hepatocytes. Fetuin-B induction by FXR agonist was abolished upon FXR knockdown by siRNA (small interfering RNA). In addition to the previously described P1 promoter, we show that the human fetuin-B gene is also transcribed from an alternative promoter, termed P2. Transcription via the P2 promoter was induced by FXR agonist treatment, whereas P1 promoter activity was not sensitive to FXR agonist treatment. Two putative FXR-response elements [IR-1 (inverted repeat-1)] were identified in the region –1.6 kb upstream of the predicted P2 transcriptional start site. Both motifs bound FXR–RXR (retinoid X receptor) complexes in vitro and were activated by FXR in transient transfection reporter assays. Mutations in the IR-1 sites abolished FXR–RXR binding and activation. Taken together, these results identify human fetuin-B as a new FXR target gene in human hepatocytes. PMID:17655523

  19. Pathway Analysis and Modeling of the Differentiation of Human Embryonic Stem Cells into Hepatocyte-like Cells

    NASA Astrophysics Data System (ADS)

    Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph

    2011-06-01

    A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.

  20. Genes for the dimerization cofactor of hepatocyte nuclear factor-1[alpha] (DCOH) are on human and murine chromsomes 10

    SciTech Connect

    Milatovich, A.; Mendel, D.B.; Crabtree, G.R.; Francke, U. )

    1993-04-01

    Hepatocyte nuclear factor-1[alpha] (HNF-1[alpha]; gene symbol, TCF1) forms dimers with itself as well as with HNF-1[beta] and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1[alpha], called DCOH, has been identified. Here, the authors report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids. 25 refs., 1 fig., 2 tabs.

  1. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

    PubMed Central

    Pfeifer, A M; Cole, K E; Smoot, D T; Weston, A; Groopman, J D; Shields, P G; Vignaud, J M; Juillerat, M; Lipsky, M M; Trump, B F

    1993-01-01

    Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685115

  2. Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

    PubMed

    Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

    2015-03-01

    Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture.

  3. Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

    PubMed

    Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

    2015-03-01

    Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. PMID:26569044

  4. Differential UGT1A1 induction by chrysin in primary human hepatocytes and HepG2 Cells.

    PubMed

    Smith, Cornelia M; Graham, Richard A; Krol, Wojciech L; Silver, Ivin S; Negishi, Masahiko; Wang, Hongbing; Lecluyse, Edward L

    2005-12-01

    Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo. PMID:16135700

  5. Detection and proteomic identification of S-nitrosated proteins in human hepatocytes.

    PubMed

    López-Sánchez, Laura M; Corrales, Fernando J; De La Mata, Manuel; Muntané, Jordi; Rodríguez-Ariza, Antonio

    2008-01-01

    The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification.

  6. A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

    SciTech Connect

    Yannam, Govardhana Rao; Han, Bing; Setoyama, Kentaro; Yamamoto, Toshiyuki; Ito, Ryotaro; Brooks, Jenna M.; Guzman-Lepe, Jorge; Galambos, Csaba; Fong, Jason V.; Deutsch, Melvin; Quader, Mubina A.; Yamanouchi, Kosho; Kabarriti, Rafi; Mehta, Keyur; Soto-Gutierrez, Alejandro; and others

    2014-02-01

    Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

  7. Augmented Inhibition of CYP3A4 in Human Primary Hepatocytes by Ritonavir Solid Drug Nanoparticles.

    PubMed

    Martin, Philip; Giardiello, Marco; McDonald, Tom O; Smith, Darren; Siccardi, Marco; Rannard, Steven P; Owen, Andrew

    2015-10-01

    Ritonavir is a protease inhibitor utilized primarily as a pharmaco-enhancer with concomitantly administered antiviral drugs including other protease inhibitors. However, poor tolerance, serious side effects, and toxicities associated with drug-drug interactions are common during exposure to ritonavir. The aim of this work was to investigate the impact of nanoformulation on ritonavir pharmacological properties. Emulsion-templated freeze-drying techniques were used to generate ritonavir (10 wt %) solid drug nanoparticle formulations. A total of 68 ritonavir formulations containing various mixtures of excipients were assessed for inhibition of CYP3A4 in baculosomes and primary human hepatocytes. Accumulation and cytotoxicity were assessed in HepG2 (hepatocytes), Caco-2 (intestinal), THP-1 (monocytes), A-THP-1 (macrophage), and CEM (lymphocytes). Transcellular permeation across Caco-2 cells was also assessed. From 68 solid drug nanoparticle formulations tested, 50 (73.5%) for baculosome and 44 (64.7%) for human primary hepatocytes exhibited enhanced CYP3A4 inhibition relative to an aqueous ritonavir solution. Sixty-one (89.7%) and 49 (72%) solid drug nanoformulations had higher apical to basal permeation across Caco-2 cells than aqueous solution of ritonavir after 60 and 120 min, respectively. No significant difference in cellular accumulation was observed for any solid drug nanoparticle for any cell type compared to aqueous ritonavir. However, incubation with the vast majority of solid drug nanoparticle formulations resulted in lower cytotoxicity of ritonavir than detected with an aqueous solution. These data provide in vitro proof of concept for improved inhibition of CYP3A4 by ritonavir through formation of solid drug nanoparticles. Nanodispersions also showed enhanced permeability across Caco-2 cells lower cytotoxicity across hepatic, intestinal, and immune cell types compared to an aqueous solution of ritonavir.

  8. Association between hepatitis B virus and MHC class I polypeptide-related chain A in human hepatocytes derived from human-mouse chimeric mouse liver.

    PubMed

    Sasaki, Reina; Kanda, Tatsuo; Wu, Shuang; Nakamoto, Shingo; Haga, Yuki; Jiang, Xia; Nakamura, Masato; Shirasawa, Hiroshi; Yokosuka, Osamu

    2015-09-01

    Due to the lack of efficient hepatitis B virus (HBV) infection systems, progress in understanding the role of innate immunity in HBV infection has remained challenging. Here we used human hepatocytes from a humanized severe combined immunodeficiency albumin promoter/enhancer driven-urokinase-type plasminogen activator mouse model for HBV infection. HBV DNA levels in culture medium from these human hepatocytes were 4.8-5.7 log IU/mL between day 16 and day 66 post-infection by HBV genotype C inoculum. HBV surface antigen (HBsAg) was also detected by chemiluminescent immunoassay from day 7 to day 66 post-infection. Western blot analysis revealed that major histocompatibility complex class I-related chain A (MICA), which plays a role in the innate immune system, was induced in HBV-infected human hepatocytes 27 days after infection compared with the uninfected control. MICA was reduced at day 62 and undetectable at day 90. Of interest, MICA expression by human hepatocytes increased after HBV infection and decreased before HBsAg loss. Human hepatocytes derived from chimeric mice with hepatocyte-humanized liver could support HBV genome replication. Further studies of the association between HBV replication and MICA induction should be conducted.

  9. Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

    PubMed

    Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

    2000-10-01

    The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

  10. Hepatocyte transplantation program: Lessons learned and future strategies.

    PubMed

    Ibars, Eugenia Pareja; Cortes, Miriam; Tolosa, Laia; Gómez-Lechón, Maria José; López, Slivia; Castell, José Vicente; Mir, José

    2016-01-14

    This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.

  11. Hepatocyte transplantation program: Lessons learned and future strategies

    PubMed Central

    Ibars, Eugenia Pareja; Cortes, Miriam; Tolosa, Laia; Gómez-Lechón, Maria José; López, Slivia; Castell, José Vicente; Mir, José

    2016-01-01

    This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used. PMID:26811633

  12. Genomic responses to hepatitis B virus (HBV) infection in primary human hepatocytes

    PubMed Central

    Ancey, Pierre-Benoit; Testoni, Barbara; Gruffaz, Marion; Cros, Marie-Pierre; Durand, Geoffroy; Le Calvez-Kelm, Florence; Durantel, David; Herceg, Zdenko; Hernandez-Vargas, Hector

    2015-01-01

    Viral infections are able to modify the host's cellular programs, with DNA methylation being a biological intermediate in this process. The extent to which viral infections deregulate gene expression and DNA methylation is not fully understood. In the case of Hepatitis B virus (HBV), there is evidence for an interaction between viral proteins and the host DNA methylation machinery. We studied the ability of HBV to modify the host transcriptome and methylome, using naturally infected primary human hepatocytes to better mimic the clinical setting. Gene expression was especially sensitive to culture conditions, independently of HBV infection. However, we identified non-random changes in gene expression and DNA methylation occurring specifically upon HBV infection. There was little correlation between expression and methylation changes, with transcriptome being a more sensitive marker of time-dependent changes induced by HBV. In contrast, a set of differentially methylated sites appeared early and were stable across the time course experiment. Finally, HBV-induced DNA methylation changes were defined by a specific chromatin context characterized by CpG-poor regions outside of gene promoters. These data support the ability of HBV to modulate host cell expression and methylation programs. In addition, it may serve as a reference for studies addressing the genome-wide consequences of HBV infection in human hepatocytes. PMID:26565721

  13. Genomic responses to hepatitis B virus (HBV) infection in primary human hepatocytes.

    PubMed

    Ancey, Pierre-Benoit; Testoni, Barbara; Gruffaz, Marion; Cros, Marie-Pierre; Durand, Geoffroy; Le Calvez-Kelm, Florence; Durantel, David; Herceg, Zdenko; Hernandez-Vargas, Hector

    2015-12-29

    Viral infections are able to modify the host's cellular programs, with DNA methylation being a biological intermediate in this process. The extent to which viral infections deregulate gene expression and DNA methylation is not fully understood. In the case of Hepatitis B virus (HBV), there is evidence for an interaction between viral proteins and the host DNA methylation machinery. We studied the ability of HBV to modify the host transcriptome and methylome, using naturally infected primary human hepatocytes to better mimic the clinical setting.Gene expression was especially sensitive to culture conditions, independently of HBV infection. However, we identified non-random changes in gene expression and DNA methylation occurring specifically upon HBV infection. There was little correlation between expression and methylation changes, with transcriptome being a more sensitive marker of time-dependent changes induced by HBV. In contrast, a set of differentially methylated sites appeared early and were stable across the time course experiment. Finally, HBV-induced DNA methylation changes were defined by a specific chromatin context characterized by CpG-poor regions outside of gene promoters.These data support the ability of HBV to modulate host cell expression and methylation programs. In addition, it may serve as a reference for studies addressing the genome-wide consequences of HBV infection in human hepatocytes.

  14. Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict a risk of drug induced hepatotoxicity.

    PubMed

    Szabo, Monika; Veres, Zsuzsa; Baranyai, Zsolt; Jakab, Ferenc; Jemnitz, Katalin

    2013-01-01

    Human hepatocytes are the gold standard for toxicological studies but they have several drawbacks, like scarce availability, high inter-individual variability, a short lifetime, which limits their applicability. The aim of our investigations was to determine, whether HepaRG cells could replace human hepatocytes in uptake experiments for toxicity studies. HepaRG is a hepatoma cell line with most hepatic functions, including a considerable expression of uptake transporters in contrast to other hepatic immortalized cell lines. We compared the effect of cholestatic drugs (bosentan, cyclosporinA, troglitazone,) and bromosulfophthalein on the uptake of taurocholate and estrone-3-sulfate in human and rat hepatocytes and HepaRG cells. The substrate uptake was significantly slower in HepaRG cells than in human hepatocytes, still, in the presence of drugs we observed a concentration dependent decrease in uptake. In all cell types, the culture time had a significant impact not only on the uptake process but on the inhibitory effect of drugs too. The most significant drug effect was measured at 4 h after seeding. Our report is among the first concerning interactions of the uptake transporters in the HepaRG, at the functional level. Results of the present study clearly show that concerning the inhibition of taurocholate uptake by cholestatic drugs, HepaRG cells are closer to human hepatocytes than rat hepatocytes. In conclusion, we demonstrated that HepaRG cells may provide a suitable tool for hepatic uptake studies. PMID:23516635

  15. Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

    PubMed Central

    Koen, Yakov M.; Sarma, Diganta; Williams, Todd D.; Galeva, Nadezhda A.; Obach, R. Scott; Hanzlik, Robert P.

    2012-01-01

    Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted

  16. Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

    SciTech Connect

    Xie, Yuchao; McGill, Mitchell R.; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson; Jaeschke, Hartmut

    2014-09-15

    Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to

  17. Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

    PubMed Central

    Chen, Yong; Li, Yanfeng; Wang, Xia; Zhang, Wei; Sauer, Vanessa; Chang, Chan-Jung; Han, Bing; Tchaikovskaya, Tatyana; Avsar, Yesim; Tafaleng, Edgar; Madhusudana Girija, Sanal; Tar, Krisztina; Polgar, Zsuzsanna; Strom, Stephen; Bouhassira, Eric E.; Guha, Chandan; Fox, Ira J.; Roy-Chowdhury, Jayanta; Roy-Chowdhury, Namita

    2015-01-01

    Summary Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases. PMID:26074313

  18. Effect of Microenvironment on Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Hepatocytes In Vitro and In Vivo

    PubMed Central

    Xue, Gai; Han, Xiaolei; Ma, Xin; Wu, Honghai; Qin, Yabin; Liu, Jianfang; Hu, Yuqin; Hong, Yang; Hou, Yanning

    2016-01-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are considered to be an ideal cell source for cell therapy of many diseases. The aim of this study was to investigate the contribution of the microenvironment to the hepatic differentiation potential of hUCMSCs in vitro and in vivo and to explore their therapeutic use in acute liver injury in rats. We established a new model to simulate the liver tissue microenvironment in vivo using liver homogenate supernatant (LHS) in vitro. This induced environment could drive hUCMSCs to differentiate into hepatocyte-like cells within 7 days. The differentiated cells expressed hepatocyte-specific markers and demonstrated hepatocellular functions. We also injected hUCMSCs into rats with CCl4-induced acute hepatic injury. The hUCMSCs were detected in the livers of recipient rats and expressed the human hepatocyte-specific markers, suggesting that hUCMSCs could differentiate into hepatocyte-like cells in vivo in the liver tissue microenvironment. Levels of biochemistry markers improved significantly after transplantation of hUCMSCs compared with the nontransplantation group (P < 0.05). In conclusion, this study demonstrated that the liver tissue microenvironment may contribute to the differentiation of hUCMSCs into hepatocytes both in vitro and in vivo. PMID:27088093

  19. Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

    PubMed Central

    Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

    2015-01-01

    Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

  20. Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

    PubMed Central

    Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

    2015-01-01

    Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

  1. Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells.

    PubMed

    Mathapati, Santosh; Siller, Richard; Impellizzeri, Agata A R; Lycke, Max; Vegheim, Karianne; Almaas, Runar; Sullivan, Gareth J

    2016-01-01

    Hepatocyte-like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small-molecule-driven protocol for in vitro differentiation of hPSCs into HLCs without the use of growth factors. hPSCs are coaxed through a developmentally relevant route via the primitive streak to definitive endoderm (DE) using the small molecule CHIR99021 (a Wnt agonist), replacing the conventional growth factors Wnt3A and activin A. The small-molecule-derived DE is then differentiated to hepatoblast-like cells in the presence of dimethyl sulfoxide. The resulting hepatoblasts are then differentiated to HLCs with N-hexanoic-Tyr, Ile-6 aminohexanoic amide (Dihexa, a hepatocyte growth factor agonist) and dexamethasone. The protocol provides an efficient and reproducible procedure for differentiation of hPSCs into HLCs utilizing small molecules. © 2016 by John Wiley & Sons, Inc. PMID:27532814

  2. PPAR{alpha} regulates the hepatotoxic biomarker alanine aminotransferase (ALT1) gene expression in human hepatocytes

    SciTech Connect

    Thulin, Petra; Rafter, Ingalill; Stockling, Kenneth; Tomkiewicz, Celine; Norjavaara, Ensio; Aggerbeck, Martine; Hellmold, Heike; Ehrenborg, Ewa; Andersson, Ulf; Cotgreave, Ian; Glinghammar, Bjoern

    2008-08-15

    In this work, we investigated a potential mechanism behind the observation of increased aminotransferase levels in a phase I clinical trial using a lipid-lowering drug, the peroxisome proliferator-activated receptor (PPAR) {alpha} agonist, AZD4619. In healthy volunteers treated with AZD4619, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were elevated without an increase in other markers for liver injury. These increases in serum aminotransferases have previously been reported in some patients receiving another PPAR{alpha} agonist, fenofibrate. In subsequent in vitro studies, we observed increased expression of ALT1 protein and mRNA in human hepatocytes after treatment with fenofibric acid. The PPAR effect on ALT1 expression was shown to act through a direct transcriptional mechanism involving at least one PPAR response element (PPRE) in the proximal ALT1 promoter, while no effect of fenofibrate and AZD4619 was observed on the ALT2 promoter. Binding of PPARs to the PPRE located at - 574 bp from the transcriptional start site was confirmed on both synthetic oligonucleotides and DNA in hepatocytes. These data show that intracellular ALT expression is regulated by PPAR agonists and that this mechanism might contribute to increased ALT activity in serum.

  3. Gene deletions and amplifications in human hepatocellular carcinomas: correlation with hepatocyte growth regulation.

    PubMed

    Nalesnik, Michael A; Tseng, George; Ding, Ying; Xiang, Guo-Sheng; Zheng, Zhong-liang; Yu, YanPing; Marsh, James W; Michalopoulos, George K; Luo, Jian-Hua

    2012-04-01

    Tissues from 98 human hepatocellular carcinomas (HCCs) obtained from hepatic resections were subjected to somatic copy number variation (CNV) analysis. Most of these HCCs were discovered in livers resected for orthotopic transplantation, although in a few cases, the tumors themselves were the reason for the hepatectomies. Genomic analysis revealed deletions and amplifications in several genes, and clustering analysis based on CNV revealed five clusters. The LSP1 gene had the most cases with CNV (46 deletions and 5 amplifications). High frequencies of CNV were also seen in PTPRD (21/98), GNB1L (18/98), KIAA1217 (18/98), RP1-1777G6.2 (17/98), ETS1 (11/98), RSU1 (10/98), TBC1D22A (10/98), BAHCC1 (9/98), MAML2 (9/98), RAB1B (9/98), and YIF1A (9/98). The existing literature regarding hepatocytes or other cell types has connected many of these genes to regulation of cytoskeletal architecture, signaling cascades related to growth regulation, and transcription factors directly interacting with nuclear signaling complexes. Correlations with existing literature indicate that genomic lesions associated with HCC at the level of resolution of CNV occur on many genes associated directly or indirectly with signaling pathways operating in liver regeneration and hepatocyte growth regulation.

  4. Construction of a differentiated human hepatocyte cell line expressing the herpes simplex virus-thymidine kinase gene.

    PubMed

    Kobayashi, N; Miyazaki, M; Westerman, K A; Noguchi, H; Sakaguchi, M; Totsugawa, T; Watanabe, T; Matsumura, T; Fujiwara, T; Leboulch, P; Tanaka, N; Namba, M

    2001-01-01

    Transient support using a hybrid artificial liver (HAL) device is a promising treatment for the patients with acute liver failure. Primary human hepatocytes are an ideal source for HAL therapy; however, the number of human livers available for hepatocyte isolation is limited by competition for use in whole organ transplantation. To overcome this problem, we previously established a highly differentiated human fetal hepatocyte cell line OUMS-29. Considering the potential risk when using these genetically engineered cells in humans, additional safeguards should be added to make the cells more clinically useful. In this work, the herpes simplex virus thymidine kinase (HSVtk) gene was retrovirally introduced into OUMS-29 cells. One of the HSVtk-expressed clones, OUMS-29/thymidine kinase (TK), grew in chemically defined serum free medium and expressed the genes of albumin, asialoglycoprotein receptor, glutamine synthetase, glutathione-S-transferase pi, and blood coagulation factor X. In vitro sensitivity of the cells to ganciclovir was evaluated. Intrasplenic transplantation of 50 x 10(6) OUMS-29/TK cells prolonged the survival of 90% hepatectomized rats compared with medium injection alone (control). In the present study, we have established highly differentiated immortalized human hepatocytes with tight regulation. The cells may be clinically useful for HAL treatment. PMID:11575821

  5. Arts & Humanities in Adult Education.

    ERIC Educational Resources Information Center

    Word's Worth: A Quarterly Newsletter of the Lifelong Learning Network, 1998

    1998-01-01

    This issue of a quarterly newsletter on lifelong learning focuses on the theme of the arts and humanities in adult literacy education. The following articles are included: (1) "In Defense of a Practical Education" (Earl Shorris); (2) "From the Program Director" (Elizabeth Bryant McCrary); (3) "Vermont Council on the Humanities: Book Discussion…

  6. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

    SciTech Connect

    Acikgoez, Ali; Karim, Najibulla; Giri, Shibashish; Schmidt-Heck, Wolfgang; Bader, Augustinus

    2009-01-15

    Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c{sub i,j} (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p{sub k} (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the

  7. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    SciTech Connect

    Marion, Tracy L.; Perry, Cassandra H.; St Claire, Robert L.; Brouwer, Kim L.R.

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  8. Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes

    SciTech Connect

    Ishihara, H.; Hara, T.; Aramaki, Y.; Tsuchiya, S.; Hosoi, K. )

    1990-05-01

    The selective delivery of human recombinant interferon (IFN)-gamma to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-gamma was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free ({sup 32}P)IFN-gamma was internalized into isolated rat hepatocytes, whereas AF-liposomes containing ({sup 32}P)IFN-gamma were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4{degrees}C) was one-fifth of the uptake (estimated at 37{degrees}C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-gamma into hepatocytes in vitro.

  9. Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

    PubMed

    Peters, Derek T; Henderson, Christopher A; Warren, Curtis R; Friesen, Max; Xia, Fang; Becker, Caroline E; Musunuru, Kiran; Cowan, Chad A

    2016-05-01

    Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754

  10. Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  11. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  12. Sex differences in liver toxicity-do female and male human primary hepatocytes react differently to toxicants in vitro?

    PubMed

    Mennecozzi, Milena; Landesmann, Brigitte; Palosaari, Taina; Harris, Georgina; Whelan, Maurice

    2015-01-01

    There is increasing amount of evidence for sex variation in drug efficiency and toxicity profiles. Women are more susceptible than men to acute liver injury from xenobiotics. In general, this is attributed to sex differences at a physiological level as well as differences in pharmacokinetics and pharmacodynamics, but neither of these can give a sufficient explanation for the diverse responses to xenobiotics. Existing data are mainly based on animal models and limited data exist on in vitro sex differences relevant to humans. To date, male and female human hepatocytes have not yet been compared in terms of their responses to hepatotoxic drugs. We investigated whether sex-specific differences in acute hepatotoxicity can be observed in vitro by comparing hepatotoxic drug effects in male and female primary human hepatocytes. Significant sex-related differences were found for certain parameters and individual drugs, showing an overall higher sensitivity of female primary hepatocytes to hepatotoxicants. Moreover, our work demonstrated that high content screening is feasible with pooled primary human hepatocytes in suspension. PMID:25849576

  13. Current status of human hepatocyte transplantation and its potential for Wilson's disease.

    PubMed

    Filippi, Celine; Dhawan, Anil

    2014-05-01

    Wilson's disease (WD) is a genetic disorder of liver copper excretion leading to its accumulation in various vital organs like the liver, brain, and kidneys. Drugs such as penicillamine, trientine, and zinc salts are the mainstay of treatment, with good outcomes; but nonresponders or a lack of compliance to the drug treatment can result in disease progression and acute liver failure (ALF). Current treatment for WD with ALF is an emergency liver transplantation and lifelong immunosuppression. Human hepatocyte transplantation (HTx) is increasingly used as treatment for liver-based metabolic defects. HTx may benefit WD patients with ALF, either as transient support until chelation treatment shows its effect or as a definitive cure through liver repopulation by healthy donor cells, as shown in animal models of WD. Although clinical trials of HTx have already proven safety and efficacy in different ALF etiologies, it remains to be demonstrated similarly in cases of WD.

  14. Activation of the farnesoid X receptor represses PCSK9 expression in human hepatocytes.

    PubMed

    Langhi, Cédric; Le May, Cédric; Kourimate, Sanae; Caron, Sandrine; Staels, Bart; Krempf, Michel; Costet, Philippe; Cariou, Bertrand

    2008-03-19

    The purpose of this study was to determine whether bile acids (BAs) modulate hepatic pro-protein convertase subtilisin/kexin 9 (PCSK9) gene expression. Immortalized human hepatocytes were treated with various BAs. Chenodeoxycholic acid (CDCA) treatment specifically decreased both PCSK9 mRNA and protein contents. Moreover, activation of the BA-activated farnesoid X receptor (FXR) by its synthetic specific agonist GW4064 also decreased PCSK9 expression. Of functional relevance, coadministration of CDCA counteracted the statin-induced PCSK9 expression, leading to a potentiation of LDL receptor activity. This study suggests that a transcriptional repression of PCSK9 by CDCA or FXR agonists may potentiate the hypolipidemic effect of statins.

  15. Markers of electrophilic stress caused by chemically reactive metabolites in human hepatocytes.

    PubMed

    Takakusa, Hideo; Masumoto, Hiroshi; Mitsuru, Ayako; Okazaki, Osamu; Sudo, Kenichi

    2008-05-01

    The metabolic activation of a drug to an electrophilic reactive metabolite and its covalent binding to cellular macromolecules is considered to be involved in the occurrence of idiosyncratic drug toxicity (IDT). As a cellular defense system against oxidative and electrophilic stress, phase II enzymes are known to be induced through a Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2/antioxidant response element system. We presumed that it is important for the risk assessment of drug-induced hepatotoxicity and IDTs to observe the biological responses evoked by exposure to reactive metabolites, and then investigated the mRNA induction profiles of phase II enzymes in human hepatocytes after exposure to problematic drugs associated with IDTs, such as ticlopidine, diclofenac, clozapine, and tienilic acid, as well as safe drugs such as levofloxacin and caffeine. According to the results, the problematic drugs exhibited inductive effects on heme oxygenase 1 (HO-1), which contrasted with the safe drugs; therefore, the induction of HO-1 mRNA seems to be correlated with the occurrence of drug toxicity, including IDT caused by electrophilic reactive metabolites. Moreover, glutathione-depletion and cytochrome P450 (P450)-inhibition experiments have shown that the observed HO-1 induction was triggered by the electrophilic reactive metabolites produced from the problematic drugs through P450-mediated metabolic bioactivation. Taken together with our present study, this suggests that HO-1 induction in human hepatocytes would be a good marker of the occurrence of metabolism-based drug-induced hepatotoxicity and IDT caused by the formation of electrophilic reactive metabolites. PMID:18227147

  16. Xeno-sensing activity of the aryl hydrocarbon receptor in human pluripotent stem cell-derived hepatocyte-like cells

    PubMed Central

    Kim, Hye-Min; Kim, Ji-Woo; Choi, Youngjun; Chun, Hang-Suk; Im, Ilkyun; Han, Yong-Mahn; Song, Chang-Woo; Yoon, Seokjoo; Park, Han-Jin

    2016-01-01

    Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity, their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor, CAR; and pregnane X receptor, PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs, whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore, ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition, an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus, AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics. PMID:26899675

  17. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes.

    PubMed

    Price, R J; Scott, M P; Giddings, A M; Walters, D G; Stierum, R H; Meredith, C; Lake, B G

    2008-06-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

  18. Disruption of mitochondrial activities in rabbit and human hepatocytes by a quinoxalinone anxiolytic and its carboxylic acid metabolite.

    PubMed

    Ulrich, R G; Bacon, J A; Cramer, C T; Petrella, D K; Sun, E L; Meglasson, M D; Holmuhamedov, E

    1998-11-01

    The quinoxalinone anxiolytic, panadiplon, was dropped from clinical development due to unexpected hepatic toxicity in human volunteers. Subsequent experimental studies in rabbits demonstrated a hepatic toxicity that resembled Reye's syndrome. In the present studies, we examined the effects of panadiplon and a metabolite, cyclopropane carboxylic acid (CPCA) on hepatic mitochondrial activities in vitro and ex vivo. Acute inhibition of beta-oidation of [14C]palmitate was observed in rabbit and human hepatocyte suspensions incubated with 100 microM panadiplon. Panadiplon (30 microM) also reduced mitochondrial uptake of rhodamine 123 (R123) in cultured rabbit and human, but not rat hepatocytes, following 18 h exposure. CPCA also impaired beta-oxidation and R123 uptake in rabbit and human hepatocytes. R123 uptake and beta-oxidation in cells from some donors was not impaired by either agent, and cell death was not observed in any experiment. Hepatocytes isolated from panadiplon-treated rabbits had reduced palmitate beta-oxidation rates and inhibited mitochondrial R123 uptake; R123 uptake remained inhibited until 48-72 h in culture. Rabbit mitochondrial respiration experiments revealed a slightly lower ratio of ATP formed/oxygen consumed in panadiplon-treated animals: direct exposure of normal rabbit liver mitochondria to panadiplon did not have this effect. Hepatocytes isolated from panadiplon-treated rabbits showed reduced respiratory control ratios and lower oxygen consumption compared to controls. Our results indicate that panadiplon induces a mitochondrial dysfunction in the liver, and suggest that this dysfunction may be attributed to the carboxylic acid metabolite.

  19. Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes.

    PubMed

    Lin, Christine; Shi, Julianne; Moore, Amanda; Khetani, Salman R

    2016-01-01

    Accurate prediction of in vivo hepatic drug clearance using in vitro assays is important to properly estimate clinical dosing regimens. Clearance of low-turnover compounds is especially difficult to predict using short-lived suspensions of unpooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been shown previously to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 enzymes in MPCCs created from unpooled cryopreserved PHH donors. MPCCs were then used to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 ml/min per kilogram). MPCCs predicted 73, 92, and 96% of drug clearance values for all tested drugs within 2-fold, 3-fold, and 4-fold of in vivo values, respectively. There was good correlation (R(2) = 0.94, slope = 1.05) of predictions between the two PHH donors. On the other hand, suspension hepatocytes and conventional monolayers created from the same donor had significantly reduced predictive capacity (i.e., 30-50% clearance values within 4-fold of in vivo), and were not able to metabolize several drugs. Finally, we modulated drug clearance in MPCCs by inducing or inhibiting P450s. Rifampin-mediated CYP3A4 induction increased midazolam clearance by 73%, while CYP3A4 inhibition with ritonavir decreased midazolam clearance by 79%. Similarly, quinidine-mediated CYP2D6 inhibition reduced clearance of dextromethorphan and desipramine by 71 and 22%, respectively. In conclusion, MPCCs created using cryopreserved unpooled PHHs can be used for drug clearance predictions and to model drug-drug interactions. PMID:26452722

  20. Association of filamin A and vimentin with hepatitis C virus proteins in infected human hepatocytes.

    PubMed

    Ghosh, S; Ahrens, W A; Phatak, S U; Hwang, S; Schrum, L W; Bonkovsky, H L

    2011-10-01

    Chronic hepatitis C (CHC) infection caused by hepatitis C virus (HCV) is a major cause of liver disease and remains a major therapeutic challenge. A variety of host proteins interact with HCV proteins. The definitive role of cytoskeletal (CS) proteins in HCV infection remains to be determined. In this study, our aim was to determine the expression profile of differentially regulated and expressed selected CS proteins and their association with HCV proteins in infected hepatocytes as possible therapeutic targets. Using proteomics, qRT-PCR, Western blot and immunofluorescence techniques, we revealed that filamin A (fila) and vimentin (vim) were prominently increased proteins in HCV-expressing human hepatoma cells compared with parental cells and in liver biopsies from patients with CHC vs controls. HCV nonstructural (NS) 3 and NS5A proteins were associated with fila, while core protein partially with fila and vim. Immunoprecipitation showed interactions among fila and NS3 and NS5A proteins. Cells treated with interferon-α showed a dose- and time-dependent decrease in CS and HCV proteins. NS proteins clustered at the perinuclear region following cytochalasin b treatment, whereas disperse cytoplasmic and perinuclear distribution was observed in the no-treatment group. This study demonstrates and signifies that changes occur in the expression of CS proteins in HCV-infected hepatocytes and, for the first time, shows the up-regulation and interaction of fila with HCV proteins. Association between CS and HCV proteins may have implications in future design of CS protein-targeted therapy for the treatment for HCV infection.

  1. Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture.

    PubMed Central

    Sugiyama, A; Arakaki, R; Ohnishi, T; Arakaki, N; Daikuhara, Y; Takada, H

    1996-01-01

    Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture. PMID:8606111

  2. Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling

    PubMed Central

    Correll, Kelly; Schiel, John A.; Finigan, Jay H.; Prekeris, Rytis; Mason, Robert J.

    2014-01-01

    There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS. PMID:24748602

  3. Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model.

    PubMed

    Xia, Lei; Hong, Xin; Sakban, Rashidah Binte; Qu, Yinghua; Singh, Nisha Hari; McMillian, Michael; Dallas, Shannon; Silva, Jose; Sensenhauser, Carlo; Zhao, Sylvia; Lim, Heng Keang; Yu, Hanry

    2016-02-01

    Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

  4. Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality.

    PubMed

    Ramasamy, Thamil Selvee; Yu, Jason S L; Selden, Clare; Hodgson, Humphery; Cui, Wei

    2013-02-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of human hepatocytes, owing to their indefinite self-renewal and pluripotent properties. Both hESC-/iPSC-derived hepatocytes hold great promise in treating liver diseases as potential candidates for cell replacement therapies or as an in vitro platform to conduct new drug trials. It has been previously demonstrated that the initiation of hESC differentiation in monolayer cultures increases the generation of definitive endoderm (DE) and subsequently of hepatocyte differentiation. However, monolayer culture may hinder the maturation of hESC-derived hepatocytes, since such two-dimensional (2D) conditions do not accurately reflect the complex nature of three-dimensional (3D) hepatocyte specification in vivo. Here, we report the sequential application of 2D and 3D culture systems to differentiate hESCs to hepatocytes. Human ESCs were initially differentiated in a monolayer culture to DE cells, which were then inoculated into Algimatrix scaffolds. Treatments of hESC-DE cells with a ROCK inhibitor before and after inoculation dramatically enhanced their survival and the formation of spheroids, which are distinct from HepG2 carcinoma cells. In comparison with monolayer culture alone, sequential 2D and 3D cultures significantly improved hepatocyte differentiation and function. Our results demonstrate that hESC-DE cells can be incorporated into Algimatrix 3D culture systems to enhance hepatocyte differentiation and function.

  5. Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

    SciTech Connect

    Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang; Cui, Guang-Hui; Wang, Xin-Hua; Chen, Xi-Gu . E-mail: xiguchen1516@yahoo.com.cn

    2007-06-15

    We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.

  6. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    PubMed

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  7. Impact of Environmental Chemicals on the Transcriptome of Primary Human Hepatocytes: Potential for Health Effects.

    PubMed

    Mitchell, Robert D; Dhammi, Anirudh; Wallace, Andrew; Hodgson, Ernest; Roe, R Michael

    2016-08-01

    New paradigms for human health risk assessment of environmental chemicals emphasize the use of molecular methods and human-derived cell lines. In this study, we examined the effects of the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels in primary human hepatocytes. These chemicals were tested individually and as a mixture. RNA-Seq showed that 100 μM DEET significantly increased transcript levels (α = 0.05) for 108 genes and lowered transcript levels for 64 genes and fipronil at 10 μM increased the levels of 2246 transcripts and decreased the levels for 1428 transcripts. Fipronil was 21-times more effective than DEET in eliciting changes, even though the treatment concentration was 10-fold lower for fipronil versus DEET. The mixture of DEET and fipronil produced a more than additive effect (levels increased for 3017 transcripts and decreased for 2087 transcripts). The transcripts affected for all chemical treatments were classified by GO analysis and mapped to chromosomes. The overall treatment responses, specific pathways, and individual transcripts affected were discussed at different levels of fold-change. Changes found in transcript levels in response to treatments will require further research to understand their importance in overall cellular, organ, and organismic function. PMID:27091632

  8. Caveolin-1 is essential in the differentiation of human adipose-derived stem cells into hepatocyte-like cells via an MAPK pathway-dependent mechanism

    PubMed Central

    GUAN, XIN; WANG, NAN; CUI, FENGGONG; LIU, YANG; LIU, PENG; ZHAO, JINGYUAN; HAN, CHAO; LI, XIAOYAN; LENG, ZHIQIAN; LI, YING; JI, XIAOFEI; ZOU, WEI; LIU, JING

    2016-01-01

    Human adipose-derived stem cells (hADSCs), widely present in the adult human body, are an emerging and attractive tool for the establishment of stem cell-based therapies for the treatment of liver disease. However, the mechanism underlying hADSCs hepatic differentiation remains to be elucidated. Caveolin-1 (Cav-1), a 21–24 kDa membrane structural protein, is important in liver regeneration and development. In the present study, fluorescence immuno-cytochemistry and western blotting were used to analyze the expression levels of Cav-1 and evaluate its effects on the hepatic differentiation of hADSCs. The results revealed that primary hADSCs preserved the ability to proliferate and differentiate into hepatocyte-like cells. As demonstrated by semiquantitative reverse transcription-polymerase chain reaction, hepatocyte-inducing factors significantly increased the expression of Cav-1 in a time-dependent manner, as indicated by increased expression levels of the albumin (ALB) and α-fetoprotein (AFP) markers. In addition the expression levels of ALB and HNF1A significantly decreased following small interfering RNA-mediated knockdown of Cav-1. The mitogen-activated protein kinase (MAPK) signaling pathway was activated during hepatic differentiation and inhibited following Cav-1 knockdown. These results suggested that Cav-1 may regulate the hepatocyte-like differentiation of hADSCs by modulating mitogen-activated protein kinase kinase/MAPK signaling. The results of the present study will provide experimental and theoretical basis for further clinical studies on stem cell transplantation in the treatment of liver disease. PMID:26717806

  9. Characterization of arsenic hepatobiliary transport using sandwich-cultured human hepatocytes.

    PubMed

    Roggenbeck, Barbara A; Carew, Michael W; Charrois, Gregory J; Douglas, Donna N; Kneteman, Norman M; Lu, Xiufen; Le, X Chris; Leslie, Elaine M

    2015-06-01

    Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.

  10. Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Kondo, Yuki; Iwao, Takahiro; Yoshihashi, Sachimi; Mimori, Kayo; Ogihara, Ruri; Nagata, Kiyoshi; Kurose, Kouichi; Saito, Masayoshi; Niwa, Takuro; Suzuki, Takayoshi; Miyata, Naoki; Ohmori, Shigeru; Nakamura, Katsunori; Matsunaga, Tamihide

    2014-01-01

    In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. PMID:25084468

  11. Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes

    SciTech Connect

    Rachek, Lyudmila I.; Yuzefovych, Larysa V.; LeDoux, Susan P.; Julie, Neil L.; Wilson, Glenn L.

    2009-11-01

    Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARgamma antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARgamma-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

  12. Induction of benzo(a)pyrene metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary cultures of adult rat hepatocytes: regulation by vitamin A

    SciTech Connect

    Steward, A.R.

    1982-01-01

    In order to develop a cellular model for studying mechanisms of enzyme induction and the effects of this induction on xenobiotic metabolism and cytotoxicity, the induction of benzo(a)pyrene (BaP) metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in primary hepatocyte cultures prepared from adult male rats and maintained in chemically-defined medium containing hormones. A derepression of induction was observed during the first 3 days in culture. Addition of 0.8 to 2.0 ..mu..g/ml of retinol acetate (RA) prevented about half of the derepression of induction occurring between 36 and 60 h in culture. Horse serum (10%) also blocked up to half of the observed derepression. Serum, however, also led to a 40% reduction in the partitioning of (/sup 3/H)TCDD from the medium into the hepatocytes. The derepresion of MFO induction in primary adult hepatocyte cultures may occur partly as a result of a deficiency of retinol. RA is hypothesized to slow the time course of induction by reducing the rate of protein turnover. RA may also partially block the shift in the dose-response curve for induction by TCDD by maintaining the normal metabolic regulation of the cytosolic receptor for TCDD. Addition of a physiological level of RA to the culture medium may therefore help to maintain the hepatocytes at a level of genetic expression more nearly representative of the intact liver.

  13. Frequent hepatocyte chimerism in long-term human liver allografts independent of graft outcome.

    PubMed

    Aini, Wulamujiang; Miyagawa-Hayashino, Aya; Ozeki, Munetaka; Tsuruyama, Tatsuaki; Tamaki, Keiji; Uemoto, Shinji; Haga, Hironori

    2013-03-01

    Microchimerism after liver transplantation is considered to promote graft tolerance or tissue repair, but its significance is controversial. By using multiplex polymerase chain reaction (PCR) of short tandem repeat (STR) loci after laser capture microdissection of hepatocyte nuclei, we compared the proportions of recipient-derived hepatocytes in long-term stable liver allografts and late dysfunctional allografts caused by chronic rejection or idiopathic post-transplantation hepatitis. Through fluorescence in situ hybridization (FISH), we also analyzed the presence of recipient-derived Y-positive hepatocytes in the biopsies of livers transplanted from female donors to male recipients. The study population comprised 24 pediatric liver transplant recipients who survived with the initial graft, whose 10-year protocol biopsy records were available, and who had normal liver function (stable graft, SG; n=13) or a late dysfunctional graft (LDG; n=11) with similar follow-up periods (mean 10.8years in the SG group and 11.2years in the LDG group). STR analysis revealed that hepatocyte chimerism occurred in 7 of 13 (54%) SGs and 5 of 11 (45%) LDGs (p=0.68). The proportion of hepatocyte chimerism was low, with a mean of 3% seen in 2 of 3 female-to-male transplanted livers (one each of SG and LDG). In conclusion, hepatocyte chimerism was a constant event. The extent of engraftment of recipient-derived hepatocytes does not seem to correlate with the degree of hepatic injury in long-term liver allografts.

  14. Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells.

    PubMed

    Lang, Jianshe; Vera, Daniel; Cheng, Yichen; Tang, Hengli

    2016-09-13

    The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology, partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study, we differentiated human pluripotent stem cells (hPSCs) into hepatocytes, one of the target cells of DENV, to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore, DENV infection activated the NF-κB pathway, which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions. PMID:27546535

  15. Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver.

    PubMed Central

    Liu, M. L.; Mars, W. M.; Zarnegar, R.; Michalopoulos, G. K.

    1994-01-01

    We have previously shown that systemically injected hepatocyte growth factor (HGF) is primarily taken up by the liver. The present study shows that HGF injected systemically or through the portal circulation is retained primarily at periportal sites. The periportal retention of HGF seems to persist longer in regenerating liver. The percentage of the total HGF injected that was retained within the liver at 1 minute after injection varied with the dose. A maximal amount of 0.157 +/- 0.012 microgram of HGF per gram liver tissue is retained by normal liver. Analysis of the circulating form of HGF in the plasma showed a relative enrichment with time for the heterodimeric form of HGF. A portion of portally injected HGF, composed of both single chain and two chain (heterodimeric) form was excreted intact in the bile. This was found in both normal and regenerating liver. These studies show that the liver can sequester large amounts of HGF and that the sequestration occurs primarily at periportal sites. Our studies support the hypothesis that a nonlysosomal processing pathway for HGF is present in the liver. Images Figure 1 Figure 3 Figure 4 Figure 6 PMID:8291602

  16. Mechanism of Vitamin D Receptor Inhibition of Cholesterol 7α-Hydroxylase Gene Transcription in Human HepatocytesS⃞

    PubMed Central

    Han, Shuxin; Chiang, John Y. L.

    2009-01-01

    Lithocholic acid (LCA) is a potent endogenous vitamin D receptor (VDR) ligand. In cholestasis, LCA levels increase in the liver and intestine. The objective of this study is to test the hypothesis that VDR plays a role in inhibiting cholesterol 7α-hydroxylase (CYP7A1) gene expression and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the nucleus of the human hepatoma cell line HepG2 and human primary hepatocytes. 1α, 25-Dihydroxy-vitamin D3 or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA to VDR completely abrogated VDR inhibition of CYP7A1 mRNA expression in HepG2 cells. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/retinoid X receptor α in the human CYP7A1 promoter. Mammalian two-hybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligand-activated VDR specifically interacts with hepatocyte nuclear factor 4α (HNF4α) to block HNF4α interaction with coactivators or to compete with HNF4α for coactivators or to compete for binding to CYP7A1 chromatin, which results in the inhibition of CYP7A1 gene transcription. This study shows that VDR is expressed in human hepatocytes and may play a critical role in the inhibition of bile acid synthesis, thus protecting liver cells during cholestasis. PMID:19106115

  17. A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes.

    PubMed

    Sogame, Yoshihisa; Kitamura, Atsushi; Yabuki, Masashi; Komuro, Setsuko

    2009-11-01

    Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations.

  18. Synergy between broccoli sprout extract and selenium in the upregulation of thioredoxin reductase in human hepatocytes.

    PubMed

    Li, Dan; Wu, Kun; Howie, A Forbes; Beckett, Geoffrey J; Wang, Wei; Bao, Yongping

    2008-09-01

    Dietary isothiocyanates and selenium (Se) can up-regulate thioredoxin reductase 1 (TR1) in cultured human HepG2 and MCF-7 cells [Zhang et al. (2003). Synergy between sulforaphane and selenium in the induction of thioredoxin reductase 1 requires both transcriptional and translational modulation. Carcinogenesis, 24, 497-503; Wang et al. (2005). Sulforaphane, erucin and iberin up-regulate thioredoxin reductase expression in human MCF-7 cells. Journal of Agricultural and Food Chemistry, 53, 1417-1421] at both the protein and mRNA levels. In this study, broccoli sprout extract (a rich source of the isothiocyanates sulforaphane and iberin) and Se interacted synergistically to induce TR1 in immortalised human hepatocytes. Broccoli sprout extracts containing 1.6, 4 and 8μM isothiocyanates were tested for their ability to induce TR1 at the protein and mRNA level. Although induction of TR1 mRNA by broccoli sprout extract (1.6-8μM) was only 1.7-2.2-fold, co-treatment with Se (0.2-1μM) enhanced the expression of TR1 mRNA (3.0-3.3-fold). Moreover, broccoli sprout extract induced the cellular concentration of TR1 and TR enzymatic activity, an induction that was augmented by Se addition. Thus, broccoli sprout extract (8μM) and Se induced cellular TR1 concentration and enzymatic activity 3.7- and 5-fold respectively, whereas, Se or broccoli sprout extract alone produced an induction of only approximately 2-fold. These data suggest that dietary isothiocyanates from broccoli sprouts and Se are important agents in the regulation of redox status in human liver cells. The synergistic effect between isothiocyanates and Se at physiologically-relevant concentrations on the induction of TR1 may play an important role in protection against oxidative stress.

  19. De Novo Donor‐Specific HLA Antibody Formation in Two Patients With Crigler‐Najjar Syndrome Type I Following Human Hepatocyte Transplantation With Partial Hepatectomy Preconditioning

    PubMed Central

    Nowak, G.; Nemeth, A.; Zemack, H.; Mörk, L.‐M.; Johansson, H.; Gramignoli, R.; Watanabe, M.; Karadagi, A.; Alheim, M.; Hauzenberger, D.; van Dijk, R.; Bosma, P. J.; Ebbesen, F.; Szakos, A.; Fischler, B.; Strom, S.; Ellis, E.; Ericzon, B.‐G.

    2015-01-01

    Clinical hepatocyte transplantation is hampered by low engraftment rates and gradual loss of function resulting in incomplete correction of the underlying disease. Preconditioning with partial hepatectomy improves engraftment in animal studies. Our aim was to study safety and efficacy of partial hepatectomy preconditioning in clinical hepatocyte transplantation. Two patients with Crigler‐Najjar syndrome type I underwent liver resection followed by hepatocyte transplantation. A transient increase of hepatocyte growth factor was seen, suggesting that this procedure provides a regenerative stimulus. Serum bilirubin was decreased by 50%, and presence of bilirubin glucuronides in bile confirmed graft function in both cases; however, graft function was lost due to discontinuation of immunosuppressive therapy in one patient. In the other patient, serum bilirubin gradually increased to pretransplant concentrations after ≈600 days. In both cases, loss of graft function was temporally associated with emergence of human leukocyte antigen donor‐specific antibodies (DSAs). In conclusion, partial hepatectomy in combination with hepatocyte transplantation was safe and induced a robust release of hepatocyte growth factor, but its efficacy on hepatocyte engraftment needs to be evaluated with additional studies. To our knowledge, this study provides the first description of de novo DSAs after hepatocyte transplantation associated with graft loss. PMID:26523372

  20. Induction of hepatocyte growth factor production in human dermal fibroblasts by caffeic acid derivatives.

    PubMed

    Kurisu, Manami; Nakasone, Rie; Miyamae, Yusaku; Matsuura, Daisuke; Kanatani, Hirotoshi; Yano, Shingo; Shigemori, Hideyuki

    2013-01-01

    Hepatocyte growth factor (HGF) has mitogenic, motogenic, and morphogenic activities in epithelial cells. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. In this study, we examined the effects of caffeic acid derivatives including 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) on HGF production in Neonatal Normal Human Dermal Fibroblasts (NHDF). Both 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) significantly induced HGF production dose-dependent manner. To know the important substructure for HGF production activity, we next investigated the effect of the partial structure of these caffeic acid derivatives. From the results, caffeic acid (3) showed strong activity on the promotion of HGF production, while hydroxytyrosol (4) and quinic acid (5) didn't show any activity. Our findings suggest that the caffeoyl moiety of caffeic acid derivatives is essential for accelerated production of HGF. The compound which has the caffeoyl moiety may be useful for the treatment of some intractable organ disease.

  1. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    SciTech Connect

    Zhou Yijun . E-mail: zhou-yijun@hotmail.com; Wang Jiahe; Zhang Jin

    2006-06-02

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-{kappa}B, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-{kappa}B, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.

  2. 3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

    SciTech Connect

    Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

    2009-02-06

    Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPAR{alpha}) signaling. Furthermore, using PPAR{alpha} agonists and antagonists, we also analyzed the effect of PPAR{alpha} signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.

  3. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    PubMed

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  4. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    PubMed Central

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  5. Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α.

    PubMed

    Theofilatos, Dimitris; Anestis, Aristomenis; Hashimoto, Koshi; Kardassis, Dimitris

    2016-01-15

    Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. PMID:26692490

  6. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells.

    PubMed

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel; Damm, Georg

    2015-05-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 10(6) KC, 2.7 × 10(5) LEC and 4.7 × 10(5) HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4-5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.

  7. Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

    PubMed Central

    Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

    1996-01-01

    OBJECTIVE: The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. SUMMARY BACKGROUND DATA: Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. METHODS: Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. RESULTS: Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. CONCLUSIONS: Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL

  8. PNPLA3 is regulated by glucose in human hepatocytes, and its I148M mutant slows down triglyceride hydrolysis.

    PubMed

    Perttilä, Julia; Huaman-Samanez, Carolina; Caron, Sandrine; Tanhuanpää, Kimmo; Staels, Bart; Yki-Järvinen, Hannele; Olkkonen, Vesa M

    2012-05-15

    Liver fat is increased in carriers of the minor G allele in rs738409 (I148M amino acid substitution) in patatin-like phospholipase domain-containing 3 (PNPLA3)/adiponutrin. We studied transcriptional regulation of PNPLA3 in immortalized human hepatocytes (IHH) and human hepatoma cells (HuH7) and the impact of PNPLA3 I148M mutant on hepatocyte triglyceride metabolism. Studies in IHH showed that silencing of the carbohydrate response element-binding protein (ChREBP) abolished induction of PNPLA3 mRNA by glucose. Glucose-dependent binding of ChREBP to a newly identified carbohydrate response element in the PNPLA3 promoter was demonstrated by chromatin immunoprecipitation. Adenoviral overexpression of mouse ChREBP in IHH failed to induce PNPLA3 mRNA. [(3)H]acetate or [(3)H]oleate incorporation with 1-h pulse labeling or 18-h [(3)H]oleate labeling in HuH7 cells showed no effect of PNPLA3 I148M on triglyceride (TG) synthesis in the absence of free fatty acid (FFA) loading. Increased [(3)H]oleate accumulation into triglycerides in I148M-expressing cells was observed after 18 h of labeling in the presence of 200 μM FFA-albumin complexes. This was accompanied by increased PNPLA3 protein levels. The rate of hydrolysis of [(3)H]TG during lipid depletion was decreased significantly by PNPLA3 I148M. Our results suggest that PNPLA3 is regulated in human hepatocytes by glucose via ChREBP. PNPLA3 I148M enhances cellular accumulation of [(3)H]TG in the presence of excess FFA, which is known to stabilize PNPLA3 protein. These data do not exclude an effect of PNPLA3 I148M on hepatocyte lipogenesis but show that the mutant increases the stability of triglycerides.

  9. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    PubMed

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

  10. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    PubMed

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  11. Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations.

    PubMed

    Neis, J M; Yap, S H; van Gemert, P J; Roelofs, H M; Bos, R P; Henderson, P T

    1986-02-01

    The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.

  12. Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity.

    PubMed

    Yan, Haoheng; Endo, Yukinori; Shen, Yi; Rotstein, David; Dokmanovic, Milos; Mohan, Nishant; Mukhopadhyay, Partha; Gao, Bin; Pacher, Pal; Wu, Wen Jin

    2016-03-01

    Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs. PMID:26712117

  13. The human beta fibrinogen promoter contains a hepatocyte nuclear factor 1-dependent interleukin-6-responsive element.

    PubMed Central

    Dalmon, J; Laurent, M; Courtois, G

    1993-01-01

    Acute-phase reactants are liver proteins whose synthesis is positively or negatively regulated during inflammation. The main mediators of this phenomenon are glucocorticoids and interleukin-6 (IL-6), a pleiotropic cytokine that also controls hematopoiesis. Functional analysis of several acute-phase reactant promoter regions has identified two major DNA motifs used by IL-6-regulated genes. The first one corresponds to a CTGG(G/A)AA sequence, and the other is a binding site for members of the C/EBP family of nuclear proteins. We have previously shown that the human beta fibrinogen (beta Fg) promoter contains an IL-6-responsive region, located between bp -150 and -67 (P. Huber, M. Laurent, and J. Dalmon, J. Biol. Chem. 265:5695-5701, 1990). In this study, using DNase I footprinting, mobility shift assays, and mutagenesis, we demonstrate that at least three subdomains of this region are necessary to observe a full response to IL-6. The most distal contains a CTGGGAA motif, and its mutation inhibits IL-6 stimulation. Another, which is able to interact with several distinct nuclear proteins, among them members of the C/EBP family, is dispensable for IL-6 induction but plays an important role in the constitutive expression of beta Fg. Finally, a proximal hepatocyte nuclear factor 1 binding site, already described as the major determinant of beta Fg tissue-specific expression, is also required for IL-6 stimulation. These results indicate a complex interplay between nuclear proteins within the beta Fg IL-6-responsive region and suggest a tight functional coupling between the tissue-specific and inducible elements. Images PMID:8423785

  14. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes

    PubMed Central

    Resseguie, Mary; Song, Jiannan; Niculescu, Mihai D.; da Costa, Kerry-Ann; Randall, Thomas A.; Zeisel, Steven H.

    2008-01-01

    Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-β-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) ∼7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.—Resseguie, M., Song, J., Niculescu, M. D., da Costa, K., Randall, T. A., Zeisel, S. H. Phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is induced by estrogen in human and mouse primary hepatocytes. PMID:17456783

  15. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

    PubMed Central

    Bell, Catherine C.; Hendriks, Delilah F. G.; Moro, Sabrina M. L.; Ellis, Ewa; Walsh, Joanne; Renblom, Anna; Fredriksson Puigvert, Lisa; Dankers, Anita C. A.; Jacobs, Frank; Snoeys, Jan; Sison-Young, Rowena L.; Jenkins, Rosalind E.; Nordling, Åsa; Mkrtchian, Souren; Park, B. Kevin; Kitteringham, Neil R.; Goldring, Christopher E. P.; Lauschke, Volker M.; Ingelman-Sundberg, Magnus

    2016-01-01

    Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI. PMID:27143246

  16. [Multipotency of adult stem cells derived from human amnion].

    PubMed

    Shi, Mingxia; Li, Weijia; Li, Bingzong; Li, Jing; Zhao, Chunhua

    2009-05-01

    Adult stem cells are drawing more and more attention due to the potential application in degenerative medicine without posing any moral problem. There is growing evidence showing that the human amnion contains various types of adult stem cell. Since amniotic tissue is readily available, it has the potential to be an important source of regenerative medicine material. In this study we tried to find multipotent adult stem cells in human amnion. We isolated stem cells from amniotic mesenchymal cells by limiting dilution assay. Similar to bone marrow derived mesenchymal stem cells, these cells displayed a fibroblast like appearance. They were positive for CD105, CD29, CD44, negative for haematopoietic (GlyA, CD31, CD34, CD45) and epithelial cell (pan-CK) markers. These stem cells had the potential to differentiate not only into osteogenic, adipogenic and endothelial lineages, but also hepatocyte-like cells and neural cells at the single-cell level depending on the culture conditions. They had the capacity for self-renewal and multilineage differentiation even after being expanded for more than 30 population doublings in vitro. So they may be an ideal stem cell source for inherited or degenerative diseases treatment.

  17. Expression of human hepatic lipase negatively impacts apolipoprotein A-I production in primary hepatocytes from Lipc-null mice.

    PubMed

    Bamji-Mirza, Michelle; Zhang, Wandong; Yao, Zemin

    2014-05-01

    This study aimed to examine whether expression of human hepatic lipase (hHL) exerted an intracellular effect on hepatic production of apolipoprotein (apo) A-I. The levels of secreted and cell-associated apoA-I were contrasted between primary hepatocytes isolated from Lipc-null and C57BL/6 mice, and between Lipc-null hepatocytes transfected with either hHL-encoding or control adenovirus. An HSPG-binding deficient hHL protein (hHLmt) was used to determine the impact of cell surface binding on HL action. Accumulation of apoA-I in conditioned media of primary hepatocytes isolated from Lipc-null mice was increased as compared to that from C57BL/6 mice. Metabolic labeling experiments showed that secretion of (35)S-apoA-I from Lipc-null cells was significantly higher than that from C57BL/6 cells. Expression of hHL in Lipc-null hepatocytes, through adenovirus-mediated gene transfer, resulted in decreased synthesis and secretion of (35)S-apoA-I, but not (35)S-apoE, as compared with cells transfected with control adenovirus. Expression of HSPG-binding deficient hHLmt in Lipc-null cells also exerted an inhibitory effect on apoA-I production, even though hHLmt displayed impaired exit from the endoplasmic reticulum as compared with hHL. Subcellular fractionation revealed that expression of hHL or hHLmt led to increased microsome-association of apoA-I relative to non-transfected control. Expression of hHL negatively impacts hepatic production of apoA-I.

  18. Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

    PubMed Central

    Sankhyan, Anurag; Sharma, Chandresh; Dutta, Durgashree; Sharma, Tarang; Chosdol, Kunzang; Wakita, Takaji; Watashi, Koichi; Awasthi, Amit; Acharya, Subrat K.; Khanna, Navin; Tiwari, Ashutosh; Sinha, Subrata

    2016-01-01

    Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants. PMID:26888694

  19. A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes

    PubMed Central

    1991-01-01

    We have identified the liver-regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte- and RLEC-iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction-mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver-specific and house-keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell-cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes. PMID:1918151

  20. Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells

    PubMed Central

    Li, Linhao; Li, Daochuan; Heyward, Scott; Wang, Hongbing

    2016-01-01

    CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population. PMID:26930610

  1. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    SciTech Connect

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  2. Water-Stable Metal-Organic Framework/Polymer Composites Compatible with Human Hepatocytes.

    PubMed

    Neufeld, Megan J; Ware, Brenton R; Lutzke, Alec; Khetani, Salman R; Reynolds, Melissa M

    2016-08-01

    Metal-organic frameworks (MOFs) have demonstrated promise in biomedical applications as vehicles for drug delivery, as well as for the ability of copper-based MOFs to generate nitric oxide (NO) from endogenous S-nitrosothiols (RSNOs). Because NO is a participant in biological processes where it exhibits anti-inflammatory, antibacterial, and antiplatelet activation properties, it has received significant attention for therapeutic purposes. Previous work has shown that the water-stable MOF H3[(Cu4Cl)3-(BTTri)8] (H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), or CuBTTri, produces NO from RSNOs and can be included within a polymeric matrix to form NO-generating materials. While such materials demonstrate potential, the possibility of MOF degradation leading to copper-related toxicity is a concern that must be addressed prior to adapting these materials for biomedical applications. Herein, we present the first cytotoxicity evaluation of an NO-generating CuBTTri/polymer composite material using 3T3-J2 murine embryonic fibroblasts and primary human hepatocytes (PHHs). CuBTTri/polymer films were prepared from plasticized poly(vinyl chloride) (PVC) and characterized via PXRD, ATR-FTIR, and SEM-EDX. Additionally, the ability of the CuBTTri/polymer films to enhance NO generation from S-nitroso-N-acetylpenicillamine (SNAP) was evaluated. Enhanced NO generation in the presence of the CuBTTri/polymer films was observed, with an average NO flux (0.90 ± 0.13 nmol cm(-2) min(-1)) within the range associated with antithrombogenic surfaces. The CuBTTri/polymer films were analyzed for stability in phosphate buffered saline (PBS) and cell culture media under physiological conditions for a 4 week duration. Cumulative copper release in both cell media (0.84 ± 0.21%) and PBS (0.18 ± 0.01%) accounted for less than 1% of theoretical copper present in the films. In vitro cell studies performed with 3T3-J2 fibroblasts and PHHs did not indicate significant toxicity, providing further

  3. Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.

    PubMed

    Lassila, Toni; Rousu, Timo; Mattila, Sampo; Chesné, Christophe; Pelkonen, Olavi; Turpeinen, Miia; Tolonen, Ari

    2015-11-10

    The objective was to compare several in vitro human liver-derived subcellular and cellular incubation systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human liver microsomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glutathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using only NADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally different compounds were used as a test set, chosen as either "positive" (ciprofloxacin, clozapine, diclofenac, ethinyl estradiol, pulegone, and ticlopidine) or "negative" (caffeine, citalopram, losartan, montelukast) compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates were observed for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram, or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relative abundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSH conjugates were different for many compounds in cellular subfractions and cell-based systems. Hepatocytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differences were minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactive metabolites do have a high impact on the results, and results between different systems are comparable only qualitatively. PMID:26263063

  4. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.

    PubMed

    Shi, Xiao-Lei; Gao, Yimeng; Yan, Yupeng; Ma, Hucheng; Sun, Lulu; Huang, Pengyu; Ni, Xuan; Zhang, Ludi; Zhao, Xin; Ren, Haozhen; Hu, Dan; Zhou, Yan; Tian, Feng; Ji, Yuan; Cheng, Xin; Pan, Guoyu; Ding, Yi-Tao; Hui, Lijian

    2016-02-01

    Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.

  5. Metabolism of RCS-8, a synthetic cannabinoid with cyclohexyl structure, in human hepatocytes by high-resolution MS

    PubMed Central

    Wohlfarth, Ariane; Pang, Shaokun; Zhu, Mingshe; Gandhi, Adarsh S; Scheidweiler, Karl B; Huestis, Marilyn A

    2015-01-01

    Background Since 2008, synthetic cannabinoids are major new designer drugs of abuse. They are extensively metabolized and excreted in urine, but limited human metabolism data are available. As there are no reports on the metabolism of RCS-8, a scheduled phenylacetylindole synthetic cannabinoid with an N-cyclohexylethyl moiety, we investigated metabolism of this new designer drug by human hepatocytes and high resolution MS. Methods After human hepatocyte incubation with RCS-8, samples were analyzed on a TripleTOF 5600+ mass spectrometer with time-of-flight survey scan and information-dependent acquisition triggered product ion scans. Data mining of the accurate mass full scan and product ion spectra employed different data processing algorithms. Results and Conclusion More than 20 RCS-8 metabolites were identified, products of oxidation, demethylation, and glucuronidation. Major metabolites and targets for analytical methods were hydroxyphenyl RCS - 8 glucuronide, a variety of hydroxycyclohexyl-hydroxyphenyl RCS-8 glucuronides, hydroxyphenyl RCS-8, as well as the demethyl-hydroxycyclohexyl RCS-8 glucuronide. PMID:24946920

  6. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.

    PubMed

    Shi, Xiao-Lei; Gao, Yimeng; Yan, Yupeng; Ma, Hucheng; Sun, Lulu; Huang, Pengyu; Ni, Xuan; Zhang, Ludi; Zhao, Xin; Ren, Haozhen; Hu, Dan; Zhou, Yan; Tian, Feng; Ji, Yuan; Cheng, Xin; Pan, Guoyu; Ding, Yi-Tao; Hui, Lijian

    2016-02-01

    Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system. PMID:26768767

  7. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

    PubMed Central

    Shi, Xiao-Lei; Gao, Yimeng; Yan, Yupeng; Ma, Hucheng; Sun, Lulu; Huang, Pengyu; Ni, Xuan; Zhang, Ludi; Zhao, Xin; Ren, Haozhen; Hu, Dan; Zhou, Yan; Tian, Feng; Ji, Yuan; Cheng, Xin; Pan, Guoyu; Ding, Yi-Tao; Hui, Lijian

    2016-01-01

    Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system. PMID:26768767

  8. Long-term culture of genome-stable bipotent stem cells from adult human liver.

    PubMed

    Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M A; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N M; Nieuwenhuis, Edward E S; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R G; van der Laan, Luc J W; Cuppen, Edwin; Clevers, Hans

    2015-01-15

    Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

  9. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

    PubMed Central

    Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

    2015-01-01

    Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

  10. Biotransformation of three phosphate flame retardants and plasticizers in primary human hepatocytes: untargeted metabolite screening and quantitative assessment.

    PubMed

    Van den Eede, Nele; de Meester, Ingrid; Maho, Walid; Neels, Hugo; Covaci, Adrian

    2016-11-01

    Tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP) and tris(1-chloro-2-propyl) phosphate (TCIPP) are current high-volume organophosphate flame retardants/plasticizers (PFRs) and are abundant in the indoor environment. While recent in vitro research has indicated potential toxic effects in the endocrine system, biotransformation of these compounds is still underexplored. In this study, we aimed to characterize the metabolite formation for three PFRs in primary human hepatocytes, an in vitro system that mimics in vivo liver metabolism more closely than hepatic subcellular fractions or cell lines. Cryopreserved human hepatocytes were thawed and suspended in media with 50 μm TBOEP or TCIPP, or 20 μm TPHP up to 2 h. Extracts were analyzed by liquid chromatography-quadrupole-time-of-flight-mass spectrometry. Quantification of biotransformation products in hepatocytes exposed for 2 h revealed that bis(1-chloro-2-propyl) phosphate and diphenyl phosphate corresponded to less than half of the depletion of TCIPP and TPHP, respectively, while bis(2-butoxyethyl) 2-hydroxyethyl phosphate compared to 40-66% of the depletion of TBOEP. Other metabolite structures of these PFRs were produced at 4- to 10-fold lower rates. These findings help interpret biological levels of the major metabolites and relate it to levels of their parent PFR. Percentage of substrate depletion was largest for TBOEP followed by comparable values for TPHP and TCIPP, indicating that hepatic clearance of TPHP and TCIPP would be slower than that of TBOEP. The resulting higher levels and longer presence of TPHP in the circulation after exposure, would allow TPHP a larger time window to exert its suspected adverse effects compared to TBOEP. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Inhibition of MDR3 Activity in Human Hepatocytes by Drugs Associated with Liver Injury.

    PubMed

    He, Kan; Cai, Lining; Shi, Qin; Liu, Hao; Woolf, Thomas F

    2015-10-19

    MDR3 dysfunction is associated with liver diseases. We report here a novel MDR3 activity assay involving in situ biosynthesis in primary hepatocytes of deuterium (d9)-labeled PC and LC-MS/MS determination of transported extracellular PC-d9. Several drugs associated with DILI such as chlorpromazine, imipramine, itraconazole, haloperidol, ketoconazole, sequinavir, clotrimazole, ritonavir, and troglitazone inhibit MDR3 activity. MDR3 inhibition may play an important role in drug-induced cholestasis and vanishing bile duct syndrome. Several lines of evidence demonstrate that the reported assay is physiologically relevant and can be used to assess the potential of chemical entities and their metabolites to modulate MDR3 activity and/or PC biosynthesis in hepatocytes. PMID:26335978

  12. Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

    PubMed

    Soldatow, Valerie; Peffer, Richard C; Trask, O Joseph; Cowie, David E; Andersen, Melvin E; LeCluyse, Edward; Deisenroth, Chad

    2016-10-01

    Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

  13. Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

    PubMed

    Soldatow, Valerie; Peffer, Richard C; Trask, O Joseph; Cowie, David E; Andersen, Melvin E; LeCluyse, Edward; Deisenroth, Chad

    2016-10-01

    Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans. PMID:27530964

  14. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  15. Spongy polyethersulfone membrane for hepatocyte cultivation: studies on human hepatoma C3A cells.

    PubMed

    Kinasiewicz, Andrzej; Smietanka, Anna; Dudziński, Konrad; Chwojnowski, Andrzej; Gajkowska, Barbara; Weryński, Andrzej

    2008-09-01

    There are different types of membranes used for hepatocyte cultivation. In our studies, spongy polyethersulfone (PES) membranes were examined as a support for hepatic cell cultivation in vitro. The extended surface of the membranes allows to introduce a high cell number especially in three-dimensional gel structure. Scanning electron microscopy analysis indicated that C3A cells used in our experiments grew well on PES membranes forming microvilli characteristic for normal hepatocytes. Analysis of cell viability proved that spongy PES membrane is well tolerated by J774 macrophages and did not stimulate nitric oxide synthesis. Bile canalicular structures were observed in fluorescence microscopy after F-actin staining with tetramethyl rhodamine iso-thiocyanate (TRITC)-phalloidin. The C3A cells showed high affinity to the PES membranes and adhered to almost 90% during the initial 24 h of incubation. Albumin production increased during static culture from the value of 805.2 +/- 284.4 (ng/24 h/initial 10(6) cells) during the first days, to 2017.6 +/- 505.9 (ng/24 h/initial 10(6) cells) after 10 days of culture. In conclusion, the spongy PES membranes can be used as scaffold for hepatocyte cultivation, especially for the creation of three-dimensional environments.

  16. Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes.

    PubMed Central

    Sanderson, J T; Letcher, R J; Heneweer, M; Giesy, J P; van den Berg, M

    2001-01-01

    We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro. PMID:11675267

  17. Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

    SciTech Connect

    Bumpus, Namandje N.

    2011-12-15

    Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibition of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death. -- Highlights: Black-Right-Pointing-Pointer 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. Black-Right-Pointing-Pointer Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. Black-Right-Pointing-Pointer JNK and Bim are required for efavirenz- and 8

  18. First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry.

    PubMed

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Liu, Hua-Fen; Huestis, Marilyn A

    2013-10-01

    Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3-carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from March 2010 to January 2013. In May 2013, the Drug Enforcement Administration listed AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB1 receptor binding affinity than CB2. These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono- and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

  19. Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

    PubMed Central

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

    2014-01-01

    Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

  20. Proteomic mapping of bezafibrate-treated human hepatocytes in primary culture using two-dimensional liquid chromatography.

    PubMed

    Alvergnas, M; Rouleau, A; Lucchi, G; Heyd, B; Ducoroy, P; Richert, L; Martin, H

    2011-03-01

    Peroxisome proliferators have been extensively studied in rodents and are known to induce liver tumors, whereas the effects of these compounds are not very clearly identified in humans when they are widely exposed to herbicides, plasticizers, solvents or drugs such as the lipid-lowering fibrate bezafibrate (BEZA). We assessed the effect of BEZA on human hepatocyte proteome. Hepatocyte proteins, including those membrane-associated, were successfully extracted and separated using 2D-liquid chromatography (PF2D, Beckman coulter). Proteins that were regulated by ≥ 1.5 fold compared to controls were identified by mass spectrometry (MALDI-TOF, Bruker Daltonics) and SwissProt bank search. BEZA modified the expression of proteins involved in various metabolic pathways as well as in cell homeostasis. No marker of peroxisome proliferation was obtained but surprisingly the expression of proteins involved in liver carcinogenicity was modulated. The co-treatment of cultures with N-acetylcysteine modified the set of proteins regulated by BEZA, either by a potentiation or an inhibition of the effects. Our study points out that the hepatocellular redox environment has to be taken into account when using fibrates in therapeutics.

  1. Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4α.

    PubMed

    Hu, Xiaojun; Xie, Peiyi; Li, Weiqiang; Li, Zhengran; Shan, Hong

    2016-09-16

    Hepatocytes from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are expected to be a useful source for cell transplantation. However, relatively low efficiency and repeatability of hepatic differentiation of human BM-MSCs remains an obstacle for clinical translation. Hepatocyte nuclear factor 4 alpha (HNF4α), a critical transcription factor, plays an essential role in the entire process of liver development. In this study, immortalized hBM-MSCs, UE7T-13 cells were transduced with a lentiviral vector containing HNF4α. The typical fibroblast-like morphology of the MSCs changed, and polygonal, epithelioid cells grew out after HNF4α transduction. In hepatocyte culture medium, HNF4α-transduced MSCs (E7-hHNF4α cells) strongly expressed the albumin (ALB), CYP2B6, alpha-1 antitrypsin (AAT), and FOXA2 mRNA and exhibited morphology markedly similar to that of mature hepatocytes. The E7-hHNF4α cells showed hepatic functions such as Indocyanine green (ICG) uptake and release, glycogen storage, urea production and ALB secretion. Approximately 28% of E7-hHNF4α cells expressed both ALB and AAT. Furthermore, these E7-hHNF4α cells via superior mesenteric vein (SMV) injection expressed human ALB in mouse chronic injured liver. In conclusion, this study represents a novel strategy by directly inducing hepatocyte-like cells from MSCs. PMID:27501760

  2. Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

    PubMed

    Yee, C J; DeFrances, M C; Bell, A; Bowen, W; Petersen, B; Michalopoulos, G K; Zarnegar, R

    1993-08-10

    A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by

  3. Activation of the Nrf2 pathway by inorganic arsenic in human hepatocytes and the role of transcriptional repressor Bach1.

    PubMed

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals.

  4. Activation of the Nrf2 pathway by inorganic arsenic in human hepatocytes and the role of transcriptional repressor Bach1.

    PubMed

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals. PMID:23738048

  5. Hypoxia and reoxygenation of primary human hepatocytes induce proteome changes of glucose metabolism, oxidative protection and peroxisomal function.

    PubMed

    Strey, Christoph W; Gestrich, Johannes; Beckhaus, Tobias; Marquez-Pinilla, Rosa Maria; Oppermann, Elsie; Mönch, Christian; Lambris, John D; Karas, Michael; Bechstein, Wolf O

    2010-10-01

    Protective hepatocellular responses to a hypoxic challenge are crucial to preserve liver function. The knowledge of affected metabolic functions could help assess and enhance hepatic ischemic tolerance. Here we studied adaptive mechanisms in human hepatocytes after hypoxia and reoxygenation using a proteomic approach. Proteins from primary hepatocytes were extracted after 6 h of hypoxia and 24 h of reoxygenation. The proteome was analyzed by 2D-electrophoresis. Densitometry and mass spectrometry (MALDI-TOF-MS) were used for protein identification. Two hundred and sixty-two spots were differentially analyzed and 33 spots displayed significant differences between hypoxic and normoxic cells. Seventeen proteins were identified by mass spectrometry. After hypoxia and reoxygenation the UTP-glucose-1-phosphate uridyltransferase, phosphoglycerate kinase1, fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, thiosulfat-sulfurtransferase, thioredoxin peroxidase, peroxiredoxin III, and annexin A2 proteins were down-regulated. An increased expression was found for carbamoyl phosphate synthetase I, heat shock 70 kDa protein5, phosphoenolpyruvate carboxy-kinase, catalase isoform2, peroxiredoxin II, glutathione S-transferase, hydroxyacid oxidase1, and F1-ATP synthase, alpha subunit1. Hepatocellular adaptation to hypoxia and reoxygenation involve glucose metabolism, peroxisomal functions, and oxidative stress protection. The identified proteins can serve as possible diagnostic targets to monitor hepatic hypoxic tolerance e.g. in the context of liver surgery and transplantation.

  6. Activation of the Nrf2 Pathway by Inorganic Arsenic in Human Hepatocytes and the Role of Transcriptional Repressor Bach1

    PubMed Central

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals. PMID:23738048

  7. Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye

    SciTech Connect

    Pernelle, K.; Le Guevel, R.; Glaise, D.; Stasio, C. Gaucher-Di; Le Charpentier, T.; Bouaita, B.; Corlu, A.; Guguen-Guillouzo, C.

    2011-08-01

    In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment. - Highlights: > We define conditions to preserve differentiation of selective pure HepaRG hepatocyte cultures. > In these conditions, CYPs

  8. A putative role of micro RNA in regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes.

    PubMed

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Chiang, John Y L

    2010-08-01

    Cholesterol 7alpha-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3'-untranslated region (3'-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or fibroblast growth factor (FGF)19. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3'-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3'-UTR reporter plasmids, and mutations of miRNA binding sites in 3'-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF19 inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

  9. Human Embryonic Stem Cell Derived Hepatocyte-Like Cells as a Tool for In Vitro Hazard Assessment of Chemical Carcinogenicity

    PubMed Central

    Yildirimman, Reha; Brolén, Gabriella; Vilardell, Mireia; Eriksson, Gustav; Synnergren, Jane; Gmuender, Hans; Kamburov, Atanas; Ingelman-Sundberg, Magnus; Castell, José; Lahoz, Agustin; Kleinjans, Jos; van Delft, Joost; Björquist, Petter; Herwig, Ralf

    2011-01-01

    Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay. PMID:21873647

  10. Hepatocyte cell therapy in liver disease.

    PubMed

    Bartlett, David Christopher; Newsome, Philip N

    2015-01-01

    Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed. PMID:26212798

  11. Cellular and molecular analyses of hprt mutation in human hepatocyte L02 cells after exposure to carbon ions

    NASA Astrophysics Data System (ADS)

    Li, Qiang; He, Jing; Jin, Xiao-Dong; Gong, Li; Li, Sha

    Mutations play an important role in carcinogenesis. The quantitative evaluation of mutation induction by heavy charged particles helps us to delineate the risks of space radiation on astronauts, as well as the risks of heavy ions on patients during tumor therapy. Hprt mutation assay, which has been used as a biological dosimeter, is an ideal gene mutation test in mammalian cells in vitro. In order to provide basic data and evidence for the risk assessment of heavy ions, the relationships between hprt mutation induction and radiation dose in human hepatocyte L02 cells were investigated for highand low-LET carbon ions and X-rays. Moreover, the carbon ion induced hprt mutation spectrum was analyzed. In our study, human hepatocyte L02 cells were irradiated with carbon ions with LET of 30keV/µm and X-rays (0.2keV/µm), respectively. The survival fraction of L02 cells was measured by means of colony-forming assay. The mutation frequency was detected by measuring 6-thioguanine-resistant clones after 10 days of incubation at the presence of 15mg/L 6-TG. To obtain the mutation spectrum, 9 10 mutant cell clones at each dose were randomly selected from the 6-TG containing medium, and were further cultured and analyzed. The deletion patterns of the 9 exons of hprt gene were analyzed with multiplex polymerase chain reactions (multiplex PCR). Our results show that the number of mutants per 106 surviving cells increased with increasing the radiation dose for both the irradiations, and the mutation frequency increased up to 1Gy while reduced with increasing dose further. Partial deletion was the most dominant deletion pattern in the hprt mutant cells, and with the increase of dose, hprt genes tended to have more total deletions and less point deletions. It can be inferred that human hepatocyte L02 cells are more radiosensitive to high-LET carbon ions than to low-LET X-rays, and carbon ions are more effective in inducing hprt mutation in L02 cells. It has been also found that the

  12. Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes.

    PubMed

    Qin, Pu; Borges-Marcucci, Lisa A; Evans, Mark J; Harnish, Douglas C

    2005-08-01

    Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.

  13. Beta-adrenergic control of phosphatidylcholine synthesis by transmethylation in hepatocytes from juvenile, adult and adrenalectomized rats.

    PubMed Central

    Marin-Cao, D; Alvarez Chiva, V; Mato, J M

    1983-01-01

    Changes in isoprenaline-sensitive phospholipid methyltransferase were studied in hepatocytes isolated from juvenile, mature and adrenalectomized rats. Isoprenaline produced greater stimulation of cyclic AMP accumulation in juvenile and mature adrenalectomized rats than in mature animals. Similarly, isoprenaline stimulated phospholipid methyltransferase in juvenile and mature adrenalectomized rats but had no effect in mature animals. Isoprenaline-mediated activation of phospholipid methyltransferase in adrenalectomized rats was time- and dose-dependent. In hepatocytes isolated from adrenalectomized rats incubated with [Me-3H]methionine or [3H]-ethanolamine the addition of isoprenaline increased the amount of radioactivity incorporated into phosphatidylcholine. The activation by isoprenaline of phospholipid methyltransferase was abolished by the beta-blocker propranolol and by insulin. These results indicate that rat liver the occupation of functional beta-receptors causes a stimulation of phospholipid methylation. It is suggested that, as reported previously, cyclic AMP activates phospholipid methyltransferase. PMID:6320796

  14. Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

    PubMed

    Ikebuchi, Fumie; Oka, Kiyomasa; Mizuno, Shinya; Fukuta, Kazuhiro; Hayata, Daichika; Ohnishi, Hiroyuki; Nakamura, Toshikazu

    2013-06-01

    Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and β-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of β-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in β-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in β-chain) may be important for accurate c-Met assembly and signalling transduction.

  15. [Effect of microRNA on proliferation caused by mutant HBx in human hepatocytes].

    PubMed

    Fu, Xiao-yu; Tan, De-ming; Hou, Zhou-hua; Hu, Zhi-liang; Liu, Guo-zhen; Ouyang, Yi; Liu, Fei

    2012-08-01

    To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein, HBx, in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect. The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation. The differential miRNA expression profiles were determined by microarray analysis and confirmed by real-time PCR. Two miRNAs, miR-338-3p and miR-551b, that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure. The cell survival rate was analyzed by MTT assay, and cell cycles were assessed by flow cytometry. Expressions of cyclinD1, cyclinG1, and E2F1 were assessed by real-time PCR and Western blotting. Compared with the microarray miRNA profile of L02/pcDNA3.0 cells, six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells, while four miRNAs were up-regulated and 12 were down-regulated in the L02/HBx cells. The microarray results were consistent with real-time PCR results. Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P less than 0.001) and induced G0/G1 phase cycle arrest. According to MTT results: for L02/HBx-d382 cells, compared with lipofectamine or non-transfected (NC) controls, the t value of miR-338-3p was 10.402, 9.133 and the t value of miR-551b was 8.763, 7.403; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 9.105, 8.074 and the t value of miR-551b was 7.673, 7.52. According to flow cytometry results: for L02/HBx-d382 cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 12.173, 11.107 and the t value of miR-551b was 15.364, 13.377; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p

  16. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes.

    PubMed

    Im, Ilkyun; Jang, Mi-Jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-12-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD(+)/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

  17. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  18. Quantitative Nuclease Protection Assays (qNPA) as Windows into Chemical-Induced Adaptive Response in Cultures of Primary Human Hepatocytes (Concentration and Time-Response)

    EPA Science Inventory

    Cultures of primary human hepatocytes have been shown to be dynamic in vitro model systems that retain liver-like functionality (e.g. metabolism, transport, induction). We have utilized these culture models to interrogate 309 ToxCast chemicals. The study design characterized both...

  19. Environmental pollutants parathion, paraquat and bisphenol A show distinct effects towards nuclear receptors-mediated induction of xenobiotics-metabolizing cytochromes P450 in human hepatocytes.

    PubMed

    Vrzal, Radim; Zenata, Ondrej; Doricakova, Aneta; Dvorak, Zdenek

    2015-10-01

    Environmental pollutants parathion, bisphenol A and paraquat were not systematically studied towards the effects on the expression of phase I xenobiotics-metabolizing cytochromes P450 (CYPs). We monitored their effects on the expression of selected CYPs in primary cultures of human hepatocytes. Moreover, we investigated their effects on the receptors regulating these CYPs, particularly arylhydrocarbon receptor (AhR), pregnane X receptor (PXR) and glucocorticoid receptor (GR) by gene reporter assays. We found that parathion and bisphenol A are the activators of AhR. Moreover, they are the inducers of CYP1A1 mRNA in hepatoma cells HepG2 as well as in human hepatocytes by AhR-dependent mechanism via formation of AhR-DNA-binding complex, as revealed by gel shift assay. All three compounds possessed anti-glucocorticoid action as revealed by GR-dependent gene reporter assay and a decline in tyrosine aminotransferase (TAT) gene expression in human hepatocytes. Moreover, parathion and bisphenol A are the activators of PXR and inducers of CYP3A4 mRNA and protein in the primary cultures of human hepatocytes. In conclusion, the studied compounds displayed distinct activities towards nuclear receptors involved in many biological processes and these findings may help us to better understand their adverse actions in pathological states followed after their exposure. PMID:26196221

  20. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: potential for gene therapy of hemophilia B.

    PubMed

    Armentano, D; Thompson, A R; Darlington, G; Woo, S L

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. We report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently gamma-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  1. Rifampin Regulation of Drug Transporters Gene Expression and the Association of MicroRNAs in Human Hepatocytes

    PubMed Central

    Benson, Eric A.; Eadon, Michael T.; Desta, Zeruesenay; Liu, Yunlong; Lin, Hai; Burgess, Kimberly S.; Segar, Matthew W.; Gaedigk, Andrea; Skaar, Todd C.

    2016-01-01

    Membrane drug transporters contribute to the disposition of many drugs. In human liver, drug transport is controlled by two main superfamilies of transporters, the solute carrier transporters (SLC) and the ATP Binding Cassette transporters (ABC). Altered expression of these transporters due to drug-drug interactions can contribute to differences in drug exposure and possibly effect. In this study, we determined the effect of rifampin on gene expression of hundreds of membrane transporters along with all clinically relevant drug transporters. Methods: In this study, primary human hepatocytes (n = 7 donors) were cultured and treated for 24 h with rifampin and vehicle control. RNA was isolated from the hepatocytes, mRNA expression was measured by RNA-seq, and miRNA expression was analyzed by Taqman OpenArray. The effect of rifampin on the expression of selected transporters was also tested in kidney cell lines. The impact of rifampin on the expression of 410 transporter genes from 19 different transporter gene families was compared with vehicle control. Results: Expression patterns of 12 clinically relevant drug transporter genes were changed by rifampin (FDR < 0.05). For example, the expressions of ABCC2, ABCB1, and ABCC3 were increased 1.9-, 1.7-, and 1.2-fold, respectively. The effects of rifampin on four uptake drug transporters (SLCO1B3, SLC47A1, SLC29A1, SLC22A9) were negatively correlated with the rifampin effects on specific microRNA expression (SLCO1B3/miR-92a, SLC47A1/miR-95, SLC29A1/miR-30d#, and SLC22A9/miR-20; r < −0.79; p < 0.05). Seven hepatic drug transporter genes (SLC22A1, SLC22A5, SLC15A1, SLC29A1, SLCO4C1, ABCC2, and ABCC4), whose expression was altered by rifampin in hepatocytes, were also present in a renal proximal tubular cell line, but in renal cells rifampin did not alter their gene expression. PXR expression was very low in the kidney cells; this may explain why rifampin induces gene expression in a tissue-specific manner. Conclusion

  2. Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

    PubMed

    Terelius, Ylva; Figler, Robert A; Marukian, Svetlana; Collado, Maria S; Lawson, Mark J; Mackey, Aaron J; Manka, David; Qualls, Charles W; Blackman, Brett R; Wamhoff, Brian R; Dash, Ajit

    2016-08-01

    Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain

  3. HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology

    PubMed Central

    Gille, Christoph; Bölling, Christian; Hoppe, Andreas; Bulik, Sascha; Hoffmann, Sabrina; Hübner, Katrin; Karlstädt, Anja; Ganeshan, Ramanan; König, Matthias; Rother, Kristian; Weidlich, Michael; Behre, Jörn; Holzhütter, Herrmann-Georg

    2010-01-01

    We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds. PMID:20823849

  4. HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology.

    PubMed

    Gille, Christoph; Bölling, Christian; Hoppe, Andreas; Bulik, Sascha; Hoffmann, Sabrina; Hübner, Katrin; Karlstädt, Anja; Ganeshan, Ramanan; König, Matthias; Rother, Kristian; Weidlich, Michael; Behre, Jörn; Holzhütter, Herrmann-Georg

    2010-09-01

    We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds. PMID:20823849

  5. Absence of oncogenic transformation despite acquisition of cytogenetic aberrations in long-term cultured telomerase-immortalized human fetal hepatocytes.

    PubMed

    Haker, Björn; Fuchs, Sigrid; Dierlamm, Judith; Brümmendorf, Tim H; Wege, Henning

    2007-10-18

    As a culture model to study hepatocarcinogenesis, telomerase-immortalized human fetal hepatocytes were monitored for karyotype changes evolving in long-term culture and development of functional defects in DNA damage response. G-banding revealed acquisition of characteristic karyotype abnormalities, e.g., trisomy 7 and monosomy X, in two independently immortalized and cultured populations after 80-100 population doublings. Interestingly, the detected aneuploidies resemble some of the genetic events observed in hepatocellular cancer. However, these genetic changes were not sufficient to induce oncogenic transformation reflected by absence of anchorage-independent growth. Furthermore, long-term cultured telomerase-immortalized cells preserved p53 expression levels and effective p53-mediated damage response. PMID:17630152

  6. Activation of the Constitutive Androstane Receptor Inhibits Gluconeogenesis without Affecting Lipogenesis or Fatty Acid Synthesis in Human Hepatocytes

    PubMed Central

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

    2014-01-01

    Objective Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods Ligand-based structure-activity models were used for virtual screening of the Specs database (www.specs.net) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. PMID:24878338

  7. Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds.

    PubMed

    Josse, Rozenn; Dumont, Julie; Fautrel, Alain; Robin, Marie-Anne; Guillouzo, André

    2012-01-15

    Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24h exposure of these hepatocyte models to 0.05 and 0.25μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. PMID:22100608

  8. An improvement in the attaching capability of cryopreserved human hepatocytes by a proteinaceous high molecule, sericin, in the serum-free solution.

    PubMed

    Miyamoto, Yoshitaka; Teramoto, Naozumi; Hayashi, Shuji; Enosawa, Shin

    2010-01-01

    The methodology of cryopreservation of human hepatocytes remains unsatisfactory. Even when the viability of thawed cells is tolerable, the cells often lose the attaching capability to a culture dish, resulting in the cells' inability to survive. Previously, we described the effectiveness of maltose on the attachment of hepatocytes. This article demonstrates that a silk-derived high molecular protein, sericin, improves the cell-attaching capability in the serum-free freezing medium. When human hepatocytes [initial viability: 60.9 ± 3.1% (mean ± SD, n = 3)] were frozen with serum-free Dulbecco's modified Eagle medium (DMEM) containing 10% dimethyl sulfoxide (DMSO), the viability was 29.4 ± 3.2% and the cell-attaching capability 20.4 ± 4.1%. On the other hand, DMEM containing 10% DMSO and 1% sericin increased the values to 45.0 ± 0.8% and 26.2 ± 3.2%. Moreover, the addition of 0.1 mol/L maltose to the sericin-containing medium improved to 42.2 ± 3.2% and 51.1 ± 1.0%, as we demonstrated in a previous report. The present results indicated that sericin combined with maltose is a novel additive in the serum-free freezing medium for human hepatocytes. PMID:20525438

  9. Effects of butylated hydroxytoluene pretreatment on the metabolism and genotoxicity of aflatoxin B1 in primary cultures of adult rat hepatocytes: selective reduction of nucleic acid binding.

    PubMed

    Salocks, C B; Hsieh, D P; Byard, J L

    1984-12-01

    To elucidate biochemical mechanisms underlying the anticarcinogenic activity of butylated hydroxytoluene (BHT), studies were undertaken to characterize the influence of BHT pretreatment on the metabolism and genotoxicity of aflatoxin B1 (AFB1) in primary cultures of rat hepatocytes. During a 10-day pretreatment period, adult male rats were fed either a control diet or a diet supplemented with 0.5% BHT. Hepatocytes were subsequently isolated from each animal and cultured in chemically defined medium. Cultures prepared from rats which had been fed BHT metabolized AFB1 more rapidly than did controls. BHT pretreatment also enhanced oxidation of AFB1 to aflatoxin M1 (AFM1), and accelerated the rate of AFM1 conjugation. Covalent binding to DNA and RNA in BHT-pretreated cultures was reduced by 91 and 82%, respectively, while protein binding decreased by only 29%. AFB1 did not stimulate detectable DNA repair synthesis in BHT-pretreated cultures, although stimulation of DNA repair was clearly evident in control cultures. In a separate experiment, consistently higher baseline concentrations of reduced glutathione were observed in BHT-pretreated cells, indicating that BHT pretreatment may enhance formation of detoxified glutathione conjugates of AFB1. These findings suggest that the anticarcinogenic activity of BHT is due in part to preferential enhancement of hepatic detoxification mechanisms, with the result that intracellular concentrations of reactive metabolites are reduced and fewer covalently bound adducts are formed.

  10. Inhibition of iron toxicity in human hepatocyte cultures by pyoverdins Pa A and Pf, the peptidic siderophores of Pseudomonas aeruginosa and fluorescens.

    PubMed

    Jégo, P; Chenoufi, N; Loréal, O; Morel, I; Pasdeloup, N; Abdallah, M A; Brissot, P; Lescoat, G

    1997-04-01

    The protective effect of pyoverdins Pa A and Pf, peptidic siderophores secreted respectively by Pseudomonas aeruginosa and fluorescens, was studied in primary cultures of human hepatocytes exposed to iron (50 or 100 microM of iron-citrate). AST, ALT and MDA releases were measured as indexes of cytotoxicity. In order to demonstrate that these chelators were able to decrease iron uptake or increase iron release from the hepatocytes, labelled cells were obtained by maintaining the cultures in the presence of 1 microM 55Fe ferric chloride plus 50 microM iron citrate. One day after iron treatment, an increase in AST, ALT and MDA release was observed with 50 or 100 microM of iron citrate; it appeared that the concentrations 50 and 100 microM of iron were highly toxic for human hepatocytes. In the presence of 50 or 100 microM of iron, the addition of 50 or 100 microM of Pa A or Pf was effective to inhibit the increase observed in the enzyme leakage and the MDA production resulting from iron exposure. In human hepatocytes cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate plus 50 or 100 microM Pa A or Pf, a net decrease of iron uptake by the cells was observed, as demonstrated by the low intracellular iron level. When the hepatocytes were cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate and then for a further day in the presence of 50 or 100 microM Pa A or Pf without additional iron, the chelators increased the extracellular iron level, indicating their iron release from the loaded cells; however, the effects of Pa A and Pf on iron release did not differ significantly. In conclusion, iron loading achieved by adding iron citrate to the culture medium is highly toxic for human hepatocytes. Pyoverdins Pa A and Pf are effective in protecting human hepatocytes against the toxic effect of iron by both decreasing the uptake of the metal and increasing its release from the loaded cells. PMID:9138275

  11. Metabolic pathways of 4-bromo-2,5-dimethoxyphenethylamine (2C-B): analysis of phase I metabolism with hepatocytes of six species including human.

    PubMed

    Carmo, Helena; Hengstler, Jan G; de Boer, Douwe; Ringel, Michael; Remião, Fernando; Carvalho, Félix; Fernandes, Eduarda; dos Reys, Lesseps A; Oesch, Franz; de Lourdes Bastos, Maria

    2005-01-01

    4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is a psychoactive designer drug of abuse that is sold under the street names "Venus", "Bromo", "Erox", "XTC" or "Nexus". Concern has been raised because only little is known about its toxicity and metabolism in humans. In the present study we incubated 2C-B with human, monkey, dog, rabbit, rat and mouse hepatocytes to identify the metabolites formed and to determine possible toxic effects as evidenced by an ATP assay. Our data allow construction of the main metabolic pathways of 2C-B. Oxidative deamination results in the 2-(4-bromo-2,5-dimethoxyphenyl)-ethanol (BDMPE) and 4-bromo-2,5-dimethoxyphenylacetic acid (BDMPAA) metabolites. Additionally, 4-bromo-2,5-dimethoxybenzoic acid (BDMBA) can be produced also by oxidative deamination. Further metabolism of BDMPE and BDMPAA may occur by demethylation. Alternatively, the later metabolites can be generated by demethylation of 2C-B followed by oxidative deamination. Two remarkable interspecies differences in metabolism of 2C-B were observed (i) a hitherto unknown metabolite, 4-bromo-2,5-dimethoxy-phenol (BDMP), was identified after incubation only with mouse hepatocytes; (ii) 2-(4-bromo-2-hydroxy-5-methoxyphenyl)-ethanol (B-2-HMPE) was produced by hepatocytes from human, monkey and rabbit but not by dog, rat and mouse. Comparing the toxic effects of 2C-B between hepatocytes of the six examined species we observed only minor interspecies differences. However, large inter-individual differences in susceptibility of hepatocytes from three human donors were observed. PMID:15590110

  12. Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

    PubMed Central

    Friborg, Jacques; Ross-Macdonald, Petra; Cao, Jian; Willard, Ryan; Lin, Baiqing; Eggers, Betsy; McPhee, Fiona

    2015-01-01

    Peginterferon lambda-1a (Lambda), a type III interferon (IFN), acts through a unique receptor complex with limited cellular expression outside the liver which may result in a differentiated tolerability profile compared to peginterferon alfa (alfa). In Phase 2b clinical studies, Lambda administered in combination with ribavirin (RBV) was efficacious in patients with hepatitis C virus (HCV) infection representing genotypes 1 through 4, and was associated with more rapid declines in HCV RNA compared to alfa plus RBV. To gain insights into potential mechanisms for this finding, we investigated the effects of HCV replication on IFN signaling in primary human hepatocytes (PHH) and in induced hepatocyte-like cells (iHLCs). HCV infection resulted in rapid down-regulation of the type I IFN-α receptor subunit 1 (IFNAR1) transcript in hepatocytes while the transcriptional level of the unique IFN-λ receptor subunit IL28RA was transiently increased. In line with this observation, IFN signaling was selectively impaired in infected cells upon stimulation with alfa but not in response to Lambda. Importantly, in contrast to alfa, Lambda was able to induce IFN-stimulated gene (ISG) expression in HCV-infected hepatocytes, reflecting the onset of innate responses. Moreover, global transcriptome analysis in hepatocytes indicated that Lambda stimulation prolonged the expression of various ISGs that are potentially beneficial to antiviral defense mechanisms. Collectively, these observed effects of HCV infection on IFN receptor expression and signaling within infected hepatocytes provide a possible explanation for the more pronounced early virologic responses observed in patients treated with Lambda compared to alfa. PMID:25826356

  13. Human recombinant vascular endothelial growth factor reduces necrosis and enhances hepatocyte regeneration in a mouse model of acetaminophen toxicity.

    PubMed

    Donahower, Brian C; McCullough, Sandra S; Hennings, Leah; Simpson, Pippa M; Stowe, Cindy D; Saad, Ali G; Kurten, Richard C; Hinson, Jack A; James, Laura P

    2010-07-01

    We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 microg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p < 0.05) compared with the APAP/vehicle mice. Hepatic glutathione (GSH) and APAP protein adduct levels were comparable between the two groups of mice, with the exception that GSH was higher at 8 h in the hrVEGF-treated mice. Subsequently, mice received two doses (before and 10 h) or three doses (before and 10 and 24 h) of hrVEGF; alanine aminotransferase values and necrosis were reduced at 24 and 36 h, respectively, in the APAP/hrVEGF mice (p < 0.05) compared with the APAP/vehicle mice. Proliferating cell nuclear antigen expression was enhanced, and interleukin-6 expression was reduced in the mice that received hrVEGF (p < 0.05) compared with the APAP/vehicle mice. In addition, treatment with hrVEGF lowered plasma hyaluronic acid levels and neutrophil counts at 36 h. Cumulatively, the data show that treatment with hrVEGF reduced toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.

  14. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    PubMed

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective.

  15. Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

    SciTech Connect

    Hu Shunying; Chen Yundai; Li Libing; Chen Jinlong; Wu Bin; Zhou, Xiao; Zhi Guang; Li Qingfang; Wang Rongliang; Duan Haifeng; Guo Zikuan; Yang Yuefeng; Xiao Fengjun; Wang Hua; Wang Lisheng

    2009-12-01

    Purpose: Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. Methods and Materials: Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. Results: Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 +- 9.01 vs. 41.94 +- 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 +- 1303 vs. 1667 +- 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 +- 1084 vs. 1566 +- 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. Conclusion: Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

  16. Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

    PubMed Central

    Yanagi, Satoshi; Kato, Chika; Takashima, Ryokichi; Kobayashi, Eiji; Hagiwara, Keitaro; Ochiya, Takahiro

    2015-01-01

    Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of

  17. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes.

    PubMed

    Xie, Yuchao; McGill, Mitchell R; Du, Kuo; Dorko, Kenneth; Kumer, Sean C; Schmitt, Timothy M; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-12-01

    3'-Hydroxyacetanilide orN-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this,we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. PMID:26431796

  18. Berberine Inhibits Human Hepatoma Cell Invasion without Cytotoxicity in Healthy Hepatocytes

    PubMed Central

    Pan, Xuediao; Yang, Zhicheng; Zang, Linquan

    2011-01-01

    Conventional chemotherapy fails to cure metastatic hepatoma mainly due to its high hepatotoxicity. Many plant-derived agents have been accepted to effectively inhibit hepatoma cell invasion. However, the investigation that whether effectual plant-derived agents against invasive hepatoma cells exert unexpected cytotoxicity in healthy hepatocytes has been ignored. This study demonstrated that berberine exhibited significant cytotoxicity in HepG2 cells mainly through upregulation of reactive oxygen species (ROS) production but was ineffective in normal Chang liver cells. Berberine exerted anti-invasive effect on HepG2 cells through suppression of matrix metalloproteinase-9 (MMP-9) expression. Moreover, berberine could significantly inhibit the activity of PI3K-AKT and ERK pathways. Combination treatment of ERK pathway inhibitor PD98059 or AKT pathway inhibitor LY294002 and berberine could result in a synergistic reduction on MMP-9 expression along with an inhibition of cell invasion. Enhancement of ROS production by berberine had no influence on its suppressive effects on the activity of PI3K-AKT and ERK pathways, as well as MMP-9 expression and HepG2 cell invasion. In conclusion, our results suggest that berberine may be a potential alternative against invasive hepatoma cells through PI3K-AKT and ERK pathways-dependent downregulation of MMP-9 expression. This study also provides a previously neglected insight into the investigation of plant-derived agents-based therapy against tumor invasion with the consideration of damage to healthy cells. PMID:21738655

  19. Analysis of intracellular reducing levels in human hepatocytes on three-dimensional focusing microchip.

    PubMed

    Xu, Chunxiu; Cai, Longfei

    2014-02-01

    A novel three-dimensional hydrodynamic focusing microfluidic device integrated with high-throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6-tetramethyl-piperidine-1-oxyl-based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath-flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three-dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath-flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home-built laser-induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59-68 cells/min.

  20. Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes.

    PubMed

    Chen, Pei-Jen; Moore, Tanya; Nesnow, Stephen

    2008-09-01

    Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen that has adverse reproductive and developmental toxicities in experimental animals. The goal of this study was to investigate the cytotoxic responses of propiconazole and its metabolites to determine if metabolism of this agent differentially affected its cytotoxic activities in hepatic tumor cell lines and in primary hepatocytes. To this end the cytotoxic effects of propiconazole and five of its metabolites were examined in three hepatic cell types: The mouse hepatoma Hepa1c1c7 cell line, the human hepatoma HepG2 cell line, and primary cultures of mouse hepatocytes. We initially compared the responses of propiconazole exposure in both Hepa1c1c7 and HepG2 cell lines over a concentration range of 0-200 microM using two assay systems: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the neutral red assay. Concentration-related cytotoxic responses were evident in both cell lines using both endpoints with the MTT assay providing enhanced sensitivity. The relative cytotoxic effects of propiconazole and five propiconazole metabolites were further assessed by the MTT assay using Hepa1c1c7 and HepG2 tumor cell lines. The cell cultures were exposed to various concentrations of propiconazole and five of its metabolites over a range of 0-400 microM. Propiconazole was cytotoxic in both cell lines in a dose-dependent manner. All five metabolites were less cytotoxic in both cell lines compared to the parent compound. The most cytotoxic metabolites in Hepa1c1c7 and HepG2 cells among the five were 3-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-1-ol and 1-(2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)propan-2-ol. Propiconazole was cytotoxic in primary mouse hepatocytes; however

  1. Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5.

    PubMed

    Stiborová, Marie; Poljaková, Jitka; Martínková, Eva; Ulrichová, Jitka; Simánek, Vilím; Dvořák, Zdeněk; Frei, Eva

    2012-12-16

    Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine-DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b(5) on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine-DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b(5) increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N(2)-oxide. CYP3A4 and 1A1 in the presence of cytochrome b(5) are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine-DNA adduct 2. These ellipticine-DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC(50) value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

  2. Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

    PubMed

    Tateno, Chise; Yamamoto, Toshinobu; Utoh, Rie; Yamasaki, Chihiro; Ishida, Yuji; Myoken, Yuka; Oofusa, Ken; Okada, Miyoko; Tsutsui, Naohisa; Yoshizato, Katsutoshi

    2015-02-01

    Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of β- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

  3. Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

    PubMed

    Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

    2013-12-01

    Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1.

  4. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    SciTech Connect

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  5. Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes.

    PubMed

    Wasiak, Sylwia; Gilham, Dean; Tsujikawa, Laura M; Halliday, Christopher; Norek, Karen; Patel, Reena G; McLure, Kevin G; Young, Peter R; Gordon, Allan; Kulikowski, Ewelina; Johansson, Jan; Sweeney, Michael; Wong, Norman C

    2016-09-01

    Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208. PMID:27570805

  6. Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

    SciTech Connect

    Josse, Rozenn; Dumont, Julie; Fautrel, Alain; Robin, Marie-Anne; Guillouzo, André

    2012-01-15

    Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered by other

  7. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    PubMed

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  8. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

    PubMed

    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  9. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    PubMed Central

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  10. Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to ZnO nanoparticles

    NASA Astrophysics Data System (ADS)

    Guan, Rongfa; Kang, Tianshu; Lu, Fei; Zhang, Zhiguo; Shen, Haitao; Liu, Mingqi

    2012-10-01

    Traces of zinc oxide nanoparticles (ZnO NPs) used may be found in the liver and kidney. The aim of this study is to determine the optimal viability assay for using with ZnO NPs and to assess their toxicity to human hepatocyte (L02) and human embryonic kidney (HEK293) cells. Cellular morphology, mitochondrial function (MTT assay), and oxidative stress markers (malondialdehyde, glutathione (GSH) and superoxide dismutase (SOD)) were assessed under control and exposed to ZnO NPs conditions for 24 h. The results demonstrated that ZnO NPs lead to cellular morphological modifications, mitochondrial dysfunction, and cause reduction of SOD, depletion of GSH, and oxidative DNA damage. The exact mechanism behind ZnO NPs toxicity suggested that oxidative stress and lipid peroxidation played an important role in ZnO NPs-elicited cell membrane disruption, DNA damage, and subsequent cell death. Our preliminary data suggested that oxidative stress might contribute to ZnO NPs cytotoxicity.

  11. Evaluation of the Endothelin Receptor Antagonists Ambrisentan, Bosentan, Macitentan, and Sitaxsentan as Hepatobiliary Transporter Inhibitors and Substrates in Sandwich-Cultured Human Hepatocytes

    PubMed Central

    Lepist, Eve-Irene; Gillies, Hunter; Smith, William; Hao, Jia; Hubert, Cassandra; St. Claire, Robert L.; Brouwer, Kenneth R.; Ray, Adrian S.

    2014-01-01

    Background Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs). Methods Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. Results The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x). Conclusions Significant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in

  12. Predictions of cytochrome P450-mediated drug-drug interactions using cryopreserved human hepatocytes: comparison of plasma and protein-free media incubation conditions.

    PubMed

    Mao, Jialin; Mohutsky, Michael A; Harrelson, John P; Wrighton, Steven A; Hall, Stephen D

    2012-04-01

    Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC(50) that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1'-hydroxylation, diclofenac 4'-hydroxylation or (R)-bufuralol 1'-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, app) method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach. PMID:22228749

  13. Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

    2016-02-01

    Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

  14. Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro.

    PubMed

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E; Manuelpillai, Ursula

    2014-04-15

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  15. Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-beta, TNF-alpha and NK cells activity.

    PubMed

    Quaresma, Juarez A S; Barros, Vera L R S; Pagliari, Carla; Fernandes, Elaine R; Guedes, Fernanda; Takakura, Cleusa F H; Andrade, Heitor F; Vasconcelos, Pedro F C; Duarte, Maria I S

    2006-02-01

    Flavivirus infection as dengue and yellow fever persists as a terrible menace to pandemics, due to Aedes prevalence in the Americas. Yellow fever is characterized by hepatocyte damage, with steatosis, apoptosis and necrosis, mainly in the midzonal region of the liver, but the injury mechanism has not been studied at the light of recent knowledge, such as the advances in cell death mechanisms, inflammatory response and cytokine cell expression tools. We studied 53 human liver paraffin embedded blocks from patients who died with yellow fever, all with histological demonstration of higher prevalence of apoptosis over necrosis and mild disproportionate inflammatory response. Viral antigens were found most frequently in hepatocytes from the midzonal area than other lobule areas, as detected by specific immunohistochemistry. Infiltrating cell subpopulations showed mainly CD4+ T lymphocytes, with small numbers of CD8+ cytotoxic lymphocytes, CD20+ B lymphocytes, NKT+ cells and S100+ dendritic cells in the sites of inflammation, as compared to normal and leptospirosis liver blocks. Some cells expressed TNF-alpha and IFN-gamma, but a much more intense proportion of TGF-beta expressing cells were found, suggesting both a Th1 and Th3 patterns of immune response in yellow fever. Most affected hepatocyte presented apoptosis markers that appear at the cell death main pathway in this infection. Viral antigens, which production could interfere in hepatocyte biology, could induce the activation of apoptosis cascade, but TGF-beta was also an apoptosis promoter. Our finding supports the key effect of the yellow fever virus in hepatocyte injury, resulting in prevalence of apoptosis over necrosis, aside from a TGF-beta action induced by the inflammatory response.

  16. Hepatocyte-Like Cells Derived from Human Amniotic Epithelial Cells Can Be Encapsulated Without Loss of Viability or Function In Vitro

    PubMed Central

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E.

    2014-01-01

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  17. Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid.

    PubMed

    Lock, Edward A; Reed, Celia J; McMillan, Joellyn M; Oatis, John E; Schnellmann, Rick G

    2007-02-12

    The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3-3mM CH for 3 days or 0.03-3mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3mM/day for 10 days, while in another cytotoxicity was seen at 1mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313-331] supporting the view that glutathione derived

  18. Hydralazine as a selective probe inactivator of aldehyde oxidase in human hepatocytes: estimation of the contribution of aldehyde oxidase to metabolic clearance.

    PubMed

    Strelevitz, Timothy J; Orozco, Christine C; Obach, R Scott

    2012-07-01

    Aldehyde oxidase (AO) metabolism could lead to significant underestimation of clearance in prediction of human pharmacokinetics as well as unanticipated exposure to AO-generated metabolites, if not accounted for early in drug research. We report a method using cryopreserved human hepatocytes and the time-dependent AO inhibitor hydralazine (K(I) = 83 ± 27 μM, k(inact) = 0.063 ± 0.007 min(-1)), which estimates the contribution of AO metabolism relative to total hepatic clearance. Using zaleplon as a probe substrate and simultaneously monitoring the AO-catalyzed formation of oxozaleplon and the CYP3A-catalyzed formation of desethyzaleplon in the presence of a range of hydralazine concentrations, it was determined that >90% inhibition of the AO activity with minimal effect on the CYP3A activity could be achieved with 25 to 50 μM hydralazine. This method was used to estimate the fraction metabolized due to AO [f(m(AO))] for six compounds with clearance attributed to AO along with four other drugs not metabolized by AO. The f(m(AO)) values for the AO substrates ranged between 0.49 and 0.83. Differences in estimated f(m(AO)) between two batches of pooled human hepatocytes suggest that sensitivity to hydralazine varies slightly with hepatocyte preparations. Substrates with a CYP2D6 contribution to clearance were affected by hydralazine to a minor extent, because of weak inhibition of this enzyme. Overall, these findings demonstrate that hydralazine, at a concentration of 25 to 50 μM, can be used in human hepatocyte incubations to estimate the contribution of AO to the hepatic clearance of drugs and other compounds.

  19. Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

    PubMed

    Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

    2014-11-01

    High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.

  20. A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes

    PubMed Central

    Qian, Xi-Jing; Zhang, Xiao-Lian; Zhao, Ping; Jin, Yong-Sheng; Chen, Hai-Sheng; Xu, Qing-Qiang; Ren, Hao; Zhu, Shi-Ying; Tang, Hai-Lin; Zhu, Yong-Zhe; Qi, Zhong-Tian

    2016-01-01

    Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations. PMID:27252043

  1. A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes.

    PubMed

    Qian, Xi-Jing; Zhang, Xiao-Lian; Zhao, Ping; Jin, Yong-Sheng; Chen, Hai-Sheng; Xu, Qing-Qiang; Ren, Hao; Zhu, Shi-Ying; Tang, Hai-Lin; Zhu, Yong-Zhe; Qi, Zhong-Tian

    2016-01-01

    Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations. PMID:27252043

  2. High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro.

    PubMed

    Shan, Jing; Logan, David J; Root, David E; Carpenter, Anne E; Bhatia, Sangeeta N

    2016-10-01

    Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable systematic interrogation of liver biology in vitro. The platform is currently configured to enable genetic knockdown screens and includes an enzyme-linked immunosorbent assay-based functional assay to quantify albumin output as a surrogate marker for hepatocyte synthetic functions as well as an image-based viability assay that counts hepatocyte nuclei. Using this platform, we identified 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research. PMID:27650791

  3. High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro.

    PubMed

    Shan, Jing; Logan, David J; Root, David E; Carpenter, Anne E; Bhatia, Sangeeta N

    2016-10-01

    Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable systematic interrogation of liver biology in vitro. The platform is currently configured to enable genetic knockdown screens and includes an enzyme-linked immunosorbent assay-based functional assay to quantify albumin output as a surrogate marker for hepatocyte synthetic functions as well as an image-based viability assay that counts hepatocyte nuclei. Using this platform, we identified 12 gene products that may be important for hepatocyte viability and/or liver identity in vitro. These results represent important first steps in the elucidation of mechanisms instrumental to the phenotypic maintenance of hepatocytes in vitro, and we hope that the tools reported here will empower additional studies in various fields of liver research.

  4. Improvement of hepatocyte viability after cryopreservation by supplementation of long-chain oligosaccharide in the freezing medium in rats and humans.

    PubMed

    Miyamoto, Yoshitaka; Suzuki, Satoshi; Nomura, Kou; Enosawa, Shin

    2006-01-01

    Factors affecting cell viability, plating efficiency, and survival of hepatocytes after cryopreservation have been investigated. We focused especially on the effect of including trehalose and related oligosaccharides in the cryopreservation fluid. This was supplemented with either glucose, trehalose, maltotriose, or other sugars, in addition to dimethyl sulfoxide (10%) and first tested with primary rat hepatocytes cooled in a controlled rate freezer. After thawing, viability by trypan blue exclusion of cells frozen in oligosaccharide-supplemented medium was significantly higher than for those cryopreserved without oligosaccharides. Use of oligosaccharides with higher molecular weights resulted in greatest improvement in viability. Moreover, attachment and survival rates in plastic dishes were approximately 1.2-1.8-fold greater after freezing in the presence of di-, tri-, and tetrasaccharides. Human hepatocytes isolated from untransplantable liver showed the same tendency regarding viability, but cell adherence was not similarly improved by the addition of oligosaccharides. Possible reasons for these differences may be prior cell damage during extended cold ischemia of the human liver, donor age, or cell degradation caused by progression of fatty liver in humans, and/or species differences.

  5. Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

    PubMed

    van Erp, Y H; Koopmans, M J; Heirbaut, P R; van der Hoeven, J C; Weterings, P J

    1992-06-01

    A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.

  6. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    PubMed

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology. PMID:27180721

  7. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  8. In vitro metabolism of rivaroxaban, an oral, direct factor Xa inhibitor, in liver microsomes and hepatocytes of rats, dogs, and humans.

    PubMed

    Lang, D; Freudenberger, C; Weinz, C

    2009-05-01

    The in vitro metabolism of rivaroxaban, a novel, oral, direct factor Xa inhibitor for the prevention and treatment of thromboembolic disorders, was investigated in several species, including humans. The objective of this study was to elucidate metabolite structures and identify the metabolic pathways to provide support for in vivo safety and clinical studies. [(14)C]Rivaroxaban was incubated with liver microsomes and hepatocytes of rats, dogs, and humans. The samples were analyzed by high-performance liquid chromatography-(14)C-tandem mass spectroscopy, to generate metabolite profiles and propose or confirm the structures of the metabolites formed. In vitro metabolite profiles showed no major differences between species. The main oxidative metabolic pathways identified for all species were hydroxylation at the morpholinone moiety (M-2, M-3, and M-8) and to a lesser extent at the oxazolidinone moiety (M-9). M-2 was the main metabolite in all microsomal incubations. M-1, a morpholinone ring-opened product formed by further oxidation of M-2, was the main metabolite in all hepatocyte incubations. Other pathways were amide hydrolysis at the morpholinone ring (M-7) and the chlorothiophene amide moiety (M-13 and M-15). In hepatocytes, M-13 was readily conjugated with glycine, leading to M-4. The metabolic fate of unlabeled M-15 was investigated separately. Incubations with human liver microsomes and hepatocytes showed that M-15 was first oxidized to the aldehyde intermediate M-16 and subsequently reduced to M-17 (alcohol) or oxidized to M-18 (carboxylic acid). No metabolism at the chlorothiophene moiety itself was found. Overall, rivaroxaban showed no species differences in metabolism, with different independent metabolic pathways and no formation of reactive metabolites. PMID:19196846

  9. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  10. Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARα in primary human hepatocytes.

    PubMed

    Kandel, Benjamin A; Thomas, Maria; Winter, Stefan; Damm, Georg; Seehofer, Daniel; Burk, Oliver; Schwab, Matthias; Zanger, Ulrich M

    2016-09-01

    The ligand-activated nuclear receptor pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two master transcriptional regulators of many important drug metabolizing enzymes and transporter genes (DMET) in response to xenobiotics including many drugs. The peroxisome proliferator-activated receptor alpha (PPARα, NR1C1), the target of lipid lowering fibrate drugs, primarily regulates fatty acid catabolism and energy-homeostasis. Recent research has shown that there are substantial overlaps in the regulated genes of these receptors. For example, both CAR and PXR also modulate the transcription of key enzymes involved in lipid and glucose metabolism and PPARα also functions as a direct transcriptional regulator of important DMET genes including cytochrome P450s CYP3A4 and CYP2C8. Despite their important and widespread influence on liver metabolism, comparative data are scarce, particularly at a global level and in humans. The major objective of this study was to directly compare the genome-wide transcriptional changes elucidated by the activation of these three nuclear receptors in primary human hepatocytes. Cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARα (WY14,643) or DMSO as vehicle control. Genomewide mRNA profiles determined with Affymetrix microarrays were analyzed for differentially expressed genes and metabolic functions. The results confirmed known prototype target genes and revealed strongly overlapping sets of coregulated but also distinctly regulated and novel responsive genes and pathways. The results further specify the role of PPARα as a regulator of drug metabolism and the role of the xenosensors PXR and CAR in lipid metabolism and energy homeostasis. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

  11. Cytotoxicity, intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells.

    PubMed

    Tlotleng, Nonhlanhla; Vetten, Melissa A; Keter, Frankline K; Skepu, Amanda; Tshikhudo, Robert; Gulumian, Mary

    2016-08-01

    Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs. PMID:27184667

  12. Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

    PubMed

    Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

    2008-01-01

    Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

  13. Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development

    PubMed Central

    Ma, Jinxia; Trop, Stefanie; Baer, Samantha; Rakhmanaliev, Elian; Arany, Zita; Dumoulin, Peter; Zhang, Hao; Romano, Julia; Coppens, Isabelle; Levitsky, Victor; Levitskaya, Jelena

    2013-01-01

    Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver. PMID:24086507

  14. Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression.

    PubMed

    Gross-Steinmeyer, Kerstin; Stapleton, Patricia L; Tracy, Julia H; Bammler, Theo K; Strom, Stephen C; Eaton, David L

    2010-08-01

    Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions. PMID:20442190

  15. Angiogenic properties of adult human thymus fat.

    PubMed

    Salas, Julián; Montiel, Mercedes; Jiménez, Eugenio; Valenzuela, Miguel; Valderrama, José Francisco; Castillo, Rafael; González, Sergio; El Bekay, Rajaa

    2009-11-01

    The endogenous proangiogenic properties of adipose tissue are well recognized. Although the adult human thymus has long been known to degenerate into fat tissue, it has never been considered as a potential source of angiogenic factors. We have investigated the expression of diverse angiogenic factors, including vascular endothelial growth factor A and B, angiopoietin 1, and tyrosine-protein kinase receptor-2 (an angiopoietin receptor), and then analyzed their physiological role on endothelial cell migration and proliferation, two relevant events in angiogenesis. The detection of the gene and protein expression of the various proteins has been performed by immunohistochemistry, Western blotting, and quantitative real-time polymerase chain reaction. We show, for the first time, that adult thymus fat produces a variety of angiogenic factors and induces the proliferation and migration of human umbilical cord endothelial cells. Based on these findings, we suggest that this fat has a potential angiogenic function that might affect thymic function and ongoing adipogenesis within the thymus.

  16. Animal models to test hiPS-derived hepatocytes in the context of inherited metabolic liver diseases.

    PubMed

    Dusséaux, Mathilde; Darche, Sylvie; Strick-Marchand, Helene

    2014-01-01

    Human induced pluripotent stem (hiPS) cells are established following reprogramming of somatic cells from a wide variety of tissues. Given the scarcity of adult human hepatocytes, hiPS-derived hepatocytes would be a valuable source of cells to study differentiation programs, model patient-specific diseases, test drug toxicities, and cell transplantation therapies. Although hiPS-derived hepatocytes are extensively characterized in cell culture assays, testing these cells in animal models is necessary to fully evaluate their differentiation profile and their lack of tumorigenicity. Immunodeficient mouse models harboring liver damage are effective hosts in which xenogeneic hepatocytes can engraft, proliferate, and participate in liver regeneration, thus constituting a stringent test of hepatocyte functionality. The in vivo evaluation of disease-specific hiPS-derived hepatocytes should broaden our understanding of the cellular and molecular processes involved in inherited metabolic liver disease phenotypes. Herein, we detail our methods to test the functions of hiPS-derived hepatocytes in the context of the immunodeficient Rag2(-/-)IL2Rγc(-/-)Alb-uPAtg mouse model.

  17. The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes

    PubMed Central

    Vadia, Stephen; Arnett, Eusondia; Haghighat, Anne-Cécile; Wilson-Kubalek, Elisabeth M.; Tweten, Rodney K.; Seveau, Stephanie

    2011-01-01

    Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell. PMID:22072970

  18. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-05-15

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  19. Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

    PubMed Central

    Ullrich, K; Basner, R; Gieselmann, V; Von Figura, K

    1979-01-01

    Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes. PMID:114170

  20. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

    SciTech Connect

    Latchoumycandane, Calivarathan; Seah, Quee Ming; Tan, Rachel C.H.; Sattabongkot, Jetsumon; Beerheide, Walter; Boelsterli, Urs A. . E-mail: phcbua@nus.edu.sg

    2006-11-15

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.

  1. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells.

    PubMed

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-08-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  2. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells

    PubMed Central

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei. This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  3. Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

    PubMed

    Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

    2015-09-01

    Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.

  4. Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project.

    PubMed

    Mueller, Stefan O; Guillouzo, André; Hewitt, Philip G; Richert, Lysiane

    2015-12-25

    The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans. PMID:25952325

  5. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

    SciTech Connect

    Dykens, James A. Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A.; Will, Yvonne

    2008-12-01

    As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

  6. GW4064, an Agonist of Farnesoid X Receptor, Represses CYP3A4 Expression in Human Hepatocytes by Inducing Small Heterodimer Partner Expression

    PubMed Central

    Zhang, Shu; Pan, Xian

    2015-01-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur. PMID:25725071

  7. GW4064, an agonist of farnesoid X receptor, represses CYP3A4 expression in human hepatocytes by inducing small heterodimer partner expression.

    PubMed

    Zhang, Shu; Pan, Xian; Jeong, Hyunyoung

    2015-05-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur.

  8. Metabolism of [2-14C]p-hydroxyphenyl acetic acid in rat, monkey and human hepatocytes and in bile-duct cannulated rats.

    PubMed

    Zhou, Lian; Liu-Kreyche, Peggy; Iyer, Ramaswamy A

    2011-04-01

    We determined the metabolism of [2-(14)C]p-hydroxyphenyl acetic acid (p-HPA) in rat (male, Sprague-Dawley), monkey (male, Cynomolgus), and human (male, Caucasian) hepatocytes, and in bile-duct cannulated (BDC) rats (male, Sprague-Dawley). Unchanged p-HPA ranged from 87.0 to 92.6% of the total radioactivity (TRA) in the extracts of rat, monkey, and human hepatocytes. Metabolites M1 (a glucuronide conjugate of p-HPA) and M2 (a glycine conjugate of p-HPA) were detected, accounting for 1-4% of TRA. After an oral dose of [2-(14)C]p-HPA to BDC rats, p-HPA-related components was predominantly excreted in urine, accounting for 83% of the dose. Bile excretion was limited, accounting for only 1.5% of the dose. Unchanged p-HPA was the predominant radioactivity in plasma (84.6% of the TRA in 1-h pooled plasma) and urine (69.6% of the dose). Metabolites M1, M2, and M3 (a glucuronide of p-HPA) were all detected in plasma, urine, and bile as minor components. In summary, p-HPA was not metabolized extensively in rat, monkey, and human hepatocytes. In rats, absorption and elimination of p-HPA were nearly complete with urinary excretion of the unchanged p-HPA as the predominant route of elimination after oral dosing. No oxidative metabolites were detected, suggesting a minimal role for P450 enzymes in its overall metabolic clearance. Therefore, p-HPA has a low potential for drug-drug interactions mediated by the concomitant inhibitors and inducers of P450 enzymes.

  9. Bridging in vitro and in vivo metabolism and transport of faldaprevir in human using a novel cocultured human hepatocyte system, HepatoPac.

    PubMed

    Ramsden, Diane; Tweedie, Donald J; Chan, Tom S; Taub, Mitchell E; Li, Yongmei

    2014-03-01

    An increased appreciation of the importance of transporter and enzyme interplay in drug clearance and a desire to delineate these mechanisms necessitates the utilization of models that contain a full complement of enzymes and transporters at physiologically relevant activities. Additionally, the development of drugs with longer half-lives requires in vitro systems with extended incubation times that allow characterization of metabolic pathways for low-clearance drugs. A recently developed coculture hepatocyte model, HepatoPac, has been applied to meet these challenges. Faldaprevir is a drug in late-stage development for the treatment of hepatitis C. Faldaprevir is a low-clearance drug with the somewhat unique characteristic of being slowly metabolized, producing two abundant hydroxylated metabolites (M2a and M2b) in feces (∼40% of the dose) without exhibiting significant levels of circulating metabolites in humans. The human HepatoPac model was investigated to characterize the metabolism and transport of faldaprevir. In human HepatoPac cultures, M2a and M2b were the predominant metabolites formed, with extents of formation comparable to in vivo. Direct glucuronidation of faldaprevir was shown to be a minor metabolic pathway. HepatoPac studies also demonstrated that faldaprevir is concentrated in liver with active uptake by multiple transporters (including OATP1B1 and Na(+)-dependent transporters). Overall, human HepatoPac cultures provided valuable insights into the metabolism and disposition of faldaprevir in humans and demonstrated the importance of enzyme and transporter interplay in the clearance of the drug. PMID:24366904

  10. Effects of chronic alcohol drinking on receptor-binding, internalization, and degradation of human immunodeficiency virus 1 envelope protein gp120 in hepatocytes.

    PubMed

    Singh, Ashok K; Jiang, Yin; Gupta, Shveta

    2007-12-01

    Although alcohol drinking increases susceptibility to human immunodeficiency virus (HIV) infection, possible mechanisms underlying the effects of alcohol are not yet known. Since the HIV envelope protein gp120 plays a key role in progression of HIV infection, the aim of the present study was to evaluate the toxicity and degradation of gp120 in hepatocytes isolated from liver of alcohol-preferring rats drinking either 15% ethanol in water or pure water for 70 days. The hypothesis was that alcohol drinking augmented the toxicity, but suppressed degradation of gp120. Hepatocytes from water-drinking rats (C-cells) or ethanol-drinking rats (Et-cells) were treated with laptacystin, anti-CD4 antibodies, CCR5 antagonist, or mannose, followed by [(125)I]gp120 or native gp120. At predetermined intervals, control (C) and ethanol exposed (Et) cells were analyzed for toxicity and degradation of gp120. In C-cells, [(125)I]gp120 binding and internalization peaked within 5-45 min and remained elevated for up to 10h and then decreased gradually. In Et-cells, [(125)I]gp120 binding peaked comparably to C-cells, but the binding remained to the peak level throughout the experimental period. C-cells exhibited the lysosomal/ubiquitin-mediated degradation of intracellular gp120, resulting in released gp120 fragments into the incubation medium that suppressed gp120-CD4 binding, improved cell viability, and inhibited gp120-induced apoptosis. Ethanol drinking suppressed gp120 degradation in and release of gp120 fragments from hepatocytes. The incubation medium of Et-cells did not suppress gp120-CD4 binding or the gp120-mediated apoptosis in hepatocytes. Thus, chronic alcohol drinking augmented the adverse effects of gp120 possibly by suppressing its degradation in hepatocytes. The present observation also suggests that a number of CCR5 or ubiquitin-based therapeutic drugs may not be effective in suppressing HIV infection in alcohol-drinking subjects.

  11. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

    SciTech Connect

    Yu, Jung Hwan; Lee, Yoo Jeong; Kim, Hyo Jung; Choi, Hyeonjin; Choi, Yoonjeong; Seok, Jo Woon; Kim, Jae-woo

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

  12. Thiamin requirement of the adult human.

    PubMed

    Sauberlich, H E; Herman, Y F; Stevens, C O; Herman, R H

    1979-11-01

    Young adult male subjects maintained on a metabolic ward were fed diets providing controlled intakes of thiamin and either 2800 or 3600 kcal. The higher level of calories was attained by an increased intake of carbohydrates. Constant weights were maintained by the subjects by adjusting daily activity and exercise schedules. Thiamin requirements were evaluated in terms of erythrocyte transketolase activity and urinary excretion of the vitamin. The results of the study revealed that a relationship exists between thiamin requirement and caloric intake and expenditure. Thus, when the calories being utilized were derived primarily from carbohydrate sources, the minimum adult male requirement for thiamin appeared to be 0.30 mg of thiamin per 1000 kcal. Urinary excretion of thiamin and erythrocyte transketolase activity appear to be reasonably reliable reflections of thiamin intakes and thiamin nutritional status. The use of these measurements in nutrition surveys appears justified. The microbiological assay (Lactobacillus viridescens) for measuring thiamin levels in urine samples appears to be a somewhat more sensitive but valid procedure as an alternate for the thiochrome method. Judged from the results of this study, the recommended intake for the adult human of 0.40 mg of thiamin per 1000 kcal by FAO/WHO and the recommended allowance of 0.5 mg per 1000 kcal by the Food and Nutrition Board of the NAS-NRC appear reasonable and amply allow for biological variations and other factors that may influence the requirement for this vitamin.

  13. Synergistic genotoxicity caused by low concentration of titanium dioxide nanoparticles and p,p'-DDT in human hepatocytes.

    PubMed

    Shi, Yun; Zhang, Jiang-Hua; Jiang, Ming; Zhu, Li-Hua; Tan, He-Qing; Lu, Bin

    2010-04-01

    The use of titanium dioxide nanoparticles (nano-TiO(2)) for the degradation of dichlorodiphenyltrichloroethane (p,p'-DDT) increases the risk of exposure to trace nano-TiO(2) and p,p'-DDT mixtures. The interaction of p,p'-DDT and nano-TiO(2) at low concentrations may alter toxic response relative to nano-TiO(2) or p,p'-DDT alone. In this work, the combined genotoxicity of trace nano-TiO(2) and p,p'-DDT on human embryo L-02 hepatocytes without photoactivation was studied. Nano-TiO(2) (0.1 g/L) was mixed with 0.01-1 mmol/L p,p'-DDT to determine adsorption isotherms. L-02 cells were exposed to different levels of p,p'-DDT (0, 0.001, 0.01, and 0.1 mumol/L) and nano-TiO(2) (0, 0.01, 0.1, and 1 microg/mL) respectively. The adsorption of p,p'-DDT by nano-TiO(2) was approximately 0.3 mmol/g. Cell viability, apoptosis, and DNA double strand breaks were similar among all test groups. Nano-TiO(2) alone (0.01-1 microg/mL) increased the levels of oxidative stress and oxidative DNA adducts (8-OHdG), but it did not induce DNA breaks or chromosome damage. Addition of trace nano-TiO(2) with trace p,p'-DDT synergistically enhanced genotoxicity via increasing oxidative stress, oxidative DNA adducts, DNA breaks, and chromosome damage in L-02 cells. Low concentrations of nano-TiO(2) and p,p'-DDT increased oxidativestress by reactive oxygen species (ROS) formation and lipid oxidation. Oxidative stress is a major pathway for DNA and chromosome damage. Dose-dependent synergistic genotoxicity induced by combined exposure of trace p,p'-DDT and nano-TiO(2) suggests a potential environmental risk of nano-TiO(2) assisted photocatalysis.

  14. Milk thistle, a herbal supplement, decreases the activity of CYP3A4 and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures.

    PubMed

    Venkataramanan, R; Ramachandran, V; Komoroski, B J; Zhang, S; Schiff, P L; Strom, S C

    2000-11-01

    Milk thistle extract is one of the most commonly used nontraditional therapies, particularly in Germany. Milk thistle is known to contain a number of flavonolignans. We evaluated the effect of silymarin, on the activity of various hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with silymarin (0.1 and 0.25 mM) significantly reduced the activity of CYP3A4 enzyme (by 50 and 100%, respectively) as determined by the formation of 6-beta-hydroxy testosterone and the activity of uridine diphosphoglucuronosyl transferase (UGT1A6/9) (by 65 and 100%, respectively) as measured by the formation of 4-methylumbelliferone glucuronide. Silymarin (0.5 mM) also significantly decreased mitochondrial respiration as determined by MTT reduction in human hepatocytes. These observations point to the potential of silymarin to impair hepatic metabolism of certain coadministered drugs in humans. Indiscriminate use of herbal products may lead to altered pharmacokinetics of certain drugs and may result in increased toxicity of certain drugs.

  15. Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes.

    PubMed

    Ghaida, J; Merker, H J

    1992-01-01

    The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18-180 mum; acrolein, 50-500 mum) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 mm) or GSH (1 mm) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

  16. Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine, and carbamazepine using human hepatocytes and HepaRG cells.

    PubMed

    Sugiyama, Ikuo; Murayama, Norie; Kuroki, Ayaka; Kota, Jagannath; Iwano, Shunsuke; Yamazaki, Hiroshi; Hirota, Takashi

    2016-09-01

    Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3-11.5) and 10.0 (6.2-13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2-19.3), 7.0 (2.5-10.8) and 14.8 (3.1-29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2-7.4), 2.7 (0.8-5.7) and 8.3 (3.5-14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes. After incubation for 72 h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine. PMID:26711482

  17. Dual effects of ketoconazole cis-enantiomers on CYP3A4 in human hepatocytes and HepG2 Cells.

    PubMed

    Novotná, Aneta; Krasulová, Kristýna; Bartoňková, Iveta; Korhoňová, Martina; Bachleda, Petr; Anzenbacher, Pavel; Dvořák, Zdeněk

    2014-01-01

    Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, Ki 0.92 µM for testosterone, IC₅₀ 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC₅₀ 0.90 µM, Ki 0.17 µM for testosterone, IC₅₀ 1.04 µM, Ki 1.51 µM for midazolam).

  18. Effect of sulfide on the cytotoxicity of arsenite and arsenate in human hepatocytes (HepG2) and human urothelial cells (UROtsa).

    PubMed

    Hinrichsen, Sinikka; Lohmayer, Regina; Zdrenka, Ricarda; Dopp, Elke; Planer-Friedrich, Britta

    2014-09-01

    Arsenic, a common poison, is known to react with sulfide in vivo, forming thioarsenates. The acute toxicity of the inorganic thioarsenates is currently unknown. Our experiments showed that a fourfold sulfide excess reduced acute arsenite cytotoxicity in human hepatocytes (HepG2) and urothelial cells (UROtsa) significantly, but had little effect on arsenate toxicity. Speciation analysis showed immediate formation of thioarsenates (up to 73 % of total arsenic) in case of arsenite, but no speciation changes for arsenate. Testing acute toxicity of mono- and trithioarsenate individually, both thioarsenates were found to be more toxic than their structural analogue arsenate, but less toxic than arsenite. Toxicity increased with the number of thio groups. The amount of cellular arsenic uptake after 24 h corresponded to the order of toxicity of the four compounds tested. The dominant to almost exclusive intracellular arsenic species was arsenite. The results imply that thiolation is a detoxification process for arsenite in sulfidic milieus. The mechanism could either be that thioarsenates regulate the amount of free arsenite available for cellular uptake without entering the cells themselves, or, based on their chemical similarity to arsenate, they could be taken up by similar transporters and reduced rapidly intracellularly to arsenite. PMID:24781333

  19. Rethinking Adult Literacy Programs: A Humanities-Based Curriculum.

    ERIC Educational Resources Information Center

    Anania, Joanne

    The Roosevelt University Humanities Enrichment Program tries to acknowledge the adult part of adult literacy. Its instructional materials are of interest and value to the adult student and, therefore, provide incentives for reading and discussion instead of serving merely as skill-building exercises. The materials are drawn from literature,…

  20. Gene transfer of human hepatocyte growth factor by the use of nanosecond pulsed laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Sato, Shunichi; Saitoh, Daizoh; Hasegawa, Makoto; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2006-05-01

    We successfully delivered a therapeutic vector construct, which carries hepatocyte growth factor (HGF) gene, to rat skin in vivo. After HGF expression vector had been intradermally injected to rat skin, LISWs were generated by irradiating the laser target put on the rat skin with nanosecond pulses from the second harmonics (532 nm) of a Q-switched Nd:YAG laser. Concentration of HGF protein increased by a factor of four by the application of LISWs when compared with that of control samples without LISW application. We also investigated the effects of LISWs on the integrity of plasmid DNA.

  1. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

    PubMed

    Broutier, Laura; Andersson-Rolf, Amanda; Hindley, Christopher J; Boj, Sylvia F; Clevers, Hans; Koo, Bon-Kyoung; Huch, Meritxell

    2016-09-01

    Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol. PMID:27560176

  2. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

    PubMed

    Broutier, Laura; Andersson-Rolf, Amanda; Hindley, Christopher J; Boj, Sylvia F; Clevers, Hans; Koo, Bon-Kyoung; Huch, Meritxell

    2016-09-01

    Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

  3. LC-MS metabolic study on quercetin and taxifolin galloyl esters using human hepatocytes as toxicity and biotransformation in vitro cell model.

    PubMed

    Vacek, Jan; Papoušková, Barbora; Vrba, Jiří; Zatloukalová, Martina; Křen, Vladimír; Ulrichová, Jitka

    2013-12-01

    Galloyl esters of quercetin and taxifolin have been recently prepared semisynthetically as part of work towards modifying the solubility and modulating the biological activity of these natural flavonoids. In this paper we focused on the liquid chromatography-mass spectrometry (LC-MS) profiling of metabolites of 3-O-galloylquercetin and 7-O-galloyltaxifolin using human hepatocytes as the in vitro cell model. A subtoxic concentration (50μM) was used for both compounds and the formation of metabolites was monitored for 2h in hepatocytes and cultivation medium separately. Using negative electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS), we identified different biotransformation patterns for the studied compounds. 3-O-Galloylquercetin is metabolized directly to glucuronides and methyl derivatives. In contrast, 7-O-galloyltaxifolin is oxidized to 7-O-galloylquercetin or cleaved to taxifolin, and consequently the products formed are sulfated or glucuronidated. The oxidative biotransformation of 3-O-galloylquercetin and 7-O-galloyltaxifolin is also accompanied by ester bond cleavage presumably by cellular enzymes (esterases) in a nonspecific manner. Our results provide fundamental insights into the biotransformation of monogalloyl esters of flavonoids and can be applied in investigations of the pharmaceutical potential of other galloylated polyphenolic substances.

  4. Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster (Crassostrea gigas) on cultured human hepatocytes under oxidative stress.

    PubMed

    Fuda, Hirotoshi; Watanabe, Mitsugu; Hui, Shu-Ping; Joko, Sae; Okabe, Hiroaki; Jin, Shigeki; Takeda, Seiji; Miki, Emiko; Watanabe, Takayuki; Chiba, Hitoshi

    2015-06-01

    The antioxidant, and hepatoprotective properties of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a natural phenolic antioxidant isolated from the Pacific oyster, were defined using cultured human hepatocyte-derived cells (C3A). DHMBA showed no cytotoxicity at 62.5-500μM, as well as chlorogenic acid (CGA), vitamin C, and vitamin E. However, butylated hydroxytoluene, eicosapentaenoic acid, docosahexaenoic acid and catechin reduced cell viability. In the presence of the prooxidant 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), DHMBA at 125-500μM improved cell viability, whereas CGA had no effect. DNA ladder formation and flow-cytometric studies indicated that DHMBA inhibited AAPH-induced apoptosis and necrosis. CGA was ineffective. Thus, DHMBA is a novel, potent antioxidant, effectively protecting cultured hepatocytes from apoptosis and necrosis caused by oxidative stress. Additionally, the concentration of DHMBA was determined by mass spectrometry to be 24.4μmol/kg wet oyster meat, and three polyphenols (gentisic acid, daidzein, and matairesinol) were newly identified in the oyster extracts.

  5. Tert-butylhydroquinone as a phenolic activator of Nrf2 antagonizes arsenic-induced oxidative cytotoxicity but promotes arsenic methylation and detoxication in human hepatocyte cell line.

    PubMed

    Duan, Xiaoxu; Liu, Dan; Xing, Xiaoyue; Li, Jinlong; Zhao, Shuo; Nie, Huifang; Zhang, Yang; Sun, Guifan; Li, Bing

    2014-08-01

    Oxidative stress plays crucial roles in exerting a variety of damages upon arsenic exposure. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator protecting cells and tissues from oxidative injuries. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a well-known synthetic Nrf2 inducer, could protect human hepatocytes against arsenic-induced cytotoxicity and oxidative injuries. Our results showed that 5 and 25 μmol/l tBHQ pretreatment suppressed the arsenic-induced hepatocellular cytotoxicity, reactive oxygen species generation, and hepatic lipid peroxidation, while relieved the arsenic-induced disturbances of intracellular glutathione balance. In addition, we also observed that tBHQ treatment promoted the arsenic biomethylation process and upregulated Nrf2-regulated downstream heme oxygenase-1 and NADPH: quinine oxidoreductase 1 mRNA expressions. Collectively, we suspected that Nrf2 signaling pathway may be involved in the protective effects of tBHQ against arsenic invasion in hepatocytes. These data suggest that phenolic Nrf2 inducers, such as tBHQ, represent novel therapeutic or dietary candidates for the population at high risk of arsenic poisoning. PMID:24970285

  6. Tert-butylhydroquinone as a phenolic activator of Nrf2 antagonizes arsenic-induced oxidative cytotoxicity but promotes arsenic methylation and detoxication in human hepatocyte cell line.

    PubMed

    Duan, Xiaoxu; Liu, Dan; Xing, Xiaoyue; Li, Jinlong; Zhao, Shuo; Nie, Huifang; Zhang, Yang; Sun, Guifan; Li, Bing

    2014-08-01

    Oxidative stress plays crucial roles in exerting a variety of damages upon arsenic exposure. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator protecting cells and tissues from oxidative injuries. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a well-known synthetic Nrf2 inducer, could protect human hepatocytes against arsenic-induced cytotoxicity and oxidative injuries. Our results showed that 5 and 25 μmol/l tBHQ pretreatment suppressed the arsenic-induced hepatocellular cytotoxicity, reactive oxygen species generation, and hepatic lipid peroxidation, while relieved the arsenic-induced disturbances of intracellular glutathione balance. In addition, we also observed that tBHQ treatment promoted the arsenic biomethylation process and upregulated Nrf2-regulated downstream heme oxygenase-1 and NADPH: quinine oxidoreductase 1 mRNA expressions. Collectively, we suspected that Nrf2 signaling pathway may be involved in the protective effects of tBHQ against arsenic invasion in hepatocytes. These data suggest that phenolic Nrf2 inducers, such as tBHQ, represent novel therapeutic or dietary candidates for the population at high risk of arsenic poisoning.

  7. Paeoniflorin protects cells from GalN/TNF-α-induced apoptosis via ER stress and mitochondria-dependent pathways in human L02 hepatocytes.

    PubMed

    Jiang, Zequn; Chen, Weiping; Yan, Xiaojing; Bi, Lei; Guo, Sheng; Zhan, Zhen

    2014-05-01

    Paeoniflorin (PF) is one of the main effective components extracted from the root of Paeonia lactiflora, which has been used clinically to treat hepatitis in traditional Chinese medicine, but the details of the underlying mechanism remain unknown. The present study was designed to investigate the mechanism of protective effect of PF on d-galactosamine (GalN) and tumor necrosis factor-α (TNF-α)-induced cell apoptosis using human L02 hepatocytes. Our results confirmed that PF could attenuate GalN/TNF-α-induced apoptotic cell death in a dose-dependent manner. The disruption of mitochondrial membrane potential and the disturbance of intracellular Ca(2+) concentration were also recovered by PF. Western blot analysis revealed that GalN/TNF-α induced the activation of a number of signature endoplasmic reticulum (ER) stress and mitochondrial markers, while PF pre-treatment had a marked dose-dependent suppression on them. Additionally, the anti-apoptotic effect of PF was further evidenced by the inhibition of caspase-3/9 activities in L02 cells. These findings suggest that PF can effectively inhibit hepatocyte apoptosis and the underlying mechanism is related to the regulating mediators in ER stress and mitochondria-dependent pathways. PMID:24777494

  8. Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen.

    PubMed

    Li, Albert P; Uzgare, Aarti; LaForge, Yumiko S

    2012-07-30

    The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.

  9. Dichloromethane metabolism to formaldehyde and reaction of formaldehyde with nucleic acids in hepatocytes of rodents and humans with and without glutathione S-transferase T1 and M1 genes.

    PubMed

    Casanova, M; Bell, D A; Heck, H D

    1997-06-01

    Metabolism of dichloromethane (DCM) to formaldehyde (HCHO) via a glutathione S-transferase (GST) pathway is thought to be required for its carcinogenic effects in B6C3F1 mice. In humans, this reaction is catalyzed primarily by the protein product of the gene GSTT1, a member of the Theta class of GST, and perhaps to a small extent by the protein product of the gene GSTM1. Humans are polymorphic with respect to both genes. Since HCHO may bind to both DNA and RNA forming DNA-protein crosslinks (DPX) and RNA-formaldehyde adducts (RFA), respectively, these products were determined in isolated hepatocytes from B6C3F1 mice, F344 rats, Syrian golden hamsters, and humans to compare species with respect to the production of HCHO from DCM and its reaction with nucleic acids. Only mouse hepatocytes formed detectable amounts of DPX, the quantities of which corresponded well with quantities of DPX formed in the livers of mice exposed to DCM in vivo [Casanova, M., Conolly, R.B., and Heck, H. d'A. (1996). Fundam. Appl. Toxicol. 31, 103-116]. Hepatocytes from all rodent species and from humans with functional GSTT1 and GSTM1 genes formed RFA. No RFA were detected in human cells lacking these genes. Yields of RFA in hepatocytes of mice were 4-fold higher than in those of rats, 7-fold higher than in those of humans, and 14-fold higher than in those of hamsters. The RFA:DPX ratio in mouse hepatocytes incubated with DCM was approximately 9.0 +/- 1.4, but it was 1.1 +/- 0.3 when HCHO was added directly to the medium, indicating that HCHO generated internally from DCM is not equivalent to that added externally to cells and that it may occupy separate pools. DPX were not detected in human hepatocytes even at concentrations equivalent to an in vivo exposure of 10,000 ppm; however, the possibility that very small amounts of DPX were produced from DCM cannot be excluded, since HCHO was formed in human cells. Maximal amounts of DPXliver that might be formed in humans were predicted from the

  10. Have you got any cholesterol? Adults' views of human nutrition

    NASA Astrophysics Data System (ADS)

    Schibeci, Renato; Wong, Khoon Yoong

    1994-12-01

    The general aim of our human nutrition project is to develop a health education model grounded in ‘everyday’ or ‘situated’ cognition (Hennessey, 1993). In 1993, we began pilot work to document adult understanding of human nutrition. We used a HyperCard stack as the basis for a series of interviews with 50 adults (25 university students, and 25 adults from offcampus). The interviews were transcribed and analysed using the NUDIST computer program. A summary of the views of these 50 adults on selected aspects of human nutrition is presented in this paper.

  11. Adult Education & Human Resource Development: Overlapping and Disparate Fields

    ERIC Educational Resources Information Center

    Watkins, Karen E.; Marsick, Victoria J.

    2014-01-01

    Adult education and human resource development as fields of practice and study share some roots in common but have grown in different directions in their histories. Adult education's roots focused initially on citizenship for a democratic society, whereas human resource development's roots are in performance at work. While they have…

  12. Effect of fulminant hepatic failure porcine plasma supplemented with essential components on encapsulated rat hepatocyte spheroids.

    PubMed

    Lee, J-H; Lee, D-H; Park, J-K; Kim, S-K; Kwon, C H D; Lee, S-K

    2012-05-01

    The development of bioartificial liver (BAL) systems has required detailed information about the functional capabilities of cultured hepatocytes during blood or plasma passage. In this study we investigated the effects of porcine plasma and various supplements on the viability and function of adult rat hepatocytes in vitro. Primary rat hepatocytes cultured in porcine plasma supplemented with various substances showed albumin synthesis rates and viability equal to or higher than those of controls. Supplementation with calcium chloride, magnesium sulfate, trace elements, amino acids, insulin, and epidermal growth factor were essential to maintain viability and high albumin synthesis. Especially, trace elements showed significantly higher and longer albumin secretion. Isolated rat hepatocytes were cultured in Spinner flasks for 24 hours to form spheroids that were harvested and encapsulated with chitosan-alginate solution before transfer to the bioreactor in the BAL system. Encapsulated rat hepatocyte spheroids cultured with porcine plasma including trace elements showed higher viability (57%) than controls (40%) after 24 hours, with ammonia removal values of 30.92 μg/10(6) cells versus the control 9.04 μg/10(6) cells. After 24 hours of operation the urea secretion value of encapsulated rat hepatocyte spheroids cultured in porcine plasma in the presence versus absence of trace elements was 76.73 μg/10(6) cells and 18.80 μg/10(6) cells, respectively. We concluded that encapsulated hepatocyte spheroids in a packed-bed bioreactor operated with human plasma including trace elements enhanced cell viability and liver function as a bases for an in vivo clinical trial of the BAL system. PMID:22564611

  13. Could adult hippocampal neurogenesis be relevant for human behavior?

    PubMed Central

    Snyder, Jason S.; Cameron, Heather A.

    2011-01-01

    Although the function of adult neurogenesis is still unclear, tools for directly studying the behavioral role of new hippocampal neurons now exist in rodents. Since similar studies are impossible to do in humans, it is important to assess whether the role of new neurons in rodents is likely to be similar to that in humans. One feature of adult neurogenesis that varies tremendously across species is the number of neurons that are generated, so a key question is whether there are enough neurons generated in humans to impact function. In this review we examine neuroanatomy and circuit function in the hippocampus to ask how many granule neurons are needed to impact hippocampal function and then discuss what is known about numbers of new neurons produced in adult rats and humans. We conclude that relatively small numbers of neurons could affect hippocampal circuits and that the magnitude of adult neurogenesis in adult rats and humans is probably larger than generally believed. PMID:21736900

  14. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D.

    PubMed

    Faulkner-Jones, Alan; Fyfe, Catherine; Cornelissen, Dirk-Jan; Gardner, John; King, Jason; Courtney, Aidan; Shu, Wenmiao

    2015-01-01

    We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine. PMID:26486521

  15. Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D.

    PubMed

    Faulkner-Jones, Alan; Fyfe, Catherine; Cornelissen, Dirk-Jan; Gardner, John; King, Jason; Courtney, Aidan; Shu, Wenmiao

    2015-01-01

    We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.

  16. Long Chain Fatty Acid Esters of Quercetin-3-O-glucoside Attenuate H₂O₂-induced Acute Cytotoxicity in Human Lung Fibroblasts and Primary Hepatocytes.

    PubMed

    Warnakulasuriya, Sumudu N; Ziaullah; Rupasinghe, H P Vasantha

    2016-01-01

    Cellular oxidative stress causes detrimental effects to macromolecules, such as lipids, nucleic acids and proteins, leading to many pathological conditions. Quercetin-3-O-glucoside (Q3G), a glycosylated derivative of quercetin (Q), is a natural polyphenolic compound known to possess antioxidant activity. The hydrophilic/lipophilic nature of an antioxidant molecule is considered as an important factor governing the accessibility to the active sites of oxidative damages in vivo. Six long chain fatty acid esters of Q3G were evaluated with comparison to Q and Q3G, for their cytoprotective activity under H₂O₂-induced oxidative stress using cell culture model systems through cell viability, lipid peroxidation and fluorescence microscopy studies. Pre-incubation of α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) esters of Q3G exhibited significantly (p ≤ 0.05) greater cell viability in both human lung fibroblast (WI-38) and human primary hepatocytes upon exposure to H₂O₂ insult when compared to the control. Cytoprotection due to oleic acid and linoleic acid esters of Q3G was observed only in human primary hepatocytes. All the derivatives, Q3G and quercetin showed ability to significantly (p ≤ 0.05) lower production of lipid hydroperoxides under induced oxidative stress, compared to the control. However, ALA and DHA esters of Q3G resulted in significantly lower lipid hydroperoxidation than Q and Q3G. Based on fluorescence microscopy study, H₂O₂-induced apoptosis was attenuated by the fatty acid derivatives of Q3G. The fatty acid derivatives of Q3G possess better cytoprotective effect than Q3G against H₂O₂-induced cytotoxicity in vitro and the concentration should be selected to avoid cytotoxicity. PMID:27058521

  17. The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells

    SciTech Connect

    Fischer, A.; Hoeger, S.J.; Stemmer, K.; Feurstein, D.J.; Knobeloch, D.; Nussler, A.; Dietrich, D.R.

    2010-05-15

    Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

  18. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    PubMed

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx. PMID:25950141

  19. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    PubMed

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

  20. EXPERIMENTAL HEPATOCYTE XENOTRANSPLANTATION – A COMPREHENSIVE REVIEW OF THE LITERATURE

    PubMed Central

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K. C.

    2015-01-01

    Background Hepatocyte transplantation is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for transplantation. Hepatocytes from other species, e.g., the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenotransplantation. Literature search and results The literature search identified 51 reports of in vivo cross-species transplantation of hepatocytes in a variety of experimental models. Most studies investigated the transplantation of human (n=23) or pig (n=19) hepatocytes. No studies explored hepatocytes from genetically-engineered pigs. The spleen was the most common site of transplantation (n=23), followed by the liver (through the portal vein [n=6]) and peritoneal cavity (n=19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. Conclusions The data provided by this literature search strengthen the hypothesis that xenotransplantation of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically-engineered pig livers may address some of the immunological problems of xenotransplantation. PMID:25950141

  1. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

    PubMed Central

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P.; Walles, Heike

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. PMID:26488607

  2. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

    PubMed

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

  3. Phytoestrogen Metabolism by Adult Human Gut Microbiota.

    PubMed

    Gaya, Pilar; Medina, Margarita; Sánchez-Jiménez, Abel; Landete, José Mᵃ

    2016-08-09

    Phytoestrogens are plant-derived polyphenols with a structure similar to human estrogens. The three main groups of phytoestrogens, isoflavones, ellagitannins, and lignans, are transformed into equol, urolithins, and enterolignans, respectively, by bacteria. These metabolites have more estrogenic/antiestrogenic and antioxidant activities than their precursors, and they are more bioavailable. The aim of this study was to analyze the metabolism of isoflavones, lignans and ellagitannins by gut microbiota, and to study the possible correlation in the metabolism of these three groups of phytoestrogens. In vitro fermentation experiments were performed with feces samples from 14 healthy adult volunteers, and metabolite formation was measured by HPLC-PAD and HPLC-ESI/MS. Only the microbiota of one subject produced equol, while most of them showed production of O-desmethylangolensin (O-DMA). Significant inter-subject differences were observed in the metabolism of dihydrodaidzein and dihydrogenistein, while the glucoside isoflavones and their aglycones showed less variability, except for glycitin. Most subjects produced urolithins M-5 and E. Urolithin D was not detected, while uroltithin B was found in half of the individuals analyzed, and urolithins A and C were detected in two and four subjects, respectively. Enterolactone was found in all subjects, while enterodiol only appeared in five. Isoflavone metabolism could be correlated with the metabolism of lignans and ellagitannins. However, the metabolism of ellagitannins and lignans could not be correlated. This the first study where the metabolism of the three groups together of phytoestrogen, isoflavones, lignans, and ellagitannins by gut microbiota is analyzed.

  4. Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice.

    PubMed

    Uchida, Takuro; Hiraga, Nobuhiko; Imamura, Michio; Yoshimi, Satoshi; Kan, Hiromi; Miyaki, Eisuke; Tsuge, Masataka; Abe, Hiromi; Hayes, C Nelson; Aikata, Hiroshi; Ishida, Yuji; Tateno, Chise; Ellis, Joan D; Chayama, Kazuaki

    2016-02-01

    We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C. PMID:26569595

  5. Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

    PubMed

    Jellali, Rachid; Bricks, Thibault; Jacques, Sébastien; Fleury, Marie-José; Paullier, Patrick; Merlier, Franck; Leclerc, Eric

    2016-07-01

    Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Establishment of In Silico Prediction Models for CYP3A4 and CYP2B6 Induction in Human Hepatocytes by Multiple Regression Analysis Using Azole Compounds.

    PubMed

    Nagai, Mika; Konno, Yoshihiro; Satsukawa, Masahiro; Yamashita, Shinji; Yoshinari, Kouichi

    2016-08-01

    Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes.

  7. Assessing the therapeutic potential of lab-made hepatocytes.

    PubMed

    Rezvani, Milad; Grimm, Andrew A; Willenbring, Holger

    2016-07-01

    Hepatocyte transplantation has potential as a bridge or even alternative to whole-organ liver transplantation. Because donor livers are scarce, realizing this potential requires the development of alternative cell sources. To be therapeutically effective, surrogate hepatocytes must replicate the complex function and ability to proliferate of primary human hepatocytes. Ideally, they are also autologous to eliminate the need for immune suppression, which can have severe side effects and may not be sufficient to prevent rejection long term. In the past decade, several methods have been developed to generate hepatocytes from other readily and safely accessible somatic cells. These lab-made hepatocytes show promise in animal models of liver diseases, supporting the feasibility of autologous liver cell therapies. Here, we review recent preclinical studies exemplifying different types of lab-made hepatocytes that can potentially be used in autologous liver cell therapies. To define the therapeutic efficacy of current lab-made hepatocytes, we compare them to primary human hepatocytes, focusing on engraftment efficiency and posttransplant proliferation and function. In addition to summarizing published results, we discuss animal models and assays effective in assessing therapeutic efficacy. This analysis underscores the therapeutic potential of current lab-made hepatocytes, but also highlights deficiencies and uncertainties that need to be addressed in future studies aimed at developing liver cell therapies with lab-made hepatocytes. (Hepatology 2016;64:287-294). PMID:27014802

  8. Adult Human Neurogenesis: From Microscopy to Magnetic Resonance Imaging

    PubMed Central

    Sierra, Amanda; Encinas, Juan M.; Maletic-Savatic, Mirjana

    2011-01-01

    Neural stem cells reside in well-defined areas of the adult human brain and are capable of generating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases. PMID:21519376

  9. Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

    PubMed Central

    Jiang, Shuying; Tanaka, Toshiya; Iwanari, Hiroko; Hotta, Hiromitsu; Yamashita, Hisahiko; Kumakura, Junko; Watanabe, Yuichiro; Uchiyama, Yasutoshi; Aburatani, Hiroyuki; Hamakubo, Takao; Kodama, Tatsuhiko; Naito, Makoto

    2003-01-01

    Background Hepatocyte nuclear factor-4α (HNF4α; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4α causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4α in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4α protein, due, in part, to the limited availability of human HNF4α-specific antibodies. Results Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4α fusion proteins as the immunizing agent. The mouse anti-human HNF4α monoclonal antibody (K9218) generated against human HNF4α1/α2/α3 amino acids 3–49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4α proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4α protein, but HEK293 showed no expression of HNF4α protein. Nuclear-specific localization of the HNF4α protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4α protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4α protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4α in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4α mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis

  10. Selective insulin resistance in hepatocyte senescence

    SciTech Connect

    Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  11. Phytoestrogen Metabolism by Adult Human Gut Microbiota.

    PubMed

    Gaya, Pilar; Medina, Margarita; Sánchez-Jiménez, Abel; Landete, José Mᵃ

    2016-01-01

    Phytoestrogens are plant-derived polyphenols with a structure similar to human estrogens. The three main groups of phytoestrogens, isoflavones, ellagitannins, and lignans, are transformed into equol, urolithins, and enterolignans, respectively, by bacteria. These metabolites have more estrogenic/antiestrogenic and antioxidant activities than their precursors, and they are more bioavailable. The aim of this study was to analyze the metabolism of isoflavones, lignans and ellagitannins by gut microbiota, and to study the possible correlation in the metabolism of these three groups of phytoestrogens. In vitro fermentation experiments were performed with feces samples from 14 healthy adult volunteers, and metabolite formation was measured by HPLC-PAD and HPLC-ESI/MS. Only the microbiota of one subject produced equol, while most of them showed production of O-desmethylangolensin (O-DMA). Significant inter-subject differences were observed in the metabolism of dihydrodaidzein and dihydrogenistein, while the glucoside isoflavones and their aglycones showed less variability, except for glycitin. Most subjects produced urolithins M-5 and E. Urolithin D was not detected, while uroltithin B was found in half of the individuals analyzed, and urolithins A and C were detected in two and four subjects, respectively. Enterolactone was found in all subjects, while enterodiol only appeared in five. Isoflavone metabolism could be correlated with the metabolism of lignans and ellagitannins. However, the metabolism of ellagitannins and lignans could not be correlated. This the first study where the metabolism of the three groups together of phytoestrogen, isoflavones, lignans, and ellagitannins by gut microbiota is analyzed. PMID:27517891

  12. Gustofacial and olfactofacial responses in human adults.

    PubMed

    Weiland, Romy; Ellgring, Heiner; Macht, Michael

    2010-11-01

    Adults' facial reactions in response to tastes and odors were investigated in order to determine whether differential facial displays observed in newborns remain stable in adults who exhibit a greater voluntary facial control. Twenty-eight healthy nonsmokers (14 females) tasted solutions of PROP (bitter), NaCl (salty), citric acid (sour), sucrose (sweet), and glutamate (umami) differing in concentration (low, medium, and high) and smelled different odors (banana, cinnamon, clove, coffee, fish, and garlic). Their facial reactions were video recorded and analyzed using the Facial Action Coding System. Adults' facial reactions discriminated between stimuli with opponent valences. Unpleasant tastes and odors elicited negative displays (brow lower, upper lip raise, and lip corner depress). The pleasant sweet taste elicited positive displays (lip suck), whereas the pleasant odors did not. Unlike newborns, adults smiled with higher concentrations of some unpleasant tastes that can be regarded as serving communicative functions. Moreover, adults expressed negative displays with higher sweetness. Except for the "social" smile in response to unpleasant tastes, adults' facial reactions elicited by tastes and odors mostly correspond to those found in newborns. In conclusion, adults' facial reactions to tastes and odors appear to remain stable in their basic displays; however, some additional reactions might reflect socialization influences.

  13. Sodium arsenite induced reactive oxygen species generation, nuclear factor (erythroid-2 related) factor 2 activation, heme oxygenase-1 expression, and glutathione elevation in Chang human hepatocytes.

    PubMed

    Li, Bing; Li, Xin; Zhu, Bo; Zhang, Xinyu; Wang, Yi; Xu, Yuanyuan; Wang, Huihui; Hou, Yongyong; Zheng, Quanmei; Sun, Guifan

    2013-07-01

    Liver is one of the major target organs of arsenic toxicity and carcinogenesis. Nuclear factor (erythroid-2 related) factor 2 (Nrf2) is a redox-sensitive transcription factor, regulating critically cellular defense responses against the toxic metallic arsenic in many cell types and tissues. This study was conducted to evaluate the hepato-cellular Nrf2 and Nrf2-regulated antioxidant reactions of sodium arsenite exposure in Chang human hepatocytes. Nrf2 and heme oxygenase-1 (HO-1) protein levels were detected by Western blot, and Nrf2-regulated HO-1 mRNA expressions were determined using semiquantitative RT-PCR by 0∼50 μmol/L of sodium arsenite exposure for 2, 6, 12, and 24 h. We also observed the changes of intracellular reactive oxygen species (ROS) and total cellular glutathione (GSH) by flow cytometry and spectrophotometry, respectively. Our results showed that intracellular ROS were both dose- and time-dependent induced by inorganic arsenic; Cellular Nrf2 protein levels increased rapidly after 2 h of exposure, elevated significantly at 6 h, and reached the maximum at 12 h. The endogenous Nrf2-regulated downstream HO-1 mRNA and protein were also induced dramatically and lasted for as long as 24 h. In addition, intracellular GSH levels elevated in consistent with Nrf2 activation. Our findings here suggest that inorganic arsenic alters cellular redox balance in hepatocytes to trigger Nrf2-regulated antioxidant responses promptly, which may represent an adaptive cell defense mechanism against inorganic arsenic induced liver injuries and hepatoxicity.

  14. Enhanced Metabolizing Activity of Human ES Cell-Derived Hepatocytes Using a 3D Culture System with Repeated Exposures to Xenobiotics.

    PubMed

    Kim, Jong Hyun; Jang, Yu Jin; An, Su Yeon; Son, Jeongsang; Lee, Jaehun; Lee, Gyunggyu; Park, Ji Young; Park, Han-Jin; Hwang, Dong-Youn; Kim, Jong-Hoon; Han, Jiyou

    2015-09-01

    Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective in vitro toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces >90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models. PMID:26089346

  15. Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

    SciTech Connect

    Chang, L.W.; Daniel, F.B. ); DeAngelo, A.B. )

    1992-01-01

    An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.

  16. Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes

    SciTech Connect

    Lim, M.S.; Lim, Priscilla L.K.; Gupta, Rashi; Boelsterli, Urs A. . E-mail: phcbua@nus.edu.sg

    2006-12-15

    Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca{sup 2+} {sub c}) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 {mu}M). At 8 h, diclofenac caused increases in [Ca{sup 2+}]{sub c} (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca{sup 2+} chelator) inhibited cell injury, indicating that Ca{sup 2+} plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, {delta}{psi}{sub m} (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca{sup 2+}]{sub c}. The CYP2C9 inhibitor sulphaphenazole (10 {mu}M) protected from diclofenac-induced cell injury and prevented increases in [Ca{sup 2+}]{sub c}, while it had no effect on the dissipation of the {delta}{psi}{sub m}. Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca{sup 2+}]{sub c}. In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca{sup 2+}]{sub c}, triggering the mPT and precipitating cell death.

  17. Adult Literacy Education and Human Rights: A View from Afghanistan

    ERIC Educational Resources Information Center

    Andersen, Susan M.; Kooij, Christina S.

    2007-01-01

    In this article, we argue that adult literacy as part of international development is an issue of both human rights and women's rights. We explore this by presenting a case study of the effects of one innovative adult literacy program in Afghanistan that places men and women, as well as various ethnicities, together in the same classroom as…

  18. Prediction of the metabolic clearance of benzophenone-2, and its interaction with isoeugenol and coumarin using cryopreserved human hepatocytes in primary culture.

    PubMed

    de Sousa, Georges; Teng, Sophie; Salle-Siri, Romain; Pery, Alexandre; Rahmani, Roger

    2016-04-01

    Benzophenone-2 (BP2) is widely used as a UV screen in both industrial products and cosmetic formulations, where it is frequently found associated with fragrance compounds, such as isoeugenol and coumarin. BP2 is now recognized as an endocrine disruptor, but to date, no information has been reported on its fate in humans. The intrinsic clearance (Clint) and metabolic interactions of BP2 were explored using cryopreserved human hepatocytes in primary cultures. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters. The substrate depletion method demonstrated that isoeugenol was cleared more rapidly than BP2 or coumarin (Clint = 259, 94.7 and 0.40 μl/min/10(6) cells respectively). This vitro model was also used to study the metabolic interactions between BP2 and isoeugenol and coumarin. Coumarin exerted no effects on either isoeugenol or BP2 metabolism, because of its independent metabolic pathway (CYP2A6). Isoeugenol appeared to be a potent competitive substrate inhibitor of BP2 metabolism, equivalent to the specific UGT1A1 substrate: estradiol. Despite the fact that inhibition of UGT by xenobiotics is not usually considered to be a major concern, the involvement of UGT1A1 in BP2 metabolism may have pharmacokinetic and pharmacological consequences, due to the its polymorphisms in humans and its pure estrogenic effect.

  19. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    PubMed

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  20. A comparative study of bifidobacteria in human babies and adults

    PubMed Central

    KHONSARI, Shadi; SUGANTHY, Mayuran; BURCZYNSKA, Beata; DANG, Vu; CHOUDHURY, Manika; PACHENARI, Azra

    2015-01-01

    The composition and diversity of the gut microbiota are known to be different between babies and adults. The aim of this project was to compare the level of bifidobacteria between babies and adults and to investigate the influence of lifestyle factors on the level of this bacterium in the gut. During this study, the levels of bifidobacteria in 10 human babies below 2 years of age were compared with that of 10 human adults above 40 years. The level of bifidobacteria proved to be significantly higher in babies in comparison with adults. This investigation concluded that a combination of several factors, such as age, diet, and BMI, has an important effect on the level of bifidobacteria in adults, while in babies, a combination of diet and age may influence the level of intestinal bifidobacteria. PMID:27200263

  1. In vitro human metabolism of permethrin isomers alone or as a mixture and the formation of the major metabolites in cryopreserved primary hepatocytes.

    PubMed

    Willemin, M-E; Kadar, A; de Sousa, G; Leclerc, E; Rahmani, R; Brochot, C

    2015-06-01

    In vitro metabolism of permethrin, a pyrethroid insecticide, was assessed in primary human hepatocytes. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters and the clearances or formation rates of the permethrin isomers (cis- and trans-) and three metabolites, cis- and trans-3-(2,2 dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis- and trans-DCCA) and 3-phenoxybenzoic acid (3-PBA). Non-specific binding and the activity of the enzymes involved in permethrin's metabolism (cytochromes P450 and carboxylesterases) were quantified. Trans-permethrin was cleared more rapidly than cis-permethrin with a 2.6-factor (25.7±0.6 and 10.1±0.3 μL/min/10(6) cells respectively). A 3-factor was observed between the formation rates of DCCA and 3-PBA obtained from trans- and cis-permethrin. For both isomers, the rate of formation of DCCA was higher than the one of 3-PBA. The metabolism of the isomers in mixture was also quantified. The co-incubation of isomers at different ratios showed the low inhibitory potential of cis- and trans-permethrin on each other. The estimates of the clearances and the formation rates in the co-incubation condition did not differ from the estimates obtained with a separate incubation. These metabolic parameters may be integrated in physiologically based pharmacokinetic (PBPK) models to predict the fate of permethrin and metabolites in the human body.

  2. Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes

    PubMed Central

    Ramanathan, Aarti; Gusarova, Viktoria; Stahl, Neil; Gurnett-Bander, Anne; Kyratsous, Christos A.

    2016-01-01

    Background Proprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab. Methods and Results Flow cytometry and Western blotting of human hepatocyte Huh-7 cells showed that, although LDLR levels were reduced when cells were exposed to increasing PCSK9 concentrations, there was no correlation between total or surface CD81 levels and the presence and amount of soluble PCSK9. Moreover, inhibiting PCSK9 with the monoclonal antibody alirocumab did not affect expression levels of CD81. In an in vitro model of HCV entry, addition of soluble PCSK9 or treatment with alirocumab had no effect on the ability of either lentiviral particles bearing the HCV glycoproteins or JFH-1 based cell culture virus to enter hepatocytes. Consistent with these in vitro findings, no differences were observed in hepatic CD81 levels using in vivo mouse models, including Pcsk9-/- mice compared with wild-type controls and hyperlipidemic mice homozygous for human Pcsk9 and heterozygous for Ldlr deletion, treated with either alirocumab or isotype control antibody. Conclusion These results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry

  3. Neural stem cells in the adult human brain

    PubMed Central

    Gonzalez-Perez, Oscar

    2012-01-01

    For decades, it was believed that the adult brain was a quiescent organ unable to produce new neurons. At the beginning of the1960's, this dogma was challenged by a small group of neuroscientists. To date, it is well-known that new neurons are generated in the adult brain throughout life. Adult neurogenesis is primary confined to the subventricular zone (SVZ) of the forebrain and the subgranular zone of the dentate gyrus within the hippocampus. In both the human and the rodent brain, the primary progenitor of adult SVZ is a subpopulation of astrocytes that have stem-cell-like features. The human SVZ possesses a peculiar cell composition and displays important organizational differences when compared to the SVZ of other mammals. Some evidence suggests that the human SVZ may be not only an endogenous source of neural precursor cells for brain repair, but also a source of brain tumors. In this review, we described the cytoarchitecture and cellular composition of the SVZ in the adult human brain. We also discussed some clinical implications of SVZ, such as: stem-cell-based therapies against neurodegenerative diseases and its potential as a source of malignant cells. Understanding the biology of human SVZ and its neural progenitors is one of the crucial steps to develop novel therapies against neurological diseases in humans. PMID:23181200

  4. Humanities and the Adult Learner in an Information Society.

    ERIC Educational Resources Information Center

    Myers, Dale; Kamholtz, Jonathan

    Humanities courses have often been given little attention in continuing education for adults, possibly because they have been viewed as not "practical" or not "job-oriented" enough in our career-oriented, technologically advanced society. However, the humanities should be an integral part of our culture and of the lives of educated persons--a…

  5. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

    PubMed Central

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J.; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  6. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

    PubMed Central

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J.; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  7. Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition.

    PubMed

    Pettinato, Giuseppe; Ramanathan, Rajesh; Fisher, Robert A; Mangino, Martin J; Zhang, Ning; Wen, Xuejun

    2016-01-01

    Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. PMID:27616299

  8. Teaching Human Rights: Grades 7 through Adult.

    ERIC Educational Resources Information Center

    Shiman, David A.

    This curriculum resource on human rights is rooted in the United Nations Universal Declaration of Human Rights and seeks to help students understand the issues involved. Using the rights categories suggested by the Universal Declaration, this book offers new ways of teaching about familiar themes. The book contains activities to encourage students…

  9. Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer.

    PubMed

    den Braver-Sewradj, Shalenie P; den Braver, Michiel W; Vermeulen, Nico P E; Commandeur, Jan N M; Richert, Lysiane; Vos, J Chris

    2016-06-01

    Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity. PMID:26921663

  10. Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway.

    PubMed

    Dai, Chongshan; Li, Daowen; Gong, Lijing; Xiao, Xilong; Tang, Shusheng

    2016-01-01

    Furazolidone (FZD), a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS) and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications. PMID:27556439

  11. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.

    PubMed

    Fujimoto, Yasuyuki; Itabe, Hiroyuki; Sakai, Jun; Makita, Minoru; Noda, Junich; Mori, Masahiro; Higashi, Yusuke; Kojima, Shinichi; Takano, Tatsuya

    2004-02-01

    Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction. PMID:14741744

  12. Combined Toxic Effects of Polar and Nonpolar Chemicals on Human Hepatocytes (HepG2) Cells by Quantitative Property-Activity Relationship Modeling

    PubMed Central

    Kim, Ki-Woong; Won, Yong Lim; Park, Dong Jin; Kim, Young Sun; Jin, Eun Sil; Lee, Sung Kwang

    2016-01-01

    We determined the toxicity of mixtures of ethyl acetate (EA), isopropyl alcohol (IPA), methyl ethyl ketone (MEK), toluene (TOL) and xylene (XYL) with half-maximal effective concentration (EC50) values obtained using human hepatocytes cells. According to these data, quantitative property-activity relationships (QPAR) models were successfully proposed to predict the toxicity of mixtures by multiple linear regressions (MLR). The leave-one-out cross validation method was used to find the best subsets of descriptors in the learning methods. Significant differences in physico-chemical properties such as boiling point (BP), specific gravity (SG), Reid vapor pressure (rVP) and flash point (FP) were observed between the single substances and the mixtures. The EC50 of the mixture of EA and IPA was significantly lower than that of contained TOL and XYL. The mixture toxicity was related to the mixing ratio of MEK, TOL and XYL (MLR equation EC50 = 3.3081 − 2.5018 × TOL − 3.2595 × XYL − 12.6596 × MEK × XYL), as well as to BP, SG, VP and FP (MLR equation EC50 = 1.3424 + 6.2250 × FP − 7.1198 × SG × FP − 0.03013 × rVP × FP). These results suggest that QPAR-based models could accurately predict the toxicity of polar and nonpolar mixtures used in rotogravure printing industries.

  13. First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry.

    PubMed

    Swortwood, Madeleine J; Ellefsen, Kayla N; Wohlfarth, Ariane; Diao, Xingxing; Concheiro-Guisan, Marta; Kronstrand, Robert; Huestis, Marilyn A

    2016-07-01

    Novel psychoactive substances (NPS) are ever changing on the drug market, making it difficult for toxicology laboratory methods to stay current with so many new drugs. Recently, PV8, a synthetic pyrrolidinophenone, was detected in seized products in Japan (2013), The Netherlands (2014), and Germany (2014). There are no controlled PV8 administration studies, and no pharmacodynamic and pharmacokinetic data. The objective was to determine PV8's metabolic stability in human liver microsome (HLM) incubation and its metabolism following human hepatocyte incubation and high-resolution mass spectrometry (HRMS) with a Thermo Scientific Q-Exactive. Data were acquired with a full-scan data-dependent mass spectrometry method. Scans were thoroughly data mined with different data processing algorithms and analyzed in WebMetaBase. PV8 exhibited a relatively short 28.8 min half-life, with an intrinsic 24.2 μL/min/mg microsomal clearance. This compound is predicted to be an intermediate clearance drug with an estimated human 22.7 mL/min/kg hepatic clearance. Metabolic pathways identified in vitro included: hydroxylation, ketone reduction, carboxylation, N-dealkylation, iminium formation, dehydrogenation, N-oxidation, and carbonylation. The top three in vitro metabolic pathways were di-hydroxylation > ketone reduction > γ-lactam formation. Authentic urine specimen analyses revealed the top three metabolic pathways were aliphatic hydroxylation > ketone reduction + aliphatic hydroxylation > aliphatic carboxylation, although the most prominent peak was parent PV8. These data provide useful urinary metabolite targets (aliphatic hydroxylation, aliphatic hydroxylation + ketone reduction, aliphatic carboxylation, and di-hydroxylation) for forensic and clinical testing, and focus reference standard companies' synthetic efforts to provide commercially available standards needed for PV8 biological specimen testing. Graphical Abstract Top four PV8 metabolites identified in vitro

  14. First metabolic profile of PV8, a novel synthetic cathinone, in human hepatocytes and urine by high-resolution mass spectrometry.

    PubMed

    Swortwood, Madeleine J; Ellefsen, Kayla N; Wohlfarth, Ariane; Diao, Xingxing; Concheiro-Guisan, Marta; Kronstrand, Robert; Huestis, Marilyn A

    2016-07-01

    Novel psychoactive substances (NPS) are ever changing on the drug market, making it difficult for toxicology laboratory methods to stay current with so many new drugs. Recently, PV8, a synthetic pyrrolidinophenone, was detected in seized products in Japan (2013), The Netherlands (2014), and Germany (2014). There are no controlled PV8 administration studies, and no pharmacodynamic and pharmacokinetic data. The objective was to determine PV8's metabolic stability in human liver microsome (HLM) incubation and its metabolism following human hepatocyte incubation and high-resolution mass spectrometry (HRMS) with a Thermo Scientific Q-Exactive. Data were acquired with a full-scan data-dependent mass spectrometry method. Scans were thoroughly data mined with different data processing algorithms and analyzed in WebMetaBase. PV8 exhibited a relatively short 28.8 min half-life, with an intrinsic 24.2 μL/min/mg microsomal clearance. This compound is predicted to be an intermediate clearance drug with an estimated human 22.7 mL/min/kg hepatic clearance. Metabolic pathways identified in vitro included: hydroxylation, ketone reduction, carboxylation, N-dealkylation, iminium formation, dehydrogenation, N-oxidation, and carbonylation. The top three in vitro metabolic pathways were di-hydroxylation > ketone reduction > γ-lactam formation. Authentic urine specimen analyses revealed the top three metabolic pathways were aliphatic hydroxylation > ketone reduction + aliphatic hydroxylation > aliphatic carboxylation, although the most prominent peak was parent PV8. These data provide useful urinary metabolite targets (aliphatic hydroxylation, aliphatic hydroxylation + ketone reduction, aliphatic carboxylation, and di-hydroxylation) for forensic and clinical testing, and focus reference standard companies' synthetic efforts to provide commercially available standards needed for PV8 biological specimen testing. Graphical Abstract Top four PV8 metabolites identified in vitro

  15. Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury.

    PubMed

    Yang, Kyunghee; Pfeifer, Nathan D; Köck, Kathleen; Brouwer, Kim L R

    2015-05-01

    The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure

  16. New neurons in the adult striatum: from rodents to humans

    PubMed Central

    Inta, Dragos; Cameron, Heather A.; Gass, Peter

    2015-01-01

    Most neurons are generated during development and are not replaced during adulthood, even if they are lost to injury or disease. It is firmly established, however, that new neurons are generated in the dentate gyrus of the hippocampus of virtually all adult mammals, including humans [1]. Many questions still remain, however, regarding adult neurogenesis in other brain regions and particularly in humans, where standard birthdating methods are not generally feasible. Exciting recent evidence indicates that calretinin-expressing interneurons are added to the adult human striatum at a substantial rate [2]. The role of new neurons is unknown, but studies in rodents will be able to further elucidate their identity and origin and then begin to understand their regulation and function. PMID:26298770

  17. Hepatocyte Isolation After Laparoscopic Liver Resection.

    PubMed

    Horner, Rosa; Kluge, Martin; Gassner, Joseph; Nösser, Maximilian; Major, Rebeka Dalma; Reutzel-Selke, Anja; Leder, Annekatrin K; Struecker, Benjamin; Morgul, Mehmet H; Pratschke, Johann; Sauer, Igor M; Raschzok, Nathanael

    2016-09-01

    Liver tissue obtained from partial hepatectomy is a common source for isolation of primary human hepatocytes. Until now, liver resections were most commonly performed by conventional open surgery. Although the laparoscopic approach is currently emerging in liver surgery, data on the outcome of hepatocyte isolation from laparoscopically resected liver tissue are not available. A total of 22 hepatocyte isolations were performed using the two-step collagenase perfusion technique from October 2015 to March 2016. Liver tissue was obtained from n = 15 open liver resections (OLRs) and n = 7 laparoscopic liver resections (LLRs). Isolation parameters (cell yield, viability, and Percoll survival) were assessed and hepatocyte function (plating efficiency, urea, albumin, and aspartate aminotransferase) was measured over a culture period of 6 days (OLR: n = 13; LLR: n = 3). Total cell yield (OLR: 36.81 ± 6.77 × 10(6) cells/g vs. LLR 16.84 ± 10.66 × 10(6) cells/g, p = 0.0318) as well as viable yield (OLR 31.70 ± 6.05 × 10(6) cells/g vs. LLR 14.70 ± 9.89 × 10(6) cells/g, p = 0.0260) was significantly higher in the OLR group. Subgroup analysis revealed that the worse outcome of isolation of laparoscopically resected liver tissue was associated with right-lateral LLRs, whereas hepatocyte isolation from left-lateral LLRs was as effective as from open surgery. Hepatocyte function did not differ between hepatocytes from openly resected versus left-lateral laparoscopically resected liver tissue. We here present the first data on hepatocyte isolation from laparoscopic liver surgery. Although the overall outcome is worse compared with open surgery, our data suggest that liver tissue from laparoscopic resection of the left lobe is an excellent source for primary human hepatocytes. PMID:27481660

  18. Adult human metapneumonovirus (hMPV) pneumonia mimicking Legionnaire's disease.

    PubMed

    Cunha, Burke A; Irshad, Nadia; Connolly, James J

    2016-01-01

    In adults hospitalized with viral pneumonias the main differential diagnostic consideration is influenza pneumonia. The respiratory viruses causing viral influenza like illnesses (ILIs), e.g., RSV may closely resemble influenza. Rarely, extrapulmonary findings of some ILIs may resemble Legionnaire's disease (LD), e.g., adenovirus, human parainfluenza virus (HPIV-3). We present a most unusual case of human metapneumonovirus pneumonia (hMPV) with some characteristic extrapulmonary findings characteristic of LD, e.g., relative bradycardia, as well as mildly elevated serum transaminases and hyphosphatemia. We believe this is the first reported case of hMPV pneumonia in a hospitalized adult that had some features of LD.

  19. Adult human metapneumonovirus (hMPV) pneumonia mimicking Legionnaire's disease.

    PubMed

    Cunha, Burke A; Irshad, Nadia; Connolly, James J

    2016-01-01

    In adults hospitalized with viral pneumonias the main differential diagnostic consideration is influenza pneumonia. The respiratory viruses causing viral influenza like illnesses (ILIs), e.g., RSV may closely resemble influenza. Rarely, extrapulmonary findings of some ILIs may resemble Legionnaire's disease (LD), e.g., adenovirus, human parainfluenza virus (HPIV-3). We present a most unusual case of human metapneumonovirus pneumonia (hMPV) with some characteristic extrapulmonary findings characteristic of LD, e.g., relative bradycardia, as well as mildly elevated serum transaminases and hyphosphatemia. We believe this is the first reported case of hMPV pneumonia in a hospitalized adult that had some features of LD. PMID:26988110

  20. Exploring the cell signalling in hepatocyte differentiation.

    PubMed

    Vasconcellos, Rebecca; Alvarenga, Érika C; Parreira, Ricardo C; Lima, Swiany S; Resende, Rodrigo R

    2016-11-01

    The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic functions and the synthesis of substances important to the body. Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesoderm, which can be obtained from various tissues. Under certain conditions, MSCs can differentiate into several cell types, including hepatocytes. Post-transcriptional regulations of liver development signalling and hepatocyte differentiation have been demonstrated. At the post-transcriptional level, microRNAs have emerged as precursors for determining cell fate during differentiation. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of gene expression. They can determine the stem cell fate by repressing the translation of target mRNAs. In this review, we outline signalling pathways involved in stem cell differentiation to hepatocytes and its interplay with liver development. Hepatic differentiation models in two-dimensional and three-dimensional cultures used to analyse signalling mechanisms will be described. We also highlight the possible miRNAs involved in this process and the transdifferentiation signalling mechanisms present in hepatocytes.

  1. Exploring the cell signalling in hepatocyte differentiation.

    PubMed

    Vasconcellos, Rebecca; Alvarenga, Érika C; Parreira, Ricardo C; Lima, Swiany S; Resende, Rodrigo R

    2016-11-01

    The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic functions and the synthesis of substances important to the body. Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesoderm, which can be obtained from various tissues. Under certain conditions, MSCs can differentiate into several cell types, including hepatocytes. Post-transcriptional regulations of liver development signalling and hepatocyte differentiation have been demonstrated. At the post-transcriptional level, microRNAs have emerged as precursors for determining cell fate during differentiation. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of gene expression. They can determine the stem cell fate by repressing the translation of target mRNAs. In this review, we outline signalling pathways involved in stem cell differentiation to hepatocytes and its interplay with liver development. Hepatic differentiation models in two-dimensional and three-dimensional cultures used to analyse signalling mechanisms will be described. We also highlight the possible miRNAs involved in this process and the transdifferentiation signalling mechanisms present in hepatocytes. PMID:27555287

  2. In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

    PubMed

    Hua, Xin; Peng, Xiao; Tan, Shengnan; Li, Chunying; Wang, Wei; Luo, Meng; Fu, Yujie; Zu, Yuangang; Smyth, Hugh

    2014-10-29

    Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA. PMID:25272989

  3. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A.

  4. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A. PMID:26341324

  5. Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes.

    PubMed

    Rasmussen, Martin Krøyer; Balaguer, Patrick; Ekstrand, Bo; Daujat-Chavanieu, Martine; Gerbal-Chaloin, Sabine

    2016-01-01

    Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway. PMID:27138278

  6. Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes

    PubMed Central

    Balaguer, Patrick; Ekstrand, Bo; Daujat-Chavanieu, Martine; Gerbal-Chaloin, Sabine

    2016-01-01

    Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway. PMID:27138278

  7. Hepatocyte growth factor increases the invasive potential of PC-3 human prostate cancer cells via an ERK/MAPK and Zeb-1 signaling pathway

    PubMed Central

    HAN, YILI; LUO, YONG; WANG, YONGXING; CHEN, YATONG; LI, MINGCHUAN; JIANG, YONGGUANG

    2016-01-01

    Hepatocyte growth factor (HGF) has been implicated in epithelial-mesenchymal transition (EMT) in numerous types of cancer. However, to the best of our knowledge, there has been no previous evidence that HGF has a role in prostate cancer. The present study aimed to investigate the effect of HGF on EMT and invasive potential, as well as the underlying molecular mechanisms, in a human prostate cancer cell line. Therefore, PC-3 cells were treated with various concentrations of HGF for varying durations. EMT-associated proteins, including E-cadherin and vimentin, were examined by western blot analysis. The effects of HGF on cell proliferation, migration, invasion and tumorigenicity were assessed using MTT, wound-healing, Transwell and soft-agar assays. Subsequently, the role of c-Met in the mediation of EMT-like changes was investigated using reverse transcription-polymerase chain reaction, western blot analysis and gene knockdown by small interfering RNA. Finally, western blot analysis was used to quantify the expression of a downstream transcription factor and extracellular signal-related kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway proteins. The results indicated that treatment with HGF induced EMT-like changes and enhanced the invasive potential of PC-3 cells. There was an increase in the expression of ERK, phosphorylated-ERK and zinc finger E-box binding homeobox-1 (Zeb-1), suggesting that EMT-like changes may be mediated through the ERK/MAPK and Zeb-1 signaling pathway. Furthermore, HGF-mediated EMT-like changes were associated with c-Met activation, and these changes were able to be blocked by c-Met knockdown. The present study demonstrated that HGF-induced EMT increased the invasive potential of PC-3 human prostate cancer cells through activating the ERK/MAPK and Zeb-1 signaling pathway. PMID:26870279

  8. Cholangiocarcinomas can originate from hepatocytes in mice

    PubMed Central

    Fan, Biao; Malato, Yann; Calvisi, Diego F.; Naqvi, Syed; Razumilava, Nataliya; Ribback, Silvia; Gores, Gregory J.; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Willenbring, Holger

    2012-01-01

    Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy. PMID:22797301

  9. Late Pleistocene adult mortality patterns and modern human establishment

    PubMed Central

    Trinkaus, Erik

    2011-01-01

    The establishment of modern humans in the Late Pleistocene, subsequent to their emergence in eastern Africa, is likely to have involved substantial population increases, during their initial dispersal across southern Asia and their subsequent expansions throughout Africa and into more northern Eurasia. An assessment of younger (20–40 y) versus older (>40 y) adult mortality distributions for late archaic humans (principally Neandertals) and two samples of early modern humans (Middle Paleolithic and earlier Upper Paleolithic) provides little difference across the samples. All three Late Pleistocene samples have a dearth of older individuals compared with Holocene ethnographic/historical samples. They also lack older adults compared with Holocene paleodemographic profiles that have been critiqued for having too few older individuals for subsistence, social, and demographic viability. Although biased, probably through a combination of preservation, age assessment, and especially Pleistocene mobility requirements, these adult mortality distributions suggest low life expectancy and demographic instability across these Late Pleistocene human groups. They indicate only subtle and paleontologically invisible changes in human paleodemographics with the establishment of modern humans; they provide no support for a life history advantage among early modern humans. PMID:21220336

  10. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    PubMed

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

  11. Profiling primaquine metabolites in primary human hepatocytes by UPLC-QTOF-MS with 13c stable isotope labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Primaquine (PQ) is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. However, hemolytic anemia is a dose-limiting side effect of primaquine therapy that limits its widespread use. The major plasma metabolite identified in humans and animals, car...

  12. Novel surface markers directed against adult human gallbladder.

    PubMed

    Galivo, Feorillo H; Dorrell, Craig; Grompe, Maria T; Zhong, YongPing; Streeter, Philip R; Grompe, Markus

    2015-07-01

    Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1(+)SOX9(+)) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.

  13. Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults

    PubMed Central

    Tong, Ann-Jay; Kollmann, Tobias R.; Smale, Stephen T.

    2015-01-01

    A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development. PMID:26147648

  14. Taraxacum mongolicum extract exhibits a protective effect on hepatocytes and an antiviral effect against hepatitis B virus in animal and human cells.

    PubMed

    Jia, Yuan-Yuan; Guan, Rong-Fa; Wu, Yi-Hang; Yu, Xiao-Ping; Lin, Wen-Yan; Zhang, Yong-Yong; Liu, Tao; Zhao, Jun; Shi, Shu-Yun; Zhao, Yu

    2014-04-01

    In order to validate the antiviral effect against hepatitis B virus (HBV) of Taraxacum mongolicum (T. mongolicum), the protective effect on hepatocytes, and antiviral properties against duck hepatitis B virus (DHBV) and HBV of T. mongolicum extract (TME) were evaluated in chemically-injured neonatal rat hepatocytes, DHBV-infected duck fetal hepatocytes and HBV-transfected HepG2.2.15 cells, respectively. The results demonstrated that TME at 50-100 µg/ml improved D-galactosamine (D-GalN), thioacetamide (TAA) and tert-butyl hydroperoxide (t-BHP)-injured rat hepatocytes, and produced protection rates of 42.2, 34.6 and 43.8% at 100 µg/ml, respectively. Furthermore, TME at 1-100 µg/ml markedly inhibited DHBV DNA replication. Additionally, TME at 25-100 µg/ml reduced HBsAg and HBeAg levels and produced inhibition rates of 91.39 and 91.72% at 100 µg/ml, respectively. TME markedly inhibited HBV DNA replication at 25-100 µg/ml. The results demonstrate the potent antiviral effect of T. mongolicum against HBV effect. The protective of TME effect on hepatocytes may be achieved by its ability to ameliorate oxidative stress. The antiviral properties of TME may contribute to blocking protein synthesis steps and DNA replication. Furthermore, major components of TME were quantificationally analyzed. These data provide scientific evidence supporting the traditional use of TME in the treatment of hepatitis.

  15. Taraxacum mongolicum extract exhibits a protective effect on hepatocytes and an antiviral effect against hepatitis B virus in animal and human cells.

    PubMed

    Jia, Yuan-Yuan; Guan, Rong-Fa; Wu, Yi-Hang; Yu, Xiao-Ping; Lin, Wen-Yan; Zhang, Yong-Yong; Liu, Tao; Zhao, Jun; Shi, Shu-Yun; Zhao, Yu

    2014-04-01

    In order to validate the antiviral effect against hepatitis B virus (HBV) of Taraxacum mongolicum (T. mongolicum), the protective effect on hepatocytes, and antiviral properties against duck hepatitis B virus (DHBV) and HBV of T. mongolicum extract (TME) were evaluated in chemically-injured neonatal rat hepatocytes, DHBV-infected duck fetal hepatocytes and HBV-transfected HepG2.2.15 cells, respectively. The results demonstrated that TME at 50-100 µg/ml improved D-galactosamine (D-GalN), thioacetamide (TAA) and tert-butyl hydroperoxide (t-BHP)-injured rat hepatocytes, and produced protection rates of 42.2, 34.6 and 43.8% at 100 µg/ml, respectively. Furthermore, TME at 1-100 µg/ml markedly inhibited DHBV DNA replication. Additionally, TME at 25-100 µg/ml reduced HBsAg and HBeAg levels and produced inhibition rates of 91.39 and 91.72% at 100 µg/ml, respectively. TME markedly inhibited HBV DNA replication at 25-100 µg/ml. The results demonstrate the potent antiviral effect of T. mongolicum against HBV effect. The protective of TME effect on hepatocytes may be achieved by its ability to ameliorate oxidative stress. The antiviral properties of TME may contribute to blocking protein synthesis steps and DNA replication. Furthermore, major components of TME were quantificationally analyzed. These data provide scientific evidence supporting the traditional use of TME in the treatment of hepatitis. PMID:24481875

  16. [The existence vomeronasal organ in adult humans].

    PubMed

    Rapiejko, Piotr; Zielnik-Jurkiewicz, Beata; Wojdas, Andrzej; Ratajczak, Jan; Jurkiewicz, Dariusz

    2007-01-01

    The influence of chemical substances (feromones) on human emotional and physical condition has fascinated psychologists, sexuologists and laryngologists since centurie. Literature conveys inconsistent information on vomeronasal organ (VNO) occurrence in humans. This organ is often called Jacobson's, and 2 symmetrical openings leading into it, located on both sides of septum, are called Ruyasch's ducts. The aim of the study was to analyze vomeronasal organ occurrence in humans in relation to age and sex. The study was conducted in a group of 634 patients, aged 18-80 years. All patients underwent routine ENT examination including rhinoscopy, nasal cavity examination with usage of 2.5x magnification lens (surgical glasses) and surgical microscope with 10x magnification. All persons had nasal cavities examined endoscopically. Every time presence of vomeronasal organ openings, along with localization, size and symmetry of these was noted. Subjects, who presented Jacobson's organ, were asked to fill a questionnaire concerning influence of smells on erotic sensations. Vomeronasal organ was fund in 312 persons, that is 49.21%. In 83.65% of cases vomeronasal organ opening size was smaller than 0.2 mm, what restricted its visibility to usage of magnifying lens, microscope, or endoscope. In 16.34% of cases only vomeronasal organ ducts openings were well visible in routine rhinoscopy without magnification. Vomeronasal organ was found more often in men than women. VNO was significantly more rare in patients with nasal septal deviation. In these cases, vomeronasal organ was usually found unilaterally, in all the cases on the concave side of deviated nasal septum. PMID:18260256

  17. Expansion of Multipotent Stem Cells from the Adult Human Brain

    PubMed Central

    Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

    2013-01-01

    The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

  18. Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

    PubMed Central

    Vuong, Linh M.; Chellappa, Karthikeyani; Dhahbi, Joseph M.; Deans, Jonathan R.; Fang, Bin; Bolotin, Eugene; Titova, Nina V.; Hoverter, Nate P.; Spindler, Stephen R.; Waterman, Marian L.

    2015-01-01

    The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways. PMID:26240283

  19. First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

    PubMed Central

    Wohlfarth, Ariane; Pang, Shaokun; Zhu, Mingshe; Gandhi, Adarsh S.; Scheidweiler, Karl B.; Liu, Hua-fen; Huestis, Marilyn A.

    2015-01-01

    BACKGROUND Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source. METHODS We incubated 10 μmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5). RESULTS XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2′-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2′-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated. CONCLUSIONS These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations. PMID:24014837

  20. Hepatocyte nuclear factor 1 and C/EBP are essential for the activity of the human apolipoprotein B gene second-intron enhancer.

    PubMed Central

    Brooks, A R; Levy-Wilson, B

    1992-01-01

    The tissue-specific transcriptional enhancer of the human apolipoprotein B gene contains multiple protein-binding sites spanning 718 bp. Most of the enhancer activity is found in a 443-bp fragment (+621 to +1064) that is located entirely within the second intron of the gene. Within this fragment, a 147-bp region (+806 to +952) containing a single 97-bp DNase I footprint exhibits significant enhancer activity. We now report that this footprint contains four distinct protein-binding sites that have the potential to bind nine distinct liver nuclear proteins. One of these proteins was identified as hepatocyte nuclear factor 1 (HNF-1), which binds with relatively low affinity to the 5' half of a 20-bp palindrome located at the 5' end of the large footprint. A binding site for C/EBP (or one of the related proteins that recognize similar sequences) was identified in the center of the 97-bp footprint. This binding site is coincident or overlaps with the binding sites for five other proteins, two of which appear to be distinct from the C/EBP-related family of proteins. The binding site for a nuclear factor designated protein I is located between the HNF-1 and C/EBP binding sites. Finally, the 3'-most 15 bp of the footprinted sequence contain a binding site for another nuclear protein, which we have called protein II. Mutations that abolish the binding of either HNF-1, protein II, or the C/EBP-related proteins severely reduce enhancer activity. However, deletion experiments demonstrated that neither the HNF-1-binding site alone, nor the combination of binding sites for HNF-1, protein I, and C/EBP, nor the C/EBP-binding site plus the protein II-binding site is sufficient to enhance transcription from a strong apolipoprotein B promoter. Rather, HNF-1 and C/EBP act synergistically with protein II to enhance transcription of the apolipoprotein B gene. Images PMID:1545795

  1. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System

    PubMed Central

    Knöspel, Fanny; Jacobs, Frank; Freyer, Nora; Damm, Georg; De Bondt, An; van den Wyngaert, Ilse; Snoeys, Jan; Monshouwer, Mario; Richter, Marco; Strahl, Nadja; Seehofer, Daniel; Zeilinger, Katrin

    2016-01-01

    Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro. PMID:27092500

  2. Human pancreatic polypeptide in children and young adults.

    PubMed

    Hanukoglu, A; Chalew, S; Kowarski, A A

    1990-01-01

    Measurement of human pancreatic polypeptide may be useful for assessment of gastrointestinal function, integrity of the parasympathetic nervous system or screening for endocrine neoplasia. In adults hPP levels have been reported to increase with age. However hPP levels throughout childhood have not been well characterized in comparison with the adult range. We studied fasting human pancreatic polypeptide (hPP) from 45 pediatric patients, from infancy - 15 years, and 18 older adolescents and adults aged 16-45 years. The mean hPP level of children (233 +/- 147 pg/ml) was significantly higher than that (113 +/- 35 pg/ml) of adults (P less than .0001). There was no difference in mean hPP levels of children with normal growth hormone secretion compared to growth hormone deficient patients. There was no effect of gender or body mass index on hPP levels. We conclude that fasting hPP levels must be interpreted with respect to the age of the subject, children particularly, in that preteens may have higher fasting levels than older teenagers and adults.

  3. Human pancreatic polypeptide in children and young adults.

    PubMed

    Hanukoglu, A; Chalew, S; Kowarski, A A

    1990-01-01

    Measurement of human pancreatic polypeptide may be useful for assessment of gastrointestinal function, integrity of the parasympathetic nervous system or screening for endocrine neoplasia. In adults hPP levels have been reported to increase with age. However hPP levels throughout childhood have not been well characterized in comparison with the adult range. We studied fasting human pancreatic polypeptide (hPP) from 45 pediatric patients, from infancy - 15 years, and 18 older adolescents and adults aged 16-45 years. The mean hPP level of children (233 +/- 147 pg/ml) was significantly higher than that (113 +/- 35 pg/ml) of adults (P less than .0001). There was no difference in mean hPP levels of children with normal growth hormone secretion compared to growth hormone deficient patients. There was no effect of gender or body mass index on hPP levels. We conclude that fasting hPP levels must be interpreted with respect to the age of the subject, children particularly, in that preteens may have higher fasting levels than older teenagers and adults. PMID:2307392

  4. Predictors of food preferences in adult humans.

    PubMed

    Logue, A W; Smith, M E

    1986-06-01

    Predictors of preferences for a wide variety of foods were examined in 303 male and female human subjects ranging from 14-68 years of age. The subjects completed questionnaires which requested information on the subject's sex, age, thinness, sensation seeking and ethnic background, as well as on the subjects' food preferences. Largely consistent with previous studies, female subjects reported higher preferences for low-calorie foods, candy and wine, and lower preferences for meat, beer, spicy foods and milk. Younger subjects reported higher preferences for sweet foods and lower preferences for foods such as chili pepper that are considered acquired tastes. Thinner subjects tended to rate both sweet foods and meat lower than did other subjects. Preferences for spicy foods or foods likely to cause illness were positively correlated with sensation seeking while preferences for sweet or bland foods or foods unlikely to cause illness were negatively correlated with sensation seeking. Subjects for whom the primary cuisine on which they were raised was Oriental cuisine preferred alcoholic beverages and non-Oriental foods less than did other subjects. A factor analysis of the food preferences yielded ten factors including those for meat and potatoes, alcohol, spices and junk food. Data on predictors of food preferences can assist research on the determinants of food preferences, however much of the variance in food preferences remains to be explained.

  5. Evaluation of calibration curve-based approaches to predict clinical inducers and noninducers of CYP3A4 with plated human hepatocytes.

    PubMed

    Zhang, J George; Ho, Thuy; Callendrello, Alanna L; Clark, Robert J; Santone, Elizabeth A; Kinsman, Sarah; Xiao, Deqing; Fox, Lisa G; Einolf, Heidi J; Stresser, David M

    2014-09-01

    Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, Cmax/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6β-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R(2) and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6β-hydroxylase activity was

  6. Human Adult Cortical Reorganization and Consequent Visual Distortion

    PubMed Central

    Dilks, Daniel D.; Serences, John T.; Rosenau, Benjamin J.; Yantis, Steven; McCloskey, Michael

    2009-01-01

    Neural and behavioral evidence for cortical reorganization in the adult somatosensory system after loss of sensory input (e.g., amputation) has been well documented. In contrast, evidence for reorganization in the adult visual system is far less clear: neural evidence is the subject of controversy, behavioral evidence is sparse, and studies combining neural and behavioral evidence have not previously been reported. Here, we report converging behavioral and neuroimaging evidence from a stroke patient (B.L.) in support of cortical reorganization in the adult human visual system. B.L.’s stroke spared the primary visual cortex (V1), but destroyed fibers that normally provide input to V1 from the upper left visual field (LVF). As a consequence, B.L. is blind in the upper LVF, and exhibits distorted perception in the lower LVF: stimuli appear vertically elongated, toward and into the blind upper LVF. For example, a square presented in the lower LVF is perceived as a rectangle extending upward. We hypothesized that the perceptual distortion was a consequence of cortical reorganization in V1. Extensive behavioral testing supported our hypothesis, and functional magnetic resonance imaging (fMRI) confirmed V1 reorganization. Together, the behavioral and fMRI data show that loss of input to V1 after a stroke leads to cortical reorganization in the adult human visual system, and provide the first evidence that reorganization of the adult visual system affects visual perception. These findings contribute to our understanding of the human adult brain’s capacity to change and has implications for topics ranging from learning to recovery from brain damage. PMID:17804619

  7. Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

    PubMed Central

    Kim, BoRa; Park, Byung Hee; Shin, Kum-Joo; Song, Seong-Won; Kim, Jung Ju; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

    2015-01-01

    The growth factors derived from the microenvironment create an environment conducive to tumor growth and survival. HGF deprivation using neutralizing antibody enhanced chemosensitivity in colorectal cancer cells (CRC). We determined secreted HGF in fibroblast conditioned medium (CM). Combination treatment of anti-HGF antibody and irinotecan (CPT-11) directly enhanced CPT-11 sensitivity in CRC. We generated xenograft in NOD/SCID mice inoculating HCT-116 human colorectal cancer cells subcutaneously with or without fibroblast. We found that the combination of CPT-11 and anti-HGF antibody induced marked suppression of tumor development. These results suggest that HGF produced by fibroblast induce CPT-11 resistance, and that anti-HGF antibody abrogate such resistance in vivo. fibroblast-derived HGF is important determinant of chemoresistance. Anti-HGF monoclonal antibody treatment confirmed the importance of this growth factor for chemoresistance in CRC. These results present new options toward the early diagnosis of chemoresistance and suggest novel combinations of chemotherapy and anti-HGF agents to prevent or significantly delay the onset of therapy resistance. PMID:26090722

  8. Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies

    PubMed Central

    Devhare, Pradip B.; Desai, Swapnil; Lole, Kavita S.

    2016-01-01

    Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20–25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies. PMID:27230536

  9. Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF.

    PubMed

    Woo, Jong Kyu; Kang, Ju-Hee; Kim, BoRa; Park, Byung Hee; Shin, Kum-Joo; Song, Seong-Won; Kim, Jung Ju; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

    2015-09-15

    The growth factors derived from the microenvironment create an environment conducive to tumor growth and survival. HGF deprivation using neutralizing antibody enhanced chemosensitivity in colorectal cancer cells (CRC). We determined secreted HGF in fibroblast conditioned medium (CM). Combination treatment of anti-HGF antibody and irinotecan (CPT-11) directly enhanced CPT-11 sensitivity in CRC. We generated xenograft in NOD/SCID mice inoculating HCT-116 human colorectal cancer cells subcutaneously with or without fibroblast. We found that the combination of CPT-11 and anti-HGF antibody induced marked suppression of tumor development. These results suggest that HGF produced by fibroblast induce CPT-11 resistance, and that anti-HGF antibody abrogate such resistance in vivo. fibroblast-derived HGF is important determinant of chemoresistance. Anti-HGF monoclonal antibody treatment confirmed the importance of this growth factor for chemoresistance in CRC. These results present new options toward the early diagnosis of chemoresistance and suggest novel combinations of chemotherapy and anti-HGF agents to prevent or significantly delay the onset of therapy resistance.

  10. Pleiotropic effects of gold(I) mixed-ligand complexes of 9-deazahypoxanthine on transcriptional activity of receptors for steroid hormones, nuclear receptors and xenoreceptors in human hepatocytes and cell lines.

    PubMed

    Kubešová, Kateřina; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-10-01

    Development of metal-based compounds is an important research avenue in anti-cancer and anti-inflammatory drug discovery. Here we examined the effects of three gold (I) mixed-ligand complexes with the general formula [Au(Ln)(PPh3)] (1, 2, 3) involving triphenylphosphine (PPh3) and a deprotonated form of O-substituted derivatives of 9-deazahypoxanthine (Ln) on the transcriptional activity of aryl hydrocarbon receptor (AhR), androgen receptor (AR), glucocorticoid receptor (GR), thyroid receptor (TR), pregnane X receptor (PXR) and vitamin D receptor (VDR), employing gene reporter assays. In addition, we measured mRNA (RT-PCR) and protein (western blot) expression of target genes for those receptors, including drug-metabolizing P450s, in primary human hepatocytes and cancer cell lines LS180 and HepG2. The tested compounds displayed anti-glucocorticoid effects, as revealed by inhibition of dexamethasone-inducible transcriptional activity of GR and down-regulation of tyrosine aminotransferase. All the compounds slightly and dose-dependently activated PXR and AhR, and moderately induced CYP3A4 and CYP1A1/2 genes in human hepatocytes and LS180 cells. The complexes antagonized basal and ligand-activated AR and VDR, indicating inverse agonist behaviour. Both basal and thyroid hormone-inducible transcriptional activity of TR was dose-dependently increased by all tested compounds. In contrast, the expression of SPOT14 mRNA was decreased by tested compounds in human hepatocytes and HepG2 cells. In conclusion, if intended for human pharmacotherapy, the potential of the complexes 1-3 to influence studied receptors should be taken in account. PMID:27318977

  11. Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

    PubMed

    Earnest-Silveira, L; Chua, B; Chin, R; Christiansen, D; Johnson, D; Herrmann, S; Ralph, S A; Vercauteren, K; Mesalam, A; Meuleman, P; Das, S; Boo, I; Drummer, H; Bock, C-T; Gowans, E J; Jackson, D C; Torresi, Joseph

    2016-08-01

    An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV. PMID:27147296

  12. SELENIUM COMPOUNDS MODULATE THE ACTIVITY OF RECOMBINANT RAT ASIII-METHYLTRANSFERASE AND THE METHYLATION OF ARSENITE BY RAT AND HUMAN HEPATOCYTES

    EPA Science Inventory

    Abstract

    Formation of methylated metabolites is a critical step in the metabolism of inorganic arsenic or selenium. We have previously shown that under conditions of a concurrent exposure selenite inhibits methylation of arsenite by cultured rat hepatocytes. Here, we com...

  13. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  14. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  15. Ultrastructural characteristics of human adult and infant cerebral cortical neurons.

    PubMed Central

    Ong, W Y; Garey, L J

    1991-01-01

    Biopsy specimens of human cerebral cortex from three adults and two infants were studied by correlating their light microscopic features in semithin sections with their ultrastructural characteristics. There was good tissue preservation, due to a minimum delay between obtaining the specimens and fixation. Pyramidal cells had a prominent apical dendrite, fine heterochromatin clumps in the nucleus and generally small numbers of cytoplasmic organelles, except for numerous free ribosomes in some of the large pyramids of Layers III to VI. Non-pyramidal cells lacked an apical dendrite and were further classified, on size and ultrastructure, into small, medium and large types. Large numbers of asymmetrical and symmetrical synapses were present in the neuropil but very few axosomatic synapses were found in the human cerebral cortex compared with subhuman primates and other mammals. Some symmetrical synapses were characterised by the presence of wide pre- and postsynaptic densities. The same general features of the adult cortex were also encountered in the infant, with certain exceptions. Many of the infant neurons had less densely packed heterochromatin, but greater numbers of free ribosomes, compared with the adult, and lipofuscin was absent. There was a total absence of myelinated fibres from the infant cortex; more large diameter dendrites were present than in the adult and axosomatic synapses were commoner. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 PMID:2050578

  16. Hepatitis B Virus (HBV) Virion and Covalently Closed Circular DNA Formation in Primary Tupaia Hepatocytes and Human Hepatoma Cell Lines upon HBV Genome Transduction with Replication-Defective Adenovirus Vectors

    PubMed Central

    Ren, Shaotang; Nassal, Michael

    2001-01-01

    Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range, there is as yet no practical small-animal system for HBV infection. The hosts of the few related animal viruses, including woodchuck hepatitis B virus and duck hepatitis B virus, are either difficult to keep or only distantly related to humans. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To bypass restrictions during the initial entry phase, we used recombinant replication-defective adenovirus vectors, either with or without a green fluorescent protein marker gene, to deliver complete HBV genomes into primary tupaia hepatocytes. Here we show that these cells, like the human hepatoma cell lines HepG2 and Huh7, are efficiently transduced by the vectors and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. We further demonstrate that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. These data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection. PMID:11152483

  17. [Generation of new nerve cells in the adult human brain].

    PubMed

    Poulsen, Frantz Rom; Meyer, Morten; Rasmussen, Jens Zimmer

    2003-03-31

    Generation of new nerve cells (neurogenesis) is normally considered to be limited to the fetal and early postnatal period. Thus, damaged nerve cells are not expected to be replaced by generation of new cells. The brain is, however, more plastic than previously assumed. This also includes neurogenesis in the adult human brain. In particular two brain regions show continuous division of neural stem and progenitor cells generating neurons and glial cells, namely the subgranular zone of the dentate gyrus and the subventricular zones of the lateral ventricles. From the latter region newly generated neuroblasts (immature nerve cells) migrate toward the olfactory bulb where they differentiate into neurons. In the dentate gyrus the newly generated neurons become functionally integrated in the granule cell layer, where they are believed to be of importance to learning and memory. It is at present not known whether neurogenesis in the adult human brain can be manipulated for specific repair after brain damage.

  18. Epidermal growth factor receptor in adult human dorsal root ganglia.

    PubMed

    Huerta, J J; Diaz-Trelles, R; Naves, F J; Llamosas, M M; Del Valle, M E; Vega, J A

    1996-09-01

    Transforming growth factor-alpha (TGFalpha) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG. However, it remains to be elucidated whether all DRG neurons express EGFr or whether differences exist among neuronal subtypes. This study was undertaken to investigate these topics in adult human DRG using immunoblotting, and combined immunohistochemistry and image analysis techniques. A mouse monoclonal antibody (clone F4) mapping within the intracytoplasmic domain of EGFr was used. Immunoblotting revealed two main proteins with estimated molecular masses of approximately/equal to 65 kDa and 170 kDa, and thus consistent with the full-length EGFr. Additional protein bands were also encountered. Light immunohistochemistry revealed specific immunoreactivity (IR) for EGFr-like proteins in most (86%) primary sensory neurons, the intensity of immunostaining being stronger in the small- and intermediate-sized ones. Furthermore, EGFr-like IR was also observed in the satellite glial cells of the ganglia as well as in the intraganglionic and dorsal root Schwann cells. Taken together, our findings demonstrate that EGFr, and other related proteins containing the epitope labeled with the antibody F4, are responsible for the EGFr IR reported in DRG. Furthermore, we demonstrated heterogeneity in the expression of EGFr-like IR in adult human primary sensory neurons, which suggests different responsiveness to their ligands.

  19. MR Assessment of Myocardial Perfusion, Viability, and Function after Intramyocardial Transfer of VM202, a New Plasmid Human Hepatocyte Growth Factor in Ischemic Swine Myocardium1

    PubMed Central

    Saeed, Maythem; Martin, Alastair; Ursell, Phillip; Do, Loi; Bucknor, Matt; Higgins, Charles B.; Saloner, David

    2008-01-01

    Purpose: VM202, a newly constructed plasmid human hepatocyte growth factor, was transferred intramyocardially after infarction for the purpose of evaluating this strategy as a therapeutic approach for protection from left ventricular (LV) remodeling. Materials and Methods: The institutional animal care and use committee approved this study. Pigs underwent coronary artery occlusion and reperfusion and served as either control (n = 8) or VM202-treated (n = 8) animals. VM202 was transferred intramyocardially into four infarcted and four periinfarcted sites. Cardiac magnetic resonance (MR) imaging (cine, perfusion, delayed enhancement) was performed in acute (3 days) and chronic (50 days ± 3 [standard error of the mean]) infarction. Histopathologic findings were used to characterize and quantify neovascularization. The t test was utilized to compare treated and control groups and to assess changes over time. Results: In acute infarction, MR imaging estimates of function, perfusion, and viability showed no difference between the groups. In chronic infarction, however, VM202 increased maximum signal intensity and upslope at first-pass perfusion imaging and reduced infarct size at perfusion and delayed-enhancement imaging. These changes were associated with a decrease in end-diastolic (2.15 mL/kg ± 0.12 to 1.73 mL/kg ± 0.10, P < .01) and end-systolic (1.33 mL/kg ± 0.07 to 0.92 mL/kg ± 0.08, P < .001) volumes and an increase in ejection fraction (38.2% ± 1.3 to 47.0% ± 1.8, P < .001). In contrast, LV function deteriorated further in control animals. Compared with control animals, VM202-treated animals revealed peninsulas and/or islands of viable myocardium in infarcted and periinfarcted regions and greater number of capillaries (218 per square millimeter ± 19 vs 119 per square millimeter ± 17, P < .05) and arterioles (21 per square millimeter ± 4 vs 3 per square millimeter ± 1, P < .001). Conclusion: Intramyocardial transfer of VM202 improved myocardial

  20. Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers

    PubMed Central

    Herter, Sylvia; Piper, Derek E.; Aaron, Wade; Gabriele, Timothy; Cutler, Gene; Cao, Ping; Bhatt, Ami S.; Choe, Youngchool; Craik, Charles S.; Walker, Nigel; Meininger, David; Hoey, Timothy; Austin, Richard J.

    2005-01-01

    Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1–P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR↓VVNG (where ↓ denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR↓VVNG or KQLQ↓VVNG, were not processed, illustrating that the P4–P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4±0.2 nM and 12±0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may

  1. Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location

    PubMed Central

    Yovchev, Mladen; Jaber, Fadi L.; Lu, Zhonglei; Patel, Shachi; Locker, Joseph; Rogler, Leslie E.; Murray, John W.; Sudol, Marius; Dabeva, Mariana D.; Zhu, Liang; Shafritz, David A.

    2016-01-01

    Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs. PMID:26763940

  2. Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5-b]pyridine in Human Plasma and Hepatocytes.

    PubMed

    Wang, Yi; Peng, Lijuan; Bellamri, Medjda; Langouët, Sophie; Turesky, Robert J

    2015-05-18

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified an adduct of PhIP formed at the Cys(34) residue of human serum albumin following reaction of albumin with the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The major adducted peptide recovered from a tryptic/chymotryptic digest was identified as the missed-cleavage peptide LQQC*([SO2PhIP])PFEDHVK, a [cysteine-S-yl-PhIP]-S-dioxide linked adduct. In this investigation, we have characterized the albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo. Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys(34) of albumin in human plasma to form LQQC*([SO2PhIP])PFEDHVK at levels that were 8-10-fold greater than the adduct levels formed with N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP forms an adduct at the sole tryptophan (Trp(214)) residue of albumin in the sequence AW*([PhIP])AVAR. However, stable adducts of PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis, suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys(34) of albumin. A stable adduct was formed at the Tyr(411) residue of albumin in hepatocytes and identified as a deaminated product of PhIP, Y(*[desaminoPhIP])TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP. PMID:25815793

  3. How long have adult humans been consuming milk?

    PubMed

    Gerbault, Pascale; Roffet-Salque, Mélanie; Evershed, Richard P; Thomas, Mark G

    2013-12-01

    Lactase is the enzyme that breaks down the milk sugar lactose, and in most mammals, including most humans, lactase activity is down-regulated after the weaning period is completed. However, in about 35% of adults worldwide, lactase continues to be expressed throughout adulthood, a feature termed lactase persistence (LP). Genetic evidence indicates that LP is a recent human adaptation, and its current geographic distribution correlates with the relative historical importance of dairying in different human populations. Investigating archaeological evidence for fresh milk consumption has proved crucial in building an account of the joint evolution of LP and dairying. A powerful technique for investigating food processing, including milk processing, in ancient populations is lipid residue analysis on archaeological pottery. We review here the archaeological and genetic evidence available that have contributed to a better understanding of the gene-culture co-evolution of LP and dairying. PMID:24339181

  4. How long have adult humans been consuming milk?

    PubMed

    Gerbault, Pascale; Roffet-Salque, Mélanie; Evershed, Richard P; Thomas, Mark G

    2013-12-01

    Lactase is the enzyme that breaks down the milk sugar lactose, and in most mammals, including most humans, lactase activity is down-regulated after the weaning period is completed. However, in about 35% of adults worldwide, lactase continues to be expressed throughout adulthood, a feature termed lactase persistence (LP). Genetic evidence indicates that LP is a recent human adaptation, and its current geographic distribution correlates with the relative historical importance of dairying in different human populations. Investigating archaeological evidence for fresh milk consumption has proved crucial in building an account of the joint evolution of LP and dairying. A powerful technique for investigating food processing, including milk processing, in ancient populations is lipid residue analysis on archaeological pottery. We review here the archaeological and genetic evidence available that have contributed to a better understanding of the gene-culture co-evolution of LP and dairying.

  5. Evaluation of multiple mechanism-based toxicity endpoints in primary cultured human hepatocytes for the identification of drugs with clinical hepatotoxicity: Results from 152 marketed drugs with known liver injury profiles.

    PubMed

    Zhang, Jie; Doshi, Utkarsh; Suzuki, Ayako; Chang, Ching-Wei; Borlak, Jürgen; Li, Albert P; Tong, Weida

    2016-08-01

    We report here the results of a collaborative research program to develop a robust and reliable in vitro system to allow an accurate definition of the drug-induced liver injury (DILI) potential of new drug entities during drug development. The in vitro hepatotoxic potential of 152 drugs with known DILI profiles were evaluated in primary cultured human hepatocytes with four mechanistically-relevant endpoints: cellular ATP depletion, reactive oxygen species (ROS), glutathione (GSH) depletion, and caspase activation for apoptosis. The drugs, 80 in the testing set and 72 in the validation set, were classified based on serious clinical/regulatory outcomes as defined by reported acute liver failure, black-box warning, and/or withdrawal. The drugs were further sub-categorized for dominant types of liver injury. Logistic regression models were performed to calculate the area under the receiver operating characteristics curve (AUROC) and to evaluate the prediction potential of the selected endpoints for serious clinical/regulatory outcomes. The ROS/ATP ratio was found to yield an excellent AUROC in both the testing (0.8989, P < 0.0001) and validation set (0.8545, P < 0.0001), and was found to distinguish drugs associated with severe from non-severe DILI cases (p < 0.0001). The results suggest that evaluation of drugs in primary human hepatocytes using the ROS/ATP ratio endpoint may aid the definition of their potential to cause severe DILI. PMID:26581450

  6. Neurons in the White Matter of the Adult Human Neocortex

    PubMed Central

    Suárez-Solá, M. Luisa; González-Delgado, Francisco J.; Pueyo-Morlans, Mercedes; Medina-Bolívar, O. Carolina; Hernández-Acosta, N. Carolina; González-Gómez, Miriam; Meyer, Gundela

    2009-01-01

    The white matter (WM) of the adult human neocortex contains the so-called “interstitial neurons”. They are most numerous in the superficial WM underlying the cortical gyri, and decrease in density toward the deep WM. They are morphologically heterogeneous. A subgroup of interstitial neurons display pyramidal-cell like morphologies, characterized by a polarized dendritic tree with a dominant apical dendrite, and covered with a variable number of dendritic spines. In addition, a large contingent of interstitial neurons can be classified as interneurons based on their neurochemical profile as well as on morphological criteria. WM- interneurons have multipolar or bipolar shapes and express GABA and a variety of other neuronal markers, such as calbindin and calretinin, the extracellular matrix protein reelin, or neuropeptide Y, somatostatin, and nitric oxide synthase. The heterogeneity of interstitial neurons may be relevant for the pathogenesis of Alzheimer disease and schizophrenia. Interstitial neurons are most prominent in human brain, and only rudimentary in the brain of non-primate mammals. These evolutionary differences have precluded adequate experimental work on this cell population, which is usually considered as a relict of the subplate, a transient compartment proper of development and without a known function in the adult brain. The primate-specific prominence of the subplate in late fetal stages points to an important role in the establishment of interstitial neurons. Neurons in the adult WM may be actively involved in coordinating inter-areal connectivity and regulation of blood flow. Further studies in primates will be needed to elucidate the developmental history, adult components and activities of this large neuronal system. PMID:19543540

  7. Methimazole slows hepatocyte streaming in rats.

    PubMed

    Oren, R; Zajicek, G; Maaravi, Y; Kenet, G; Karmely, F; Hubert, A; Raanani, P; Arber, N

    1997-07-01

    Hepatocytes are hypothesized to continually stream from the portal tract to the terminal hepatic vein. By this model, when a cell divides, one of its progeny replaces the dividing ancestor and the other is displaced into a more remote location. The present experiment aims to demonstrate that hypothyroidism affects liver cell turnover. Thirty male adult rats were divided into two groups. One received methimazole for two weeks and the other served as control. Each rat was injected intraperitoneally with 18.5 KBq [3H]thymidine/g body weight. Rats were killed after 1 hr and two and four weeks. Autoradiography was done. The distance of the labeled cells from the portal tract was measured. The mean TSH levels of the methimazole-treated group and controls were 1.45 and 0.25 mM/liter, respectively (P < 0.01). Hepatocyte streaming was lower in hypothyroid (1.8 microns/day) than in untreated rats (2.5 microns/day) (P < 0.01). The respective labeling indices 1 hr after labeling were 0.9% and 1.24% (P < 0.05). We conclude that hypothyroidism diminishes hepatocyte and littoral cell turnover and slows down their streaming.

  8. Hepatocyte innervation in primates

    PubMed Central

    1977-01-01

    The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed. PMID:406265

  9. Ontogeny of morningness-eveningness across the adult human lifespan

    NASA Astrophysics Data System (ADS)

    Randler, Christoph

    2016-02-01

    Sleep timing of humans can be classified alongside a continuum from early to late sleepers, with some people (larks) having an early activity, early bed, and rise times and others (owls) with a more nocturnally orientated activity. Only a few studies reported that morningness-eveningness changes significantly during the adult lifespan based on community samples. Here, I applied a different methodological approach to seek for evidence for the age-related changes in morningness-eveningness preferences by using a meta-data from all available studies. The new aspect of this cross-sectional approach is that only a few studies themselves address the age-related changes of the adult lifespan development, but that many studies are available that provide exactly the data needed. The studies came from 27 countries and included 36,939 participants. Age was highly significantly correlated with scores on the Composite Scale of Morningness ( r = 0.70). This relationship seems linear, because a linear regression explained nearly the same amount of variance compared to other models such as logarithmic, quadratic, or cubic models. The standard deviation of age correlated with the standard deviation of CSM scores ( r = 0.55), suggesting when there is much variance in age in a study; in turn, there is much variance in morningness. This meta-analytical approach shows that morningness-eveningness changes across the adult lifespan and that older age is related to higher morningness.

  10. Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

    PubMed

    Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

    2016-09-01

    Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans. PMID:27372715

  11. Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

    SciTech Connect

    Qadri, Ishtiaq Hu, L.-J.; Iwahashi, Mieko; Al-Zuabi, Subhi; Quattrochi, Linda C.; Simon, Francis R.

    2009-02-01

    Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from - 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1{alpha}. HNF4{alpha} induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1{alpha} while HNF4{alpha} induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1{alpha} and HNF4{alpha} playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

  12. Multipotent progenitor cells isolated from adult human pancreatic tissue.

    PubMed

    Todorov, I; Nair, I; Ferreri, K; Rawson, J; Kuroda, A; Pascual, M; Omori, K; Valiente, L; Orr, C; Al-Abdullah, I; Riggs, A; Kandeel, F; Mullen, Y

    2005-10-01

    The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus. PMID:16298614

  13. Modeling Nonalcoholic Fatty Liver Disease with Human Pluripotent Stem Cell-Derived Immature Hepatocyte-Like Cells Reveals Activation of PLIN2 and Confirms Regulatory Functions of Peroxisome Proliferator-Activated Receptor Alpha

    PubMed Central

    Graffmann, Nina; Ring, Sarah; Kawala, Marie-Ann; Wruck, Wasco; Ncube, Audrey; Trompeter, Hans-Ingo

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD/steatosis) is a metabolic disease characterized by the incorporation of fat into hepatocytes. In this study, we developed an in vitro model for NAFLD based on hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells. We induced fat storage in these HLCs and detected major expression changes of metabolism-associated genes, as well as an overall reduction of liver-related microRNAs. We observed an upregulation of the lipid droplet coating protein Perilipin 2 (PLIN2), as well as of numerous genes of the peroxisome proliferator-activated receptor (PPAR) pathway, which constitutes a regulatory hub for metabolic processes. Interference with PLIN2 and PPARα resulted in major alterations in gene expression, especially affecting lipid, glucose, and purine metabolism. Our model recapitulates many metabolic changes that are characteristic for NAFLD. It permits the dissection of disease-promoting molecular pathways and allows us to investigate the influences of distinct genetic backgrounds on disease progression. PMID:27308945

  14. Potential interactions between HIV drugs, ritonavir and efavirenz and anticancer drug, nilotinib--a study in primary cultures of human hepatocytes that is applicable to HIV patients with cancer.

    PubMed

    Pillai, Venkateswaran C; Parise, Robert A; Christner, Susan M; Rudek, Michelle A; Beumer, Jan H; Venkataramanan, Raman

    2014-11-01

    Nilotinib is used to treat chronic myeloid leukemia (CML), and is metabolized by CYP3A. With a black-box warning for QT prolongation, which is exposure dependent, controlling for drug interactions is clinically relevant. Treatments of HIV patients with CML are with HAART drugs, ritonavir and efavirenz, may cause complex drug interactions through CYP3A inhibition or induction. We evaluated the interactions of ritonavir or efavirenz on nilotinib using human hepatocytes and compared these interactions with those of ketoconazole or rifampin, classical CYP3A inhibitor and inducer, respectively. Hepatocytes were treated with vehicle, ritonavir (10 μM), ketoconazole (10 μM), efavirenz (10 μM), or rifampin (10 μM) for 5 days. On day 5, nilotinib (3 μM) was coincubated for an additional 24-48 hours. The concentrations of nilotinib were quantitated in collected samples (combined lysate and medium) by LC-MS. Apparent intrinsic clearance (CL(int,app)) of nilotinib was lowered 5.8- and 3.1-fold, respectively, by ritonavir and ketoconazole. Efavirenz and rifampin increased the CL(int,app) of nilotinib by 2.1- and 4.1-fold, respectively. The clinically recommended dose (300 mg twice daily) of nilotinib may have to be reduced substantially (150 mg once daily) or increased (400 mg thrice daily), respectively, to achieve desired drug exposure, when ritonavir or efavirenz is co-administered.

  15. Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat

    PubMed Central

    1990-01-01

    Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u- globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u- globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes. PMID:1688854

  16. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    SciTech Connect

    Mota, Linda C.; Barfield, Christina; Hernandez, Juan P.; Baldwin, William S.

    2011-05-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

  17. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    PubMed Central

    Mota, Linda C; Barfield, Christina; Hernandez, Juan P; Baldwin, William S.

    2011-01-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating Phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, and mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP. PMID:21376070

  18. A Hedgehog Survival Pathway in ‘Undead’ Lipotoxic Hepatocytes

    PubMed Central

    Kakisaka, Keisuke; Cazanave, Sophie C.; Werneburg, Nathan W.; Razumilava, Nataliya; Mertens, Joachim C.; Bronk, Steve F.; Gores, Gregory J.

    2012-01-01

    Background & Aims Ballooned hepatocytes in nonalcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine if ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro. Methods Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using Huh-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3−/− mice. Results Ballooned hepatocytes in NASH display diminished expression of the caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which via AP-1 induces SHH expression. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3−/− cells during lipotoxic stress. Conclusion Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway. PMID:22641094

  19. Novel Mechanism of Impaired Function of Organic Anion-Transporting Polypeptide 1B3 in Human Hepatocytes: Post-Translational Regulation of OATP1B3 by Protein Kinase C Activation

    PubMed Central

    Powell, John; Farasyn, Taleah; Köck, Kathleen; Meng, Xiaojie; Pahwa, Sonia; Brouwer, Kim L. R.

    2014-01-01

    The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [3H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [3H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti–phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds. PMID:25200870

  20. Hepatocyte behavior within three-dimensional porous alginate scaffolds.

    PubMed

    Glicklis, R; Shapiro, L; Agbaria, R; Merchuk, J C; Cohen, S

    2000-02-01

    A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100-150 microm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 microm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 microg albumin/10(6) cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 microg/10(6) cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation.

  1. Neuropeptide Y in the adult and fetal human pineal gland.

    PubMed

    Møller, Morten; Phansuwan-Pujito, Pansiri; Badiu, Corin

    2014-01-01

    Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

  2. MicroRNA-194 Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1

    PubMed Central

    Jung, Kwang Hwa; McCarthy, Ryan L.; Zhou, Chong; Uprety, Nadima; Barton, Michelle Craig; Beretta, Laura

    2015-01-01

    MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 over expression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. PMID:26731713

  3. MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

    PubMed

    Jung, Kwang Hwa; McCarthy, Ryan L; Zhou, Chong; Uprety, Nadima; Barton, Michelle Craig; Beretta, Laura

    2016-05-01

    MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296.

  4. Gustatory reaction time to various sweeteners in human adults.

    PubMed

    Yamamoto, T; Kato, T; Matsuo, R; Kawamura, Y; Yoshida, M

    1985-09-01

    Reaction times to recognize the sweet taste of 12 sweeteners at various concentrations were measured in 48 human adults. The reaction time (T) decreased with increasing concentration (C) of each sweetener applied to the anterior dorsal tongue. The relationships between T and C, and T and logC were well described by a rectangular hyperbola formula for each of the 12 sweeteners. Reaction times to discriminate sweet taste quality between pairs of sweeteners were measured, then a similarity index was calculated. Factor analysis based on correlation coefficients between pairs of sweeteners which were obtained by the similarity indices has indicated classification of the sweeteners. Sucrose, fructose, glucose, maltose, sorbitol and aspartame tend to group together. Na-cyclamate and Na-saccharin form another group. DL-alanine, stevioside and neohesperidin dihydrochalcone are rather independent and do not belong to any group.

  5. Hepatocyte cryopreservation: is it time to change the strategy?

    PubMed

    Stéphenne, Xavier; Najimi, Mustapha; Sokal, Etienne M

    2010-01-01

    Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative, or at least as a bridge, to orthotopic liver transplantation. The success of such a therapeutic approach remains limited by the quality of the transplanted cells. Cryopreservation remains the best option for long-term storage of hepatocytes, providing a permanent and sufficient cell supply. However, isolated adult hepatocytes are poorly resistant to such a process, with a significant alteration both at the morphological and functional levels. Hence, the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols, as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes. Intracellular ice formation or exposure to hyperosmotic solutions remains the main phenomenon of cryopreservation process, and its effects on cell quality and cell death induction will be discussed. The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing. Such strategies, such as vitrification, will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies. PMID:20039443

  6. In vitro metabolism of the glucagon-like peptide-1 (GLP-1)-derived metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in mouse and human hepatocytes.

    PubMed

    Sharma, Raman; McDonald, Thomas S; Eng, Heather; Limberakis, Chris; Stevens, Benjamin D; Patel, Sheena; Kalgutkar, Amit S

    2013-12-01

    Previous studies have revealed that the glucoincretin hormone glucagon-like peptide-1 (GLP-1)(7-36)amide is metabolized by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) to yield GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, as the principal metabolites. Contrary to the previous notion that GLP-1(7-36)amide metabolites are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with both GLP-1(9-36)amide and GLP-1(28-36)amide in animals and humans. In the present work, we examined the metabolic stability of the two GLP-1(7-36)amide metabolites in cryopreserved hepatocytes, which have been used to demonstrate the in vitro insulin-like effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis. To examine the metabolic stability of the GLP-1(7-36)amide metabolites, a liquid chromatography-tandem mass spectrometry assay was developed for the quantitation of the intact peptides in hepatocyte incubations. GLP-1(9-36)amide and GLP-1(28-36)amide were rapidly metabolized in mouse [GLP-1(9-36)amide: t(1/2) = 52 minutes; GLP-1(28-36)amide: t(1/2) = 13 minutes] and human hepatocytes [GLP-1(9-36)amide: t(1/2) = 180 minutes; GLP-1(28-36)amide: t(1/2) = 24 minutes), yielding a variety of N-terminal cleavage products that were characterized using mass spectrometry. Metabolism at the C terminus was not observed for either peptides. The DPP-IV and NEP inhibitors diprotin A and phosphoramidon, respectively, did not induce resistance in the two peptides toward proteolytic cleavage. Overall, our in vitro findings raise the intriguing possibility that the insulinomimetic effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds.

  7. Human hepatocyte growth factor in alcoholic liver disease: a comparison with change in alpha-fetoprotein. Department of Veterans Affairs Cooperative Study Group 275.

    PubMed

    Mendenhall, C L; Roos, F; Moritz, T E; Roselle, G A; Chedid, A; Grossman, C J; Rouster, S D; Bennett, G L; Lake, J R

    1996-12-01

    To evaluate the hepatic regenerative response in patients with alcoholic liver disease, sera from 263 patients with severe alcoholic hepatitis and/or cirrhosis were analyzed for hepatocyte growth factor (HGF) and alpha-fetoprotein (AFP). HGF concentration was elevated above healthy controls in 95% of the patients (median level = 2.4 ng/ml), whereas AFP tended to be depressed below controls (median level = 4.1 ng/ml). Correlations with parameters of liver injury (i.e., ascites, encephalopathy, AST bilirubin, and protime) all showed a more significant correlation with HGF concentrations than those of AFP. Patients with HGF levels below the mean (4 ng/ml) exhibited significantly better survival (median survival = 35 months vs. 8.5 months for those with HGF > or = 4 ng/ml; p = 0.007). Serum HGF levels were associated with various specific histologic features of alcoholic hepatitis that included, but were not exclusively related to, necrosis. PMID:8986214

  8. Current status of hepatocyte xenotransplantation.

    PubMed

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  9. Development of an intact hepatocyte activation system for routine use with the mouse lymphoma assay

    SciTech Connect

    Brock, K.H.; Moore, M.M.; Oglesby, L.A.

    1987-01-01

    The authors have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK/sup +/-/-3.7.2C mouse lymphoma cells. This method should provide a means of simulating more closely in-vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200-250 gm adult male Sprague-Dawley rats; 1 x 10/sup 6/ viable hepatocytes were seeded per flask. Rapid attachment of the hepatocytes (2 hr) was obtained by using fibronectin-coated 25-cm/sup 2/ tissue culture flasks. Cocultivated cultures were incubated at 37/sup 0/C on a platform rocker at 32 oscillations per minute. A 16-hr cocultivated period was selected. With this hepatocyte activation methodology, CP, DMN, DMBA, and B(a)P, genotoxins that require metabolic activation, could be detected as mutagens in L5178Y/TK/sup +/-/ cells.

  10. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

    PubMed Central

    Yin, Hao; Xue, Wen; Chen, Sidi; Bogorad, Roman L; Benedetti, Eric; Grompe, Markus; Koteliansky, Victor; Sharp, Phillip A; Jacks, Tyler; Anderson, Daniel G

    2014-01-01

    We demonstrate CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9–mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases. PMID:24681508

  11. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.

    PubMed

    Yin, Hao; Xue, Wen; Chen, Sidi; Bogorad, Roman L; Benedetti, Eric; Grompe, Markus; Koteliansky, Victor; Sharp, Phillip A; Jacks, Tyler; Anderson, Daniel G

    2014-06-01

    We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

  12. A biokinetic model for systemic technetium in adult humans

    DOE PAGES

    Leggett, Richard Wayne; Giussani, Augusto

    2015-04-10

    The International Commission on Radiological Protection (ICRP) currently is updating its biokinetic and dosimetric models for internally deposited radionuclides. Technetium (Tc), the lightest element that exists only in radioactive form, has two important isotopes from the standpoint of potential risk to humans: the long-lived isotope 99Tm(T1/2=2.1x105 y) is present in high concentration in nuclear waste, and the short-lived isotope 99mTc (T1/2=6.02 h) is the most commonly used radionuclide in diagnostic nuclear medicine. This paper reviews data on the biological behavior of technetium and proposes a biokinetic model for systemic technetium in the adult human body for use in radiation protection.more » Compared with the ICRP s current occupational model for systemic technetium, the proposed model provides a more realistic description of the paths of movement of technetium in the body; provides greater consistency with experimental and medical data; and, for most radiosensitive organs, yields substantially different estimates of cumulative activity (total radioactive decays within the organ) following uptake of 99Tm or 99mTc to blood.« less