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Sample records for human hepatocyte cultures

  1. Rifampicin induction of lidocaine metabolism in cultured human hepatocytes.

    PubMed

    Li, A P; Rasmussen, A; Xu, L; Kaminski, D L

    1995-08-01

    In our laboratory, cultured human hepatocytes are being evaluated as an experimental system to study drug interactions. We report the effect of a known cytochrome P450 (CYP) inducer, rifampicin, on the metabolism of lidocaine by primary human hepatocytes. Rifampicin has been shown to induce CYP3A4, a major human hepatic CYP isozyme that is known to metabolize lidocaine to its primary metabolite, monoethylglycinexylidide. Human hepatocytes were cultured on collagen-coated plates in serum-free, hormone-supplemented Waymouth medium for a 3-day period before they were treated with rifampicin at 50 microM for 1 to 3 days. Hepatocytes isolated from five individuals were studied, and, in all cases, lidocaine metabolism was found to be induced by rifampicin, as demonstrated by a higher rate of monoethylglycinexylidide formation than concurrent controls. For three of the hepatocyte cultures, lidocaine metabolism was evaluated at various times after treatment. Induction was observed at 1 day after treatment, and reached higher levels at day 2 or 3. The level of induction was found to be approximately 100% for two hepatocyte isolations and approximately 600% for one isolation. In a separate experiment, hepatocytes were treated with rifampicin for a 2-day period. Rate of lidocaine metabolism at multiple substrate concentrations (10-120 microM) were then studied. Rifampicin induction of lidocaine metabolism (approximately 100%) was observed at all the lidocaine concentrations studied. Lineweaver-Burk plot of the data showed an increase in Vmax and a less significant change in Km. Induction of lidocaine metabolism by rifampicin (concentrations of 0.1-50 microM) was found to be dose-dependent, with significant induction observed at 1 microM and higher concentrations. (ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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

  3. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

    PubMed

    Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

    2016-09-06

    Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug.

  4. 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.

  5. 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 ...

  6. Ultrastructural and biochemical alterations induced in human, rat and mouse hepatocytes in primary culture exposed to selected carcinogens

    SciTech Connect

    Cole, K.H.

    1987-01-01

    Aflatoxin B1 (AFB{sub 1}), dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-AAF) and actinomycin D are all potential human liver carcinogens. In order to investigate carcinogenic susceptibility of human liver to these agents, primary cultures of normal human hepatocytes were exposed to the four carcinogens. In the first series of experiments, human, rat, and mouse hepatocytes in primary culture were exposed to actinomycin D, AFB{sub 1}, and DMN for 24 h and examined for ultrastructural alterations. In an effort to relate the ultrastructural effects with total covalent binding of the carcinogen to DNA, human, rat and mouse hepatocytes were exposed to 2.0 {times} 10{sup {minus}7} M ({sup 3}H)AFB{sub 1} for 24 h. Hepatocytes from male rats had the highest degree of ({sup 3}H)AFB{sub 1}-DNA binding. Human hepatocytes contained the next highest binding level, while hepatocytes from female rats bound 38 pmoles/mg DNA. The AFB{sub 1}-DNA binding level in mouse hepatocytes was 1.4 pmoles/mg DNA. In similar experiments, human, and male and female rat hepatocytes in primary culture were exposed to the carcinogen 2-acetylamino (9-{sup 14}C)fluorene for 24 h. It was determined that male rat hepatocytes had the highest amount of radiolabeled 2-AAF bound to their DNA, female rats contained 0.57 nmoles/mg DNA, while human hepatocytes contained 0.29 nmoles/mg DNA.

  7. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  8. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  9. 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

  10. Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyte culture intoxicated with alpha-amanitin.

    PubMed

    Magdalan, Jan; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Sozański, Tomasz; Szeląg, Adam; Dziegięl, Piotr

    2011-01-01

    α-Amanitin (α-AMA) is the main toxin of Amanita phalloides and its subspecies (A. virosa and A. verna). The primary mechanism of α-AMA toxicity is associated with protein synthesis blocking in hepatocytes. Additionally, α-AMA exhibits prooxidant properties that may contribute to its severe hepatotoxicity. The aim of the present study was to assess the effect of α-AMA on lipid peroxidation and the activities of superoxide dismutase (SOD) and catalase (CAT) in human hepatocyte culture. The effects of benzylpenicillin (BPCN), N-acetyl-L-cysteine (ACC), and silibinin (SIL) on SOD and CAT activities and on lipid peroxidation in human hepatocyte culture intoxicated with α-AMA were also examined. In human hepatocyte culture, 48-hour exposure to α-AMA at a 2-μM concentration caused an increase in SOD activity, a reduction of CAT activity, and a significant increase in lipid peroxidation. Changes in SOD and CAT activity caused by α-AMA could probably enhance lipid peroxidation by increased generation of hydrogen peroxide combined with reduced detoxification of that oxygen radical. The addition of antidotes (ACC or SIL) to the culture medium provided more effective protection against lipid peroxidation in human hepatocytes intoxicated with α-AMA than the addition of BPCN, possessing no antioxidant properties.

  11. 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.

  12. 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

  13. Drug-induced cholestasis risk assessment in sandwich-cultured human hepatocytes.

    PubMed

    Oorts, Marlies; Baze, Audrey; Bachellier, Philippe; Heyd, Bruno; Zacharias, Thomas; Annaert, Pieter; Richert, Lysiane

    2016-08-01

    Drug-induced cholestasis (DIC) is recognized as one of the prime mechanisms for DILI. Hence, earlier detection of drug candidates with cholestatic signature is crucial. Recently, we introduced an in vitro model for DIC and evaluated its performance with several cholestatic drugs. We presently expand on the validation of this model by 14 training compounds (TCs) of the EU-EFPIA IMI project MIP-DILI. Several batches of human hepatocytes in sandwich-culture were qualified for DIC assessment by verifying the bile acid-dependent increase in sensitivity to the toxic effects of cyclosporin A. The cholestatic potential of the TCs was expressed by determining the drug-induced cholestasis index (DICI). A safety margin (SM) was calculated as the ratio of the lowest TC concentration with a DICI≤0.80 to the Cmax,total. Nefazodone, bosentan, perhexiline and troglitazone were flagged for cholestasis (SM<30). The hepatotoxic (but non-cholestatic) compounds, amiodarone, diclofenac, fialuridine and ximelagatran, and all non-hepatotoxic compounds were cleared as "safe" for DIC. Tolcapone and paracetamol yielded DICI-based SM values equal to or higher than those based on cytotoxicity, thus excluding DIC as a DILI mechanism. This hepatocyte-based in vitro assay provides a unique tool for early and reliable identification of drug candidates with cholestasis risk.

  14. 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.

  15. Expression profiling of interindividual variability following xenobiotic exposures in primary human hepatocyte cultures

    SciTech Connect

    Goyak, Katy M.O.; Johnson, Mary C.; Strom, Stephen C.; Omiecinski, Curtis J.

    2008-09-01

    To examine the magnitude of human variability across the entire transcriptome after chemical challenge, we profiled gene expression responses to three different prototypic chemical inducers in primary human hepatocyte cultures from ten independent donors. Correlation between basal expression in any two hepatocyte donors ranged from r{sup 2} values of 0.967 to 0.857, and chemical treatment tended to negatively impact correlation between donors. Including anticipated target genes, 10,812, 8373, and 7847 genes were changed in at least one donor by Aroclor 1254 (A1254), di(2-ethylhexyl) phthalate (DEHP), and phenobarbital (PB), respectively. A subset of these gene targets (n = 41) were altered with a high level of reproducibility in at least 9 donors, gene responses that correlated well with literature-reported mechanism of action. Filtering responses to the level of gene subsets clarified the biological impact associated with the respective chemical effectors, in lieu of substantial interindividual variation among donor responses. In these respects, the use of hierarchical clustering methods successfully grouped seven of the ten donors into chemical-specific rather than donor-specific clusters. However, at the whole-genome level, the magnitude of conserved gene expression changes among donors was surprisingly small, with fewer than 50% of the gene responses altered by a single chemical conserved in more than one donor. The use of higher level descriptors, such as those defined by the PANTHER classification system, may enable more consistent categorization of gene expression changes across individuals, as increased reproducibility was identified using this method.

  16. 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.

  17. Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.

    PubMed

    Birk, Richard; Sommer, Ulrich; Faber, Anne; Aderhold, Christoph; Schulz, Johannes D; Hörmann, Karl; Goessler, Ulrich Reinhart; Stern-Straeter, Jens

    2014-06-01

    Tissue engineering is a promising research field, which aims to create new functional muscle tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal muscle tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.

  18. Hepatocyte Culture in Autologous Decellularized Spleen Matrix

    PubMed Central

    Gao, Rui; Wu, Wanquan; Xiang, Junxi; Lv, Yi; Zheng, Xinglong; Chen, Qian; Wang, Haohua; Wang, Bo; Liu, Zhengwen; Ma, Feng

    2015-01-01

    abstract Background and Aims: Using decellularized scaffold to reengineer liver tissue is a promising alternative therapy for end-stage liver diseases. Though the decellularized human liver matrix is the ideal scaffold for reconstruction of the liver theoretically, the shortage of liver donors is still an obstacle for potential clinical application. Therefore, an appropriate alternative scaffold is needed. In the present study, we used a tissue engineering approach to prepare a rat decellularized spleen matrix (DSM) and evaluate the effectiveness of this DSM for primary rat hepatocytes culture. Methods: Rat decellularized spleen matrix (DSM) was prepared by perfusion of a series of detergents through spleen vasculature. DSM was characterized by residual DNA and specific extracellular matrix distribution. Thereafter, primary rat hepatocytes were cultured in the DSM in a 3-dimensional dynamic culture system, and liver cell survival and biological functions were evaluated by comparison with 3-dimensional sandwich culture and also with cultured in decellularized liver matrix (DLM). Results: Our research found that DSM did not exhibit any cellular components, but preserved the main extracellular matrix and the intact vasculature evaluated by DNA detection, histology, immunohistochemical staining, vessel corrosion cast and upright metallurgical microscope. Moreover, the method of DSM preparation procedure was relatively simple with high success rate (100%). After seeding primary hepatocytes in DSM, the cultured hepatocytes survived inside DSM with albumin synthesis and urea secretion within 10 d. Additionally, hepatocytes in dynamic culture medium had better biological functions at day 10 than that in sandwich culture. Albumin synthesis was 85.67 ± 6.34 μg/107cell/24h in dynamic culture in DSM compared to 62.43 ± 4.59 μg/107cell/24h in sandwich culture (P < 0.01) and to 87.54 ± 5.25 μg/107cell/24h in DLM culture (P > 0.05); urea release was 32.14 ± 8.62

  19. Co-culturing human prostate carcinoma cells with hepatocytes leads to increased expression of E-cadherin

    PubMed Central

    Yates, C C; Shepard, C R; Stolz, D B; Wells, A

    2007-01-01

    Metastasis is a multi-step process wherein tumour cells detach from the primary mass, migrate through barrier matrices, gain access to conduits to disseminate, and subsequently survive and proliferate in an ectopic site. During the initial invasion stage, prostate carcinoma cells undergo epithelial–mesenchymal-like transition with gain of autocrine signalling and loss of E-cadherin, hallmarks that appear to enable invasion and dissemination. However, some metastases express E-cadherin, and we found close connections between prostate carcinoma cells and hepatocytes in a liver microtissue bioreactor. We hypothesise that phenotypic plasticity occurs late in prostate cancer progression at the site of ectopic seeding. Immunofluorescence staining for E-cadherin in co-cultures of hepatocytes and DU-145 prostate cancer cells revealed E-cadherin upregulation at peripheral sites of contact by day 2 of co-culture; E-cadherin expression also increased in PC-3 cells in co-culture. These carcinoma cells bound to hepatocytes in an E-cadherin-dependent manner. Although the signals by which the hepatocytes elicited E-cadherin expression remain undetermined, it appeared related to downregulation of epidermal growth factor receptor (EGFR) signalling. Inhibition of autocrine EGFR signalling increased E-cadherin expression and cell–cell heterotypic adhesion; further, expression of a downregulation-resistant EGFR variant prevented E-cadherin upregulation. These findings were supported by finding E-cadherin and catenins but not activated EGFR in human prostate metastases to the liver. We conclude that the term epithelial–mesenchymal transition only summarises the transient downregulation of E-cadherin for invasion with re-expression of E-cadherin being a physiological consequence of metastatic seeding. PMID:17406365

  20. 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.

  1. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  2. 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

  3. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells.

    PubMed

    Asplund, Annika; Pradip, Arvind; van Giezen, Mariska; Aspegren, Anders; Choukair, Helena; Rehnström, Marie; Jacobsson, Susanna; Ghosheh, Nidal; El Hajjam, Dorra; Holmgren, Sandra; Larsson, Susanna; Benecke, Jörg; Butron, Mariela; Wigander, Annelie; Noaksson, Karin; Sartipy, Peter; Björquist, Petter; Edsbagge, Josefina; Küppers-Munther, Barbara

    2016-02-01

    Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using one standardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.

  4. 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...

  5. 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.

  6. Effect of rifampin and nelfinavir on the metabolism of methadone and buprenorphine in primary cultures of human hepatocytes.

    PubMed

    Moody, David E; Fang, Wenfang B; Lin, Shen-Nan; Weyant, Denise M; Strom, Stephen C; Omiecinski, Curtis J

    2009-12-01

    We tested the hypothesis that primary cultures of human hepatocytes could predict potential drug interactions with methadone and buprenorphine. Hepatocytes (five donors) were preincubated with dimethyl sulfoxide (DMSO) (vehicle), rifampin, or nelfinavir before incubation with methadone or buprenorphine. Culture media (0-60 min) was analyzed by liquid chromatography-tandem mass spectrometry for R- and S-methadone and R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) or for buprenorphine, norbuprenorphine, and their glucuronides [buprenorphine-3-glucuronide (B-3-G) and norbuprenorphine-3-glucuronide (N-3-G)]. R- and S-EDDP were detected in three of five, four of five, and five of five media from cells pretreated with DMSO, nelfinavir, and rifampin. R-EDDP increased 3.1- and 26.5-fold, and S-EDDP increased 2.5- and 21.3-fold after nelfinavir and rifampin. The rifampin effect was significant. B-3-G production was detected in media of all cells incubated with buprenorphine and accounted for most of the buprenorphine loss from culture media; it was not significantly affected by either pretreatment. Norbuprenorphine and N-3-G together were detected in three of five, four of five, and five of five donors pretreated with DMSO, nelfinavir and rifampin, and norbuprenorphine in one of five, one of five, and two of five donors. Although there was a trend for norbuprenorphine (2.8- and 4.9-fold) and N-3-G (1.7- and 1.9-fold) to increase after nelfinavir and rifampin, none of the changes were significant. To investigate low norbuprenorphine production, buprenorphine was incubated with human liver and small intestine microsomes fortified to support both N-dealkylation and glucuronidation; N-dealkylation predominated in small intestine and glucuronidation in liver microsomes. These studies support the hypothesis that methadone metabolism and its potential for drug interactions can be predicted with cultured human hepatocytes, but for buprenorphine the combined

  7. Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William’s E Medium followed by Hepatocyte Differentiation Inducer Treatment

    PubMed Central

    Tomizawa, Minoru; Shinozaki, Fuminobu; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Ishige, Naoki

    2016-01-01

    Background Hepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival. Materials and Methods 201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William’s E (WE) medium, followed by a 2-day culture in HDI. Results Expression levels of α-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition). Conclusion 201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression. PMID:27073925

  8. Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells.

    PubMed

    Harris, Angela J; Dial, Stacey L; Casciano, Daniel A

    2004-05-18

    Toxicogenomics is a relatively new discipline of toxicology. Microarrays and bioinformatics tools are being used successfully to understand the effects of toxicants on in vivo and in vitro model systems, and to gain a better understanding of the relevance of in vitro models commonly used in toxicological studies. In this study, cDNA filter arrays were used to determine the basal expression patterns of human cultured primary hepatocytes from different male donors; compare the gene expression profile of HepG2 to that of primary hepatocytes; and analyze the effects of three genotoxic hepatocarcinogens; aflatoxin B(1) (AFB(1)), 2-acetylaminofluorene (2AAF), and dimethylnitrosamine (DMN), as well as one non-gentoxic hepatotoxin, acetaminophen (APAP) on gene expression in both in vitro systems. Real-time PCR was used to verify differential gene expression for selected genes. Of the approximately 31,000 genes screened, 3-6% were expressed in primary hepatocytes cultured on matrigel for 16 h. Of these genes, 867 were expressed in cultured hepatocytes from all donors. HepG2 cells expressed about 98% of the genes detectable in cultured primary hepatocytes, however, 31% of the HepG2 transcriptome was unique to the cell line. A number of these genes are expressed in human liver but expression is apparently lost during culture. There was considerable variability in the response to chemical carcinogen exposure in primary hepatocytes from different donors. The transcription factors, E2F1 and ID1 mRNA were increased three-fold and six-fold (P < 0.05, P < 0.01), respectively, in AFB(1) treated primary human hepatocytes but were not altered in HepG2. ID1 expression was also increased by dimethylnitrosamine, acetylaminofluorene and acetaminophen in both primary hepatocytes and HepG2. Identification of genes that are expressed in primary hepatocytes from most donors, as well as those genes with variable expression, will aid in understanding the variability in human reactions to drugs

  9. Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes.

    PubMed

    Zucchini, Nathalie; de Sousa, Georges; Bailly-Maitre, Béatrice; Gugenheim, Jean; Bars, Rémi; Lemaire, Géraldine; Rahmani, Roger

    2005-08-15

    The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.

  10. Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes

    PubMed Central

    Rondini, Elizabeth A.; Duniec-Dmuchowski, Zofia

    2016-01-01

    Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR–wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism. PMID:26798158

  11. 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...

  12. 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...

  13. Constrained spheroids for prolonged hepatocyte culture.

    PubMed

    Tong, Wen Hao; Fang, Yu; Yan, Jie; Hong, Xin; Hari Singh, Nisha; Wang, Shu Rui; Nugraha, Bramasta; Xia, Lei; Fong, Eliza Li Shan; Iliescu, Ciprian; Yu, Hanry

    2016-02-01

    Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.

  14. 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.

  15. Secretion of albumin and induction of CYP1A2 and CYP3A4 in novel three-dimensional culture system for human hepatocytes using micro-space plate.

    PubMed

    Nishimura, Masuhiro; Hagi, Mieko; Ejiri, Yoko; Kishimoto, Sanae; Horie, Toru; Narimatsu, Shizuo; Naito, Shinsaku

    2010-01-01

    We evaluated a novel primary three-dimensional culture system for human hepatocytes using micro-space plates. The functional activity of human hepatocytes in primary culture was determined by measuring albumin secretion from hepatocytes to medium and measuring expression levels of albumin, CYP1A2 and CYP3A4 mRNA. Albumin secretion was higher in micro-space plates compared with traditional plates after 72 h of culture; the levels of albumin secretion from hepatocytes to medium in culture using micro-space plates after 96 h of culture were 2.7-fold higher than those in culture using traditional plates, and secretion of albumin in micro-space plate culture subsequently remained constant. Expression levels of albumin, CYP1A2 and CYP3A4 mRNA in the culture of hepatocytes were significantly higher using micro-space plates than using traditional plates. The inducibility of CYP1A2 and CYP3A4 mRNA after exposure to inducers in hepatocyte culture on micro-space plates was comparable to that in culture on traditional plates, while expression of CYP1A2 and CYP3A4 mRNA after exposure to inducers was higher on micro-space plates than on traditional plates. The present study demonstrates that a novel primary three-dimensional culture system of cryopreserved human hepatocytes using micro-space plates could be used for evaluating the induction of drug-metabolizing enzymes in humans. This in vitro method may thus be useful for screening the induction potency of new drug candidates.

  16. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

    PubMed

    Vorrink, Sabine U; Ullah, Shahid; Schmidt, Staffan; Nandania, Jatin; Velagapudi, Vidya; Beck, Olof; Ingelman-Sundberg, Magnus; Lauschke, Volker M

    2017-03-06

    Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.-Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

  17. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    SciTech Connect

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-05-15

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to

  18. 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

  19. 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...

  20. Coculture with mesenchymal stem cells results in improved viability and function of human hepatocytes.

    PubMed

    Fitzpatrick, Emer; Wu, Yue; Dhadda, Paramjeet; Hughes, Robin D; Mitry, Ragai R; Qin, Hong; Lehec, Sharon C; Heaton, Nigel D; Dhawan, Anil

    2015-01-01

    Hepatocyte transplantation is becoming an accepted therapy for acute liver failure, either as a bridge to liver regeneration or to organ transplantation. Hepatocytes provide liver function in place of the failing organ. The maintenance of sufficient viability and function of the transplanted hepatocytes is a concern. There is a lot of recent interest in mesenchymal stem cells (MSCs) for the provision of structural and trophic support to hepatocytes, but few studies currently use primary human hepatocytes. The aim of this study was to investigate if coculture of human MSCs with cryopreserved human hepatocytes may improve their function and viability, thus with potential for cellular therapy of liver disease. MSCs were isolated from human umbilical cord or adipose tissue. Hepatocytes were isolated from donor organs unsuitable for transplantation. MSCs and hepatocytes were cocultured in both direct and indirect contact. Conditioned medium (CM) from cocultured MSCs and hepatocytes was also used on hepatocytes. Viability and liver-specific function were compared between test and controls. Human hepatocytes that were cocultured directly with MSCs demonstrated improved production of albumin from day 5 to day 25 of culture. This effect was most prominent at day 15. Likewise, urea production was improved in coculture from day 5 to 25. Indirect coculture demonstrated improved albumin production by day 4 (1,107 ng/ml) versus hepatocyte monoculture (940 ng/ml). Hepatocytes in CM demonstrated a nonsignificant improvement in function. The viability of cocultured hepatocytes was superior to that of monocultured cells with up to a 16% improvement. Thus, coculture of human hepatocytes with MSCs demonstrates both improved function and viability. The effect is seen mainly with direct coculture but can also be seen in indirect culture and with CM. Such coculture conditions may convey major advantages in hepatocyte survival and function for cell transplantation.

  1. 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.

  2. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

    PubMed

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-11-30

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  3. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    PubMed Central

    Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

    2016-01-01

    The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

  4. 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.

  5. Production of hepatocyte like cells from human pluripotent stem cells

    PubMed Central

    Hannan, Nicholas R.F; Segeritz, Charis-Patricia; Touboul, Thomas; Vallier, Ludovic

    2013-01-01

    Large scale production of hepatocytes from a variety of genetic backgrounds would be beneficial for drug screening and to provide a source of cells to be used as a substitute for liver transplantation. However, fully functional primary hepatocytes remain difficult to expand in vitro and circumventing this problem by using an alternative source of cells is desirable. Here, we describe a 25 day protocol to direct the differentiation of human pluripotent stem cells into a near homogenous population of hepatocyte-like cells. As cells progress through this protocol they express genes in a chronological manner similar to that described during in-vivo hepatic development. The protocol relies on culture systems devoid of serum, feeders or complex extra-cellular matrices enabling molecular analyses without interference from unknown factors. This approach works efficiently with human embryonic stem cells and human induced pluripotent stem cells and was recently used to model liver diseases in vitro. PMID:23424751

  6. Cyclosporin A drug interactions. Screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes.

    PubMed

    Pichard, L; Fabre, I; Fabre, G; Domergue, J; Saint Aubert, B; Mourad, G; Maurel, P

    1990-01-01

    In previous papers we demonstrated that cyclosporin A (CsA) was specifically oxidized in rabbit and human liver by cytochrome P-450IIIA. We therefore anticipated that any drug that is an inducer or an inhibitor of this cytochrome should lead to interaction with CsA when given in association with it. In order to confirm this hypothesis, primary cultures of human hepatocytes and human liver microsomes were used to "reproduce" in vitro clinically significant interactions observed between CsA and drugs known either as specific inducers (i.e., rifampicin) or as specific inhibitors (i.e., erythromycin) of P-450IIIA. Our results were in close agreement with the clinical reports. Human hepatocytes maintained in primary cultures for 72 hr in the presence of 50 microM rifampicin exhibited increased levels of P-450IIIA, determined by Western blot using specific antibodies, and concomitant increase in CsA oxidase activity, determined by HPLC analysis of extra and intracellular media. Conversely, these cultures exhibited erythromycin concentration-dependent decreases in CsA oxidase activity when incubated in the presence of 5, 20, and 100 microM erythromycin. In addition, a Lineweaver-Burk analysis of the erythromycin-mediated inhibition of CsA oxidase activity in human liver microsomes revealed competitive inhibition (with Ki of 75 microM) as expected, this macrolide being a specific substrate of P-450IIIA. Using this experimental approach, 59 molecules representative of 17 different therapeutic classes were screened for inducers and inhibitors of CsA oxidase activity. Our results allowed us to elucidate the molecular mechanism of previously observed, but unexplained, drug interactions involving CsA, and to detect drugs that should interfere with CsA metabolism as inducers or inhibitors. Drugs detected as potential inducers of CsA oxidase included: rifampicin, sulfadimidine, phenobarbital, phenytoin, phenylbutazone, dexamethasone, sulfinpyrazone, and carbamazepine. Drugs

  7. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    PubMed

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined.

  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. 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.

  10. Dipeptide Phe-Cys derived from in silico thermolysin-hydrolysed RuBisCO large subunit suppresses oxidative stress in cultured human hepatocytes.

    PubMed

    Je, Jae-Young; Cho, Young-Sook; Gong, Min; Udenigwe, Chibuike C

    2015-03-15

    A dipeptide (Phe-Cys) was predicted to be bioactive following bioinformatics analysis of the large subunit of plant and microalgae ribulose-1,5-bisphosphate carboxylase (RuBisCO), which was hydrolysed in silico with thermolysin. The peptide was synthesised and found to possess in vitro reducing potential and inhibitory activity against lipid peroxidation, comparable to the activity of glutathione. In cultured Chang human hepatocytes, 2.5-10 μM Phe-Cys was found to induce the suppression of reactive oxygen species formation and membrane lipid peroxidation in oxidative stressed cells. Intracellular glutathione levels were found to increase in the peptide-treated cells under normal condition, which can potentially contribute in protecting the cells from oxidative damage. Furthermore, Western blot analysis showed that the levels of antioxidant enzymes, catalase and superoxide dismutase-1, increased in the hepatic cells when treated with Phe-Cys in the presence of the oxidant. The results show that this peptide has great potential to be used against oxidative stress-induced health conditions.

  11. 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.

  12. Acute accumulation of free cholesterol induces the degradation of perilipin 2 and Rab18-dependent fusion of ER and lipid droplets in cultured human hepatocytes

    PubMed Central

    Makino, Asami; Hullin-Matsuda, Françoise; Murate, Motohide; Abe, Mitsuhiro; Tomishige, Nario; Fukuda, Mitsunori; Yamashita, Shizuya; Fujimoto, Toyoshi; Vidal, Hubert; Lagarde, Michel; Delton, Isabelle; Kobayashi, Toshihide

    2016-01-01

    Dysregulated hepatic cholesterol homeostasis with free cholesterol accumulation in the liver is relevant to the pathogenesis of nonalcoholic steatohepatitis, contributing to the chronicity of liver toxicity. Here we examined the effect of free cholesterol accumulation on the morphology and biochemical properties of lipid droplets (LDs) in cultured hepatocytes. Acute free cholesterol accumulation induced the fusion of LDs, followed by degradation of the coat protein of LDs, perilipin 2 (PLIN2; also called adipophilin or adipose differentiation–related protein), and association of apolipoprotein B 100 (ApoB 100) to LDs. The degradation of PLIN2 was inhibited by inhibitors of ubiquitination, autophagy, and protein synthesis. The results indicate that association of ApoB 100 with LDs is dependent on the activity of low–molecular weight GTP-binding protein Rab18 and highlight the role of LDs as targets of free cholesterol toxicity in hepatocytes. PMID:27582390

  13. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-04

    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.

  14. Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytes.

    PubMed

    Hartman, J Craig; Brouwer, Kenneth; Mandagere, Arun; Melvin, Lawrence; Gorczynski, Richard

    2010-06-01

    To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [(3)H]estradiol-17beta-D-glucuronide (for OATP), and [2-D-penicillamine, 5-D-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 micromol*L-1 for inhibition and at 2 micromol*L-1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%-100% of control), whereas bosentan was most affected (32%-58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

  15. Zearalenone activates pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor and corresponding phase I target genes mRNA in primary cultures of human hepatocytes.

    PubMed

    Ayed-Boussema, Imen; Pascussi, Jean Marc; Maurel, Patrick; Bacha, Hassen; Hassen, Wafa

    2011-01-01

    The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and β-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 μM.

  16. Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.

    PubMed

    Ito, T; Yasui, K; Mukaigawa, J; Katsume, A; Kohara, M; Mitamura, K

    2001-09-01

    Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

  17. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    PubMed Central

    Liu, Lie-Gang; Yan, Hong; Yao, Ping; Zhang, Wen; Zou, Li-Jun; Song, Fang-Fang; Li, Ke; Sun, Xiu-Fa

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage. METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively. Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA). RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of cellular MDA level, LDH, and AST activities in supernatants. Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes. CONCLUSION: A positive relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage. PMID:16052683

  18. Hepatocyte differentiation.

    PubMed

    Olsavsky Goyak, Katy M; Laurenzana, Elizabeth M; Omiecinski, Curtis J

    2010-01-01

    Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

  19. 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. PMID:27600213

  20. Analysis of the Metabolic Pathway of Bosentan and of the Cytotoxicity of Bosentan Metabolites Based on a Quantitative Modeling of Metabolism and Transport in Sandwich-Cultured Human Hepatocytes.

    PubMed

    Matsunaga, Norikazu; Kaneko, Naomi; Staub, Angelina Yukiko; Nakanishi, Takeo; Nunoya, Ken-ichi; Imawaka, Haruo; Tamai, Ikumi

    2016-01-01

    To quantitatively understand the events in the human liver, we modeled a hepatic disposition of bosentan and its three known metabolites (Ro 48-5033, Ro 47-8634, and Ro 64-1056) in sandwich-cultured human hepatocytes based on the known metabolic pathway. In addition, the hepatotoxicity of Ro 47-8634 and Ro 64-1056 was investigated because bosentan is well known as a hepatotoxic drug. A model illustrating the hepatic disposition of bosentan and its three metabolites suggested the presence of a novel metabolic pathway(s) from the three metabolites. By performing in vitro metabolism studies on human liver microsomes, a novel metabolite (M4) was identified in Ro 47-8634 metabolism, and its structure was determined. Moreover, by incorporating the metabolic pathway of Ro 47-8634 to M4 into the model, the hepatic disposition of bosentan and its three metabolites was successfully estimated. In hepatocyte toxicity studies, the cell viability of human hepatocytes decreased after exposure to Ro 47-8634, and the observed hepatotoxicity was diminished by pretreatment with tienilic acid (CYP2C9-specific inactivator). Pretreatment with 1-aminobenzotriazole (broad cytochrome P450 inactivator) also tended to maintain the cell viability. Furthermore, Ro 64-1056 showed hepatotoxicity in a concentration-dependent manner. These results suggest that Ro 64-1056 is directly involved in bosentan-induced liver injury partly because CYP2C9 specifically mediates hydroxylation of the t-butyl group of Ro 47-8634. Our findings demonstrate the usefulness of a quantitative modeling of hepatic disposition of drugs and metabolites in sandwich-cultured hepatocytes. In addition, the newly identified metabolic pathway may be an alternative route that can avoid Ro 64-1056-induced liver injury.

  1. Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

    PubMed

    Ostrowska, A; Bode, D C; Pruss, J; Bilir, B; Smith, G D; Zeisloft, S

    2000-01-01

    There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly

  2. The comparative effects of diethyldithiocarbamate-copper complex with established proteasome inhibitors on expression levels of CYP1A2/3A4 and their master regulators, aryl hydrocarbon and pregnane X receptor in primary cultures of human hepatocytes.

    PubMed

    Vrzal, Radim; Dvorak, Zdenek

    2016-12-01

    In the recent years, a therapeutic potential of disulfiram (Antabuse) complex with copper, as an anticancer drug, was recognized towards several cancer cell lines. The proteasome was suggested as one of the cellular targets for this compound. As the therapeutic use of diethyldithiocarbamate-copper complex (CuET) is expected to increase, it is of great interest to know whether this compound may be the source of drug-drug interactions via the induction of biotransformation enzymes, especially cytochromes P450 (CYPs). To this purpose, we examined the effect of CuET and compared it with typical inducers (rifampicin and dioxin) of CYPs and with well-established proteasome inhibitors (MG132 and bortezomib). Diethyldithiocarbamate-copper complex revealed inconsistent and rather modulatory effect on the expression of CYP1A2 and CYP3A4 in several cultures of human hepatocytes. Moreover, it was able to cause neither ubiquitin accumulation nor significant and dose-dependent inhibition of proteasome activity. It had no effect on essential transcription factors involved in regulation of selected CYPs, aryl hydrocarbon (AhR) nor pregnane X receptor (PXR). However, the AhR protein was increased in majority of examined hepatocyte cultures. The main finding of this study is that: (i) disulfiram-copper complex is not the cause of drug-drug interactions via CYP1A2/3A4 induction; (ii) proteasome inhibitors may have different impact on studied parameters in given in vitro system.

  3. 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

    2016-08-05

    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.

  4. Morphological behaviour and metabolic capacity of cryopreserved human primary hepatocytes cultivated in a perfused multiwell device.

    PubMed

    Vivares, Aurelie; Salle-Lefort, Sandrine; Arabeyre-Fabre, Catherine; Ngo, Robert; Penarier, Geraldine; Bremond, Michele; Moliner, Patricia; Gallas, Jean-François; Fabre, Gerard; Klieber, Sylvie

    2015-01-01

    1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.

  5. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.

    PubMed

    Huang, Pengyu; Zhang, Ludi; Gao, Yimeng; He, Zhiying; Yao, Dan; Wu, Zhitao; Cen, Jin; Chen, Xiaotao; Liu, Changcheng; Hu, Yiping; Lai, Dongmei; Hu, Zhenlei; Chen, Li; Zhang, Ying; Cheng, Xin; Ma, Xiaojun; Pan, Guoyu; Wang, Xin; Hui, Lijian

    2014-03-06

    The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded in vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.

  6. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

    PubMed

    Ji, LiLi; Liu, TianYu; Wang, ZhengTao

    2010-04-01

    Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

  7. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

    PubMed

    March, Sandra; Ramanan, Vyas; Trehan, Kartik; Ng, Shengyong; Galstian, Ani; Gural, Nil; Scull, Margaret A; Shlomai, Amir; Mota, Maria M; Fleming, Heather E; Khetani, Salman R; Rice, Charles M; Bhatia, Sangeeta N

    2015-12-01

    The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

  8. Helicobacter hepaticus Induces an Inflammatory Response in Primary Human Hepatocytes

    PubMed Central

    Kleine, Moritz; Worbs, Tim; Schrem, Harald; Vondran, Florian W. R.; Kaltenborn, Alexander; Klempnauer, Jürgen; Förster, Reinhold; Josenhans, Christine; Suerbaum, Sebastian; Bektas, Hüseyin

    2014-01-01

    Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytomety. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a

  9. Environmental carcinogens in human target tissues in culture. Progress report

    SciTech Connect

    Hsu, I.C.

    1986-02-19

    Cells from different organ or animal species have shown diverse activities in activation and detoxification of chemical carcinogens. Based on the mutation assays, human hepatocytes were more effective than animal hepatocytes in detoxification of aromatic nitrogen compounds. The adduct formation was also different in human and rodent hepatocytes exposed to aminofluorene (AF) or acetylaminofluorene (AAF). Both AF and AAF adduct DNA were observed in rat liver cells exposed to AF or AAF. However, very little acetylation or deacetyl of the DNA adducts occurred in the human hepatocytes. Human hepatocytes treated with AF in primary culture produced mainly AF adducted DNA while AAF treated cells formed AAF adduct DNA. 2 figs., 1 tab.

  10. Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

    PubMed Central

    Ishii, Masayuki; Kino, Junichi; Ichinohe, Norihisa; Tanimizu, Naoki; Ninomiya, Takafumi; Suzuki, Hiromu; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2017-01-01

    The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

  11. Gas-permeable membranes and co-culture with fibroblasts enable high-density hepatocyte culture as multilayered liver tissues.

    PubMed

    Evenou, Fanny; Hamon, Morgan; Fujii, Teruo; Takeuchi, Shoji; Sakai, Yasuyuki

    2011-07-01

    To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three-dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell-cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate-based format, enabling high density hepatocyte culture as a stable 3D-multilayer. Multilayered co-cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen-conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co-cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate-based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co-cultures were maintained for at least 1 week; the so-cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate-based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening.

  12. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

    PubMed

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A

    2015-01-01

    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

  13. Human foreskin fibroblast-like stromal cells can differentiate into functional hepatocytic cells.

    PubMed

    Huang, Hsing-I; Chen, Shao-Kuan; Wang, Robert Y-L; Shen, Chia-Rui; Cheng, Yu-Che

    2013-12-01

    Foreskin fibroblast-like stromal cells (FDSCs) are progenitors isolated from human tissue that can differentiate into diverse cell types. Many types of stem cells can differentiate into hepatocyte-like cells, which could be used for drug testing or in liver regeneration therapy, but whether FDSCs can be converted into functional hepatocytes is unknown. FDSCs show divergent properties when cultured in distinct media, forming spheres in Dulbecco's modified Eagle's medium (DMEM) containing F12, epidermal growth factor (EGF), and basic fibroblast growth factor (b-FGF), but have fibroblast-like morphology when cultured in DMEM-based growth medium. Both cell populations express the typical mesenchymal stem cell markers CD90, CD105, and CD73, but the p75 neurotrophin receptor (p75NTR) was detected only in FDSC spheres. Both types of FDSCs can differentiate into hepatocyte-like cells, which express typical liver markers, including albumin and hepatocyte paraffin 1 (Hep Par1), along with liver-specific biological activities. When plasmids containing the human hepatitis B virus (HBV) genome were transfected transiently into FDSCs, differentiated hepatocyte-like cells secrete large amounts of HBe and HBs antigens. FDSCs could be used for clinical hepatic therapy and/or serve as a model of HBV.

  14. Benzylpenicillin, acetylcysteine and silibinin as antidotes in human hepatocytes intoxicated with alpha-amanitin.

    PubMed

    Magdalan, Jan; Ostrowska, Alina; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Podhorska-Okołów, Marzena; Patrzałek, Dariusz; Szelag, Adam; Dziegiel, Piotr

    2010-07-01

    Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to alpha-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to alpha-AMA at concentration 2microM. Exposure of the cells to alpha-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to alpha-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in alpha-AMA toxicity.

  15. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    PubMed

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  16. High Content Analysis of Human Pluripotent Stem Cell Derived Hepatocytes Reveals Drug Induced Steatosis and Phospholipidosis

    PubMed Central

    Pradip, Arvind; Steel, Daniella; Jacobsson, Susanna; Holmgren, Gustav; Ingelman-Sundberg, Magnus; Sartipy, Peter; Björquist, Petter; Johansson, Inger; Edsbagge, Josefina

    2016-01-01

    Hepatotoxicity is one of the most cited reasons for withdrawal of approved drugs from the market. The use of nonclinically relevant in vitro and in vivo testing systems contributes to the high attrition rates. Recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into pure cultures of hepatocyte-like cells expressing functional drug metabolizing enzymes open up possibilities for novel, more relevant human cell based toxicity models. The present study aimed to investigate the use of hiPSC derived hepatocytes for conducting mechanistic toxicity testing by image based high content analysis (HCA). The hiPSC derived hepatocytes were exposed to drugs known to cause hepatotoxicity through steatosis and phospholipidosis, measuring several endpoints representing different mechanisms involved in drug induced hepatotoxicity. The hiPSC derived hepatocytes were benchmarked to the HepG2 cell line and generated robust HCA data with low imprecision between plates and batches. The different parameters measured were detected at subcytotoxic concentrations and the order of which the compounds were categorized (as severe, moderate, mild, or nontoxic) based on the degree of injury at isomolar concentration corresponded to previously published data. Taken together, the present study shows how hiPSC derived hepatocytes can be used as a platform for screening drug induced hepatotoxicity by HCA. PMID:26880940

  17. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  18. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    SciTech Connect

    Shen Chong; Meng Qin Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 {mu}M which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 {mu}M. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to {beta}-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  19. Arsenite decreases CYP3A23 induction in cultured rat hepatocytes by transcriptional and translational mechanisms

    SciTech Connect

    Noreault, Trisha L.; Nichols, Ralph C.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Peter R.; Evans, Ronald M.; Sinclair, Jacqueline F. . E-mail: JSINC@dartmouth.edu

    2005-12-01

    Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 {mu}M arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.

  20. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.

    PubMed

    Igoudjil, Anissa; Massart, Julie; Begriche, Karima; Descatoire, Véronique; Robin, Marie-Anne; Fromenty, Bernard

    2008-06-01

    The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c

  1. Dynamic Interplay of Flow and Collagen Stabilizes Primary Hepatocytes Culture in a Microfluidic Platform

    PubMed Central

    Hegde, Manjunath; Jindal, Rohit; Bhushan, Abhinav; Bale, Shyam Sundhar; McCarty, William J; Golberg, Inna; Usta, O. Berk; Yarmush, Martin L.

    2014-01-01

    The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or ‘sandwich’ configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretion, and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures result in the formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain well-connected cellular network and differentiated function. PMID:24770663

  2. Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform.

    PubMed

    Hegde, Manjunath; Jindal, Rohit; Bhushan, Abhinav; Bale, Shyam Sundhar; McCarty, William J; Golberg, Inna; Usta, O Berk; Yarmush, Martin L

    2014-06-21

    The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or in 'sandwich' configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretions and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures show formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete a higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain a well-connected cellular network and a differentiated function.

  3. Applicability of second-generation upcyte® human hepatocytes for use in CYP inhibition and induction studies

    PubMed Central

    Ramachandran, Sarada D; Vivarès, Aurélie; Klieber, Sylvie; Hewitt, Nicola J; Muenst, Bernhard; Heinz, Stefan; Walles, Heike; Braspenning, Joris

    2015-01-01

    Human upcyte® hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte® hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC50 values for each compound correctly classified them as potent inhibitors. Upcyte® hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte® hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUCu/F2, and Cmax,u/Ind50. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte® hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte® hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations. PMID:26516577

  4. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    PubMed

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  5. Circadian Rhythms of PER2::LUC in Individual Primary Mouse Hepatocytes and Cultures

    PubMed Central

    Molyneux, Penny C.; Yu, Jimmy K.; Li, Alexander S.; Leise, Tanya L.; Harrington, Mary E.

    2014-01-01

    Background Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. Results In this study we isolated primary hepatocytes from transgenic Per2Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2−/− Per2Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. Conclusions Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals. PMID:24498336

  6. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  7. Optimizing human hepatocyte models for metabolic phenotype and function: effects of treatment with dimethyl sulfoxide (DMSO).

    PubMed

    Nikolaou, Nikolaos; Green, Charlotte J; Gunn, Pippa J; Hodson, Leanne; Tomlinson, Jeremy W

    2016-11-01

    Primary human hepatocytes are considered to be the "gold standard" cellular model for studying hepatic fatty acid and glucose metabolism; however, they come with limitations. Although the HepG2 cell line retains many of the primary hepatocyte metabolic functions they have a malignant origin and low rates of triglyceride secretion. The aim of this study was to investigate whether dimethyl sulfoxide supplementation in the media of HepG2 cells would enhance metabolic functionality leading to the development of an improved in vitro cell model that closely recapitulates primary human hepatocyte metabolism. HepG2 cells were cultured in media containing 1% dimethyl sulfoxide for 2, 4, 7, 14, and 21 days. Gene expression, protein levels, intracellular triglyceride, and media concentrations of triglyceride, urea, and 3-hydroxybutyrate concentrations were measured. Dimethyl sulfoxide treatment altered the expression of genes involved in lipid (FAS, ACC1, ACC2, DGAT1, DGAT2, SCD) and glucose (PEPCK, G6Pase) metabolism as well as liver functionality (albumin, alpha-1-antitrypsin, AFP). mRNA changes were paralleled by alterations at the protein level. DMSO treatment decreased intracellular triglyceride content and lactate production and increased triglyceride and 3-hydroxybutyrate concentrations in the media in a time-dependent manner. We have demonstrated that the addition of 1% dimethyl sulfoxide to culture media changes the metabolic phenotype of HepG2 cells toward a more primary human hepatocyte phenotype. This will enhance the currently available in vitro model systems for the study of hepatocyte biology related to pathological processes that contribute to disease and their response to specific therapeutic interventions.

  8. Species-specific interaction of HIV protease inhibitors with accumulation of cholyl-glycylamido-fluorescein (CGamF) in sandwich-cultured hepatocytes.

    PubMed

    Ye, Zhi-wei; Van Pelt, Jos; Camus, Sandrine; Snoeys, Jan; Augustijns, Patrick; Annaert, Pieter

    2010-06-01

    Using sandwich-cultured hepatocytes from rat, dog, pig, and human, we investigated the species-specificity of interaction of HIV protease inhibitors (PI) with in vitro hepatic accumulation of the bile salt analogue cholyl-glycylamido-fluorescein (CGamF). Extracellular sodium depletion or coincubation with the OATP/Oatp inhibitors rifampicin and digoxin revealed that about 35% of active CGamF accumulation was mediated by Ntcp/NTCP in rat and human hepatocytes, while the contribution of this sodium-dependent transporter reached 50-60% in dog and pig hepatocytes. One or more sodium-independent transporters, likely belonging to the Oatp/OATP family, constitute a major transport mechanism for CGamF accumulation. Various HIV PI (0.5, 5, 25 microM) exhibited pronounced species differences in their interaction with active CGamF accumulation (1 microM), although some similarity was observed between the dog and human interaction profiles when HIV PI were tested at 0.5 microM. Atazanavir, indinavir, and darunavir were the most potent inhibitors of CGamF accumulation in human hepatocytes. Potent inhibition of CGamF accumulation by ritonavir in rat hepatocytes contrasted with a weak effect in human hepatocytes. Thorough characterization of in vitro disposition of probe substrates in preclinical species compared to human hepatocytes will ultimately support a better insight in species-specific mechanisms underlying drug interactions and drug-mediated toxicity.

  9. Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

    PubMed

    Deharde, Daniela; Schneider, Christin; Hiller, Thomas; Fischer, Nicolas; Kegel, Victoria; Lübberstedt, Marc; Freyer, Nora; Hengstler, Jan G; Andersson, Tommy B; Seehofer, Daniel; Pratschke, Johann; Zeilinger, Katrin; Damm, Georg

    2016-10-01

    Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of

  10. Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

    PubMed Central

    Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

    2009-01-01

    Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

  11. 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

  12. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  13. 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.

  14. Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development.

    PubMed

    Hoshiba, Takashi; Otaki, Takayuki; Nemoto, Eri; Maruyama, Hiroka; Tanaka, Masaru

    2015-08-19

    The development of bioartificial liver (BAL) is expected because of the shortage of donor liver for transplantation. The substrates for BAL require the following criteria: (a) blood compatibility, (b) hepatocyte adhesiveness, and (c) the ability to maintain hepatocyte-specific functions. Here, we examined blood-compatible poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (PTHFA) as the substrates for BAL. HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrates. The spreading of HepG2 cells was suppressed on PMEA substrates because integrin contribution to cell adhesion on PMEA substrate was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substrate, whereas the expression decreased on PTHFA substrates due to the nuclear localization of Yes-associated protein (YAP). These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate cell functions for blood-contacting tissue engineering.

  15. Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

    PubMed

    Mezey, E; Potter, J J; Diehl, A M

    1986-01-15

    Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

  16. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  17. Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle.

    PubMed

    Wu, Kun Lieh; Chang, Siou Han; Manousakas, Ioannis; Huang, Han Hsiang; Teong, Benjamin; Chuang, Chin Wen; Kuo, Shyh Ming

    2015-03-13

    Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.

  18. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  19. Differential effects of long-term exposure to Aroclor 1254 on lipid secretion by primary cultures of adult rat hepatocytes

    SciTech Connect

    Mendoza-Figueroa, T.; Hernandez, A.; Lopez, L.

    1992-06-01

    PCBs produce hepatic triglyceride (TG) accumulation (fatty liver) in experimental animals and humans exposed accidentally and occupationally. It has been suggested that this effect could be due to a block in TG secretion. On the other hand, increased levels of plasmatic TG and cholesterol have been described in rats after dietary exposure to Aroclor 1254 (Aro) and other PCBs; hypertriglyceridemia and hypertension have been also described in humans exposed for long periods to low concentrations of PCBs. Since the study of hepatic lipid metabolism and its alteration by toxic chemicals is complicated in the whole animal, short term cultures of adult rat hepatocytes have been used. We have described a system for the long term culture of adult rat hepatocytes which for several weeks maintain differentiated functions, like fatty acid and TG synthesis and their export to the culture medium. In this paper we used this culture system to study the effect of long-term exposure to micromolar concentrations of Aro on the secretion of lipids by cultured hepatocytes. 27 refs., 4 figs., 1 tab.

  20. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  1. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  2. Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction.

    PubMed

    Ringel, M; von Mach, M A; Santos, R; Feilen, P J; Brulport, M; Hermes, M; Bauer, A W; Schormann, W; Tanner, B; Schön, M R; Oesch, F; Hengstler, J G

    2005-01-05

    An important application of hepatocyte cultures is identification of drugs acting as inducers of biotransformation enzymes that alter metabolic clearance of other therapeutic agents. In the present study we optimized an in vitro system with hepatocytes cultured in alginate microspheres that allow studies of enzyme induction with excellent sensitivity. Induction factors obtained with standard inducers, such as 3-methylcholanthrene or phenobarbital, were higher compared to those with conventional hepatocyte co-cultures on collagen coated dishes. This is illustrated by activities of 7-ethoxyresorufin-O-deethylase (EROD) after incubation with 5 microM 3-methylcholanthrene (3-MC), a standard inducer for cytochrome P4501A1 and 1A2. Mean activities for solvent controls and 3-MC exposed cells were 2.99 and 449 pmol/min/mg protein (induction factor: 150) for hepatocytes cultured in microspheres compared to 2.72 and 80.6 pmol/min/mg (induction factor: 29.6) for hepatocytes on collagen coated dishes. To compare these in vitro data to the in vivo situation male Sprague Dawley rats, the same strain that was used also for the in vitro studies, were exposed to 3-MC in vivo using a protocol that guarantees maximal induction. Activities were 29.2 and 1656 pmol/min/mg in liver homogenate of solvent and 3-MC treated animals (induction factor: 56.7). Thus, the absolute activities of 3-MC exposed hepatocytes in microspheres are lower compared to the in vivo situation. However, the induction factor in vitro was even higher compared to the in vivo situation (150-fold versus 56.7-fold). A similar scenario was observed using phenobarbital (0.75 mM) for induction of CYP2B and 3A isoenzymes: induction factors for testosterone hydroxylation in position 16beta were 127.5- and 50.4-fold for hepatocytes in microspheres and conventionally cultured hepatocytes, respectively. The new in vitro system with hepatocytes embedded in solid alginate microspheres offers several technical advantages: (i

  3. A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

    PubMed

    Strick-Marchand, Helene; Dusséaux, Mathilde; Darche, Sylvie; Huntington, Nicholas D; Legrand, Nicolas; Masse-Ranson, Guillemette; Corcuff, Erwan; Ahodantin, James; Weijer, Kees; Spits, Hergen; Kremsdorf, Dina; Di Santo, James P

    2015-01-01

    Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

  4. A thin-walled polydimethylsiloxane bioreactor for high-density hepatocyte sandwich culture.

    PubMed

    Tan, Guo-Dong Sean; Toh, Guoyang William; Birgersson, Erik; Robens, Jeffrey; van Noort, Danny; Leo, Hwa Liang

    2013-06-01

    In vitro drug testing requires long-term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24-well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long-term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days.

  5. Methamphetamine enhances Hepatitis C virus replication in human hepatocytes.

    PubMed

    Ye, L; Peng, J S; Wang, X; Wang, Y J; Luo, G X; Ho, W Z

    2008-04-01

    Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease.

  6. Enhancement of proliferation in a rat hepatocyte co-culture model after mitogenic stimulation.

    EPA Science Inventory

    Primary mouse and rat hepatocyte cultures have long been the gold standard for assessment of cellular changes following chemical exposure. While helpful for assessing proliferative and responses in vitro, these cultures are limited to 1 or 2 days of incubation. Our motivation was...

  7. Liver-specific RNA metabolism in hepatocytes in long-term culture

    SciTech Connect

    Georgoff, I.; Isom, H.C.; Salditt-Georgieff, M.; Darnell, J.E. Jr.

    1986-05-01

    The authors have examined the transcription of liver-specific and housekeeping genes in rat hepatocytes maintained in culture for up to 40 days in chemically defined medium. RNA extracted from these cells was analyzed using northern and dot blot methods. RNA in nuclei extracted from parallel cultures was analyzed by nascent chain extension experiments. Autoradiography of northern and dot blots indicated that in vitro hepatocyte gene expression relative to in vivo liver gene expression can be divided into four classes: (1) liver-specific genes that show loss of expression with time in culture; (2) liver-specific genes that are expressed for up to 40 days in culture at levels approximately 30-100% of those found in liver, e.g., albumin; (3) housekeeping genes that are expressed for up to 40 days in culture at levels approximately equivalent to those found in liver; and (4) housekeeping genes that are expressed at higher levels in cells in culture than liver. The results of nascent chain extension experiments indicated that the rate of synthesis of albumin message was many fold lower in hepatocytes than in liver, whereas rates of synthesis of messages for housekeeping genes were equal to or slightly higher in nuclei from hepatocytes in culture than in nuclei extracted from liver. Molecular analyses of nuclear RNA suggest that the high levels of albumin mRNA in cultured cells are due to an increased stability probably in the cytoplasm.

  8. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis?

    PubMed

    Vinken, Mathieu; Vanhaecke, Tamara; Rogiers, Vera

    2012-04-01

    Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative process and thus for the generation of a long-term hepatic in vitro tool. Such experimental system is urgently needed, especially in the light of the stringent European legislative modifications that are currently encountered by the pharmaceutical, chemical and, particularly, the cosmetic industry.

  9. [An experimental study on the protective effect of hepatocyte growth factor (HGF) for the primary cultured hepatocytes obtained from iron-loaded rats].

    PubMed

    Yoshida, J

    1995-01-01

    Pathological iron deposition in liver is often found in various liver diseases. The deposited iron is thought to be one of the most important factor of liver cell injury, not only in hemochromotosis but also in cirrhosis following hepatitis virus B or C infection. To investigate the influence of the deposited iron on damage and regeneration of hepatocyte, primary cultured hepatocytes obtained from carbonyl iron-loaded rats were treated with carbon tetrachloride (CCl4) in the presence or absence of hepatocyte growth factor (HGF). Although the section of liver from carbonyl iron-loaded rats showed no necrosis and fibrosis, iron-loaded hepatocytes contained about twofold more iron than control. The damage of iron-loaded hepatocytes induced by CCl4 was more serious than that of control, and HGF decreased this injury only in iron-loaded hepatocytes but not in control. There is no difference in DNA synthesis stimulated by HGF between iron-loaded hepatocytes and control. These findings suggest that the pathological iron deposition induces the fragility of hepatocyte and that cytoprotective effect of HGF is induced by this pathological iron.

  10. Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

    PubMed Central

    Gieseck III, Richard L.; Hannan, Nicholas R. F.; Bort, Roque; Hanley, Neil A.; Drake, Rosemary A. L.; Cameron, Grant W. W.; Wynn, Thomas A.; Vallier, Ludovic

    2014-01-01

    Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard. PMID:24466060

  11. Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model.

    PubMed

    Bissig, Karl-Dimiter; Le, Tam T; Woods, Niels-Bjarne; Verma, Inder M

    2007-12-18

    We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah(-/-)) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse liver is repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene can be observed. We further report the development of a hepatocyte transplantation method involving a transcutaneous, intrahepatic injection in neonatal mice. Human hepatocytes engraft over the entire injected lobe with an expansion pattern similar to those observed with intrasplenic transplantation.

  12. 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

  13. Sulodexide induces hepatocyte growth factor release in humans.

    PubMed

    Borawski, Jacek; Dubowski, Miroslaw; Pawlak, Krystyna; Mysliwiec, Michal

    2007-03-08

    Heparin influences numerous pleiotropic growth factors, including hepatocyte growth factor (HGF), partially by their release from endothelial and extracellular matrix stores. The effects of sulodexide, a heparin-like glycosaminoglycan medication of growing importance in medicine, on HGF liberation are not known. We performed a 2-week open-label sulodexide trial in healthy male volunteers. The drug was initially administered intravenously (i.v.) in a single dose of 1200 Lipoprotein Lipase Releasing Units (LRU), then -- orally for 12 days (500 LRU twice a day), and -- again by i.v. route (1200 LRU) on day 14. Intravenous sulodexide injections were repeatedly found to induce marked and reproducible increases in immunoreactive plasma HGF levels (more than 3500% vs baseline after 10 min, and more than 1200% after 120 min), and remained unchanged when measured 120 min following oral sulodexide administration. The percentage increments in plasma HGF evoked by i.v. sulodexide at both time points and on both days inversely correlated with baseline levels of the growth factor. On day 14, the HGF levels after 120 min and their percentage increase vs baseline were strongly and directly dependent on i.v. sulodexide dose per kg of body weight. This study shows that sulodexide has a novel, remarkable and plausibly biologically important stimulating effect on the release of pleiotropic hepatocyte growth factor in humans.

  14. In vitro biocompatibility of polypyrrole/PLGA conductive nanofiber scaffold with cultured rat hepatocytes

    NASA Astrophysics Data System (ADS)

    Chu, Xue-Hui; Xu, Qian; Feng, Zhang-Qi; Xiao, Jiang-Qiang; Li, Qiang; Sun, Xi-Tai; Cao, Yang; Ding, Yi-Tao

    2014-09-01

    To intruduce conductive biomaterial into liver tissue engineering, a conductive nanofiber scaffold, polypyrrole/poly(lactic-co-glycolic)acid(PLGA), was designed and prepared via electro-spinning and oxidative polymerization. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. SEM revealed pseudopodium formation and abundant extracellular matrix on the surface of PLGA membrane and polypyrrole/PLGA membrane. The adhesion rate, cellular activity, urea synthesis and albumin secretion of the hepatocytes cultured on polypyrrole/PLGA group were similar to those on the PLGA group, but were significantly higher than those on the control group. There were no significant differences in concentrations of LDH and TNF-α among three groups. These results suggested the potential application of this conductive nanofiber scaffold as a suitable substratum for hepatocyte culturing in liver tissue engineering.

  15. Comparison of S9 mix and hepatocytes as external metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1

    SciTech Connect

    Madle, E.; Tiedemann, G.; Madle, S.; Oett, A.; Kaufmann, G.

    1986-01-01

    Two external metabolizing systems, S9 mix from Aroclor-induced rat livers and freshly isolated hepatocytes, were used for activation in cultures of human lymphocytes and V79 cells. 7, 12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) were employed as indirectly acting reference mutagens. Mutagenic effects were measured by induction of sister chromatid exchange (SCE). With DMBA, SCE-inducing effects were found to be quite similar after activation by S9 mix and activation by hepatocytes. In contrast with AFB1, S9 activation led to a stronger SCE induction than hepatocyte activation in both target cells. The induction of chromosomal aberrations by AFB1 after activation by the two metabolizing systems was also analyzed in V79 cells. This experiment again revealed that AFB1 was more efficiently activated by S9 mix than by hepatocytes. The experiments have shown that the suitability of hepatocytes as an activation system is not restricted to microbial or eukaryotic point mutation assays, but that hepatocyte metabolism can also be successfully included in cytogenetic tests with short- and long-term cultures of mammalian target cells.

  16. Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture.

    PubMed

    Goral, Vasiliy N; Au, Sam H; Faris, Ronald A; Yuen, Po Ki

    2014-07-01

    In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.

  17. Differentiation of human foreskin fibroblast-derived induced pluripotent stem cells into hepatocyte-like cells.

    PubMed

    Wang, Jianjun; Zhao, Ping; Wan, Zhihong; Jin, Xueyuan; Cheng, Yongqian; Yan, Tao; Qing, Song; Ding, Ning; Xin, Shaojie

    2016-10-01

    The aim of this study was to investigate the differentiation potential of induced pluripotent stem cells (iPSCs) derived from human foreskin fibroblasts (HFFs) into hepatocyte-like cells (HLCs). The iPSCs were firstly induced by transduction of OCT4, SOX2, KLF4, and c-MYC into HFFs using retrovirus. Afterwards, expressions of pluripotency factors were identified by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence staining, and karyotype, embryoid, and teratoma were observed by microscope. Then, iPSCs were gradually differentiated into endoderm cells, hepatic progenitor cells, and mature HLCs by special culture medium. During this process, differentiation efficiency into each kind of cells was evaluated by detecting SOX17, HNF4a, and ALB using flow cytometry, respectively. Besides, enzyme-linked immunosorbent assay was conducted to detect the secretion of ALB in iPSC-induced HLCs and quantitative reverse transcription-polymerase chain reaction was performed to detect the expression levels of hepatocyte-specific genes. The iPSCs were successfully induced by HFFs, which exhibited typical embryonic stem cells morphology, positive alkaline phosphatase staining, normal diploid karyotype, and positive expression of various pluripotency factors. Meanwhile, spherical embryoid and teratoma with 3 germ layers were formed by iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells and mature HLCs, and the differentiation efficiency was 55.7 ± 2.9%, 45.7 ± 4.8%, and 35.0 ± 3.9%, respectively. Besides, the secretion of ALB and expression of various hepatocyte-specific genes was highly detected in iPSC-induced HLCs. The iPSCs were successfully derived from HFFs and then differentiated into HLCs, which proved a new source for hepatocyte transplantation.

  18. 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.

  19. Effect of Dimethyl Sulfoxide and Melatonin on the Isolation of Human Primary Hepatocytes.

    PubMed

    Solanas, Estela; Sostres, Carlos; Serrablo, Alejandro; García-Gil, Agustín; García, Joaquín J; Aranguren, Francisco J; Jiménez, Pilar; Hughes, Robin D; Serrano, María T

    2015-01-01

    The availability of fully functional human hepatocytes is critical for progress in human hepatocyte transplantation and the development of bioartificial livers and in vitro liver systems. However, the cell isolation process impairs the hepatocyte status and determines the number of viable cells that can be obtained. This study aimed to evaluate the effects of using dimethyl sulfoxide (DMSO) and melatonin in the human hepatocyte isolation protocol. Human hepatocytes were isolated from liver pieces resected from 10 patients undergoing partial hepatectomy. Each piece was dissected into 2 equally sized pieces and randomized, in 5 of 10 isolations, to perfusion with 1% DMSO-containing perfusion buffer or buffer also containing 5 mM melatonin using the 2-step collagenase perfusion technique (experiment 1), and in the other 5 isolations to standard perfusion or perfusion including 1% DMSO (experiment 2). Tissues perfused with DMSO yielded 70.6% more viable hepatocytes per gram of tissue (p = 0.076), with a 26.1% greater albumin production (p < 0.05) than those perfused with control buffer. Melatonin did not significantly affect (p > 0.05) any of the studied parameters, but cell viability, dehydrogenase activity, albumin production, urea secretion, and 7-ethoxycoumarin O-deethylase activity were slightly higher in cells isolated with melatonin-containing perfusion buffer compared to those isolated with DMSO. In conclusion, addition of 1% DMSO to the hepatocyte isolation protocol could improve the availability and functionality of hepatocytes for transplantation, but further studies are needed to clarify the mechanisms involved.

  20. Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress.

    PubMed

    Bhogal, Ricky H; Weston, Christopher J; Curbishley, Stuart M; Adams, David H; Afford, Simon C

    2012-04-01

    The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.

  1. Interactions of inhibitors of carnitine palmitoyltransferase I and fibrates in cultured hepatocytes.

    PubMed Central

    Gerondaes, P; Alberti, K G; Agius, L

    1988-01-01

    Culture of rat hepatocytes with etomoxir, an inhibitor of carnitine palmitoyltransferase I (CPT I), for 48 h, resulted in increased carnitine acetyltransferase (CAT) activity (74%), a marked decrease in CPT activity (82%) measured in detergent extracts, and increased activities of glucose-6-phosphate dehydrogenase (227%) and fructose-1,6-bisphosphatase (65%). Changes in CAT and CPT activities were not observed after 4 h culture with etomoxir. When hepatocytes were cultured with etomoxir and benzafibrate (a hypolipidaemic analogue of clofibrate) for 48 h, etomoxir prevented the 5-fold increase in CAT activity caused by bezafibrate, whereas bezafibrate suppressed the increase in glucose-6-phosphate dehydrogenase and fructose-bisphosphatase caused by etomoxir. However, bezafibrate did not prevent the suppression of CPT activity by etomoxir. Etomoxir inhibited palmitate beta-oxidation and ketogenesis after short-term (0-4 h) and long-term (48 h) exposure, but it caused accumulation of triacylglycerol in hepatocytes only after short-term exposure (0-4 h). These effects of etomoxir on fatty acid metabolism and suppression of CPT (after 48 h) were similar in periportal and perivenous hepatocytes, but the increases in CAT and glucose-6-phosphate dehydrogenase activities were higher in periportal than in perivenous cells. The effects of CPT I inhibitors on CAT activity and long-term suppression of CPT activity are probably mediated by independent mechanisms. PMID:3421940

  2. Perifusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro.

    PubMed

    Gebhardt, R; Wegner, H; Alber, J

    1996-04-01

    The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 micromol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including

  3. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  4. Treatment of surgically induced acute liver failure with transplantation of highly differentiated immortalized human hepatocytes.

    PubMed

    Kobayashi, N; Miyazaki, M; Fukaya, K; Inoue, Y; Sakaguchi, M; Noguchi, H; Matsumura, T; Watanabe, T; Totsugawa, T; Tanaka, N; Namba, M

    2000-01-01

    Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.

  5. Morphology and albumin secretion of adult rat hepatocytes cultured on a hydrophobic porous expanded polytetrafluoroethylene membrane.

    PubMed

    Kurosawa, Hiroshi; Yuminamochi, Eri; Yasuda, Ruri; Amano, Yoshifumi

    2003-01-01

    Primary culture of rat hepatocytes was performed on a hydrophobic porous expanded polytetrafluoroethylene (ePTFE) membrane incorporated into the base of a culture dish. Two types of ePTFE membranes, a uniaxially expanded type (ePTFE-1) and a biaxially expanded type (ePTFE-2), could be used as the culture surfaces for hepatocytes. The formation of multicellular aggregates was observed in the culture dish when each membrane type was used. A pore size of 1 mum or higher was adequate for cell adhesion and albumin secretion for both membrane types. The activity of albumin secretion in the dish with the ePTFE membrane was markedly higher than that in the polystyrene dish. Spheroidal multicellular aggregates (spheroids) were observed when hepatocytes were cultured on the ePTFE-1 membrane. The ePTFE-1 membrane maintained the albumin secretion activity for a longer period than the non-expanded PTFE film. It was assumed that the cooperative action of membrane structure and oxygen permeability promoted the formation of cell aggregates and increased the albumin secretion activity.

  6. Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

    PubMed

    Viollon-Abadie, C; Bigot-Lasserre, D; Nicod, L; Carmichael, N; Richert, L

    2000-12-01

    Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T(4)-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), beta-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T(4)-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T(4)-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T(4)-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T(4)-UGT activities.

  7. 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.

  8. Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties.

    PubMed

    Wilkening, Stefan; Stahl, Frank; Bader, Augustinus

    2003-08-01

    Cultures of primary hepatocytes and hepatoma cell line HepG2 are frequently used in in vitro models for human biotransformation studies. In this study, we characterized and compared the capacity of these model systems to indicate the presence of different classes of promutagens. Genotoxic sensitivity, enzyme activity, and gene expression were monitored in response to treatment with food promutagens benzo[a]pyrene, dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA damage could be detected reliably with the comet assay in primary human hepatocytes, which were maintained in sandwich culture. All three promutagens caused DNA damage in primary cells, but in HepG2 no genotoxic effects of DMN and PhIP could be detected. We supposed that the lack of specific enzymes accounts for their inability to process these promutagens. Therefore, we quantified the expression of a broad range of genes coding for drug-metabolizing enzymes with real-time reverse transcription-polymerase chain reaction. The genes code for cytochromes p450 and, in addition, for a series of important phase II enzymes. The expression level of these genes in human hepatocytes was similar to those previously reported for human liver samples. On the other hand, expression levels in HepG2 differed significantly from that in human. Activity and expression, especially of phase I enzymes, were demonstrated to be extremely low in HepG2 cells. Up-regulation of specific genes by test substances was similar in both cell types. In conclusion, human hepatocytes are the preferred model for biotransformation in human liver, whereas HepG2 cells may be useful to study regulation of drug-metabolizing enzymes.

  9. Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures.

    PubMed

    Cable, E E; Isom, H C

    1997-12-01

    We previously showed that primary rat hepatocytes plated on a rat-tail collagen coated dish and fed a chemically-defined medium supplemented with 2% dimethylsulfoxide (DMSO) can be maintained in a well-differentiated, non-replicating state for periods of several months. In this study, we show that the addition of copper, iron, and zinc to the DMSO-containing chemically defined medium induced DNA synthesis and cell replication during the first two months in culture without loss of hepatic differentiation. DNA synthesis occurred throughout the hepatocyte population without regard to cellular size. No changes were observed in properties indicative of well-differentiated hepatocytes, including cellular morphology, ultrastructure, albumin, or cytokeratin-8 expression. During the third month in culture, after the hepatocytes had become confluent, pseudoduct structures became apparent. Examination of cells lining the ducts by immunohistochemistry showed that these cells lost the ability to express albumin and stained more intensely for cytokeratin 19 and laminin. The ultrastructure of the cells lining the ducts was altered and became more characteristic of bile duct cells. Immunoelectron microscopy revealed that connexin 43, a marker of bile-duct proliferation, was expressed in the duct-like cells. We conclude that under these specific nutritive conditions, primary rat hepatocytes proliferate and, with time, begin to form duct-like structures with altered gene expression and ultrastructural properties.

  10. Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures.

    PubMed

    Thompson, Rodney W; Valentine, Holly L; Valentine, William M

    2003-06-30

    Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H(2)S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H(2)S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H(2)S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H(2)S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

  11. 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

  12. 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...

  13. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

    NASA Astrophysics Data System (ADS)

    Otsuka, Hidenori; Sasaki, Kohei; Okimura, Saya; Nagamura, Masako; Nakasone, Yuichi

    2013-12-01

    Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell-matrix interactions, soluble stimuli and cell-cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

  14. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  15. Enrichment of hepatocytes in a HepaRG culture using spatially selective photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Bednarkiewicz, Artur; Rodrigues, Robim M.; Whelan, Maurice P.

    2010-03-01

    The human hepatoma HepaRG cell line is an in vitro cell model that is becoming an important tool in drug metabolism, hepatotoxicity, genotoxicity, and enzyme induction studies. The cells are highly proliferative during their undifferentiated state but once committed, they differentiate into two distinctly different cell types, namely, hepatocyte-like and biliary epithelial-like cells. The presence of the latter in the cell culture is considered to be a drawback of the cell model. Since the proliferating undifferentiated HepaRG cells have a bipotent character, the only way to improve the content ratio of hepatic versus biliary cells of differentiated HepaRG cells is to eradicate biliary cells in situ, in a way that free surface space does not become available and thus no transdifferentiation can occur. Spatially selective photodynamic therapy has proven to be effective for that purpose. First, all the cells were administered aminolevulinic acid (δ-ALA) to stimulate the synthesis of protoporphyrin IX (PpIX), a naturally occurring photosensitizer. Then, the biliary cells were automatically identified and outlined by bright-field image processing. Last, UV light patterns were projected onto the epithelial cells alone by a spatial light modulation device connected to an optical microscope; therefore, only these cells were destroyed by photodynamic therapy.

  16. Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts.

    PubMed

    Dirkx, Ruud; Meyhi, Els; Asselberghs, Stanny; Reddy, Janardan; Baes, Myriam; Van Veldhoven, Paul P

    2007-06-08

    Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.

  17. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

    PubMed Central

    Ruch, R J; Klaunig, J E; Schultz, N E; Askari, A B; Lacher, D A; Pereira, M A; Goldblatt, P J

    1986-01-01

    Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes. PMID:3816733

  18. Dependence of the carbon-tetrachloride--induced death of cultured hepatocytes on the extracellular calcium concentration

    SciTech Connect

    Casini, A.F.; Farber, J.L.

    1981-01-01

    The role of extracellular Ca/sup 2/+ ions in the killing of liver cells by CCl/sub 4/ was studied in primary cultures of rat hepatocytes. The dependence of in vitro cell killing on the metabolism of CCl/sub 4/ was first examined in order to document the similarity between the action of CCl/sub 4/ on cultured hepatocytes and the action of CCl/sub 4/ on liver cells in the intact animal. Cells prepared from male rats pretreated with phenobarbital were more sensitive to CCl/sub 4/ than cells prepared from either male or female rats. The killing of hepatocytes by CCl/sub 4/ was prevented by addition of SKF 525A to the culture medium. This protection was accompanied by evidence of decreased CCl/sub 4/ metabolism as assessed by the extent of covalent binding of 14C-CCl/sub 4/ metabolites to total cellular lipids and proteins, and by the extent of formation of conjugated dienes accompanying the peroxidation of phospholipids isolated from total cell lipids. The extent of killing of the hepatocytes by CCl/sub 4/ was dependent on the Ca/sup 2/+ concentration in the tissue culture medium. The results of this study indicate that it is the presence of extracellular Ca/sup 2/+ that converts initially nonlethal cell injury into irreversible cell injury in CCl/sub 4/-treated cells. This action of Ca/sup 2/+ most likely represents an influx into the cell across an injured permeability barrier at the plasma membrane, in accord with the accumulation of large quantities of Ca/sup 2/+ in CCl/sub 4/-intoxicated liver cells in the intact animal. The relation between this alteration in Ca/sup 2/+ homeostasis and the metabolism of CCl/sub 4/ is discussed.

  19. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes

    SciTech Connect

    Ruch, R.J.; Klaunig, J.E.; Schultz, N.E.; Askari, A.B.; Lacher, D.A.; Pereira, M.A.; Goldblatt, P.J.

    1986-11-01

    Mechanisms of chloroform (CHCl/sub 3/) and carbon tetrachloride (CCl/sub 4/) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl/sub 3/ and CCl/sub 4/ was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl/sub 3/ (5 mM) and CCl/sub 4/ (2.5 mM) used and with the longest duration of treatment (20 hr). CCl/sub 4/ was approximately 16 times more toxic than CHCl/sub 3/ to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25..mu..M) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl/sub 3/ and CCl/sub 4/ toxicity. The toxicity of CHCl/sub 3/ and CCl/sub 4/ could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25..mu..M), ..cap alpha..-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl/sub 3/ and CCl/sub 4/ are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl/sub 3/ and CCl/sub 4/; and free radicals may be important mediators of the toxicity of these two halomethanes.

  20. A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency.

    PubMed

    Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

    2015-01-01

    Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.

  1. Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.

    PubMed

    Broeders, Jessica J W; Parmentier, Céline; Truisi, Germaine L; Jossé, Rozenn; Alexandre, Eliane; Savary, Camille C; Hewitt, Philip G; Mueller, Stefan O; Guillouzo, André; Richert, Lysiane; van Eijkeren, Jan C H; Hermens, Joop L M; Blaauboer, Bas J

    2015-12-25

    Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 μM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

  2. HBV life cycle is restricted in mouse hepatocytes expressing human NTCP

    PubMed Central

    Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

    2014-01-01

    Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1–6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1–6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created. PMID:24509445

  3. Human but Not Mouse Hepatocytes Respond to Interferon-Lambda In Vivo

    PubMed Central

    Hermant, Pascale; Demarez, Céline; Mahlakõiv, Tanel; Staeheli, Peter; Meuleman, Philip; Michiels, Thomas

    2014-01-01

    The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-λ) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-λ. In contrast, the IFN-λ response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-λ response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-λ. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-λ response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-λ. The lack of mouse hepatocyte response to IFN-λ was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-λ. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-λ in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-λ in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment. PMID:24498220

  4. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    PubMed

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.

  5. 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.

  6. In vitro drug metabolism of green tea catechins in human, monkey, dog, rat and mouse hepatocytes.

    PubMed

    Chen, Wendy W; Qin, Geng-Yao; Zhang, Ting; Feng, Wan-Yong

    2012-06-01

    The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and

  7. 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

  8. Comparative Metabolism, Cytotoxicity, and Genotoxicity of Chemical Carcinogens in Primary Cultures of Hepatocytes

    DTIC Science & Technology

    1985-05-01

    death and regeneration. Cytotoxicity is easily assessed in primary hepatocyte cultures by light microscopic observation (Green ott al., 1981) and/or the...The recent development of a sensitive scintillation detection of repair provides a three day assay (Loury and Byard, 1983). A UV light positive control...RATS FED 0.5% BUTYLATED HYDROXYTOLUENEa Amount of ascron.•,cut./cultur-b P10o0. Al’"I boumd/t ,acrtmooletuloc Ratlo of bound producto k’ontro I WN

  9. Generation of major human excretory and circulating drug metabolites using a hepatocyte relay method.

    PubMed

    Ballard, T Eric; Orozco, Christine C; Obach, R Scott

    2014-05-01

    The prediction of human drug metabolites using in vitro experiments containing human-derived reagents is an important approach in modern drug research; however, this can be challenging for drugs that are slowly metabolized. In this report, we describe the use of a recently developed human hepatocyte relay method for the purpose of predicting human drug metabolite profiles. Five compounds for which in vivo human metabolism data were available were selected for the investigation of this method, and the results were compared with data gathered in hepatocyte suspensions as well as previous data from a micropatterned hepatocyte coculture method. The hepatocyte relay method demonstrated an improved performance (generation of 75% of human in vivo metabolites) for those drugs for which previous methods showed a relatively low rate of success (50% of human in vivo metabolites). Metabolites included those arising from both oxidative and conjugative reactions and metabolites that required sequential reactions. Two 4-hour relays were shown to adequately generate metabolites, and no further benefit was derived from more relays. Overall, it can be concluded that the hepatocyte relay assay method can be successfully used in the generation of relevant human metabolites, even for challenging drugs.

  10. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  11. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    PubMed

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  12. Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

    PubMed

    Badolo, Lassina; Jensen, Bente; Säll, Carolina; Norinder, Ulf; Kallunki, Pekka; Montanari, Dino

    2015-02-01

    1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

  13. 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.

  14. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  15. Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels.

    PubMed

    Haque, Amranul; Gheibi, Pantea; Gao, Yandong; Foster, Elena; Son, Kyung Jin; You, Jungmok; Stybayeva, Gulnaz; Patel, Dipali; Revzin, Alexander

    2016-09-29

    The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.

  16. Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels

    PubMed Central

    Haque, Amranul; Gheibi, Pantea; Gao, Yandong; Foster, Elena; Son, Kyung Jin; You, Jungmok; Stybayeva, Gulnaz; Patel, Dipali; Revzin, Alexander

    2016-01-01

    The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs–this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function. PMID:27681582

  17. Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells.

    PubMed

    Langenbach, R; Rudo, K

    1988-12-01

    The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.

  18. 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

  19. S-Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes

    PubMed Central

    Tomasi, Maria Lauda; Ramani, Komal; Lopitz-Otsoa, Fernando; Rodríguez, Manuel S.; Li, Tony W. H.; Ko, Kwangsuk; Yang, Heping; Bardag-Gorce, Fawzia; Iglesias-Ara, Ainhoa; Feo, Francesco; Pascale, Maria Rosa; Mato, José M.; Lu, Shelly C.

    2010-01-01

    Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. PMID:20196119

  20. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T. )

    1991-09-01

    Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

  1. Applying Stable Isotope Labeled Amino Acids in Micropatterned Hepatocyte Co-Culture to Directly Determine the Degradation Rate Constant for CYP3A4.

    PubMed

    Takahashi, Ryan H; Shahidi-Latham, Sheerin; Wong, Susan; Chang, Jae H

    2017-03-13

    The rate of enzyme degradation (kdeg) is an important input parameter for the prediction of clinical drug-drug-interactions (DDI) that result from mechanism-based inactivation or induction of cytochrome P450s. Currently, a large range of reported estimates for CYP3A4 enzyme degradation exists, and consequently, large uncertainty exists in steady-state predictions for DDI. In the current investigations, stable isotope labeled amino acids in culture (SILAC) was applied to a long-lived primary human hepatocyte culture, HepatoPac, to directly monitor the degradation of CYP3A4. This approach allowed selective isotope labeling of a population of de novo synthesized CYP3A4, and specific quantification of proteins with mass spectrometry to determine the CYP3A4 degradation within the hepatocytes. The kdeg estimate was 0.026 ± 0.005 h- 1. This value was reproduced by cultures derived across four individual donors. For these cultures, data indicated that CYP3A4 mRNA and total protein expression (i.e. labeled and not labeled P450s), and activity were stable over the period where degradation had been determined. This kdeg value for CYP3A4 was in good agreement with recently reported values that used alternate analytical approaches, but also employed micropatterned primary human hepatocytes as the in vitro model.

  2. 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.

  3. 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

  4. 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.

  5. Adenosine 3',5'-cyclic monophosphate involvement in hepatic triacylglyceride lipase release from prazosin-stimulated primary cultured rat hepatocytes.

    PubMed

    Nakamura, Tetsuya; Morita, Tetsuo

    2014-01-01

    We recently found that hepatic triglyceride lipase (HTGL) was released from primary cultured rat hepatocytes after treatment with prazosin, an antagonist of alpha-1 adrenoceptors. However, the details of prazosin-induced HTGL release remain uncertain. Here we investigated whether changes in cAMP levels in hepatocytes were related to HTGL release from prazosin-stimulated hepatocytes. When hepatocytes were treated with prazosin, cAMP levels during stimulated release of HTGL increased in a time- and dose-dependent manner. Stimulated release of HTGL was suppressed by the adenylate cyclase inhibitors MDL-12,330A and 2',5'-dideoxyadenosine. Further, cAMP-dependent protein kinase A (PKA) activity in prazosin-stimulated hepatocytes also increased in a time- and dose-dependent manner. Moreover, prazosin-stimulated HTGL release was suppressed by the PKA inhibitors H-89 and KT5720. These results suggest that prazosin-stimulated HTGL release from hepatocytes was due to cAMP production and partly due to subsequent PKA activation in hepatocytes.

  6. Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

    PubMed Central

    Ryu, Hyun Mi; Park, Sung Gyoo; Yea, Sung Su; Jang, Won Hee; Yang, Young-Il; Jung, Guhung

    2006-01-01

    AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2×, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK. PMID:16937494

  7. Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

    SciTech Connect

    Vance, J.E.; Vance, D.E.

    1986-05-01

    The role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine (PC) is made both by the CDP-choline pathway and by the methylation of phosphatidyl-ethanolamine (PE), which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanol-amine). Monolayer cultures of rat hepatocytes were incubated in the presence of (methyl-/sup 3/H)choline, (2-/sup 3/H)ethanolamine or (3-/sup 3/H)serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the culture medium. The specific radioactivities of PC and PE derived from (1-/sup 3/H)ethanolamine were markedly lower (approximately 1/2 and less than 1/10, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of PC made from (methyl-/sup 3/H)choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with (3-/sup 3/H)serine, the specific radioactivities of PC and PE were significantly higher in the lipoproteins than in the cells. These data indicate that specific pools of phospholipids are selected on the basis of their routes of biosynthesis, for secretion into lipoproteins.

  8. [Use of hepatocytes in primary cultures to predict potential hepatocarcinogenicity of compounds

    PubMed

    Bieri, F.; Muakkassah-Kelly, S.; Bentley, P.

    1990-01-01

    Short-term tests for genotoxic activity will detect a variety of potential carcinogens. However, experience over the last few years has shown that many chemical carcinogens are not detected in these tests. A large proportion of these non-mutagenic (non-genotoxic) carcinogens induce tumors in the rodent liver after life-long feeding. One class of such carcinogens are the hypolipidemic drugs many of which induce a characteristic hepatomegaly upon subchronic treatment. Typically this liver enlargement is accompanied by increased mitotic activity and a proliferation of the hepatic peroxisome compartment. Results also suggest a correlation between the degree and the nature of the hepatomegalic response to a compound, and its hepatocarcinogenic potency, but it remains unclear whether the growth stimulation and/or the peroxisome proliferation is correlated with the carcinogenicity. Both, the induction of peroxisomal enzyme and of replicative DMA synthesis may be measured in primary cultures of adult rat hepatocytes following in vitro exposition. Moreover, the ability of the compounds tested to induce in vitro replicative DNA synthesis in primary cultures of adult rat hepatocytes correlated well with the potency of the hepatomegalic response induced in vivo in treated rodents. Consequently, studies using such cultures might be useful to characterize and possibly predict in vitro the hepatocarcinogenic potency of some new compounds. The early recognition of the possible carcinogenic property of a new substance might accordingly contribute to the reduction of a number of life-long studies.

  9. Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies.

    PubMed

    Zavan, B; Brun, P; Vindigni, V; Amadori, A; Habeler, W; Pontisso, P; Montemurro, D; Abatangelo, G; Cortivo, R

    2005-12-01

    Tissue engineering is a promising approach to developing hepatic tissue suitable for the functional replacement of a failing liver. The aim of the present study was to investigate whether an extracellular cell matrix obtained from fibroblasts-cultured within scaffolds of hyaluronic acid (HYAFF) could influence the proliferation rate and survival of rat hepatocytes both during long-term culture and after in vivo transplantation. Cultures were evaluated by histological and morphological analysis, a proliferation assay and metabolic activity (albumin secretion). Hepatocytes cultured in extracellular matrix-enriched scaffolds exhibited a round cellular morphology and re-established cell-cell contacts, growing into aggregates of several cells along and/or among fibers in the fabric. Hepatocytes were able to secrete albumin up to 14 days in culture. In vivo results demonstrated the biocompatibility of HYAFF-11 implanted in nude mice, in which hepatocytes maintained small well-organised aggregates until the 35th day. In conclusion, the presence of a fibroblast-secreted extracellular matrix improved the biological properties of the hyaluronan scaffold, favoring the survival and morphological integrity of hepatocytes in vitro and in vivo.

  10. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

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

    PubMed Central

    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

  12. Methanethiol and dimethylsulfide formation from 3-methylthiopropionate in human and rat hepatocytes.

    PubMed

    Blom, H J; van den Elzen, J P; Yap, S H; Tangerman, A

    1988-11-18

    This study was designed to investigate the metabolism of methanethiol, and the involvement of methanethiol and its metabolites in the transamination pathway of methionine. Gaseous methanethiol, methanethiol-mixed disulfides and dimethylsulfide were formed from 3-methylthiopropionate, a metabolite in the transamination pathway of methionine, during incubation with human and rat hepatocytes. An increase of the 3-methylthiopropionate concentration resulted in an increased formation of the products, up to a substrate concentration of 4.4 mM. Higher substrate levels resulted in a decreased methanethiol formation, probably due to poisoning of the system. However, in human hepatocytes the formation of dimethylsulfide increased up to a 3-methylthiopropionate concentration of 12.5 mM. The formation of methanethiol, dimethylsulfide and methanethiol-mixed disulfides from 3-methylthiopropionate in hepatocytes of both human and rat support the hypothesis that methanethiol can be formed from methionine via the transamination pathway.

  13. 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

  14. Photolytic degradation of benorylate: effects of the photoproducts on cultured hepatocytes

    SciTech Connect

    Castell, J.V.; Gomez, M.J.; Mirabet, V.; Miranda, M.A.; Morera, I.M.

    1987-05-01

    The photodegradation of benorylate (4'-(acetamido)phenyl-2-acetoxybenzoate), a drug frequently used in rheumatoid arthritis therapy, has been examined under different sets of experimental conditions. Several photoproducts have been isolated and identified on the basis of their IR, NMR, and MS spectra. The most significant photochemical process is the photo-Fries rearrangement of benorylate, leading to 5-acetamido-2'-acetoxy-2-hydroxybenzophenone (1). This compound undergoes a rapid transacylation to the isomeric 5'-acetamido-2'-acetoxy-2-hydroxybenzophenone (2). A primary culture of rat hepatocytes has been used to evaluate the possible toxicity of these two benzophenones, keeping in mind the following criteria: leakage of cytosolic enzymes, attachment index to culture plates, gluconeogenesis from lactate and fructose, glycogen balance, and albumin synthesis. At the concentrations assayed, neither of the two major photoproducts of benorylate (benzophenones 1 and 2) had significant toxic effects on liver cells in culture.

  15. Photolytic degradation of benorylate: effects of the photoproducts on cultured hepatocytes.

    PubMed

    Castell, J V; Gomez, M J; Mirabet, V; Miranda, M A; Morera, I M

    1987-05-01

    The photodegradation of benorylate [4'-(acetamido)phenyl-2-acetoxybenzoate], a drug frequently used in rheumatoid arthritis therapy, has been examined under different sets of experimental conditions. Several photoproducts have been isolated and identified on the basis of their IR, NMR, and MS spectra. The most significant photochemical process is the photo-Fries rearrangement of benorylate, leading to 5-acetamido-2'-acetoxy-2-hydroxybenzophenone (1). This compound undergoes a rapid transacylation to the isomeric 5'-acetamido-2'-acetoxy-2-hydroxybenzophenone (2). A primary culture of rat hepatocytes has been used to evaluate the possible toxicity of these two benzophenones, keeping in mind the following criteria: leakage of cytosolic enzymes, attachment index to culture plates, gluconeogenesis from lactate and fructose, glycogen balance, and albumin synthesis. At the concentrations assayed, neither of the two major photoproducts of benorylate (benzophenones 1 and 2) had significant toxic effects on liver cells in culture.

  16. Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

    PubMed

    Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

    2011-05-01

    α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

  17. Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells

    PubMed Central

    Yanagihara, Kana; Liu, Yujung; Kanie, Kei; Takayama, Kazuo; Kokunugi, Minako; Hirata, Mitsuhi; Fukuda, Takayuki; Suga, Mika; Nikawa, Hiroki; Mizuguchi, Hiroyuki; Kato, Ryuji

    2016-01-01

    Abstract Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However, the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study, we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line, H9, which is known to differentiate into hepatocytes, and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs, hPSC-derived hepatoblast-like differentiated cells, and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus, our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines. PMID:27733097

  18. Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

    PubMed

    Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2007-10-01

    We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

  19. Effect of curcumin analog on gamma-radiation-induced cellular changes in primary culture of isolated rat hepatocytes in vitro.

    PubMed

    Srinivasan, M; Sudheer, A Ram; Rajasekaran, K N; Menon, Venugopal P

    2008-10-22

    The present study was aimed to evaluate the radioprotective effect of curcumin analog, on gamma-radiation-induced toxicity in primary cultures of isolated rat hepatocytes. Hepatocytes were isolated from the liver of rats by collagenase perfusion. The DNA damage was analysed by single cell gel electrophoresis (comet assay). An increase in the severity of DNA damage was observed with the increase in gamma-radiation dose at 1-4 Gy in cultured rat hepatocytes. The levels of lipid peroxidative indices like thiobarbituric acid reactive substances (TBARSs) were increased significantly, whereas the levels of reduced glutathione (GSH) and antioxidant enzymes were significantly decreased in gamma-irradiated groups. The maximum damage to hepatocytes was observed at 4Gy gamma-irradiation. Pretreatment with different concentrations of curcumin analog (1.38, 6.91 and 13.82 microM) shows a significant decrease in the levels of TBARS and DNA damage. Pretreatment with curcumin analog prevents the loss of enzymic and non-enzymic antioxidants like GSH upon gamma-irradiation. The maximum protection of hepatocytes was observed at 6.91 microM of curcumin analog pretreatment. Thus, our result shows that pretreatment with curcumin analog protects the hepatocytes against gamma-radiation-induced cellular damage.

  20. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

    PubMed

    Caperna, T J; Shannon, A E; Stoll, M; Blomberg, L A; Ramsay, T G

    2015-07-01

    Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

  1. Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

    PubMed Central

    Chowdhury, M H; Agius, L

    1987-01-01

    Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action. PMID:2827626

  2. A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture.

    PubMed

    McCarty, William J; Usta, O Berk; Luitje, Martha; Bale, Shyam Sundhar; Bhushan, Abhinav; Hegde, Manjunath; Golberg, Inna; Jindal, Rohit; Yarmush, Martin L

    2014-03-01

    The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen "double gel" or "overlay". To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.

  3. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin

    PubMed Central

    1992-01-01

    Nitric oxide (NO) is a short-lived biologic mediator that is shown to be induced in various cell types and to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of the inducible type of the NO synthase (NOS). However, there is limited evidence for the existence of such inducible NOS in a human cell type. We show here the induction of NO biosynthesis in freshly isolated human hepatocytes (HC) after stimulation with interleukin 1, tumor necrosis factor (TNF), IFN- gamma, and endotoxin. Increased levels of nitrite (NO2-) and nitrate (NO3-) in culture supernatants were associated with NADPH-dependent NOS activity in the cell lysates. The production of NO2- and NO3- was inhibited by NG-monomethyl L-arginine and was associated with an increase in cyclic guanylate monophosphate release. The data presented here provide evidence for the existence of typical inducible NO biosynthesis in a human cell type. PMID:1377225

  4. Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells

    PubMed Central

    Lammi, Carmen; Zanoni, Chiara; Ferruzza, Simonetta; Ranaldi, Giulia; Sambuy, Yula; Arnoldi, Anna

    2016-01-01

    Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion. PMID:27455315

  5. Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2004-10-01

    Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.

  6. Screening for oestrogenic activity of plant and food extracts using in vitro trout hepatocyte cultures.

    PubMed

    Bennetau-Pelissero, C; Latonnelle, K Gontier; Lamothe, V; Shinkaruk-Poix, S; Kaushik, S J

    2004-01-01

    The use of in vitro trout hepatocyte cultures is shown to provide a simple and effective way to screen plant and food products for oestrogenic activity. The relative oestrogenic activities of 0.1 g each of extracts of phytosterol, soy isoflavone, red clover, kudzu and soybean extracts were determined using this assay and found to be equivalent to 212, 1, 3.2, 132 and 1025 nM of 17beta-estradiol, respectively. Controls were performed on soybean and kudzu extracts using specific ELISAs for isoflavones and these confirmed the validity of the cell culture assay. The method described offers an advantage over current methods in that it can detect increased oestrogenic activity that may occur as a result of metabolic activation of pre- or pro-oestrogens liver cells.

  7. Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells.

    PubMed

    Wang, Yu; Alhaque, Sharmin; Cameron, Kate; Meseguer-Ripolles, Jose; Lucendo-Villarin, Baltasar; Rashidi, Hassan; Hay, David C

    2017-03-02

    Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines promising advances in cell-based modelling and therapies.

  8. Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.

    PubMed

    Sanoh, Seigo; Santoh, Masataka; Takagi, Masashi; Kanayama, Tatsuya; Sugihara, Kazumi; Kotake, Yaichiro; Ejiri, Yoko; Horie, Toru; Kitamura, Shigeyuki; Ohta, Shigeru

    2014-09-01

    Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

  9. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides

    PubMed Central

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

  10. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides.

    PubMed

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs.

  11. Cultures of human liver cells in simulated microgravity environment

    NASA Astrophysics Data System (ADS)

    Yoffe, B.; Darlington, G. J.; Soriano, H. E.; Krishnan, B.; Risin, D.; Pellis, N. R.; Khaoustov, V. I.

    1999-01-01

    We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

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

    PubMed Central

    Peng, H; Armentano, D; MacKenzie-Graham, L; Shen, R F; Darlington, G; Ledley, F D; Woo, S L

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We 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 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 form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 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. Images PMID:3186716

  13. 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...

  14. Transcriptional and metabolic flux profiling of triadimefon effects on cultured hepatocytes

    SciTech Connect

    Iyer, Vidya V.; Ovacik, Meric A.; Androulakis, Ioannis P.; Roth, Charles M.; Ierapetritou, Marianthi G.

    2010-11-01

    Conazoles are a class of azole fungicides used to prevent fungal growth in agriculture, for treatment of fungal infections, and are found to be tumorigenic in rats and/or mice. In this study, cultured primary rat hepatocytes were treated to two different concentrations (0.3 and 0.15 mM) of triadimefon, which is a tumorigenic conazole in rat and mouse liver, on a temporal basis with daily media change. Following treatment, cells were harvested for microarray data ranging from 6 to 72 h. Supernatant was collected daily for three days, and the concentrations of various metabolites in the media and supernatant were quantified. Gene expression changes were most significant following exposure to 0.3 mM triadimefon and were characterized mainly by metabolic pathways related to carbohydrate, lipid and amino acid metabolism. Correspondingly, metabolic network flexibility analysis demonstrated a switch from fatty acid synthesis to fatty acid oxidation in cells exposed to triadimefon. It is likely that fatty acid oxidation is active in order to supply energy required for triadimefon detoxification. In 0.15 mM triadimefon treatment, the hepatocytes are able to detoxify the relatively low concentration of triadimefon with less pronounced changes in hepatic metabolism.

  15. Effects of metal ions on lipid peroxidation in cultured rat hepatocytes loaded with alpha-linolenic acid.

    PubMed

    Furono, K; Suetsuga, T; Sugihara, N

    1996-06-07

    We investigated the ability of various redox-active metal ions to induce lipid peroxidation in normal and alpha-linolenic acid-loaded (LNA-loaded) cultured rat hepatocytes. Lipid peroxidation was estimated by the accumulation of malondialdehyde (MDA) in the culture medium. At low concentrations induction was highest with ferrous ions (Fe), whereas at high concentrations, vanadium (V) and copper ions (Cu) had the greatest effect on both groups of hepatocytes. With any one of the three metal ions, the extent of lipid peroxidation in LNA-loaded hepatocytes was several times greater compared to normal cells. In addition, upon the addition of Fe or V, LNA-loaded hepatocytes were injured whereas normal cells were not. The addition of Cu caused substantial cell injury in normal hepatocytes, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant like N,N'-diphenyl-p-phenylene-diamine (DPPD) almost completely prevented Fe- and V-induced cell injury, and reduced Cu-induced cell injury. alpha-Tocopherol behaved in a way similar to but less effective than DPPD. .OH radical scavengers such as mannitol and dimethyl sulfoxide (DMSO) had no effect on lipid peroxidation induced by any metal ions in LNA-loaded hepatocytes. Addition of cadmium ions (Cd), which required the lowest concentration to cause cell injury, induced a slight increase in lipid peroxidation in normal hepatocytes, but did not induce lipid peroxidation to the same extent as seen in LNA-loaded cells treated with any of the three metal ions already mentioned. The inhibition of lipid peroxidation by DPPD scarcely protected LNA-loaded hepatocytes from Cd-induced cell injury. None of the other metal ions including aluminum (Al), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and tin (Sn) ions, effectively induced lipid peroxidation in either group of hepatocytes, except cobalt ions (Co), which had a peroxidative effect in LNA

  16. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  17. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.

    PubMed

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

  18. Celsior solution compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia-reperfusion injury.

    PubMed

    Janssen, Hermann; Janssen, Petra H E; Broelsch, Christoph E

    2003-07-01

    Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.

  19. Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

    PubMed Central

    Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

    1996-01-01

    Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

  20. 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.

  1. 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.

  2. 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.

  3. Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes

    PubMed Central

    Ghini, Veronica; Di Nunzio, Mattia; Tenori, Leonardo; Valli, Veronica; Danesi, Francesca; Capozzi, Francesco; Luchinat, Claudio; Bordoni, Alessandra

    2017-01-01

    Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA) supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR) in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect—propionic acid (PRO) and protocatechuic acid (PCA)—has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination) and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action. PMID:28208746

  4. Regulation of adiponectin receptor 1 in human hepatocytes by agonists of nuclear receptors

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas; Kirchner, Stefan; Laberer, Sabine; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2005-09-02

    The adiponectin receptors AdipoR1 and AdipoR2 have been identified to mediate the insulin-sensitizing effects of adiponectin. Although AdipoR2 was suggested to be the main receptor for this adipokine in hepatocytes, AdipoR1 protein is highly abundant in primary human hepatocytes and hepatocytic cell lines. Nuclear receptors are main regulators of lipid metabolism and activation of peroxisome proliferator-activated receptor {alpha} and {gamma}, retinoid X receptor (RXR), and liver X receptor (LXR) by specific ligands may influence AdipoR1 abundance. AdipoR1 protein is neither altered by RXR or LXR agonists nor by pioglitazone. In contrast, fenofibric acid reduces AdipoR1 whereas hepatotoxic troglitazone upregulates AdipoR1 protein in HepG2 cells. Taken together this work shows for the first time that AdipoR1 protein is expressed in human hepatocytes but that it is not a direct target gene of nuclear receptors. Elevated AdipoR1 induced by hepatotoxic troglitazone may indicate a role of this receptor in adiponectin-mediated beneficial effects in liver damage.

  5. 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.

  6. Human Cord Blood Stem Cells Generate Human Cytokeratin 18-Negative Hepatocyte-Like Cells in Injured Mouse Liver

    PubMed Central

    Sharma, Amar Deep; Cantz, Tobias; Richter, Rudolf; Eckert, Klaus; Henschler, Reinhard; Wilkens, Ludwig; Jochheim-Richter, Andrea; Arseniev, Lubomir; Ott, Michael

    2005-01-01

    Differentiation of adult bone marrow (BM) cells into nonhematopoietic cells is a rare phenomenon. Several reports, however, suggest that human umbilical cord blood (hUCB)-derived cells give rise to hepatocytes after transplantation into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, we analyzed the hepatic differentiation potential of hUCB cells and compared the frequency of newly formed hepatocyte-like cells in the livers of recipient NOD-SCID mice after transplantation of hUCB versus murine BM cells. Mononuclear cell preparations of hUCB cells or murine BM from enhanced green fluorescent protein transgenic or wild-type mice were transplanted into sublethally irradiated NOD-SCID mice. Liver regeneration was induced by carbon tetrachloride injury with and without sub-sequent hepatocyte growth factor treatment. By immunohistochemistry and reverse transcriptasepolymerase chain reaction, we detected clusters of hepatocyte-like cells in the livers of hUCB-transplanted mice. These cells expressed human albumin and Hep Par 1 but mouse CK18, suggesting the formation of chimeric hepatocyte-like cells. Native fluorescence microscopy and double immunofluorescence failed to detect single hepatocytes derived from transplanted enhanced green fluorescent protein-transgenic mouse BM. Fluorescent in situ hybridization rarely revealed donor-derived hepatocyte-like cells after cross-gender mouse BM transplantation. Thus, hUCB cells have differentiation capabilities different from murine BM cells after transplantation into NOD-SCID mice, demonstrating the importance of further testing before hUCB cells can be used therapeutically. PMID:16049339

  7. Nitric oxide-mediated antiplasmodial activity in human and murine hepatocytes induced by gamma interferon and the parasite itself: enhancement by exogenous tetrahydrobiopterin.

    PubMed Central

    Mellouk, S; Hoffman, S L; Liu, Z Z; de la Vega, P; Billiar, T R; Nussler, A K

    1994-01-01

    Expression of inducible nitric oxide (NO) synthase has been shown to inhibit the development of several pathogens, including fungi, bacteria, parasites, and viruses. However, there is still controversy as to whether this effector mechanism can inhibit the development of human pathogens. We now report that gamma interferon (IFN-gamma) induces the elimination of Plasmodium falciparum-infected primary human hepatocytes from cultures and that the antimalarial activity is dependent on NO. Infection with the parasite alone in the absence of added IFN-gamma caused a 10-fold increase in NO formation. Both spontaneous inhibition and IFN-gamma-induced inhibition of Plasmodium yoelii-infected murine hepatocytes were increased with the addition of the NO synthase cofactor tetrahydrobiopterin, or sepiapterin, which is converted to tetrahydrobiopterin. These results indicate that under in vitro conditions the parasite itself provides a signal that triggers induction of the NO pathway in human and murine hepatocytes and that NO formation in infected hepatocytes is limited by tetrahydrobiopterin availability. PMID:8063424

  8. Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury

    PubMed Central

    YANG, KYUNGHEE; GUO, CEN; WOODHEAD, JEFFREY L; CLAIRE, ROBERT LST.; WATKINS, PAUL B; SILER, SCOTT Q; HOWELL, BRETT A; BROUWER, KIM LR

    2016-01-01

    Sandwich-cultured hepatocytes (SCH) are metabolically competent and have proper localization of basolateral and canalicular transporters with functional bile networks. Therefore, this cellular model is a unique tool that can be used to estimate biliary excretion of compounds. SCH have been used widely to assess hepatobiliary disposition of endogenous and exogenous compounds and metabolites. Mechanistic modeling based on SCH data enables estimation of metabolic and transporter-mediated clearances, which can be employed to construct physiologically-based pharmacokinetic models for prediction of drug disposition and drug-drug interactions in humans. In addition to pharmacokinetic studies, SCH also have been employed to study cytotoxicity and perturbation of biological processes by drugs and hepatically-generated metabolites. Human SCH can provide mechanistic insights underlying clinical drug-induced liver injury (DILI). In addition, data generated in SCH can be integrated into systems pharmacology models to predict potential DILI in humans. In this review, applications of SCH in studying hepatobiliary drug disposition and bile acid-mediated DILI are discussed. An example is presented to show how data generated in the SCH model was used to establish a quantitative relationship between intracellular bile acids and cytotoxicity, and how this information was incorporated into a systems pharmacology model for DILI prediction. PMID:26869411

  9. Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury.

    PubMed

    Yang, Kyunghee; Guo, Cen; Woodhead, Jeffrey L; St Claire, Robert L; Watkins, Paul B; Siler, Scott Q; Howell, Brett A; Brouwer, Kim L R

    2016-02-01

    Sandwich-cultured hepatocytes (SCH) are metabolically competent and have proper localization of basolateral and canalicular transporters with functional bile networks. Therefore, this cellular model is a unique tool that can be used to estimate biliary excretion of compounds. SCH have been used widely to assess hepatobiliary disposition of endogenous and exogenous compounds and metabolites. Mechanistic modeling based on SCH data enables estimation of metabolic and transporter-mediated clearances, which can be used to construct physiologically based pharmacokinetic models for prediction of drug disposition and drug-drug interactions in humans. In addition to pharmacokinetic studies, SCH also have been used to study cytotoxicity and perturbation of biological processes by drugs and hepatically generated metabolites. Human SCH can provide mechanistic insights underlying clinical drug-induced liver injury (DILI). In addition, data generated in SCH can be integrated into systems pharmacology models to predict potential DILI in humans. In this review, applications of SCH in studying hepatobiliary drug disposition and bile acid-mediated DILI are discussed. An example is presented to show how data generated in the SCH model were used to establish a quantitative relationship between intracellular bile acids and cytotoxicity, and how this information was incorporated into a systems pharmacology model for DILI prediction.

  10. In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and a dynamic three-dimensional bioreactor system.

    PubMed

    Darnell, Malin; Ulvestad, Maria; Ellis, Ewa; Weidolf, Lars; Andersson, Tommy B

    2012-10-01

    Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes. (14)C-diclofenac and a candidate from AstraZeneca's drug development program, (14)C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.

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

    PubMed Central

    Bachour-El Azzi, Pamela; Sharanek, Ahmad; Burban, Audrey; Li, Ruoya; Guével, Rémy Le; Abdel-Razzak, Ziad; Stieger, Bruno; Guguen-Guillouzo, Christiane; Guillouzo, André

    2015-01-01

    The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na+-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis. PMID:25690737

  12. Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid.

    PubMed

    Kang, Young Bok Abraham; Rawat, Siddhartha; Cirillo, Joseph; Bouchard, Michael; Noh, Hongseok Moses

    2013-12-01

    This paper presents a novel liver model that mimics the liver sinusoid where most liver activities occur. A key aspect of our current liver model is a layered co-culture of primary rat hepatocytes (PRHs) and primary rat liver sinusoidal endothelial cells (LSECs) or bovine aortic endothelial cells (BAECs) on a transwell membrane. When a layered co-culture was attempted with a thin Matrigel layer placed between hepatocytes and endothelial cells to mimic the space of Disse, the cells did not form completely separated monolayers. However, when hepatocytes and endothelial cells were cultured on the opposite sides of a transwell membrane, PRHs co-cultured with LSECs or BAECs maintained their viability and normal morphology for 39 and 57 days, respectively. We assessed the presence of hepatocyte-specific differentiation markers to verify that PRHs remained differentiated in the long-term co-culture and analyzed hepatocyte function by monitoring urea synthesis. We also noted that the expression of cytochrome P-450 remained similar in the co-cultured system from day 1 to day 48. Thus, our novel liver model system demonstrated that primary hepatocytes can be cultured for extended times and retain their hepatocyte-specific functions when layered with endothelial cells.

  13. 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.

  14. Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

    PubMed

    Rodeiro, I; Donato, M T; Martínez, I; Hernández, I; Garrido, G; González-Lavaut, J A; Menéndez, R; Laguna, A; Castell, J V; Gómez-Lechón, M J

    2008-08-01

    The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.

  15. Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes.

    PubMed

    Jeon, Yu Jin; Kim, Hyoung Seok; Song, Kyung-Sik; Han, Ho Jae; Park, Soo Hyun; Chang, Woochul; Lee, Min Young

    2015-04-01

    Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2-induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.

  16. 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.

  17. 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.

  18. 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

  19. Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis.

    PubMed

    Huang, Hsiu-Chen; Chen, Chun-Chi; Chang, Wen-Cheng; Tao, Mi-Hua; Huang, Cheng

    2012-09-01

    The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.

  20. Generation of Endoderm derived Human iPS cells from Primary Hepatocytes

    PubMed Central

    Liu, Hua; Ye, Zhaohui; Kim, Yong-Hak; Sharkis, Saul; Jang, Yoon-Young

    2010-01-01

    Recent advances in induced pluripotent stem (iPS) cell research significantly changed our perspective on regenerative medicine. Patient specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to hES cells or safer than hES cells. There are several important issues which need to be addressed and foremost are the safety and efficacy of human iPS cells from different origins. Human iPS cells have been derived mostly from cells originated from mesoderm, with a few cases from ectoderm. So far there has been no report of endoderm derived human iPS cells, preventing comprehensive comparative investigations on the quality of human iPS cells from different origins. Here we show for the first time reprogramming of human endoderm derived cells (i.e. primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. The technology to develop endoderm derived human iPS cell lines, together with other established cell lines, will provide a foundation to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells for cell therapy. For studying liver disease pathogenesis, this technology also provides a potentially more amenable system to generate liver disease specific iPS cells. PMID:20432258

  1. New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

    PubMed Central

    Holtzinger, Audrey; Streeter, Philip R.; Sarangi, Farida; Hillborn, Scott; Niapour, Maryam; Ogawa, Shinichiro; Keller, Gordon

    2015-01-01

    The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. PMID:26493401

  2. 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.

  3. Challenging small human hepatocytes with opiates: further characterization of a novel prototype bioartificial liver.

    PubMed

    Wurm, Martin; Woess, Claudia; Libiseller, Kathrin; Beer, Beate; Pavlic, Marion

    2010-03-01

    Bioartificial liver (BAL) systems can take over liver functions in patients undergoing liver failure until transplantation. Recently, a novel prototype rotary BAL has been developed using small human hepatocytes (SH). This study investigated the metabolism of opiates morphine and methadone in the BAL and their influence on the basic cell culture parameters, viability, and growth of SH. Opiates may be present in patients due to pain therapy, anticancer treatment, or drug abuse. Cells were cultivated in the BAL for a total of 12 days and exposed twice to 100 microg/L of morphine or methadone. Morphine and methadone concentrations were analyzed using gas chromatography with a mass spectrometry detector. Further, the production of albumin, lactate dehydrogenase release, lactate release, urea production, and glucose consumption were measured. Cell viability and growth were determined by confocal microscopy. Cytochrome P 3A4 and uridindiphosphat (UDP) glucuronosyl transferase 2B7 in SH were analyzed by western blot. The mean cell density during treatment was 5.5 +/- 0.7 x 10(6) cells/mL (n = 6) and was not altered significantly by the opiates. Cell viability stayed above 90%. Morphine was not reduced by SH and was a stress factor as determined by decreased metabolic activity. On the other hand, SH metabolized methadone showing first-order kinetics: the first-order rate constant k = 0,019, half-life t(1/2) = 36 h. Methadone metabolism led to decreased urea and albumin production. The expression of cytochrome P 3A4, mainly responsible for methadone metabolism, was proved in SH. The prototype BAL is basically suited to support liver functions, provided patients receive therapy with methadone.

  4. Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema.

    PubMed

    Plantier, Laurent; Marchand-Adam, Sylvain; Marchal-Sommé, Joëlle; Lesèche, Guy; Fournier, Michel; Dehoux, Monique; Aubier, Michel; Crestani, Bruno

    2005-04-01

    Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of 2.50-47.62 pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.2 pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of 20.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(2). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of 2.2-9.3 pg/mug) was similar to that of controls at baseline (2.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.

  5. Explanted diseased livers - a possible source of metabolic competent primary human hepatocytes.

    PubMed

    Kleine, Moritz; Riemer, Marc; Krech, Till; DeTemple, Daphne; Jäger, Mark D; Lehner, Frank; Manns, Michael P; Klempnauer, Jürgen; Borlak, Jürgen; Bektas, Hueseyin; Vondran, Florian W R

    2014-01-01

    Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5'-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent

  6. Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies.

    PubMed

    Pedersen, Jenny M; Shim, Yoo-Sik; Hans, Vaibhav; Phillips, Martin B; Macdonald, Jeffrey M; Walker, Glenn; Andersen, Melvin E; Clewell, Harvey J; Yoon, Miyoung

    2016-01-01

    Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices - RealBio(®) and QuasiVivo(®) (QV) - and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental

  7. Fluid Dynamic Modeling to Support the Development of Flow-Based Hepatocyte Culture Systems for Metabolism Studies

    PubMed Central

    Pedersen, Jenny M.; Shim, Yoo-Sik; Hans, Vaibhav; Phillips, Martin B.; Macdonald, Jeffrey M.; Walker, Glenn; Andersen, Melvin E.; Clewell, Harvey J.; Yoon, Miyoung

    2016-01-01

    Accurate prediction of metabolism is a significant outstanding challenge in toxicology. The best predictions are based on experimental data from in vitro systems using primary hepatocytes. The predictivity of the primary hepatocyte-based culture systems, however, is still limited due to well-known phenotypic instability and rapid decline of metabolic competence within a few hours. Dynamic flow bioreactors for three-dimensional cell cultures are thought to be better at recapitulating tissue microenvironments and show potential to improve in vivo extrapolations of chemical or drug toxicity based on in vitro test results. These more physiologically relevant culture systems hold potential for extending metabolic competence of primary hepatocyte cultures as well. In this investigation, we used computational fluid dynamics to determine the optimal design of a flow-based hepatocyte culture system for evaluating chemical metabolism in vitro. The main design goals were (1) minimization of shear stress experienced by the cells to maximize viability, (2) rapid establishment of a uniform distribution of test compound in the chamber, and (3) delivery of sufficient oxygen to cells to support aerobic respiration. Two commercially available flow devices – RealBio® and QuasiVivo® (QV) – and a custom developed fluidized bed bioreactor were simulated, and turbulence, flow characteristics, test compound distribution, oxygen distribution, and cellular oxygen consumption were analyzed. Experimental results from the bioreactors were used to validate the simulation results. Our results indicate that maintaining adequate oxygen supply is the most important factor to the long-term viability of liver bioreactor cultures. Cell density and system flow patterns were the major determinants of local oxygen concentrations. The experimental results closely corresponded to the in silico predictions. Of the three bioreactors examined in this study, we were able to optimize the experimental

  8. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.

    PubMed

    Moravcová, A; Červinková, Z; Kučera, O; Mezera, V; Rychtrmoc, D; Lotková, H

    2015-01-01

    In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone.

  9. Toxicity monitoring with primary cultured hepatocytes underestimates the acetaminophen-induced inflammatory responses of the mouse liver.

    PubMed

    Tachibana, Shinjiro; Shimomura, Akiko; Inadera, Hidekuni

    2011-01-01

    In vitro gene expression profiling with isolated hepatocytes has been used to assess the hepatotoxicity of certain chemicals because of animal welfare issues. However, whether an in vitro system can completely replace the in vivo system has yet to be elucidated in detail. Using a focused microarray established in our laboratory, we examined gene expression profiles in the mouse liver and primary cultured hepatocytes after treatment with different doses of acetaminophen, a widely used analgesic that frequently causes liver injury. The acute hepatotoxicity of acetaminophen was confirmed by showing the induction of an oxidative stress marker, heme oxygenase-1, elevated levels of serum transaminase, and histopathological findings. In vivo microarray and network analysis showed that acetaminophen treatment provoked alterations in relation to the inflammatory response, and that tumor necrosis factor-α plays a central role in related pathway alterations. By contrast, pathway analyses in in vitro isolated hepatocytes did not find such prominent changes in the inflammation-related networks compared with the in vivo situation. Thus, although in vitro gene expression profiles are useful for evaluating the direct toxicity of chemicals, indirect toxicities including inflammatory responses mediated by cell-cell interactions or secondary toxicity due to pathophysiological changes in the whole body may be overlooked. Our results indicate that the in vitro hepatotoxicity prediction system using isolated hepatocytes does not fully reflect the in vivo cellular response. An in vitro system may be appropriate, therefore, for high throughput screening to detect the direct hepatotoxicity of a test compound.

  10. 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.

  11. Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes.

    PubMed

    Szabó, Mónika; Veres, Zsuzsa; Bátai-Konczos, Attila; Kékesi, Orsolya; Kis, Emese; Szabó, Kitti; Jemnitz, Katalin

    2014-09-01

    Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects.

  12. Relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

    SciTech Connect

    Kindberg, G.M.; Refsnes, M.; Christoffersen, T.; Norum, K.R.; Berg, T.

    1987-05-25

    The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with /sup 125/I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

  13. Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells

    SciTech Connect

    Marra, C.A.; de Alaniz, M.J.; Brenner, R.R.

    1986-03-01

    The effect of dexamethasone on the incorporation and conversion of (1-14C)eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10(-6) M) also caused a marked decrease in the biosynthesis of this polyunsaturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the delta 5 desaturation of labeled 20:3 omega 6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells.

  14. Evaluation of transcriptomic signature as a valuable tool to study drug-induced cholestasis in primary human hepatocytes.

    PubMed

    Parmentier, Céline; Couttet, Philippe; Wolf, Armin; Zaccharias, Thomas; Heyd, Bruno; Bachellier, Philippe; Uteng, Marianne; Richert, Lysiane

    2017-02-10

    Primary human hepatocyte (PHH) sandwich cultures from five different donors were daily exposed to cyclosporine A (CsA), ibuprofen (IBU), chlorpromazine (CPZ), amiodarone (AMI) and paracetamol (APAP) at their respective Cmax (total) for short-term (1-3 days) and long-term treatment (14 days). Whole genome mRNA profiles (34,693 genes in total) were conducted using an Illumina microarray platform. The impact of compound treatments on gene signatures involved in liver differentiation, cholestasis and in bile acid homeostasis was evaluated. Notably, PHH from the five donors showed a highly comparable phenotype of terminally differentiated hepatocytes. As expected, PHH exposed to 100 µM APAP showed no signs of hepatotoxicity both after short- and long-term treatment. CsA at 0.7 µM, IBU at 100 µM, AMI at 2.5 µM and CPZ at 0.1-0.2 µM presented, in line with their cholestatic syndromes reported at therapeutic doses, transcriptomic signatures of cholestasis in PHH cultures; deregulation of genes involved in bile acid homeostasis further confirmed this finding. The strength of the cholestasis signature obtained after treatment with CsA, IBU and AMI could be directly related to the basal expression of the respective drug metabolizing enzymes in the various PHH cultures from different individuals. Our data show that the PHH model system combined with transcriptomics carries the future promise to identify individual gene expression profiles predictive of increased cholestasis risk. As the present work suggests possible correlation between mRNA levels of ADME relevant genes and a transcriptomic signature of cholestasis, particular focus on this research question could be the emphasis of additional data collection.

  15. 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.

  16. Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes.

    PubMed Central

    Baqué, S; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin. Images Figure 3 PMID:7998979

  17. Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

    PubMed Central

    Rose, Kelly A.; Holman, Natalie S.; Green, Angela M.; Andersen, Melvin E.; LeCluyse, Edward L.

    2017-01-01

    Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxinand model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses. PMID:26869439

  18. Mechanistic insights from comparing intrinsic clearance values between human liver microsomes and hepatocytes to guide drug design.

    PubMed

    Di, Li; Keefer, Christopher; Scott, Dennis O; Strelevitz, Timothy J; Chang, George; Bi, Yi-An; Lai, Yurong; Duckworth, Jonathon; Fenner, Katherine; Troutman, Matthew D; Obach, R Scott

    2012-11-01

    Metabolic stability of drug candidates are often determined in both liver microsome and hepatocyte assays. Comparison of intrinsic clearance values between the two assays provides additional information to guide drug design. Intrinsic clearance values from human liver microsomes and hepatocytes were compared for a set of commercial drugs with known metabolic pathways and transporter characteristics. The results showed that for compounds that were predominately metabolized by CYP mediated mechanisms, the intrinsic clearance values from the two assays were comparable. For compounds with non-CYP pathways, such as UGT and AO, intrinsic clearance was faster in hepatocytes than in microsomes. Substrates of uptake or efflux transporters in this study did not have significant differences of intrinsic clearance between microsomes and hepatocytes, when uptake into the hepatocytes was not the rate-limiting step. When hepatic uptake was rate limiting, intrinsic clearance in microsomes was faster than that in hepatocytes, which was more prevalent for compounds with rapid metabolism. Low passive permeability can limit the exposure to drug molecules to the metabolizing enzymes in the hepatocytes in relationship to the rate of metabolism. The faster the rate of metabolism, the higher permeability is needed for molecule to enter the cells and not becoming rate-limiting. The findings are very useful for drug discovery programs to gain additional insights on mechanistic information to help drug design without added experiments. Follow-up studies can then be designed to address specific questions.

  19. Insulin resistance in uremia. Characterization of lipid metabolism in freshly isolated and primary cultures of hepatocytes from chronic uremic rats.

    PubMed Central

    Caro, J F; Lanza-Jacoby, S

    1983-01-01

    We have studied the mechanism(s) of hyperlipidemia and liver insulin sensitivity in a rat model of severe chronic uremia (U). Basal lipid synthesis was decreased in freshly isolated hepatocytes from U when compared with sham-operated ad lib.-fed controls (alfC). Basal lipid synthesis in pair-fed controls (pfC) was in between U and alfC. Similarly, the activity of liver acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase was diminished in U. Muscle and adipose tissue lipoprotein lipase was also decreased. Insulin stimulated lipid synthesis in freshly isolated hepatocytes from alfC. Hepatocytes from U and pfC were resistant to this effect of insulin. To ascertain if the insulin resistance in U was due to starvation (chow intake 50% of alfC) or to uremia itself, the U and pfC were intragastrically fed an isocaloric diet via a Holter pump the last week of the experimental period. Hepatocytes from orally fed U and pfC were also cultured for 24 h in serum-free medium. While freshly isolated and cultured U hepatocytes remained insulin resistant, those from pfC normalized, in vivo and in vitro, when they were provided with enough nutrients. Conclusions: (a) Hyperlipidemia in uremia is not due to increased synthesis, but to defect(s) in clearance. (b) Insulin does not stimulate lipid synthesis in uremia. This finding, along with our recent demonstration that insulin binding and internalization are not decreased in the uremic liver, suggests that a post-binding defect(s) in the liver plays an important role in the mechanism(s) of insulin resistance in uremia. (c) Cultured hepatocytes from uremic rats remain insulin resistant. This quality renders these cells useful in studying the postinsulin binding events responsible for the insulin-resistant state in the absence of complicating hormonal and substrate changes that occur in vivo. PMID:6350367

  20. 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

  1. 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

  2. Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection

    PubMed Central

    Sakurai, Fuminori; Mitani, Seiji; Yamamoto, Tatsuro; Takayama, Kazuo; Tachibana, Masashi; Watashi, Koichi; Wakita, Takaji; Iijima, Sayuki; Tanaka, Yasuhito; Mizuguchi, Hiroyuki

    2017-01-01

    In order to understand the life cycle of hepatitis B virus (HBV) and to develop efficient anti-HBV drugs, a useful in vitro cell culture system which allows HBV infection and recapitulates virus-host interactions is essential; however, pre-existing in vitro HBV infection models are often problematic. Here, we examined the potential of human induced-pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPS-HLCs) as an in vitro HBV infection model. Expression levels of several genes involved in HBV infection, including the sodium taurocholate cotransporting polypeptide (NTCP) gene, were gradually elevated as the differentiation status of human iPS cells proceeded to iPS-HLCs. The mRNA levels of these genes were comparable between primary human hepatocytes (PHHs) and iPS-HLCs. Following inoculation with HBV, we found significant production of HBV proteins and viral RNAs in iPS-HLCs. The three major forms of the HBV genome were detected in iPS-HLCs by Southern blotting analysis. Anti-HBV agents entecavir and Myrcludex-B, which are a nucleoside analogue reverse transcriptase inhibitor and a synthetic pre-S1 peptide, respectively, significantly inhibited HBV infection in iPS-HLCs. These data demonstrate that iPS-HLCs can be used as a promising in vitro HBV infection model. PMID:28374759

  3. Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

    PubMed

    Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

    2017-02-01

    We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

  4. HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis

    PubMed Central

    Silberstein, Erica; Ulitzky, Laura; Lima, Livia Alves; Cehan, Nicoleta; Teixeira-Carvalho, Andréa; Roingeard, Philippe; Taylor, Deborah R.

    2016-01-01

    Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets. PMID:27280444

  5. Metabolic Rate Constants for Hydroquinone in F344 Rat and Human Liver Isolated Hepatocytes: Application to a PBPK model.

    SciTech Connect

    Poet, Torka S.; Wu, Hong; English, J C.; Corley, Rick A.

    2004-11-15

    Hydroquinone (HQ) is an important industrial chemical that also occurs naturally in foods and in the leaves and bark of a number of plant species. Exposure of laboratory animals to HQ may result in a species-, sex-, and strain-specific nephrotoxicity. The sensitivity of male F344 vs. female F344 and Sprague-Dawley rats or B6C3F1 mice appears to be related to differences in the rates of formation and further metabolism of key nephrotoxic metabolites. Metabolic rate constants for the conversion of HQ through several metabolic steps to the mono-glutathione conjugate and subsequent detoxification via mercapturic acid were measured in suspension cultures of hepatocytes isolated from male F344 rats and humans. An in vitro mathematic kinetic model was used to analyze each metabolic step by simultaneously fitting the disappearance of each substrate and the appearance of subsequent metabolites. An iterative, nested approach was used whereby downstream metabolites were considered first and the model was constrained by the requirement that rate constants determined during analysis of individual metabolic steps must also satisfy the complete, integrated metabolism scheme, including competitive pathways. The results from this study indicated that the overall capacity for metabolism of HQ and its mono-glutathione conjugate is greater in hepatocytes from humans than those isolated from rats, suggesting a greater capacity for detoxification of the glutathione conjugates. Metabolic rate constants were applied to an existing physiologically based pharmacokinetic model and the model was used to predict total glutathione metabolites produced in the liver. The results showed that body burdens of these metabolites will be much higher in rats than humans.

  6. Fabrication of 3D-culture platform with sandwich architecture for preserving liver-specific functions of hepatocytes using 3D bioprinter.

    PubMed

    Arai, Kenichi; Yoshida, Toshiko; Okabe, Motonori; Goto, Mitsuaki; Mir, Tanveer Ahmad; Soko, Chika; Tsukamoto, Yoshinari; Akaike, Toshihiro; Nikaido, Toshio; Zhou, Kaixuan; Nakamura, Makoto

    2016-09-19

    The development of new three-dimensional (3D) cell culture system that maintains the physiologically relevant signals of hepatocytes is essential in drug discovery and tissue engineering research. Conventional two-dimensional (2D) culture yields cell growth, proliferation, and differentiation. However, gene expression and signaling profiles can be different from in vivo environment. Here, we report the fabrication of a 3D culture system using an artificial scaffold and our custom-made inkjet 3D bioprinter as a new strategy for studying liver-specific functions of hepatocytes. We built a 3D culture platform for hepatocytes-attachment and formation of cell monolayer by interacting the galactose chain of galactosylated alginate gel (GA-gel) with asialoglycoprotein receptor (ASGPR) of hepatocytes. The 3D geometrical arrangement of cells was controlled by using 3D bioprinter, and cell polarity was controlled with the galactosylated hydrogels. The fabricated GA-gel was able to successfully promote adhesion of hepatocytes. To observe liver-specific functions and to mimic hepatic cord, an additional parallel layer of hepatocytes was generated using two gel sheets. These results indicated that GA-gel biomimetic matrices can be used as a 3D culture system that could be effective for the engineering of liver tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2016.

  7. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  8. 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

  9. Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes.

    PubMed

    Turncliff, Ryan Z; Meier, Peter J; Brouwer, Kim L R

    2004-08-01

    Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.

  10. Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

    SciTech Connect

    Rajaraman, Ganesh; Chen, Jie; Chang, Thomas K.H. . E-mail: tchang@interchange.ubc.ca

    2006-12-01

    The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations {>=} 75 {mu}g/ml and {>=} 750 {mu}g/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 {mu}g/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [{sup 14}C]-leucine incorporation. At the level present in a modulating concentration (50 {mu}g/ml) of the extract, ginkgolide A (0.55 {mu}g/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.

  11. Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

    SciTech Connect

    Mundy, Lukas J.; Jones, Stephanie P.; Crump, Doug; Herve, Jessica C.; Konstantinov, Alex; Utley, Fiona; Potter, David; Kennedy, Sean W.

    2010-11-01

    Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared the EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono-ortho substituted polychlorinated biphenyls (PCBs), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and 2,3'4,4',5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC{sub 50} and EC{sub threshold} values for EROD and CYP1A4/5 mRNA concentration-response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.

  12. Acetaminophen cytotoxicity is ameliorated in a human liver organotypic co-culture model

    PubMed Central

    Nelson, Leonard J.; Navarro, Maria; Treskes, Philipp; Samuel, Kay; Tura-Ceide, Olga; Morley, Steven D.; Hayes, Peter C.; Plevris, John N.

    2015-01-01

    Organotypic liver culture models for hepatotoxicity studies that mimic in vivo hepatic functionality could help facilitate improved strategies for early safety risk assessment during drug development. Interspecies differences in drug sensitivity and mechanistic profiles, low predictive capacity, and limitations of conventional monocultures of human hepatocytes, with high attrition rates remain major challenges. Herein, we show stable, cell-type specific phenotype/cellular polarity with differentiated functionality in human hepatocyte-like C3A cells (enhanced CYP3A4 activity/albumin synthesis) when in co-culture with human vascular endothelial cells (HUVECs), thus demonstrating biocompatibility and relevance for evaluating drug metabolism and toxicity. In agreement with in vivo studies, acetaminophen (APAP) toxicity was most profound in HUVEC mono-cultures; whilst in C3A:HUVEC co-culture, cells were less susceptible to the toxic effects of APAP, including parameters of oxidative stress and ATP depletion, altered redox homeostasis, and impaired respiration. This resistance to APAP is also observed in a primary human hepatocyte (PHH) based co-culture model, suggesting bidirectional communication/stabilization between different cell types. This simple and easy-to-implement human co-culture model may represent a sustainable and physiologically-relevant alternative cell system to PHHs, complementary to animal testing, for initial hepatotoxicity screening or mechanistic studies of candidate compounds differentially targeting hepatocytes and endothelial cells. PMID:26632255

  13. Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

    PubMed

    Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

    2013-01-01

    We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

  14. 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.

  15. A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture

    PubMed Central

    McCarty, William J.; Usta, O. Berk; Luitje, Martha; Bale, Shyam Sundhar; Bhushan, Abhinav; Hegde, Manjunath; Golberg, Inna; Jindal, Rohit; Yarmush, Martin L.

    2014-01-01

    The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen “double gel” or “overlay”. To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues. PMID:24932459

  16. 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.

  17. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

    PubMed

    Schmidt, M; Schmitz, H-J; Baumgart, A; Guédon, D; Netsch, M I; Kreuter, M-H; Schmidlin, C B; Schrenk, D

    2005-02-01

    Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key

  18. 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

    2016-08-05

    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

  19. 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

  20. 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.

  1. Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

    PubMed

    Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

    2014-11-01

    The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.

  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

    2016-08-03

    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. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    PubMed

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca(2+) level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca(2+) sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca(2+) imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca(2+) oscillation and sustained Ca(2+) signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca(2+) signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

  4. Natural pyrethrins induces apoptosis in human hepatocyte cells via Bax- and Bcl-2-mediated mitochondrial pathway.

    PubMed

    Yang, Yun; Zong, Mimi; Xu, Wenping; Zhang, Yang; Wang, Bo; Yang, Mingjun; Tao, Liming

    2017-01-25

    Natural pyrethrins have been widely used for pest control in organic farming and for residential indoor pest managements. Although the specific mechanisms underlying their activity are incompletely understood, natural pesticides are considered the safest based on their target specificity and rapid degradation in the environment. Here, we used in vitro bioassays to characterize the cytotoxic effects of natural pyrethrins and attempted to delineate the cellular and molecular mechanisms of their cytotoxicity against human hepatocytes. The results demonstrate that natural pyrethrins reduce cell viability and enhance apoptosis in HepG2 cells. In addition, the current data indicate that natural pyrethrins cause a reduction in the mitochondrial membrane potential (Δψm), increase reactive oxygen species production, and up-regulate the Bax/Bcl-2 expression, leading to the release of cytochrome-c into the cytosol, activation of caspase-9 and caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, the results indicate that natural pyrethrins has potentially exert adverse effects on human health by inducing caspase-dependent apoptosis in hepatocytes through Bax- and Bcl-2-mediated mitochondrial pathway.

  5. Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

    PubMed

    Jeong, Hyeon-Uk; Kim, Ju-Hyun; Kong, Tae Yeon; Choi, Won Gu; Lee, Hye Suk

    2016-04-01

    Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

  6. MPTP and MPTP analogs induced cell death in cultured rat hepatocytes involving the formation of pyridinium metabolites.

    PubMed

    Singh, Y; Swanson, E; Sokoloski, E; Kutty, R K; Krishna, G

    1988-11-01

    MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) which has been shown to produce a Parkinson-like syndrome in humans and monkeys also causes cell death in cultures of rat hepatocytes. Treatment of cells with MPTP or its metabolite MPP+ (1-methyl-4-phenyl pyridinium ion), resulted in leakage of lactic acid dehydrogenase and 14C-labeled adenine nucleotides, as well as marked depletion of ATP and glutathione. Deprenyl, a specific inhibitor of monoamine oxidase-B, the enzyme catalyzing the oxidation of MPTP into MPP+, blocked the lethal effect of MPTP, but gave no protection from MPP+-induced cell death. The 4'-fluoro and 4'-chloro analogs of MPTP evoked toxicities similar to that of the parent compound, while N-butyl-PTP, 4'-amino-MPTP, and 2'-methyl-MPTP were relatively less toxic. N-Acetylamino-MPTP was found virtually nontoxic. The cell death produced by these analogs was also associated with leakage of [14C]adenine nucleotides, which is an indicator of loss of ATP from cells. All these compounds except the N-acetylamino analog were converted to corresponding pyridinium metabolites by liver cells when analyzed by high-pressure liquid chromatography and plasma desorption mass spectrometry. MPTP and its analogs also served as substrates for rat liver mitochondrial monoamine oxidase to varying degrees. Toxicity of various analogs, with the noticeable exception of 2'-methyl-MPTP, was inhibited by deprenyl. These findings indicate that the conversion of MPTP and its analogs to corresponding pyridinium metabolites is essential for the expression of toxicity.

  7. Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat HepatocytesS⃞

    PubMed Central

    Houghton, Jessica S.; Uyeda, Craig; Grillo, Mark P.; Jin, Lixia

    2011-01-01

    Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na+-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction

  8. Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 induce apoptosis in primary cultured rat hepatocytes.

    PubMed

    Ashida, H; Shiotani, B; Adachi, H; Hashimoto, T; Kanazawa, K; Danno, G

    1998-11-01

    The cytotoxicity of heterocyclic amines, dietary carcinogens derived from cooked foods, to primary cultured rat hepatocytes was studied. A tryptophan pyrolysis product, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was the most cytotoxic of 11 compounds tested. Trp-P-1 was found to induce apoptosis as measured by morphological changes in nuclear chromatin and internucleosomal DNA fragmentation. 3-Amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) showed a moderate apoptotic effect, and other compounds had a similar but weaker effect.

  9. 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

  10. Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice.

    PubMed

    Czaya, Brian; Singh, Saurav; Yanucil, Christopher; Schramm, Karla; Faul, Christian; Grabner, Alexander

    2017-03-10

    The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

  11. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies.

    PubMed

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, André; Cocquerel, Laurence; Allet, Cécile; Dumont, Patrick; Loyens, Anne; Leteurtre, Emmanuelle; Omary, M Bishr; Dubuisson, Jean; Rouillé, Yves; Wychowski, Czeslaw

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  12. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

    SciTech Connect

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, Andre; Cocquerel, Laurence; Allet, Cecile; Dumont, Patrick; Loyens, Anne; Leteurtre, Emmanuelle; Omary, M. Bishr; Dubuisson, Jean; Rouille, Yves; Wychowski, Czeslaw

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  13. 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

  14. Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes.

    PubMed

    Gardner, Anne M; Cook, Matthew R; Gardner, Paul R

    2010-07-30

    Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K(m) values for ascorbate, cytochrome b(5), NO, and O(2) of 0.25 mm, 0.3 microm, 40 nm, and approximately 20 microm and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m(-1) s(-1) and 3 x 10(6) m(-1) s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k(cat) of 1.2 s(-1), a K(m)(NO) of 40 nm, and a k(cat)/K(m)(NO) (k'(NOD)) value of 3 x 10(7) m(-1) s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O(2)] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b(5) as reductants.

  15. 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.

  16. Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes

    PubMed Central

    Davidson, Matthew D.; Ballinger, Kimberly R.; Khetani, Salman R.

    2016-01-01

    Hyperglycemia in type 2 diabetes mellitus has been linked to non-alcoholic fatty liver disease, which can progress to inflammation, fibrosis/cirrhosis, and hepatocellular carcinoma. Understanding how chronic hyperglycemia affects primary human hepatocytes (PHHs) can facilitate the development of therapeutics for these diseases. Conversely, elucidating the effects of hypoglycemia on PHHs may provide insights into how the liver adapts to fasting, adverse diabetes drug reactions, and cancer. In contrast to declining PHH monocultures, micropatterned co-cultures (MPCCs) of PHHs and 3T3-J2 murine embryonic fibroblasts maintain insulin-sensitive glucose metabolism for several weeks. Here, we exposed MPCCs to hypo-, normo- and hyperglycemic culture media for ~3 weeks. While albumin and urea secretion were not affected by glucose level, hypoglycemic MPCCs upregulated CYP3A4 enzyme activity as compared to other glycemic states. In contrast, hyperglycemic MPCCs displayed significant hepatic lipid accumulation in the presence of insulin, while also showing decreased sensitivity to insulin-mediated inhibition of glucose output relative to a normoglycemic control. In conclusion, we show for the first time that PHHs exposed to hypo- and hyperglycemia can remain highly functional, but display increased CYP3A4 activity and selective insulin resistance, respectively. In the future, MPCCs under glycemic states can aid in novel drug discovery and mechanistic investigations. PMID:27312339

  17. Long-term exposure to abnormal glucose levels alters drug metabolism pathways and insulin sensitivity in primary human hepatocytes

    NASA Astrophysics Data System (ADS)

    Davidson, Matthew D.; Ballinger, Kimberly R.; Khetani, Salman R.

    2016-06-01

    Hyperglycemia in type 2 diabetes mellitus has been linked to non-alcoholic fatty liver disease, which can progress to inflammation, fibrosis/cirrhosis, and hepatocellular carcinoma. Understanding how chronic hyperglycemia affects primary human hepatocytes (PHHs) can facilitate the development of therapeutics for these diseases. Conversely, elucidating the effects of hypoglycemia on PHHs may provide insights into how the liver adapts to fasting, adverse diabetes drug reactions, and cancer. In contrast to declining PHH monocultures, micropatterned co-cultures (MPCCs) of PHHs and 3T3-J2 murine embryonic fibroblasts maintain insulin-sensitive glucose metabolism for several weeks. Here, we exposed MPCCs to hypo-, normo- and hyperglycemic culture media for ~3 weeks. While albumin and urea secretion were not affected by glucose level, hypoglycemic MPCCs upregulated CYP3A4 enzyme activity as compared to other glycemic states. In contrast, hyperglycemic MPCCs displayed significant hepatic lipid accumulation in the presence of insulin, while also showing decreased sensitivity to insulin-mediated inhibition of glucose output relative to a normoglycemic control. In conclusion, we show for the first time that PHHs exposed to hypo- and hyperglycemia can remain highly functional, but display increased CYP3A4 activity and selective insulin resistance, respectively. In the future, MPCCs under glycemic states can aid in novel drug discovery and mechanistic investigations.

  18. Sensitivity of bald eagle (Haliaeetus leucocephalus) hepatocyte cultures to induction of cytochrome P4501A by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Kennedy, Sean W; Jones, Stephanie P; Elliott, John E

    2003-01-01

    Graded doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were added to primary hepatocyte cultures of bald eagle (Haliaeetus leucocephalus) embryos to determine their sensitivity to induction of cytochrome P4501A (CYP1A) and porphyrin accumulation. No porphyrin accumulation was observed, but both CYP1A catalytic activity (using the ethoxyresorufin-O-deethylase (EROD) assay) and immunodetectable CYP1A were induced by relatively high concentrations of TCDD. Bald eagle hepatocytes were less sensitive to CYP1A induction than hepatocytes from any other avian species that we have studied to date. These in vitro results are in general agreement with recent assessments of field data, which indicate that bald eagles are relatively insensitive to some of the effects of TCDD and related compounds. Preparation of bald eagle hepatocytes was challenging because existing methods did not yield monolayers of cells. Here we describe details of a new method that was successful for bald eagle hepatocytes. This new method is used routinely in our laboratory to prepare hepatocyte cultures from birds for examination of various biochemical responses to environmental contaminants.

  19. Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

    PubMed

    Xie, Cheng-Rong; Sun, Hongguang; Wang, Fu-Qiang; Li, Zhao; Yin, Yi-Rui; Fang, Qin-Liang; Sun, Yu; Zhao, Wen-Xiu; Zhang, Sheng; Zhao, Wen-Xing; Wang, Xiao-Min; Yin, Zhen-Yu

    2015-09-01

    Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and non‑tumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF‑induced DNA hypermethylation.

  20. The Role of Constitutive Androstane Receptor in Oxazaphosphorine-mediated Induction of Drug-metabolizing Enzymes in Human Hepatocytes

    PubMed Central

    Wang, Duan; Li, Linhao; Fuhrman, Jennifer; Ferguson, Stephen; Wang, Hongbing

    2013-01-01

    Purpose The objective of this study was to investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME). Methods Induction of DMEs was evaluated using real-time RT-PCR and Western blotting analysis in human primary hepatocyte (HPH) cultures. Activation of CAR, pregnane X receptor (PXR), and aryl hydrocarbon receptor by CPA and IFO was assessed in cell-based reporter assays in HepG2 cells and/or nuclear translocation assays in HPHs. Results CYP2B6 reporter activity was significantly enhanced by CPA and IFO in HepG2 cells co-transfected with CYP2B6 reporter plasmid and a chemical-responsive human CAR variant (CAR1+A) construct. Real-time RT-PCR and Western blotting analysis in HPHs showed that both CPA and IFO induced the expressions of CYP2B6 and CYP3A4. Notably, treatment of HPHs with CPA but not IFO resulted in significant nuclear accumulation of CAR, which represents the initial step of CAR activation. Further studies in HPHs demonstrated that selective inhibition of PXR by sulforaphane preferentially repressed IFO- over CPA-mediated induction of CYP2B6. Conclusion These results provide novel insights into the differential roles of CAR in the regulation of CPA- and IFO-induced DME expression and potential drug-drug interactions. PMID:21487929

  1. Metabolic Consequences of TGFβ Stimulation in Cultured Primary Mouse Hepatocytes Screened from Transcript Data with ModeScore

    PubMed Central

    Hoppe, Andreas; Ilkavets, Iryna; Dooley, Steven; Holzhütter, Hermann-Georg

    2012-01-01

    TGFβ signaling plays a major role in the reorganization of liver tissue upon injury and is an important driver of chronic liver disease. This is achieved by a deep impact on a cohort of cellular functions. To comprehensively assess the full range of affected metabolic functions, transcript changes of cultured mouse hepatocytes were analyzed with a novel method (ModeScore), which predicts the activity of metabolic functions by scoring transcript expression changes with 987 reference flux distributions, which yielded the following hypotheses. TGFβ multiplies down-regulation of most metabolic functions occurring in culture stressed controls. This is especially pronounced for tyrosine degradation, urea synthesis, glucuronization capacity, and cholesterol synthesis. Ethanol degradation and creatine synthesis are down-regulated only in TGFβ treated hepatocytes, but not in the control. Among the few TGFβ dependently up-regulated functions, synthesis of various collagens is most pronounced. Further interesting findings include: down-regulation of glucose export is postponed by TGFβ, TGFβ up-regulates the synthesis capacity of ketone bodies only as an early response, TGFβ suppresses the strong up-regulation of Vanin, and TGFβ induces re-formation of ceramides and sphingomyelin. PMID:24957771

  2. Suppression of CYP2B Induction by Alendronate-Mediated Farnesyl Diphosphate Synthase Inhibition in Primary Cultured Rat Hepatocytes

    PubMed Central

    Jackson, Nancy M.; Kocarek, Thomas A.

    2008-01-01

    We previously reported that squalestatin 1-mediated induction of CYP2B expression is attributable to squalene synthase inhibition and accumulation of an endogenous isoprenoid(s) that is capable of activating the constitutive androstane receptor. To determine whether squalestatin 1-mediated CYP2B induction is strictly dependent upon the biosynthesis of farnesyl pyrophosphate (FPP), the substrate for squalene synthase, the effects of alendronate, a nitrogen-containing bisphosphonate inhibitor of farnesyl diphosphate synthase, were determined on basal, squalestatin 1-inducible, and phenobarbital-inducible CYP2B expression in primary cultured rat hepatocytes. Alendronate treatment alone had no effect on CYP2B or CYP3A mRNA expression in the hepatocyte cultures, but alendronate co-treatment completely suppressed squalestatin 1-mediated CYP2B mRNA induction at concentrations (60 and 100 μM) that effectively inhibited cellular farnesyl diphosphate synthase activity, as assessed by reductions of squalestatin 1-mediated FPP accumulation, and that were not toxic to the cells, as indicated by a lack of effect on MTT activity. Alendronate co-treatment also partially suppressed phenobarbital-inducible CYP2B expression, and this suppressive effect was attenuated by additional co-treatment with the upstream pathway inhibitor, pravastatin. These findings demonstrate that squalestatin 1-mediated CYP2B induction cannot occur in the absence of FPP biosynthesis, but also indicate that one or more upstream isoprenoids, possibly isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate, function to antagonize the CYP2B induction process. PMID:18617600

  3. A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues.

    PubMed

    Hart, Steven N; Li, Ye; Nakamoto, Kaori; Subileau, Eva-anne; Steen, David; Zhong, Xiao-bo

    2010-06-01

    HepaRG cells, derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes. More importantly, differentiated HepaRG cells are able to maintain activities of many xenobiotic-metabolizing enzymes, and expression of the metabolizing enzyme genes can be induced by xenobiotics. The ability of these cells to express and induce xenobiotic-metabolizing enzymes is in stark contrast to the frequently used HepG2 cells. The previous studies have mainly focused on a set of selected genes; therefore, it is of significant interest to know the extent of similarity of gene expression at whole genome levels in HepaRG cells and HepG2 cells compared with primary human hepatocytes and human liver tissues. To accomplish this objective, we used Affymetrix (Santa Clara, CA) U133 Plus 2.0 arrays to characterize the whole genome gene expression profiles in triplicate biological samples from HepG2 cells, HepaRG cells (undifferentiated and differentiated cells), freshly isolated primary human hepatocytes, and frozen liver tissues. After using similarity matrix, principal components, and hierarchical clustering methods, we found that HepaRG cells globally transcribe genes at levels more similar to human primary hepatocytes and human liver tissues than HepG2 cells. In particular, many genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 cells compared with primary human hepatocytes and liver samples. The transcriptomic similarity of HepaRG with primary human hepatocytes is encouraging for use of HepaRG cells in the study of xenobiotic metabolism, hepatotoxicology, and hepatocyte differentiation.

  4. Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: A novel in vitro cholestatic model

    PubMed Central

    Ni, Xuan; Gao, Yimeng; Wu, Zhitao; Ma, Leilei; Chen, Chen; Wang, Le; Lin, Yunfei; Hui, Lijian; Pan, Guoyu

    2016-01-01

    Drug-induced cholestasis is a leading cause of drug withdrawal. However, the use of primary human hepatocytes (PHHs), the gold standard for predicting cholestasis in vitro, is limited by their high cost and batch-to-batch variability. Mature hepatocyte characteristics have been observed in human induced hepatocytes (hiHeps) derived from human fibroblast transdifferentiation. Here, we evaluated whether hiHeps could biosynthesize and excrete bile acids (BAs) and their potential as PHH alternatives for cholestasis investigations. Quantitative real-time PCR (qRT-PCR) and western blotting indicated that hiHeps highly expressed BA synthases and functional transporters. Liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that hiHeps produced normal intercellular unconjugated BAs but fewer conjugated BAs than human hepatocytes. When incubated with representative cholestatic agents, hiHeps exhibited sensitive drug-induced bile salt export pump (BSEP) dysfunction, and their response to cholestatic agent-mediated cytotoxicity correlated well with that of PHHs (r2 = 0.8032). Deoxycholic acid (DCA)-induced hepatotoxicity in hiHeps was verified by elevated aspartate aminotransferase (AST) and γ-glutamyl-transferase (γ-GT) levels. Mitochondrial damage and cell death suggested DCA-induced toxicity in hiHeps, which were attenuated by hepatoprotective drugs, as in PHHs. For the first time, hiHeps were reported to biosynthesize and excrete BAs, which could facilitate predicting cholestatic hepatotoxicity and screening potential therapeutic drugs against cholestasis. PMID:27934920

  5. 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.

  6. 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.

  7. Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures.

    PubMed Central

    Hirsch-Ernst, K I; Kietzmann, T; Ziemann, C; Jungermann, K; Kahl, G F

    2000-01-01

    P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2). Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2). Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2). Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2). In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2). These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile. PMID:10947958

  8. In vitro metabolism of [(14)C]-benalaxyl in hepatocytes of rats, dogs and humans.

    PubMed

    Nallani, Gopinath C; ElNaggar, Shaaban F; Shen, Li; Chandrasekaran, Appavu

    2017-03-01

    The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [(14)C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [(14)C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

  9. Effect of simvastatin on the synthesis and secretion of lipoproteins in relation to the metabolism of cholesterol in cultured hepatocytes.

    PubMed

    Ribeiro, A; Mangeney, M; Loriette, C; Thomas, G; Pepin, D; Janvier, B; Chambaz, J; Bereziat, G

    1991-11-27

    In primary culture of rat hepatocytes, simvastatin, a powerful HMGCoA reductase inhibitor, inhibited acetate incorporation into cellular and secreted cholesterol and cholesteryl-esters, without any significant effect on triacylglycerol synthesis and secretion. When applied to the culture for 24 h at 10(-7) M, a concentration shown to inhibit cholesterol synthesis by 61%, simvastatin increased apolipoprotein BH and BL synthesis and secretion and strongly decreased apolipoprotein AI synthesis and secretion whereas apolipoprotein AIV remained unaffected. The synthesis and secretion of apolipoprotein E was only slightly affected in contrast with other situations where cholesterol synthesis decreased. All of these modifications occurred at a post-transcriptional level, as the corresponding messenger RNAs of the apolipoproteins did not vary. These results suggest that either the drug itself or variations in cholesterol synthesis might be involved in apo B and apo AI synthesis and secretion.

  10. Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates

    PubMed Central

    Bell, Peter; Wang, Lili; Gao, Guangping; Haskins, Mark E.; Tarantal, Alice F.; McCarter, Robert J.; Zhu, Yanqing; Yu, Hongwei; Wilson, James M.

    2011-01-01

    Gene transfer vectors based on adeno-associated virus 8 (AAV8) are highly efficient in liver transduction and can be easily administered by intravenous injection. In mice, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in periportal regions. This transduction bias has important implications for gene therapy that aims to correct metabolic liver enzymes because metabolic zonation along the porto-central axis requires the expression of therapeutic proteins within the zone where they are normally localized. In the present study we compared the expression pattern of AAV8 expressing green fluorescent protein (GFP) in liver between mice, dogs, and non-human primates. We confirmed the pericentral dominance in transgene expression in mice with AAV8 when the liver-specific thyroid hormone-binding globulin (TBG) promoter was used but also observed the same expression pattern with the ubiquitous chicken β-actin (CB) and cytomegalovirus (CMV) promoters, suggesting that transduction zonation is not caused by promoter specificity. Predominantly pericentral expression was also found in dogs injected with AAV8. In contrast, in cynomolgus and rhesus macaques the expression pattern from AAV vectors was reversed, i.e. transgene expression was most intense around portal areas and less intense or absent around central veins. Infant rhesus macaques as well as newborn mice injected with AAV8 however showed a random distribution of transgene expression with neither portal nor central transduction bias. Based on the data in monkeys, adult humans treated with AAV vectors are predicted to also express transgenes predominantly in periportal regions whereas infants are likely to show a uniform transduction pattern in liver. PMID:21778099

  11. A 96-well plate assay for CYP4503A induction using cryopreserved human hepatocytes.

    PubMed

    Kamiguchi, Naomi; Aoyama, Eiji; Okuda, Teruaki; Moriwaki, Toshiya

    2010-11-01

    A reliable and practical CYP3A induction assay with cryopreserved human hepatocytes in a 96-well format was developed. Various 96-well plates with different basement membrane were evaluated using prototypical inducers, rifampicin, phenytoin, and carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6β-hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC(50) values of 0.36 ± 0.28 μM from four batches of human hepatocytes using the 96-well plate with TL Matrigel. The rank order of induction potency for nine inducers or noninducers at a concentration of 10 μM were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 μM rifampicin. Cotreatment of avasimibe or efavirenz with 10 μM rifampicin was found to reduce CYP3A activities induced by rifampicin at a lower rate than treatment with rifampicin alone, whereas treatment with phenobarbital and carbamazepine had no effect. From a comparison of induced CYP3A activities and gene expression levels, there were compounds that would cause induction of CYP3A4 mRNA but not activity, presumably due to their inhibitory effect on CYP3A activity. The cotreatment assay of test compound with rifampicin allows us to exclude the false-negative results caused by the cytotoxicity and/or the mechanism-based inactivation, when the drug candidate's ability for CYP3A induction is evaluating the enzyme activity. This 96-well plate assay, which is robust, reproducible, and convenient, has demonstrated the paramount applicability to the early drug discovery stage.

  12. Stimulation of fatty acid oxidation by a 3-thia fatty acid reduces triacylglycerol secretion in cultured rat hepatocytes.

    PubMed

    Skrede, S; Bremer, J; Berge, R K; Rustan, A C

    1994-08-01

    The present work shows that when mitochondrial beta-oxidation is stimulated by the hypolipemic, non-beta-oxidizable fatty acid analogue tetradecylthioacetic acid, there is a decrease in the secretion of triacylglycerol in cultured rat hepatocytes. In order to study the effects of tetradecylthioacetic acid in cells with different fatty acid oxidation rates, cells were grown without or with L-carnitine supplement or with addition of the beta-oxidation inhibitor L-aminocarnitine. In cells grown without and with L-carnitine in the medium, the oxidation of [1-14C]oleic acid was stimulated by tetradecylthioacetic acid, whereas it was not significantly changed by palmitic acid. In cells grown with L-aminocarnitine, oxidation of [1-14C]oleic acid was almost abolished both in the absence and in presence of tetradecylthioacetic acid. The effect of tetradecylthioacetic acid and palmitic acid on incorporation of [1-14C]oleic acid into triacylglycerol was similar under all conditions. In the presence of L-carnitine, secretion of oleic acid-labeled triacylglycerol was reduced significantly more by tetradecylthioacetic acid than by palmitic acid. The effects of tetradecylthioacetic acid and palmitic acid on secretion of oleic acid-labeled triacylglycerol were reversed in cells grown with L-aminocarnitine, where palmitic acid was the stronger inhibitor. These results were substantiated by determination of mass of triacylglycerol secreted. It is concluded that tetradecylthioacetic acid reduces secretion of triacylglycerol from rat hepatocytes mainly by acutely stimulating fatty acid oxidation.

  13. P21waf-1/cip-1/sdi-1 is expressed at G1 phase in primary culture of hepatocytes from old rats, presumably preventing the cells from entering the S phase of the cell cycle.

    PubMed

    Sawada, N; Kojima, T; Obata, H; Saitoh, M; Isomura, H; Kokai, Y; Satoh, M; Mori, M

    1996-11-21

    To elucidate whether p21waf-1/cip-1/sdi-1 expression is associated with loss of growth potential of hepatocytes of old rats, we determined p21waf-1/cip-1/sdi-1 expression of hepatocytes from old (30 months) rats during the cell cycle in primary culture. A high level of expression of p21waf-1/cip-1/sdi-1 was detected at the G1 phase in old-rat hepatocytes, but after the S phase in young-rat hepatocytes. Consistently, the incidence of the cells positive for p21waf-1/cip-1/sdi-1 in nuclei before entering the S phase was significantly higher in old-rat hepatocytes than in young-rat hepatocytes. These results account for the loss of growth potential of old-rat hepatocytes in vitro and the marked retardation of regeneration of liver in old rats in vivo.

  14. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas.

    PubMed Central

    Olivero, M.; Rizzo, M.; Madeddu, R.; Casadio, C.; Pennacchietti, S.; Nicotra, M. R.; Prat, M.; Maggi, G.; Arena, N.; Natali, P. G.; Comoglio, P. M.; Di Renzo, M. F.

    1996-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8980383

  15. Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7.

    PubMed

    Fujimoto, Yasuyuki; Itabe, Hiroyuki; Kinoshita, Tetsuaki; Homma, Koichi J; Onoduka, Jun; Mori, Masahiro; Yamaguchi, Shinji; Makita, Minoru; Higashi, Yusuke; Yamashita, Atsushi; Takano, Tatsuya

    2007-06-01

    Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

  16. Protective effects of HGF gene-expressing human mesenchymal stem cells in acetaminophen-treated hepatocytes.

    PubMed

    Jang, Yun Ho; You, Dong Hun; Nam, Myeong Jin

    2015-01-01

    Mesenchymal stem cells (MSC) secrete a great variety of cytokines that have beneficial paracrine actions. Hepatocyte growth factor (HGF) promotes proliferation in several cell types. The aim of the present study was to investigate the protective effect of HGF gene-transfected MSC (HGF-MSC) in acetaminophen (AAP)-treated hepatocytes. We transfected the HGF gene into MSCs and confirmed HGF expression by RT-PCR and western blot. The concentration of HGF in HGF-MSC conditioned media (HGFCM) was upregulated compared with that in control MSCCM samples. Cell viability was increased in HGFCM-treated hepatocytes. Expression of Mcl-1, an anti-apoptosis protein, was increased and expression of pro-apoptosis proteins (Bad, Bik and Bid) was decreased in HGFCM-treated hepatocytes. HGF-MSC had protective effects on AAP-induced hepatocyte damage by enhancing proliferation. These results suggest that HGF-expressing MSCs may provide regenerative potential for liver cell damage.

  17. 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.

  18. Identification and characterization of a Nuclear Factor Kappa B p65 proteolytic fragment in nuclei of porcine hepatocytes in monolayer culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatocytes prepared from suckling pigs, and maintained in monolayer culture were used to investigate transcription factor activity at the cellular level. The hepatic response to proinflammatory signals is controlled by the activation of several transcription factors, including, Nuclear Factor-Kapp...

  19. INTEGRATED DISINFECTION BY-PRODUCTS (DBP) MIXTURES RESEARCH: GENE EXPRESSION ALTERATIONS IN PRIMARY RAT HEPATOCYTE CULTURES EXPOSED TO DBP MIXTURES FORMED BY CHLORINATION AND OZONATION/POSTCHLORINATION

    EPA Science Inventory

    What is the study?
    This study was designed to provide data on the in vitro toxicity of water concentrates containing complex mixtures of DBPs. Rat hepatocytes in primary culture were exposed for 24 hr to full strength, 1:10 or 1:20 dilutions of chlorination or ozonation/chl...

  20. EROD induction in microsomes and primary hepatocyte cultures prepared from individual double-crested cormorant embryo livers

    SciTech Connect

    Davis, J.A.; Fry, D.M.; Wilson, B.W.

    1994-12-31

    Double-crested cormorants (Phalacrocorax auritus) are being studied as a high trophic level indicator species for toxic contamination in San Francisco Bay and other locations on the California coast. In 1993, median ethoxyresorufin-o-deethylase (EROD) activities in embryo liver microsomes sampled from San Francisco Bay colonies were 4.2-fold and 2.3-fold higher than two coastal locations. In 1994 cormorant embryo livers from these same locations were again analyzed for liver microsomal EROD activity. In addition, basal and maximal EROD activity were measured in primary hepatocyte cultures from portions of the same livers used for microsomal EROD analysis to determine whether factors other than contaminant burdens in the egg contribute to the observed differences in activity. Differences in basal activity and induction capacity among the populations are discussed in the context of the hypothesis that polluted habitats select for increased metabolic capacity in affected populations.

  1. Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes.

    PubMed

    Kasuya, Junichi; Sudo, Ryo; Masuda, Genta; Mitaka, Toshihiro; Ikeda, Mariko; Tanishita, Kazuo

    2015-03-01

    In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.

  2. 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.

  3. Hepatitis C virus infection impairs IRF-7 translocation and Alpha interferon synthesis in immortalized human hepatocytes.

    PubMed

    Raychoudhuri, Amit; Shrivastava, Shubham; Steele, Robert; Dash, Srikanta; Kanda, Tatsuo; Ray, Ranjit; Ray, Ratna B

    2010-11-01

    Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces beta interferon (IFN-β) expression (T. Kanda, R. Steele, R. Ray, and R. B. Ray, J. Virol. 81:12375-12381, 2007). However, the regulation of the downstream signaling pathway for IFN-α production by HCV is not clearly understood. In this study, the regulation of the IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1 but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces IFN-stimulated response element activation, as evidenced by upregulation of 2',5'-oligoadenylate synthetase 1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV-infected IHH, IFN-α expression initially increased (up to 24 h) and then decreased at later time points, and IFN-α-inducible protein 27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly(I-C) or IFN-α treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-α signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

  4. Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells.

    PubMed

    Dai, Chongshan; Tang, Shusheng; Li, Daowen; Zhao, Kena; Xiao, Xilong

    2015-01-01

    Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2-16 μg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 μM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p < 0.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.

  5. 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.

  6. Changes in ascorbic acid content in primary cultured rat hepatocytes exposed to 2,2'-azobis (2-amidinopropane) dihydrochloride, a radical initiator.

    PubMed

    Sasakii, K; Kimura, K; Kitaguchi, Y; Onoue, T; Ogura, H; Aoyagi, Y

    2001-08-01

    Changes in ascorbate content in primary cultured rat hepatocytes exposed to oxidative stress derived from water soluble radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were examined. Cells were exposed to 0.05 and 5 mg/ml of AAPH as 'mild' and severe' oxidative stresses, respectively. Lipid peroxidation in hepatocytes was induced by 'severe' oxidative stress, but not by 'mild' oxidative stress. Ascorbate decreased at 6 hr after administration of both mild' and severe' oxidative stresses, and recovered to the control level after a further 6 hr. In cells treated with 'severe oxidative stress, however, total ascorbate (reduced form plus oxidized form) had increased 24 hr after administration. These results indicated that consumption alone did not account for the increase of ascorbate in hepatocytes under oxidative stress.

  7. Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

    PubMed

    Noyer, C M; Immenschuh, S; Liem, H H; Muller-Eberhard, U; Wolkoff, A W

    1998-07-01

    Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

  8. Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes.

    PubMed

    Park, Jungmi; Jang, Kyung Lib

    2014-04-04

    Hepatitis C virus (HCV) core protein is known to induce promoter hypermethylation of tumor suppressor genes including E-cadherin to repress their expression when overexpressed in human hepatocytes; however, its actual role during HCV infection is still unknown. Here, we report that infection with HCV derived from pJFH-1 replicon system that mimics natural infection elevates protein levels of DNA methyltransferase 1 and 3b to enhance DNMT activity in human hepatocytes. As a consequence, HCV induced promoter hypermethylation of E-cadherin, resulting in repression of its expression. In addition down-regulation of E-cadherin by HCV led to epithelial-mesenchymal transition that is known to be a critical event during the late stage of tumorigenesis.

  9. Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

    PubMed

    Papageorgiou, Ioannis; Grepper, Susan; Unadkat, Jashvant D

    2013-02-01

    CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

  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. Inhibition of cholesterol biosynthesis by a water-soluble garlic extract in primary cultures of rat hepatocytes.

    PubMed

    Gebhardt, R

    1991-08-01

    Cultured rat hepatocytes continually synthesize cholesterol form radiolabeled acetate during a 24 h incubation period and export it, presumably as VLDL (very low density lipoprotein) to the culture medium. Mevastatin inhibits cholesterol biosynthesis by 90%. Incubation of the cultures with water-soluble extracts of garlic powder (Kwai, Sapec) diminish cholesterol biosynthesis (20-25%) as well as its export into the medium (30-35%). The IC50-value is 90 micrograms/ml. Between about 0.25 and 10 mg/ml the average maximal inhibition amounts to about 23%. Cytotoxicity of the extracts is apparent at concentrations above 125 mg/ml only. Pure alliin alone, or after incubation with alliinase (conversion to allicin) in concentrations corresponding to its content in the extracts does not exert any inhibition. Replacement of 14C-acetate by 14C-mevalonate omits the inhibitory effect. The activity of HMGCoA (hydroxymethylglutaryl-CoA) reductase is significantly reduced by garlic extracts at 50 micrograms/ml. At higher concentrations fatty acid synthetase, cholesterol 7 alpha-hydroxylase and cholesterol acyltransferase are slightly inhibited. Fatty acid synthetase is the only one of these enzymes which is inhibited by alliin at very high concentrations. These results demonstrate that water-soluble garlic extracts diminish hepatic cholesterol biosynthesis, thus contributing to the reduction of blood cholesterol. The main target site seems to be HMGCoA-reductase. The actual active principle(s) is still unknown. Alliin, however, does not seem to be of major significance.

  12. The methyl ester of okadaic acid is more potent than okadaic acid in disrupting the actin cytoskeleton and metabolism of primary cultured hepatocytes

    PubMed Central

    Espiña, Begoña; Louzao, MCarmen; Cagide, Eva; Alfonso, Amparo; Vieytes, Mercedes R; Yasumoto, Takeshi; Botana, Luis M

    2010-01-01

    Background and purpose: Okadaic acid (OA) and microcystins (MCs) are structurally different toxins with the same mechanism of action, inhibition of serine/threonine protein phosphatases (PPs). Methyl okadaate (MeOk), a methyl ester derivative of OA, was considered almost inactive due to its weak inhibition of PP1 and PP2A. Here, we have investigated the activity and potency of MeOk in hepatic cells in comparison with that of OA and MCs. Experimental approach: We tested the effects of MeOK, OA and microcystin-leucine and arginine (MC-LR) on the metabolic rate, the actin cytoskeleton and glucose uptake in a rat hepatocyte cell line (Clone 9) and in primary cultured rat hepatocytes. PP2A was assayed to compare OA and MeOk activity. Key results: MeOk disrupted the actin cytoskeleton and depressed the metabolic rate of both types of rat hepatocytes, being six-fold less potent than OA in Clone 9 cells but nearly six-fold more potent in primary cultured hepatocytes. However, unlike OA, MeOk did not change glucose uptake in these cells, suggesting a weak inhibition of PP2A, as confirmed in direct assays of PP2A activity. Conclusions and implications: Although MeOk was originally described as a weakly bioactive molecule, it clearly depressed the metabolic rate and disrupted the cytoskeleton in primary and immortalized rat hepatocytes. Furthermore, MeOk affected primary hepatocytes at much lower concentrations than those affecting immortalized cells. These effects were unrelated to PP2A inhibition. Our results suggest the risk to public health from MeOk in foodstuffs should be re-evaluated. PMID:20015092

  13. Kavalactones Yangonin and Methysticin induce apoptosis in human hepatocytes (HepG2) in vitro.

    PubMed

    Tang, J; Dunlop, R A; Rowe, A; Rodgers, K J; Ramzan, I

    2011-03-01

    While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ~ 40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved.

  14. The cytological effects of HBCDs on human hepatocyte L02 and the potential molecular mechanism.

    PubMed

    An, Jing; Zou, Wen; Chen, Cen; Zhong, Fang Y; Yu, Qiang Z; Wang, Qi J

    2013-01-01

    The concentration of hexabromocyclododecanes (HBCDs) in the environment media and organism samples are gradually rising with the increase of HBCDs usage. This study is designed to investigate the cytological effects of HBCDs on human hepatocyte L02 and explore the potential molecular mechanism. The results of CCK-8 assay showed that high concentration of HBCDs (>20 μM) significantly suppressed cell survival, while comparatively lower dose of HBCDs (10(-13)-10(-7)M) slightly stimulated cell proliferation (P < 0.05). In the mean time, high concentration HBCDs markedly induced cell apoptosis and DNA damage, accompanying with increase of intracellular Ca(2+)level and decrease of mitochondrial membrane potential (P < 0.05). ROS level induced by low concentration of HBCDs was comparatively lower than that by high concentration of HBCDs. In addition, low concentration HBCDs exposure (10(-13)-10(-7)M) resulted in up-regulation of PCNA protein expression level in a time-dependent manner. However, high concentration HBCDs exposure led to increase of Apaf-1 expression level. In conclusion, loss of mitochondrial membrane potential and activation of Apaf-1 mediated pathway involve the L02 cell apoptosis induced by high concentration HBCDs exposure. However, low concentration HBCDs exposure could stimulate cell proliferation of L02 cells, which might be associated with enhancement of PCNA expression.

  15. Therapeutic role of human hepatocyte growth factor (HGF) in treating hair loss

    PubMed Central

    2016-01-01

    Hepatocyte growth factor (HGF) is a paracrine hormone that plays an important role in epithelial-mesenchymal transition. HGF secreted by mesenchymal cells affects many properties of epithelial cells, such as proliferation, motility, and morphology. HGF has been reported to promote follicular growth. The purpose of the present study is to investigate the therapeutic role of HGF in hair loss treatment. A recombinant vector containing the human HGF (hHGF) gene (pTARGET-hHGF) was constructed, and the expression of hHGF in vitro was quantitatively and qualitatively evaluated. The effect of hHGF on hair growth was tested in mice, and results demonstrated that pTARGET-hHGF was successfully delivered into fibroblasts in vitro leading to a high expression of hHGF. Local injections of the pTARGET-hHGF recombinant vector into mice resulted in multiple beneficial effects compared to placebo, including faster hair regeneration, improved follicle development, and significantly increased HGF receptor (HGF-R). In conclusion, we have established a nonviral vector of hHGF which could be utilized to manipulate the sheath fibroblasts surrounding hair follicles (HF), thereby stimulating hair regeneration. PMID:27833804

  16. 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.

  17. 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.

  18. Therapeutic role of human hepatocyte growth factor (HGF) in treating hair loss.

    PubMed

    Qi, Yonghao; Li, Miao; Xu, Lian; Chang, Zhijing; Shu, Xiong; Zhou, Lijun

    2016-01-01

    Hepatocyte growth factor (HGF) is a paracrine hormone that plays an important role in epithelial-mesenchymal transition. HGF secreted by mesenchymal cells affects many properties of epithelial cells, such as proliferation, motility, and morphology. HGF has been reported to promote follicular growth. The purpose of the present study is to investigate the therapeutic role of HGF in hair loss treatment. A recombinant vector containing the human HGF (hHGF) gene (pTARGET-hHGF) was constructed, and the expression of hHGF in vitro was quantitatively and qualitatively evaluated. The effect of hHGF on hair growth was tested in mice, and results demonstrated that pTARGET-hHGF was successfully delivered into fibroblasts in vitro leading to a high expression of hHGF. Local injections of the pTARGET-hHGF recombinant vector into mice resulted in multiple beneficial effects compared to placebo, including faster hair regeneration, improved follicle development, and significantly increased HGF receptor (HGF-R). In conclusion, we have established a nonviral vector of hHGF which could be utilized to manipulate the sheath fibroblasts surrounding hair follicles (HF), thereby stimulating hair regeneration.

  19. Bosentan and Rifampin Interactions Modulate Influx Transporter and Cytochrome P450 Expression and Activities in Primary Human Hepatocytes.

    PubMed

    Han, Kyoung-Moon; Ahn, Sun-Young; Seo, Hyewon; Yun, Jaesuk; Cha, Hye Jin; Shin, Ji-Soon; Kim, Young-Hoon; Kim, Hyungsoo; Park, Hye-Kyung; Lee, Yong-Moon

    2017-02-06

    The incidence of polypharmacy-which can result in drug-drug interactions-has increased in recent years. Drug-metabolizing enzymes and drug transporters are important polypharmacy modulators. In this study, the effects of bosentan and rifampin on the expression and activities of organic anion-transporting peptide (OATP) and cytochrome P450 (CYP450) 2C9 and CYP3A4 were investigated in vitro. HEK293 cells and primary human hepatocytes overexpressing the target genes were treated with bosentan and various concentrations of rifampin, which decreased the uptake activities of OATP transporters in a dose-dependent manner. In primary human hepatocytes, CYP2C9 and CYP3A4 gene expression and activities decreased upon treatment with 20 μM bosentan+200 μM rifampin. Rifampin also reduced gene expression of OATP1B1, OATP1B3, and OATP2B1 transporter, and inhibited bosentan influx in human hepatocytes at increasing concentrations. These results confirm rifampin- and bosentan-induced interactions between OATP transporters and CYP450.

  20. Glucuronidation versus oxidation of the flavonoid galangin by human liver microsomes and hepatocytes.

    PubMed

    Otake, Yoko; Hsieh, Faye; Walle, Thomas

    2002-05-01

    In a previous study, we used human liver microsomes for the first time to study cytochrome P450 (P450)-mediated oxidation of the flavonoid galangin. The combination of CYP1A2 and CYP2C9 produced a V(max)/K(m) value of 13.6 +/- 1.1 microl/min/mg of protein. In the present extended study, we determined glucuronidation rates for galangin with the same microsomes. Two major and one minor glucuronide were identified by liquid chromatography/mass spectrometry. The V(max)/K(m) values for the two major glucuronides conjugated in the 7- and 3-positions were 155 +/- 30 and 427 +/- 26 microl/min/mg of protein, thus, exceeding that of oxidation by 11 and 31 times, respectively. This highly efficient glucuronidation appeared to be catalyzed mainly by the UDP-glucuronosyltransferase (UGT)1A9 isoform but also by UGT1A1 and UGT2B15. Sulfation of galangin by the human liver cytosol, mediated mainly but not exclusively by sulfotransferase (SULT) 1A1, also appeared to be efficient. These conclusions were strongly supported by experiments using the S9 fraction of the human liver, in which all three metabolic pathways could be directly compared. When galangin metabolism was examined in fresh plated hepatocytes from six donors, glucuronidation clearly predominated followed by sulfation. Oxidation occurred only to a minor extent in two of the donors. This study for the first time establishes that glucuronidation and sulfation of galangin, and maybe other flavonoids, are more efficient than P450-mediated oxidation, clearly being the metabolic pathways of choice in intact cells and therefore likely also in vivo.

  1. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes.

    PubMed

    Priore, Paola; Siculella, Luisa; Gnoni, Gabriele Vincenzo

    2014-07-01

    Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

  2. Evaluation of the cytotoxicity of 10 chemicals in human and rat hepatocytes and in cell lines: Correlation between in vitro data and human lethal concentration.

    PubMed

    Ponsoda, X; Jover, R; Núñez, C; Royo, M; Castell, J V; Gómez-Lechón, M J

    1995-12-01

    The cytotoxicity of 10 chemicals from the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) list (nos 21-30) was evaluated in human and rat cultured hepatocytes and in two established cell lines (HepG2 and 3T3) according to the MEIC programme organized by the Scandinavian Society of Cell Toxicology. The MTT test was used as the endpoint of cytotoxicity after 24hr of exposure to the chemicals. Theophylline, phenobarbital and paraquat were the least cytotoxic compounds in the cellular systems (IC(50) = 450-17,000 mum) except for the 3T3 cells. The seven remaining chemicals (dextropropoxyphene, propranolol, arsenic trioxide, cupric sulfate, mercuric chloride, thioridazine and thallium sulfate) showed a similar relative cytotoxic ranking in the four in vitro systems in the lower range of concentrations (IC(50) = 2-350 mum). The data suggest that these 10 chemicals have a basal cytotoxic effect common to the four in vitro systems, and probably none of these compounds could be considered either hepatotoxic or species specific. The correlation between in vitro data and human lethal blood concentrations showed that the predictability of the in vitro systems was similar to that of in vivo rodent tests (LD(50)) only when low cytotoxic concentrations (IC(10)) were used for correlation.

  3. 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.

  4. Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

    PubMed

    Elphick, Lucy M; Pawolleck, Nadine; Guschina, Irina A; Chaieb, Leila; Eikel, Daniel; Nau, Heinz; Harwood, John L; Plant, Nick J; Williams, Robin S B

    2012-03-01

    Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of β-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

  5. Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

    PubMed Central

    He, Yong; Zhou, Jun; Dou, Ke-Feng; Chen, Yong; Yan, Qing-Guo; Li, Hai-Min

    2004-01-01

    AIM: To construct pEGFP-hepatocyte growth factor (HGF) expression vector, the to detect its expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocytes. METHODS: Human HGF cDNA was ligated to the pEGFP vector. Recombinant plasmid was transfected into human hepatocyte line QZG with liposome. Expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 h after transfection to detect the number of [3H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry. Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions. RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment was cloned into BamH I and Sal I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy. The [3H]-TdR uptake became 7 times as many as in the control group 96 h after transfection. After HGF transfection, the survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P < 0.05), and the leakage of intracellular alanine transaminase and potassium ions decreased (586 nkat/L vs 1089 nkat/L, P < 0.01; and 5.59 mmol/L vs 6.02 mmol/L, P < 0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other non-transfected cells. CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning. PMID:15334679

  6. Lycopene as a natural protector against gamma-radiation induced DNA damage, lipid peroxidation and antioxidant status in primary culture of isolated rat hepatocytes in vitro.

    PubMed

    Srinivasan, M; Sudheer, A Ram; Pillai, K Raveendran; Kumar, P Raghu; Sudhakaran, P R; Menon, V P

    2007-04-01

    The present study was designed to evaluate the radioprotective effect of lycopene, a naturally occurring dietary carotenoid, on gamma-radiation induced toxicity in cultured rat hepatocytes. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), ceruloplasmin, vitamins A, E, C and uric acid. The DNA damage was analysed by single cell gel electrophoresis (comet assay). The increase in the severity of DNA damage was observed with the increase in gamma-radiation dose (1, 2 and 4 Gy) in cultured rat hepatocytes. TBARS were increased significantly whereas the levels of GSH, vitamins C, E and A, ceruloplasmin, uric acid and antioxidant enzymes were significantly decreased in gamma-irradiated groups. The maximum damage to hepatocytes was observed at 4 Gy irradiation. Pretreatment with lycopene (1.86, 9.31 and 18.62 microM) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH, vitamins A, E, C, uric acid and ceruloplasmin. The maximum protection of hepatocytes was observed at 9.31 muM of lycopene pretreatment. Thus, our results show that pretreatment with lycopene offers protection against gamma-radiation induced cellular damage and can be developed as an effective radioprotector during radiotherapy.

  7. Na(+)-Ca sup 2+ exchange in cultured rat hepatocytes: Evidence against a role in cytosolic Ca sup 2+ regulation or signaling

    SciTech Connect

    Lidofsky, S.D.; Xie, M.H.; Scharschmidt, B.F. )

    1990-07-01

    Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ((Ca2+)i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of (Ca2+)i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of (Ca2+)i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates (Ca2+)i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. (Ca2+)i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The (Ca2+)i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in (Ca2+)i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of (Ca2+)i in hepatocytes.

  8. Transplantation of Porcine Hepatocytes Cultured with Polylactic Acid-O-Carboxymethylated Chitosan Nanoparticles Promotes Liver Regeneration in Acute Liver Failure Rats

    PubMed Central

    Chen, Zhong; Chang, Renan; Guan, Weijun; Cai, Hongyu; Tang, Fei; Zhu, Wencai; Chen, Jiahui

    2011-01-01

    In this study, free porcine hepatocytes suspension (Group A), porcine hepatocytes embedded in collagen gel (Group B), porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel (Group C), and PLA-O-CMC nanoparticles alone (Group D) were transplanted into peritoneal cavity of ALF rats, respectively. The result showed that plasma HGF levels were elevated post-transplantation with a peak at 12 hr. The rats in Group C showed highest plasma HGF levels at 2, 6, 12, 24 and 36 hr post-transplantation and lowest HGF level at 48 hr. Plasma VEGF levels were elevated at 48 hr post-transplantation with a peak at 72 hr. The rats in Group C showed highest plasma HGF levels at 48, 72, and 96 hr post-transplantation. The liver functions in Group C were recovered most rapidly. Compared with Group B, Group C had significant high liver Kiel 67 antigen labeling index (Ki-67 LI) at day 1 post-HTx (P < .05). Ki-67 LI in groups B and C was higher than that in groups A and D at days 5 and 7 post-HTx. In conclusion, intraperitoneal transplantation of porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel can promote significantly liver regeneration in ALF rats. PMID:21603218

  9. Networks of Human Culture

    NASA Astrophysics Data System (ADS)

    Csermely, Peter

    Now that you are back from the zoo, where you tried in vain to shake the six hands of all the ants for several hours,1 it is time to start our fifth trip into Netland. Let us go and see what those macroscopic ants known as human beings can achieve. I will show you what type of networks we have figured out to support the last variety of social networks from the previous chapter.

  10. In Silico Models for Dynamic Connected Cell Cultures Mimicking Hepatocyte-Endothelial Cell-Adipocyte Interaction Circle

    PubMed Central

    Andreoni, Chiara; Orsi, Gianni; De Maria, Carmelo; Montemurro, Francesca; Vozzi, Giovanni

    2014-01-01

    The biochemistry of a system made up of three kinds of cell is virtually impossible to work out without the use of in silico models. Here, we deal with homeostatic balance phenomena from a metabolic point of view and we present a new computational model merging three single-cell models, already available from our research group: the first model reproduced the metabolic behaviour of a hepatocyte, the second one represented an endothelial cell, and the third one described an adipocyte. Multiple interconnections were created among these three models in order to mimic the main physiological interactions that are known for the examined cell phenotypes. The ultimate aim was to recreate the accomplishment of the homeostatic balance as it was observed for an in vitro connected three-culture system concerning glucose and lipid metabolism in the presence of the medium flow. The whole model was based on a modular approach and on a set of nonlinear differential equations implemented in Simulink, applying Michaelis-Menten kinetic laws and some energy balance considerations to the studied metabolic pathways. Our in silico model was then validated against experimental datasets coming from literature about the cited in vitro model. The agreement between simulated and experimental results was good and the behaviour of the connected culture system was reproduced through an adequate parameter evaluation. The developed model may help other researchers to investigate further about integrated metabolism and the regulation mechanisms underlying the physiological homeostasis. PMID:25502576

  11. In silico models for dynamic connected cell cultures mimicking hepatocyte-endothelial cell-adipocyte interaction circle.

    PubMed

    Andreoni, Chiara; Orsi, Gianni; De Maria, Carmelo; Montemurro, Francesca; Vozzi, Giovanni

    2014-01-01

    The biochemistry of a system made up of three kinds of cell is virtually impossible to work out without the use of in silico models. Here, we deal with homeostatic balance phenomena from a metabolic point of view and we present a new computational model merging three single-cell models, already available from our research group: the first model reproduced the metabolic behaviour of a hepatocyte, the second one represented an endothelial cell, and the third one described an adipocyte. Multiple interconnections were created among these three models in order to mimic the main physiological interactions that are known for the examined cell phenotypes. The ultimate aim was to recreate the accomplishment of the homeostatic balance as it was observed for an in vitro connected three-culture system concerning glucose and lipid metabolism in the presence of the medium flow. The whole model was based on a modular approach and on a set of nonlinear differential equations implemented in Simulink, applying Michaelis-Menten kinetic laws and some energy balance considerations to the studied metabolic pathways. Our in silico model was then validated against experimental datasets coming from literature about the cited in vitro model. The agreement between simulated and experimental results was good and the behaviour of the connected culture system was reproduced through an adequate parameter evaluation. The developed model may help other researchers to investigate further about integrated metabolism and the regulation mechanisms underlying the physiological homeostasis.

  12. 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.

  13. Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

    PubMed Central

    Takahashi, M; Ota, S; Shimada, T; Hamada, E; Kawabe, T; Okudaira, T; Matsumura, M; Kaneko, N; Terano, A; Nakamura, T

    1995-01-01

    Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism. Images PMID:7738166

  14. Synthetic peptides from Plasmodium falciparum apical membrane antigen 1 (AMA-1) specifically interacting with human hepatocytes.

    PubMed

    Valbuena, J; Rodríguez, L; Vera, R; Puentes, A; Curtidor, H; Cortés, J; Rosas, J; Patarroyo, M E

    2006-10-01

    Plasmodium falciparum apical membrane antigen 1 (AMA-1) is expressed during both the sporozoite and merozoite stage of the parasite's life cycle. The role placed by AMA-1 during sporozoite invasion of hepatocytes has not been made sufficiently clear to date. Identifying the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for sporozoite-hepatocyte interaction. Binding assays between P. falciparum AMA-1 peptides and HepG2 cell were performed in this study to identify possible AMA-1 functional regions. Four AMA-1 high activity binding peptides (HABPs) bound specifically to hepatocytes: 4310 ((74)QHAYPIDHEGAEPAPQEQNL(93)), 4316 ((194)TLDEMRHFYKDNKYVKNLDE(213)), 4321 ((294)VVDNWEKVCPRKNLQNAKFGY(313)) and 4332 ((514)AEVTSNNEVVVKEEYKDEYA(533)). Their binding to these cells became saturable and resistant to treatment with neuraminidase. Most of these peptides were located in AMA-1 domains I and III, these being target regions for protective antibody responses. These peptides interacted with 36 and 58 kDa proteins on the erythrocyte surface. Some of the peptides were found in exposed regions of the AMA-1 protein, thereby facilitating their interaction with host cells. It is thus probable that AMA-1 regions defined by the four peptides mentioned above are involved in sporozoite-hepatocyte interaction.

  15. Co-culture of hepatoma cells with hepatocytic precursor (stem-like) cells inhibits tumor cell growth and invasion by downregulating Akt/NF-κB expression

    PubMed Central

    Sui, Cheng-Jun; Xu, Miao; Li, Wei-Qing; Yang, Jia-Mei; Yan, Hong-Zhu; Liu, Hui-Min; Xia, Chun-Yan; Yu, Hong-Yu

    2016-01-01

    Hepatocytic stem cells (HSCs) have inhibitory effects on hepatocarcinoma cells. The present study investigated the effects of HSC activity in hepatocarcinoma cells in vitro. A Transwell co-culture system of hepatocytic precursor (stem-like) WB-F344 cells and hepatoma CBRH-7919 cells was used to assess HSC activity in metastasized hepatoma cells in vitro. Nude mouse xenografts were used to assess HSC activity in vivo. Co-culture of hepatoma CBRH-7919 cells with WB-F344 cells suppressed the growth and colony formation, tumor cell migration and invasion capacity of CBRH-7919 cells. The nude mouse xenograft assay demonstrated that the xenograft size of CBRH-7919 cells following co-culture with WB-F344 cells was significantly smaller compared with that of control cells. Furthermore, the expression levels of the epithelial markers E-cadherin and β-catenin were downregulated, while the mesenchymal markers α-SMA and vimentin were upregulated. Co-culture of CBRH-7919 cells with WB-F344 cells downregulated NF-κB and phospho-Akt expression. In conclusion, hepatocytic precursor (stem-like) WB-F344 cells inhibited the growth, colony formation and invasion capacity of metastasized hepatoma CBRH-7919 cells in vitro and in vivo by downregulating Akt/NF-κB signaling. PMID:27895771

  16. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  17. Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine.

    PubMed

    Fang, Jia-Long; Beland, Frederick A

    2013-01-01

    Nevirapine is a non-nucleoside reverse transcriptase (RT) inhibitor used for the treatment of AIDS and the prevention of mother-to-child transmission of HIV-1. Despite its therapeutic benefits, treatment with nevirapine has been associated with significant incidences of liver and dermal toxicity. The present study examined the effects of nevirapine on cell growth and death in human hepatocyte HepG2 cells and THLE2 cells and the possible pathways involved in these effects. The concentrations of nevirapine inhibiting 50% cell growth were similar for both cell lines. Nevirapine (0-250 µM) treatment caused a slight increase in the amount of lactate dehydrogenase released into the medium. Apoptotic cell death did not contribute to the decrease in viable cells. Exposing of HepG2 cells to nevirapine caused G2/M phase arrest, and the activity of senescence-associated β-galactosidase was not altered. In THLE2 cells, the percentage of cells in G1/G0 phase was increased and cellular senescence was induced in a concentration-dependent manner. Endogenous non-telomeric RT activity was not detected in either cell line. Western blot analysis indicated lower levels of p53 and phospho-p53 (ser15) in HepG2 cells as compared to THLE2 cells; no significant changes in p53 or phospho-p53 (ser15) were noted with nevirapine treatment. These data demonstrate that nevirapine inhibits cell growth, induces cell cycle arrest at different phases, and has different effects on cellular senescence in HepG2 cells and THLE2 cells. The differential responses appear to be related to differences in the basal levels of p53 in the HepG2 cells and THLE2 cells.

  18. Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes.

    PubMed

    Yang, Ming; Chu, Eugene M; Caslake, Muriel J; Edelstein, Celina; Scanu, Angelo M; Hill, John S

    2010-02-01

    We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30-40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 microM) and 87% (0.7 microM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.

  19. 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

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

    PubMed

    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

    2016-02-18

    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.

  1. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

    SciTech Connect

    Noreault-Conti, Trisha L.; Jacobs, Judith M.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Jacqueline F.; Nichols, Ralph C. . E-mail: ralph.c.nichols@dartmouth.edu

    2006-12-15

    Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

  2. The quantitative prediction of in vivo enzyme-induction caused by drug exposure from in vitro information on human hepatocytes.

    PubMed

    Kato, Motohiro; Chiba, Koji; Horikawa, Masato; Sugiyama, Yuichi

    2005-08-01

    There have been no reports of the quantitative prediction of induction for drug-metabolizing enzymes in humans. We have tried to predict such enzyme induction in humans from in vitro data obtained using human hepatocytes. The in vitro and in vivo data on enzyme induction by inducers, such as rifampicin, phenobarbital and omeprazole, were collected from the published literature. The degree of enzyme induction in humans was compared with that predicted from in vitro data on human hepatocytes. Using the in vivo data, we calculated the hepatic intrinsic clearance of typical CYP substrates, such as midazolam and caffeine, before and after inducer treatment and estimated the induction ratios of hepatic intrinsic clearance following treatment. In the in vitro studies, the amount of mRNA or enzyme and enzyme activity in human hepatocytes, with or without an inducer, were compared and the induction ratios were estimated. The unbound mean concentration was taken as an index of drug exposure and the induction ratios in the in vivo and in vitro studies were compared. The unbound mean concentrations of inducers used in the in vitro studies were higher than those in the in vivo studies. The maximum induction ratios by inducers in the in vitro studies were higher than those in the in vivo studies. The induction ratio for rifampicin, omeprazole, troglitazone, dexamethasone and phenobarbital increased as the unbound mean concentration increased to reach a constant value. The induction of CYP3A and 1A was analyzed by the Emax model. The maximum induction ratio (Emax) and the concentration at half maximum induction (EC50) for rifampicin, omeprazole, troglitazone, dexamethasone and phenobarbital were 12.3, 0.847 micromol/L, 2.36, 0.225 micromol/L, 6.86, 0.002 micromol/L, 8.30, 9.32 micromol/L, and 7.62, 58.4 micromol/L, respectively. The Emax and EC50 of omeprazole for CYP1A were 12.02 and 0.075 micromol/L, respectively. The predicted induction ratio of all those inducers, except for

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

    PubMed

    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.

  4. Hepatoprotective Activity of Water Extracts from Chaga Medicinal Mushroom, Inonotus obliquus (Higher Basidiomycetes) Against Tert-Butyl Hydroperoxide-Induced Oxidative Liver Injury in Primary Cultured Rat Hepatocytes.

    PubMed

    Hong, Ki Bae; Noh, Dong Ouk; Park, Yooheon; Suh, Hyung Joo

    2015-01-01

    We examined the hepatoprotective activity of Inonotus obliquus water extract (IO-W) against tert-butyl hydroperoxide (t-BHP)-induced oxidative liver injury in the primary cultured rat hepatocyte. The 50% radical scavenging concentrations (SC50s) of IO-W for radical-scavenging activity against 2,2'-azino-bis-(3-ethylbenzothi- azoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were 5.19 mg/mL and 0.39 mg/mL, respectively. IO-W pretreatment to the primary cultured hepatocytes significantly (p<0.05) protected the cells from t-BHP-induced cytotoxic injury even at a low concentration of IO-W (10 µg/mL). The cellular leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as malondialdehyde (MDA) formation caused by t-BHP were significantly (p<0.05) suppressed by IO-W pretreatment (>100 µg/ mL). In conclusion, this study demonstrates that IO-W exhibited hepatoprotective activity against t-BHP-induced oxidative liver injury in the primary cultured hepatocyte probably via its abilities of quenching free radicals, inhibiting the leakage of ALT, AST, and LDH, and decreasing MDA formation.

  5. Toxic effects of wastewaters collected at upstream and downstream sites of a purification station in cultures of rainbow trout hepatocytes.

    PubMed

    Risso-de Faverney, C; Devaux, A; Lafaurie, M; Girard, J P; Rahmani, R

    2001-08-01

    The toxic effects of wastewater samples, collected in December 1998, from upstream (U) and downstream (D) sites of the purification station of the town of Nice (South-East France on the coast of the Mediterranean Sea) were assessed undiluted and at various dilutions (75%, 50%, and 25% of collected water sample), on trout hepatocyte cultures treated for 48 or 72 h. Chemical contamination (PCBs, PAHs, Cd, Cu, Pb, and Zn) was also evaluated by chemical analysis. The water samples from the upstream site were more cytotoxic than those from the downstream site. The induction of CYP1A enzyme and metallothioneins (MTs) were selected as specific indicators of exposure to organic contaminants and metals, respectively. CYP1A-related EROD activity as well as protein expression were found to be greatly induced after 72 h exposure of the hepatocytes to the undiluted water samples (U(100%) and D(100%)), but CYP1A1 mRNA was significantly overexpressed only by samples from the upstream site. Maximal MT levels were reached after 48 h of treatment with the least concentrated water samples (U(25%) and D(25%)). Glutathione S-transferase (GST) activities were similarly increased under the same conditions. On the other hand, there was no significant glutathione peroxidase (GPx) activity response. Induction of apoptosis was analyzed by using as markers both the fragmentation of the nuclear DNA into oligonucleosomal-length fragments recognized as a "DNA ladder" and the activation of DEVD (Asp-Glu-Val-Asp)-dependent protease considered as the central mediator of programmed cell death. Significant DNA cleavage was only detectable after 72-h exposure to the most concentrated water samples from upstream sites (U(75%) and U(100%)). DEVD-dependent protease activities were significantly increased, mainly in cells exposed to U(75%) and D(25%) for 72 h. In addition, pollution-related DNA damage assessed by using the Comet assay was approximatively 1.5 times greater than that of the control level

  6. Human neuroblastoma cultures for biorobotics.

    PubMed

    Ferrández, J M; Lorente, V; de Santos, D; Cuadra, J M; de la Paz, F; Alvarez, J R; Fernández, E

    2011-01-01

    This paper introduces a new biorobotic system using human neuroblastoma cultures and centre of area learning for basic robotic guidance. Multielectrode Arrays Setups have been designed for direct culturing neural cells over silicon or glass substrates, providing the capability to stimulate and record simultaneously populations of neural cells. The main objective of this work will be to control a robot using this biological neuroprocessor and a new simple centre of area learning scheme. The final system could be applied for testing how chemicals affect the behaviour of the robot or to establish the basis for new hybrid optogenetic neuroprostheses based on stimulating optically genetic-modified neurons.

  7. Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

    PubMed Central

    Huang, Jieqiong; Guo, Xinyue; Li, Weihong; Zhang, Haiyan

    2017-01-01

    The generation of hepatocytes that are derived from human adipose stem cells (hASCs) represents an alternative to human hepatocytes for individualized therapeutic and pharmaceutical applications. However, the mechanisms facilitating hepatocyte differentiation from hASCs are not well understood. Here, we show that upon exposure to glycogen synthase kinase 3 (GSK3) inhibitors alone, the expression of definitive endoderm specific genes GATA4, FOXA2, and SOX17 in hASCs significantly increased in a manner with activation of Wnt/β-catenin signalling. Down regulation of the β-catenin expression attenuates the effect of GSK3 inhibitors on the induction of these specific genes. The cells induced using GSK3 inhibitors were directed to differentiate synchronously into hepatocyte-like cells (HLCs) after further combinations of soluble factors by a reproducible three-stage method. Moreover, hASC-HLCs induced using GSK3 inhibitors possess low-density lipoprotein uptake, albumin secretion, and glycogen synthesis ability, express important drug-metabolizing cytochrome P450 (CYP450) enzymes, and demonstrate CYP450 activity. Therefore, our findings suggest that activation of Wnt/β-catenin signalling via GSK3 inhibitors in definitive endoderm specification may represent an important mechanism mediating hASCs differentiated to functional hepatocyte. Furthermore, development of similar compounds may be useful for robust, potentially scalable and cost-effective generation of functional hepatocytes for drug screening and predictive toxicology platforms. PMID:28094799

  8. 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

  9. Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes.

    PubMed

    Mundy, Lukas J; Crump, Doug; Jones, Stephanie P; Konstantinov, Alex; Utley, Fiona; Potter, David; Kennedy, Sean W

    2012-04-01

    Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 concentration-response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.

  10. BOLISM OF ARSENITE IN CULTURED PRIMARY HEPATOCYTES FROM SIX MAMMALIAN SPECIES

    EPA Science Inventory

    Inorganic arsenic (iAs) is an environmental toxin and carcinogen. Biomethylation is the major pathway for the metabolism of iAs in many mammalian species, including the human. The liver is considered the primary site for iAs methylation and As (+3 oxidation state) methyltransfera...

  11. 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

    2016-02-02

    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.

  12. 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.

  13. Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Nagaoka, Masato; Kobayashi, Motohiro; Kawai, Chie; Mallanna, Sunil K.; Duncan, Stephen A.

    2015-01-01

    Maintenance and differentiation of human pluripotent stem cells (hPSCs) usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth—Holm—Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp) motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs. PMID:26308339

  14. Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response

    PubMed Central

    Christudoss, Pamela; Mickey, Kristen; Tessman, Robert; Ni, Hong-min; Swerdlow, Russell

    2017-01-01

    Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis. PMID:28163822

  15. Xanthohumol induces phase II enzymes via Nrf2 in human hepatocytes in vitro.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Baer-Dubowska, Wanda

    2013-02-01

    The aim of this study was to investigate whether xanthohumol may exert chemoprotective activity through the modulation of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in immortalized normal THLE-2 hepatocytes and a hepatocellular carcinoma HepG2 cell line. Cells were incubated in the presence of xanthohumol and the activation of Nrf2 and expression of genes controlled by this transcription factor were evaluated. Additionally, p53 level was assessed. Xanthohumol increased the expression and led to the activation of Nrf2 in both cell lines. However, in contrast to normal cells the expression of genes controlled by this transcription factor was not affected in HepG2 cells, except for GSTA and GSTP. Xanthohumol, beside the induction of GSTs and HO-1, significantly elevated NQO1 expression in concert with p53 level in normal hepatocytes. The activation of Nrf2 pathway and subsequently phase II enzymes in concert with p53 induction in normal hepatocytes may account for the molecular mechanism of the chemopreventive activity of xanthohumol. On the other hand its cytotoxicity towards HCC cells shown in this study indicates that it may also be considered as potentially chemotherapeutic.

  16. Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

    PubMed

    Kocarek, T A; Reddy, A B

    1996-11-01

    It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of

  17. 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.

  18. Differentiated properties of hepatocytes induced from pancreatic cells.

    PubMed

    Tosh, David; Shen, Chia-Ning; Slack, Jonathan M W

    2002-09-01

    Transdifferentiation of pancreas to liver is a well-recognized phenomenon and has been described in animal experiments and human pathology. We recently produced an in vitro model for the transdifferentiation (or conversion) of the pancreatic cell line AR42J-B13 to hepatocytes based on culture with dexamethasone (Dex). To determine whether the hepatocytes express markers of hepatic intermediary metabolism and detoxification, we investigated the patterns of expression of glucokinase, cytochrome P450s CYP3A1 and CYP2B1/2, testosterone/4-nitrophenol uridine diphosphate glucuronosyltransferase (UDPGT), and aryl sulfotransferase. All were expressed. We also determined the expression of 2 enzymes involved in ammonia detoxification: carbamoylphosphate synthetase I (CPS I) and glutamine synthetase (GS). These enzymes are normally strictly compartmentalized in liver in a wide periportal pattern and the last downstream perivenous hepatocytes, respectively. Following culture with Dex, CPS I and GS are expressed in 2 different cell populations, suggesting that both periportal and perivenous hepatocytes are induced. We also produced a reporter assay based on the activation of green fluorescent protein (GFP) by the transthyretin (TTR) promoter or glucose-6-phosphatase (G6Pase) promoter. After culture with Dex, transfected cells begin to express GFP, showing that hepatic promoters are activated in concert with the induction of the hepatocyte phenotype. Lastly, we examined the stability of the hepatic phenotype and found that some cells still express liver markers (transferrin or albumin) up to 14 days after removal of Dex. In conclusion, these results suggest that pancreatic hepatocytes produced by this method may offer an alternative model to primary cultures of hepatocytes for the study of liver function.

  19. Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

    PubMed

    Nims, R W; Lubet, R A; Fox, S D; Jones, C R; Thomas, P E; Reddy, A B; Kocarek, T A

    1998-03-27

    In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

  20. Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

    SciTech Connect

    Okayasu, T.; Hasegawa, K.; Ishibashi, T.

    1987-07-01

    Addition of platelet-activating factor (PAF) to cells doubly labeled with (/sup 14/C)glycerol plus (/sup 3/H)arachidonic acid resulted in a transient decrease of (/sup 14/C)glycerol-labeled phosphatidylinositol (PI) and a transient increase of (/sup 14/C)glycerol-labeled lysophosphatidylinositol (LPI). (/sup 3/H)Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The /sup 3/H//sup 14/C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of (/sup 14/C)glycerol-labeled DG paralleled the loss of triacyl (/sup 14/C)glycerol and the /sup 3/H//sup 14/C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- (/sup 3/H)inositol-prelabeled cells, PAF induced a transient decrease of (/sup 3/H)phosphatidylinositol-4,5-bis-phosphate (TPI) and (/sup 3/H)phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with /sup 32/Pi induced a transient decrease of (/sup 32/P)polyphosphoinositides at 20 sec to 1 min. (/sup 32/P)LPI appeared within 10 sec after stimulation and paralleled the loss of (/sup 32/P)PI. (/sup 3/H)Inositol triphosphate, (/sup 3/H)inositol diphosphate, and (/sup 3/H)inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.

  1. Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures.

    PubMed

    Battaglia, Valentina; Compagnone, Alessandra; Bandino, Andrea; Bragadin, Marcantonio; Rossi, Carlo Alberto; Zanetti, Filippo; Colombatto, Sebastiano; Grillo, Maria Angelica; Toninello, Antonio

    2009-03-01

    It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

  2. 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...

  3. Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

    PubMed

    Xu, Jimin; Capezzone, Marco; Xu, Xiao; Hershman, Jerome M

    2005-02-01

    We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT

  4. Excretory/secretory products of Fasciola hepatica but not recombinant phosphoglycerate kinase induce death of human hepatocyte cells.

    PubMed

    Bąska, Piotr; Norbury, Luke J; Wiśniewski, Marcin; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-06-01

    The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts' liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein - FhPGK, had no impact on cell survival.

  5. 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

  6. 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

  7. A three-dimensional collagen-sponge-based culture system coated with simplified recombinant fibronectin improves the function of a hepatocyte cell line.

    PubMed

    Nishida, Yuuki; Taniguchi, Akiyoshi

    2016-03-01

    Hepatocytes are widely used in pharmaceutical drug discovery tests, but their hepatic functions decrease rapidly during in vitro culture. Many culture systems have been devised to address this problem. We here report that a three-dimensional (3D) collagen-based scaffold coated with simplified recombinant fibronectin (FN) enhanced the function of a hepatocyte cell line. The developed culture system uses a honeycomb collagen sponge coated with collagen-binding domain (CBD)-cell attachment site (CAS), a chimeric protein comprising the CBD and CAS of FN. The function of HepG2 cells grown on honeycomb collagen sponge coated with CBD-CAS was investigated by determining the messenger RNA (mRNA) expression levels of several genes. The mRNA expression level of albumin increased 3.25 times in cells grown on CBD-CAS-coated honeycomb collagen sponge for 3 days; the expression level of CCAAT/enhancer binding protein (C/EBPα) increased 40-fold after 1 d and up to 150-fold after 3 d. These results suggested that CBD-CAS-coated honeycomb collagen sponge could improve the functions of hepatocytes by inducing C/EBPα expression. The activation of cytochrome P450 (CYP) enzymes in HepG2 cells grown on CBD-CAS-coated honeycomb collagen sponge was measured at the mRNA level and was found to increase between two and six times compared to cells grown without the CBD-CAS coating, showing that this culture system induced CYP gene expression and thus may be useful in drug metabolism assays.

  8. Recombinant human hepatocyte growth factor (HGF), but not rat HGF, elicits glomerular injury and albuminuria in normal rats via an immune complex-dependent mechanism.

    PubMed

    Mizuno, Shinya; Ikebuchi, Fumie; Fukuta, Kazuhiro; Kato, Takashi; Matsumoto, Kunio; Adachi, Kiichi; Abe, Tetsushi; Nakamura, Toshikazu

    2011-03-01

    1. Hepatocyte growth factor (HGF) has the therapeutic potential to improve renal fibrosis and proteinuria in rodents with chronic kidney disease. In contrast, long-term administration of human HGF to normal rats reportedly elicits proteinuria. Thus, the role of HGF during proteinuria remains contentious. The aim of the present study was to demonstrate that human HGF is antigenic to rodents and that immune complex formation causes proteinuria. 2. We administered either human or rat HGF to normal rats for 28 days. Albuminuria was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The renal phenotypes of the two HGF treatments were examined using histological techniques. 3. Administration of human HGF (1 mg/kg per day, i.v.) to rats led to severe albuminuria and glomerular hypertrophy in association with increased blood levels of anti-human HGF IgG and IgG deposition in mesangial areas. Furthermore, an immune complex between human HGF and anti-human HGF IgG stimulated the production of proteinuric cytokines (including transforming growth factor-β) in rat cultured mesangial cells. In contrast, treatment of healthy rats with rat HGF for 4 weeks caused neither mesangial IgG deposition nor elevated anti-HGF IgG in the blood. Overall, rat HGF did not provoke albuminuria. 4. We conclude that human HGF produces pseudotoxic effects in normal rat kidneys via an immune complex-mediated pathway, whereas syngenic HGF is safe due to less deposition of glomerular IgG. Our results affirm the safety of the repeated use of syngenic HGF for the treatment of chronic organ diseases, such as renal fibrosis and liver cirrhosis.

  9. Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors.

    PubMed

    Desbans, C; Hilgendorf, C; Lutz, M; Bachellier, P; Zacharias, T; Weber, J C; Dolgos, H; Richert, L; Ungell, A-L

    2014-01-01

    1.  It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2.  The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3.  We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4.  It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

  10. Effect of L-carnitine on glycogen synthesis and ATP production in cultured hepatocytes of the newborn rat.

    PubMed

    Nishida, N; Sugimoto, T; Takeuchi, T; Kobayashi, Y

    1989-11-01

    Changes in intracellular carnitine, ATP and glycogen concentration were studied when hepatocytes of newborn or adult rats were incubated with oleate, lactate and/or carnitine. Hepatocytes of adult rats appeared to be able to maintain cellular carnitine concentration without exogenous carnitine supplementation. Hepatocytes of newborn rats appeared to be unable to maintain cellular carnitine concentration without carnitine supplementation. Moreover, ATP concentration and glycogen concentration were significantly increased by the carnitine supplement with oleate and/or lactate compared to the unsupplemented group. Increases in both intracellular ATP and carnitine concentration depended on the concentration of carnitine added to the medium. These results suggest that carnitine may be an important factor in glycogen synthesis and ATP production of newborn infants.

  11. Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

    PubMed Central

    Bell, Catherine C.; Vorrink, Sabine U.; Palmgren, Henrik; Duffin, Rodger; Andersson, Tommy B.; Ingelman-Sundberg, Magnus

    2017-01-01

    Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems—hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids—at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans. PMID:28137721

  12. Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

    PubMed Central

    Li, Xiaoming; Xu, Min; Wang, Fei; Ji, Yong; DavidsoN, W. Sean; Li, Zongfang; Tso, Patrick

    2015-01-01

    We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis. PMID:26556724

  13. Superior performance of co-cultured mesenchymal stem cells and hepatocytes in poly(lactic acid-glycolic acid) scaffolds for the treatment of acute liver failure.

    PubMed

    Liu, Mingying; Yang, Jiacai; Hu, Wenjun; Zhang, Shichang; Wang, Yingjie

    2016-02-02

    Recently, cell-based therapies have attracted attention as promising treatments for acute liver failure (ALF). Bone marrow-derived mesenchymal stem cells (MSCs) are potential candidates for co-culture with hepatocytes in poly(lactic acid-glycolic acid) (PLGA) scaffolds to support hepatocellular function. However, the mechanism of culturing protocol using PLGA scaffolds for MSC differentiation into hepatocyte-like cells as well as the therapeutic effect of cell seeded PLGA scaffolds on ALF remain unsatisfactory in clinical application. Here, MSCs and hepatocytes were co-cultured at ratios of 1:2.5 (MSCs: Hep), 1:5 and 1:10, respectively. The proliferation abilities of these co-cultured cells were detected by CCK8, MTT, EdU and by scanning electron microscopy (SEM), and the ability of MSCs to differentiate into hepatocytes was detected by PCR, western blot and immunofluorescence staining. Therapeutic trials of cell seeded PLGA scaffolds were conducted through mouse abdominal cavity transplantation. Results showed that the 1:5 group showed significantly higher cellular proliferation than the 1:2.5 and 1:10 groups, supernatant albumin and urea nitrogen levels were also significantly higher in the 1:5 group than in other two groups. Similarly, the 1:5 group demonstrated better DNA transcription and liver-specific protein (albumin, CK18 and P450) production. Meanwhile, the GalN-stimulated levels of ALT, AST and TBil in mouse serum were down-regulated significantly more by (MSC  +  Hep)-PLGA scaffold treatment than MSC-PLGA or Hep-PLGA scaffold treatments. Furthermore, the (MSC  +  Hep)-PLGA scaffold-treated ALF mice showed a lower immunogenic response level than the other two groups. These data suggested that the ratio of 1:5 (MSC:Hep) co-cultures was the optimal ratio for MSCs to support hepatocellular metabolism and function in PLGA scaffolds in vitro, the (MSC  +  Hep)-PLGA scaffold treatment could perform better restoration for damaged liver

  14. Hepatocyte Polarity

    PubMed Central

    Treyer, Aleksandr; Müsch, Anne

    2013-01-01

    Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

  15. Constitutive gp130 activation rapidly accelerates the transformation of human hepatocytes via an impaired oxidative stress response

    PubMed Central

    Herden, Johannes; Parplys, Ann Christin; Borgmann, Kerstin; Schmidt-Arras, Dirk; Lohse, Ansgar W.; Rose-John, Stefan; Wege, Henning

    2016-01-01

    Pro-inflammatory signaling pathways, especially interleukin 6 (IL-6), and reactive oxygen species (ROS) promote carcinogenesis in the liver. In order to elucidate the underlying oncogenic mechanism, we activated the IL-6 signal transducer glycoprotein 130 (gp130) via stable expression of a constitutively active gp130 construct (L-gp130) in untransformed telomerase-immortalized human fetal hepatocytes (FH-hTERT). As known from hepatocellular adenomas, forced gp130 activation alone was not sufficient to induce malignant transformation. However, additional challenge of FH-hTERT L-gp130 clones with oxidative stress resulted in 2- to 3-fold higher ROS levels and up to 6-fold more DNA-double strand breaks (DSB). Despite increased DNA damage, ROS-challenged FH-hTERT L-gp130 clones displayed an enhanced proliferation and rapidly developed colony growth capabilities in soft agar. As driving gp130-mediated oncogenic mechanism, we detected a decreased expression of antioxidant genes, in particular glutathione peroxidase 3 and apolipoprotein E, and an absence of P21 upregulation following ROS-conferred induction of DSB. In summary, an impaired oxidative stress response in hepatocytes with gp130 gain-of-function mutations, as detected in dysplastic intrahepatic nodules and hepatocellular adenomas, is one of the central oncogenic mechanisms in chronic liver inflammation. PMID:27489351

  16. A mutation of MET, encoding hepatocyte growth factor receptor, is associated with human DFNB97 hearing loss

    PubMed Central

    Mujtaba, Ghulam; Schultz, Julie M; Imtiaz, Ayesha; Morell, Robert J; Friedman, Thomas B; Naz, Sadaf

    2015-01-01

    Background Hearing loss is a heterogeneous neurosensory disorder. Mutations of 56 genes are reported to cause recessively inherited nonsyndromic deafness. Objective We sought to identify the genetic lesion causing hearing loss segregating in a large consanguineous Pakistani family. Methods and Results Mutations of GJB2 and all other genes reported to underlie recessive deafness were ruled out as the cause of the phenotype in the affected members of the participating family. Homozygosity mapping with a dense array of one million SNP markers allowed us to map the gene for recessively inherited severe hearing loss to chromosome 7q31.2, defining a new deafness locus designated DFNB97 (maximum LOD score of 4.8). Whole-exome sequencing revealed a novel missense mutation c.2521T>G (p.F841V) in MET, which encodes the receptor for hepatocyte growth factor. The mutation co-segregated with the hearing loss phenotype in the family and was absent from 800 chromosomes of ethnically matched control individuals as well as from 136,602 chromosomes in public databases of nucleotide variants. Analyses by multiple prediction programs indicated that p.F841V is likely damaging to MET function. Conclusion We identified a missense mutation of MET, encoding the hepatocyte growth factor receptor, as a likely cause of hearing loss in humans. PMID:25941349

  17. Therapeutic Efficacy of Human Hepatocyte Transplantation in a SCID/uPA Mouse Model with Inducible Liver Disease

    PubMed Central

    Douglas, Donna N.; Kawahara, Toshiyasu; Sis, Banu; Bond, David; Fischer, Karl P.; Tyrrell, D. Lorne J.; Lewis, Jamie T.; Kneteman, Norman M.

    2010-01-01

    Background Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. Methodology/Principal Findings In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32–87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. Conclusions/Significance Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against v

  18. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  19. PPO/PEO modified hollow fiber membranes improved sensitivity of 3D cultured hepatocytes to drug toxicity via suppressing drug adsorption on membranes.

    PubMed

    Shen, Chong; Meng, Qin; He, Wenjuan; Wang, Qichen; Zhang, Guoliang

    2014-11-01

    The three dimensional (3D) cell culture in polymer-based micro system has become a useful tool for in vitro drug discovery. Among those polymers, polysulfone hollow fiber membrane (PSf HFM) is commonly used to create a microenvironment for cells. However, the target drug may adsorb on the polymeric surface, and this elicits negative impacts on cell exposure due to the reduced effective drug concentration in culture medium. In order to reduce the drug adsorption, PSf membrane were modified with hydrophilic Pluronic (PEO-b-PPO-b-PEO) copolymers, L121, P123 and F127 (PEO contents increase from 10%, 30% to 70%), by physical adsorption. As a result, the hydrophilicity of HFMs increased at an order of PSfF127>P123>L121 HFMs. The three modified membrane all showed significant resistance to adsorption of acid/neutral drugs. More importantly, the adsorption of base drugs were largely reduced to an average value of 11% on the L121 HFM. The improved resistance to drug adsorption could be attributed to the synergy of hydrophobic/neutrally charged PPO and hydrophilic PEO. The L121 HFM was further assessed by evaluating the drug hepatotoxicity in 3D culture of hepatocytes. The base drugs, clozapine and doxorubicin, showed more sensitive hepatotoxicity on hepatocytes in L121 HFM than in PSf HFM, while the acid drug, salicylic acid, showed the similar hepatotoxicity to hepatocytes in both HFMs. Our finding suggests that PSf HFM modified by PEO-b-PPO-b-PEO copolymers can efficiently resist the drug adsorption onto polymer membrane, and consequently improve the accuracy and sensitivity of in vitro hepatotoxic drug screening.

  20. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    PubMed

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-05

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune

  1. Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization.

    PubMed

    Park, Joon-Hong; Na, Hee-Jun; Kwon, Young-Guen; Ha, Kwon-Soo; Lee, Seon-Jin; Kim, Chun-Ki; Lee, Kwang-Soon; Yoneyama, Toshie; Hatakeyama, Kazuyuki; Kim, Peter K M; Billiar, Timothy R; Kim, Young-Myeong

    2002-12-06

    Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.

  2. [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

  3. Cell sources for in vitro human liver cell culture models.

    PubMed

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

  4. Cell sources for in vitro human liver cell culture models

    PubMed Central

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

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

    PubMed Central

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

    2015-01-01

    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

  6. Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice

    PubMed Central

    Takahara, Ikuko; Akazawa, Yuko; Tabuchi, Maiko; Matsuda, Katsuya; Miyaaki, Hisamitsu; Kido, Youko; Kanda, Yasuko; Taura, Naota; Ohnita, Ken; Takeshima, Fuminao; Sakai, Yusuke; Eguchi, Susumu; Nakashima, Masahiro; Nakao, Kazuhiko

    2017-01-01

    Background and aims A high serum level of saturated free fatty acids (FFAs) is associated with the development of nonalcoholic fatty liver disease (NAFLD). X-box binding protein-1 (XBP-1) is activated by FFA treatment upon splicing. XBP-1 is a transcription factor induced by the endoplasmic reticulum (ER) stress sensor endoribonuclease inositol-requiring enzyme 1 alpha (IRE1α). However, the role of XBP-1 in NAFLD remains relatively unexplored. Toyocamycin was recently reported to attenuate the activation of XBP-1, possibly by inducing a conformational change in IRE1α. In this study, we examined the effect of toyocamycin on hepatocyte lipoapoptosis and steatosis. We also explored the effects of toyocamycin in a mouse model of NAFLD. Methods Huh-7 cells and isolated rat primary hepatocytes were treated with palmitic acid (PA), which is a saturated FFA, in the presence or absence of toyocamycin. In addition, male C57BL/6J mice were fed a diet rich in saturated fat, fructose, and cholesterol (FFC) for 4 months, after which the effect of toyocamycin was assessed. Results Toyocamycin attenuated FFA-induced steatosis. It also significantly reduced PA-induced hepatocyte lipoapoptosis. In addition, toyocamycin reduced the expression of cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein (CHOP), which is a key player in ER stress-mediated apoptosis, as well as its downstream cell death modulator, death receptor 5. In the in vivo study, toyocamycin ameliorated the liver injury caused by FFC-induced NAFLD. It also reduced hepatic steatosis and the expression of lipogenic genes. Conclusions The data we obtained suggest that toyocamycin attenuates hepatocyte lipogenesis and ameliorates NAFLD in vivo and may therefore be beneficial in the treatment of NAFLD in humans. PMID:28278289

  7. 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.

  8. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  9. Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity

    SciTech Connect

    Kiang, Tony K.L.; Teng Xiaowei; Surendradoss, Jayakumar; Karagiozov, Stoyan; Abbott, Frank S.; Chang, Thomas K.H.

    2011-05-01

    The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.

  10. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    PubMed

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  11. Changes in levels of argininosuccinate lyase mRNA during induction by glucagon and cyclic AMP in cultured foetal-rat hepatocytes.

    PubMed Central

    Renouf, S; Buquet, C; Fairand, A; Benamar, M; Husson, A

    1993-01-01

    During the perinatal period, the activity of the urea-cycle enzyme argininosuccinate lyase (ASL) is regulated by glucocorticoids, glucagon and insulin. In this study, the effects of glucagon and cyclic AMP (cAMP) analogues were examined on the synthesis of ASL and on the level of its corresponding mRNA in cultured foetal hepatocytes. Northern-blot analysis revealed that these agents only gave a transient induction of ASL mRNA amount, which reached a peak at 6 h and declined thereafter. This induction preceded the increase in enzyme activity and amount which could be observed for 2 or 3 days of culture. Stimulation of ASL mRNA accumulation by a combination of cAMP analogues and dexamethasone was additive, indicating that glucocorticoids and cAMP are both necessary to promote hepatocyte differentiation and that inductions could occur via independent pathways. Induction by cAMP analogues could be abolished by actinomycin D, suggesting a control mechanism at the transcriptional level. Puromycin was without effect on ASL mRNA induction by cAMP, indicating that no ongoing protein synthesis was required in the stimulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8387274

  12. Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo

    PubMed Central

    Schrader, Charlotte; Ernst, Insa M A; Sinnecker, Heike; Soukup, Sebastian T; Kulling, Sabine E; Rimbach, Gerald

    2012-01-01

    A number of cardioprotective effects, including the reduced oxidation of the low-density lipoprotein (LDL) particles, have been attributed to dietary soy isoflavones. Paraoxonase 1 (PON1), an enzyme mainly synthesized in the liver, may exhibit anti-atherogenic activity by protecting LDL from oxidation. Thus, dietary and pharmacological inducers of PON1 may decrease cardiovascular disease risk. Using a luciferase reporter gene assay we screened different flavonoids for their ability to induce PON1 in Huh7 hepatocytes in culture. Genistein was the most potent flavonoid with regard to its PON1-inducing activity, followed by daidzein, luteolin, isorhamnetin and quercetin. Other flavonoids such as naringenin, cyanidin, malvidin and catechin showed only little or no PON1-inducing activity. Genistein-mediated PON1 transactivation was partly inhibited by the oestrogen-receptor antagonist fulvestrant as well as by the aryl hydrocarbon receptor antagonist 7-ketocholesterol. In contrast to genistein, the conjugated genistein metabolites genistein-7-glucuronide, genistein-7-sulfate and genistein-7,4′-disulfate were only weak inducers of PON1 transactivation. Accordingly, dietary genistein supplementation (2 g/kg diet over three weeks) in growing rats did not increase hepatic PON1 mRNA and protein levels as well as plasma PON1 activity. Thus, genistein may be a PON1 inducer in cultured hepatocytes in vitro, but not in rats in vivo. PMID:22304296

  13. Environmental carcinogens in human target tissues in culture. Progress report

    SciTech Connect

    Hsu, I.C.

    1984-10-19

    The metabolism and mutagenicity of 1-nitropyrene, 1-aminopyrene, 2-aminofluorene and acetylaminofluarene in hepatic cell cultures is investigated. Metabolic products were assayed by thin-layer chromatography. Ancillary studies on Ames' assary mutagencity screening, unscheduled DNA synthesis by hepatocytes and binding to nuclear structures are also reported. 1 fig., 1 tab.

  14. Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes.

    PubMed

    Srovnalova, Alzbeta; Svecarova, Michaela; Zapletalova, Michaela Kopecna; Anzenbacher, Pavel; Bachleda, Petr; Anzenbacherova, Eva; Dvorak, Zdenek

    2014-01-22

    Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 μM with IC50 = 32 μM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 μM with IC50 = 44 μM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected.

  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. 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

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

    PubMed Central

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

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated 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 adducts formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves formation of mitochondrial protein adducts and mitochondrial dysfunction. PMID:26431796

  18. Metabolic hormones regulate basal and growth hormone-dependent igf2 mRNA level in primary cultured coho salmon hepatocytes: effects of insulin, glucagon, dexamethasone, and triiodothyronine.

    PubMed

    Pierce, A L; Dickey, J T; Felli, L; Swanson, P; Dickhoff, W W

    2010-03-01

    Igf1 and Igf2 stimulate growth and development of vertebrates. Circulating Igfs are produced by the liver. In mammals, Igf1 mediates the postnatal growth-promoting effects of growth hormone (Gh), whereas Igf2 stimulates fetal and placental growth. Hepatic Igf2 production is not regulated by Gh in mammals. Little is known about the regulation of hepatic Igf2 production in nonmammalian vertebrates. We examined the regulation of igf2 mRNA level by metabolic hormones in primary cultured coho salmon hepatocytes. Gh, insulin, the glucocorticoid agonist dexamethasone (Dex), and glucagon increased igf2 mRNA levels, whereas triiodothyronine (T(3)) decreased igf2 mRNA levels. Gh stimulated igf2 mRNA at physiological concentrations (0.25x10(-9) M and above). Insulin strongly enhanced Gh stimulation of igf2 at low physiological concentrations (10(-11) M and above), and increased basal igf2 (10(-8) M and above). Dex stimulated basal igf2 at concentrations comparable to those of stressed circulating cortisol (10(-8) M and above). Glucagon stimulated basal and Gh-stimulated igf2 at supraphysiological concentrations (10(-7) M and above), whereas T(3) suppressed basal and Gh-stimulated igf2 at the single concentration tested (10(-7) M). These results show that igf2 mRNA level is highly regulated in salmon hepatocytes, suggesting that liver-derived Igf2 plays a significant role in salmon growth physiology. The synergistic regulation of igf2 by insulin and Gh in salmon hepatocytes is similar to the regulation of hepatic Igf1 production in mammals.

  19. Culture Representation in Human Reliability Analysis

    SciTech Connect

    David Gertman; Julie Marble; Steven Novack

    2006-12-01

    Understanding human-system response is critical to being able to plan and predict mission success in the modern battlespace. Commonly, human reliability analysis has been used to predict failures of human performance in complex, critical systems. However, most human reliability methods fail to take culture into account. This paper takes an easily understood state of the art human reliability analysis method and extends that method to account for the influence of culture, including acceptance of new technology, upon performance. The cultural parameters used to modify the human reliability analysis were determined from two standard industry approaches to cultural assessment: Hofstede’s (1991) cultural factors and Davis’ (1989) technology acceptance model (TAM). The result is called the Culture Adjustment Method (CAM). An example is presented that (1) reviews human reliability assessment with and without cultural attributes for a Supervisory Control and Data Acquisition (SCADA) system attack, (2) demonstrates how country specific information can be used to increase the realism of HRA modeling, and (3) discusses the differences in human error probability estimates arising from cultural differences.

  20. Visual Culture, Art History and the Humanities

    ERIC Educational Resources Information Center

    Castaneda, Ivan

    2009-01-01

    This essay will discuss the need for the humanities to address visual culture studies as part of its interdisciplinary mission in today's university. Although mostly unnoticed in recent debates in the humanities over historical and theoretical frameworks, the relatively new field of visual culture has emerged as a corrective to a growing…

  1. Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

    PubMed

    Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

    2017-03-15

    Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.

  2. Human nature, cultural diversity and evolutionary theory.

    PubMed

    Plotkin, Henry

    2011-02-12

    Incorporating culture into an expanded theory of evolution will provide the foundation for a universal account of human diversity. Two requirements must be met. The first is to see learning as an extension of the processes of evolution. The second is to understand that there are specific components of human culture, viz. higher order knowledge structures and social constructions, which give rise to culture as invented knowledge. These components, which are products of psychological processes and mechanisms, make human culture different from the forms of shared knowledge observed in other species. One serious difficulty for such an expanded theory is that social constructions may not add to the fitness of all humans exposed to them. This may be because human culture has existed for only a relatively short time in evolutionary terms. Or it may be that, as some maintain, adaptation is a limited, even a flawed, aspect of evolutionary theory.

  3. Human nature, cultural diversity and evolutionary theory

    PubMed Central

    Plotkin, Henry

    2011-01-01

    Incorporating culture into an expanded theory of evolution will provide the foundation for a universal account of human diversity. Two requirements must be met. The first is to see learning as an extension of the processes of evolution. The second is to understand that there are specific components of human culture, viz. higher order knowledge structures and social constructions, which give rise to culture as invented knowledge. These components, which are products of psychological processes and mechanisms, make human culture different from the forms of shared knowledge observed in other species. One serious difficulty for such an expanded theory is that social constructions may not add to the fitness of all humans exposed to them. This may be because human culture has existed for only a relatively short time in evolutionary terms. Or it may be that, as some maintain, adaptation is a limited, even a flawed, aspect of evolutionary theory. PMID:21199849

  4. HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes

    PubMed Central

    Zhu, Zhaowen; Tran, Huy; Mathahs, M. Meleah; Moninger, Thomas O.; Schmidt, Warren N.

    2017-01-01

    Introduction Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. Results Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. Conclusions HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT

  5. 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

  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. Control of glycolysis in cultured chick embryo hepatocytes. Fructose 2,6-bisphosphate content and phosphofructokinase-1 activity are stimulated by insulin and epidermal growth factor.

    PubMed Central

    Hamer, M J; Dickson, A J

    1990-01-01

    Chick embryo hepatocytes were maintained in monolayer culture in a serum-free chemically defined medium for periods of up to 2 days. Over this time period, insulin provoked selective increases (up to 5-fold) in factors relevant to the control of glycolysis: the activities of phosphofructokinase-1 (PFK-1), phosphofructokinase-2 (PFK-2) and hexokinase isoenzymes and the content of fructose 2,6-bisphosphate (F26BP). Half-maximal effects of insulin on pFK-1 activity were in the physiological range (0.1 nM). Changes in enzyme activities and F26BP content in response to insulin were correlated with stimulation of glycolytic flux as estimated by radioisotopic flux. These data are discussed in relation to known changes which occur in hepatic glycolytic activity and PFK-1 activity in the intact chick around hatching. The effects of insulin on F26BP content, PFK-1 activity and glycolytic flux were mimicked by epidermal growth factor (EGF). In contrast, phorbol esters produced minimal actions on any of the above parameters. Our data indicate that protein kinase C is not involved in the actions of insulin or EGF in control of F26BP content or PFK-1 activity. This work indicates that the related tyrosyl kinase receptors of insulin and EGF may provoke identical responses within hepatocytes, but through the utilization of different transduction systems which merge to common control points. Images Fig. 1 PMID:2143894

  8. Involvement of leukocyte function-associated antigen-1 (LFA-1) in the invasion of hepatocyte cultures by lymphoma and T-cell hybridoma cells

    PubMed Central

    1987-01-01

    We studied the interaction of MB6A lymphoma and TAM2D2 T cell hybridoma cells with hepatocyte cultures as an in vitro model for in vivo liver invasion by these tumor cells. A monoclonal antibody against leukocyte function-associated antigen-1 (LFA-1) inhibited adhesion of the tumor cells to the surface of hepatocytes and consequently strongly reduced invasion. This effect was specific since control antibodies, directed against Thy.1 and against T200, of the same isotype, similar affinity, and comparable binding to these cells, did not inhibit adhesion. This suggests that LFA-1 is involved in the formation of liver metastases by lymphoma cells. TAM2D2 T cell hybridoma cells were agglutinated by anti- LFA-1, but not by control antibodies. Reduction of adhesion was not due to this agglutination since monovalent Fab fragments inhibited adhesion as well, inhibition was also seen under conditions where agglutination was minimal, and anti-LFA-1 similarly affected adhesion of MB6A lymphoma cells that were not agglutinated. The two cell types differed in LFA-1 surface density. TAM2D2 cells exhibited 400,000 surface LFA-1 molecules, 10 times more than MB6A cells. Nevertheless, the level of adhesion and the extent of inhibition by the anti-LFA-1 antibody were only slightly larger for the TAM2D2 cells. PMID:3301869

  9. Nimesulide-induced electrophile stress activates Nrf2 in human hepatocytes and mice but is not sufficient to induce hepatotoxicity in Nrf2-deficient mice.

    PubMed

    Kale, Vijay M; Hsiao, Chin-ju J; Boelsterli, Urs A

    2010-05-17

    Nimesulide is a widely prescribed nitroaromatic sulfoanilide-type nonsteroidal anti-inflammatory drug that, despite its favorable safety profile, has been associated with rare cases of idiosyncratic drug-induced liver injury (DILI). Because reactive metabolites have been implicated in DILI, we aimed at investigating whether hepatic bioactivation of nimesulide produces a protein-reactive intermediate in hepatocytes. Also, we explored whether nimesulide can activate the transcription factor Nrf2 that would protect from drug-induced hepatocyte injury. We found that [(14)C]-nimesulide covalently bound to human liver microsomes (<50 pmol/mg under standard conditions) or immortalized human hepatocytes in a sulfaphenazole-sensitive, rifampicin-inducible manner; yet the overall extent of binding was modest. Although exposure of hepatocytes to nimesulide was not associated with increased net levels of superoxide anion, nimesulide (100 microM, 24 h) caused nuclear translocation of Nrf2 in a sulfaphenazole-sensitive manner, indicating a role of electrophilic metabolites. However, knockdown of Nrf2 with siRNA did not make the cells more sensitive to nimesulide-induced cell injury. Similarly, exposure of wild-type C57BL/6x129 Sv mice to nimesulide (100 mg/kg/day, po, for 5 days) was associated with nuclear translocation of immunoreactive Nrf2 in a small number of hepatocytes and induced >2-fold the expression levels of the Nrf2-target gene Nqo1 in wild-type but not Nrf2-null mice. Nimesulide administered to Nrf2(-/-) knockout mice did not cause increases in serum ALT activity or any apparent histopathological signs of liver injury. In conclusion, these data indicate that nimesulide is bioactivated by CYP2C to a protein-reactive electrophilic intermediate that activates the Nrf2 pathway even at nontoxic exposure levels.

  10. 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

  11. 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.

  12. 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.

  13. Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug Candidates.

    PubMed

    Lauschke, Volker M; Hendriks, Delilah F G; Bell, Catherine C; Andersson, Tommy B; Ingelman-Sundberg, Magnus

    2016-12-19

    The liver is an organ with critical importance for drug treatment as the disposition and response to a given drug is often determined by its hepatic metabolism. Patient-specific factors can entail increased susceptibility to drug-induced liver injury, which constitutes a major risk for drug development programs causing attrition of promising drug candidates or costly withdrawals in postmarketing stages. Hitherto, mainly animal studies and 2D hepatocyte systems have been used for the examination of human drug metabolism and toxicity. Yet, these models are far from satisfactory due to extensive species differences and because hepatocytes in 2D cultures rapidly dedifferentiate resulting in the loss of their hepatic phenotype and functionality. With the increasing comprehension that 3D cell culture systems more accurately reflect in vivo physiology, in the recent decade more and more research has focused on the development and optimization of various 3D culture strategies in an attempt to preserve liver properties in vitro. In this contribution, we critically review these developments, which have resulted in an arsenal of different static and perfused 3D models. These systems include sandwich-cultured hepatocytes, spheroid culture platforms, and various microfluidic liver or multiorgan biochips. Importantly, in many of these models hepatocytes maintain their phenotype for prolonged times, which allows probing the potential of newly developed chemical entities to cause chronic hepatotoxicity. Moreover, some platforms permit the investigation of drug action in specific genetic backgrounds or diseased hepatocytes, thereby significantly expanding the repertoire of tools to detect drug-induced liver injuries. It is concluded that the development of 3D liver models has hitherto been fruitful and that systems are now at hand whose sensitivity and specificity in detecting hepatotoxicity are superior to those of classical 2D culture systems. For the future, we highlight the