Construction and evaluation of drug-metabolizing cell line for bioartificial liver support system

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.     

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.     

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.     

Induction of zone-like liver function gradients in HepG2 cells by varying culture medium height

In most laboratory-scale mammalian cell cultures, the primary mode of oxygen delivery to cultured cells is by passive diffusion through a thin layer of culture medium, and the height of culture medium chosen may therefore have a significant effect on the phenotype of oxygen-sensitive cell types. Many of the liver functions performed by hepatocytes are thought to be regulated into zones by the local oxygen concentration; of particular interest to in vitro toxicologists, the cytochrome P450 family of detoxification enzymes is known to be preferentially expressed by hepatocytes at low (perivenous) oxygen concentrations. Using an array of different medium heights in a 12-well plate format, we show that the height of culture medium has a significant effect on cytochrome P450 1A1 detoxification activity, glucose metabolism, and cell morphology of HepG2 hepatocellular carcinoma cultures. In particular, cytochrome P450 activity exhibits a maximum at medium heights corresponding to perivenous oxygen concentrations. This work demonstrates that optimizing cell culture performance is not always the same as maximizing oxygen delivery.     

A transcriptomic study suggesting human iPSC-derived hepatocytes potentially offer a better in vitro model of hepatotoxicity than most hepatoma cell lines

Hepatocytes derived from human induced pluripotent stem cells (iPSCs) hold great promise as an in vitro liver model by virtue of their unlimited long-term supply, stability and consistency in functionality, and affordability of donor diversity. However, the suitability of iPSC-derived hepatocytes (iPSC-Heps) for toxicology studies has not been fully validated. In the current study, we characterized global gene expression profiles of iPSC-Heps in comparison to those of primary human hepatocytes (PHHs) and several human hepatoma cell lines (HepaRG, HuH-7, HepG2, and HepG2/C3A). Furthermore, genes associated with hepatotoxicity, drug-metabolizing enzymes, transporters, and nuclear receptors were extracted for more detailed comparisons. Our results showed that iPSC-Heps correlate more closely to PHHs than hepatoma cell lines, suggesting that iPSC-Heps had a relatively mature hepatic phenotype that more closely resembles that of adult hepatocytes. HepaRG was the sole exception but nonetheless suffers from lack of donor diversity and poor prediction of hepatotoxicity. The effects of sex differences and DMSO treatment on gene expression of the cellular models were also investigated. Overall, the results presented in the current study suggest that iPSC-Heps represent a reproducible source of human hepatocytes and a promising in vitro model for hepatotoxicity evaluation. Further studies are needed to develop a robust protocol for hepatocyte differentiation towards a more mature adult phenotype.     

Differential sensitivity of human hepatoma cell line and primary rat hepatocyte culture to benzo(a)pyrene-induced unscheduled DNA synthesis and adduct formation.

We studied the genotoxic potential of a carcinogen in the human hepatoma cell line, HepG2 and in primary rat hepatocyte culture. HepG2 is a well differentiated human hepatoblastoma cell line with biotransforming capacity. Rat hepatocytes were obtained by the standard two-step in situ perfusion technique. Following benzo(a)pyrene treatment, both HepG2 and primary rat hepatocyte culture showed unscheduled DNA synthesis with different sensitivity. In ³²P-postlabelling analysis, the chromatogram revealed quantitative and qualitative differences between HepG2 and primary rat hepatocyte cultures when treated with 10 μM benzo(a)pyrene for 18 hr. The results have demonstrated that the HepG2 cell line may be used in addition to primary rat hepatocytes in risk assessment for detection of environmental carcinogens.     

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.     

Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such, as β-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype is present. This study was supported by grants to B. Christ from the German Ministry of Education and Research (01 ZZ 0109 and NBL3-NG4) as well as by grants from the Federal State of Saxonia-Anhalt through the Wilhelm-Roux-Program at the Medical Faculty of the Martin-Luther-University of Halle-Wittenberg to B. Christ (09/07 and 04/03).     

A fluorescent cell-based assay for cytochrome P-450 isozyme 1A2 induction and inhibition

High throughput lead optimization requires simple, homogeneous cell-based assays capable of defining the druglike properties of first-round screening hits. Induction and inhibition of the Phase I drug-metabolizing enzymes are central to this process. We report here an assay for induction and inhibition of cytochrome P-450 (CYP) isozyme 1A2 that meets these requirements. It utilizes HepG2/C3A, a human liver cell line, and ethoxyresorufin. Using methylcholanthrene, CYP1A2 can be induced dramatically, and it is inhibited by furafylline, a mechanism-based inhibitor of this enzyme.     

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Co-cultures of hepatocytes with epithelial-like cell lines: Expression of drug-biotransformation activities by hepatocytes

To improve long-term expression of drug biotransformation activities in hepatocytes, we have examined the suitability of several epithelial-like cell lines (MDCK, MS and L-132) for supporting functional co-cultures with rat hepatocytes. Cells were selected on the basis of their compatibility with hepatocytes, formation of stable monolayers in the absence of serum and lack of drug biotransformation activities. The expression of individual elements of the biotransformation system was evaluated in these co-cultures. Co-cultured hepatocytes remained viable and showed a characteristic polygonal shape for more than a week. Depending on the cell line used, levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities of co-cultured hepatocytes oscillated between 24–47% of their initial value after 4 days in culture. The highest levels of monooxygenase activity were found in hepatocytes co-cultured with MS cells (41–47%). In contrast, these activities decreased to 6% when hepatocytes were maintained in pure culture for the same period. The activities of the conjugating enzymes UDP-glucuronyltransferase and glutathione S-transferase were maintained at nearly the initial levels during the complete period of study, both in pure and mixed-cultures, regardless of the cell line used. MS cells adapted themselves much better to serum-free culture conditions, and the co-culture with rat hepatocyte was technically easier. After one week, total cytochrome P450 and reduced glutathione in rat hepatocytes/MS co-cultures were 31% and 127% respectively of the day O values, whereas they were undetectable in pure culture. A clear induction of monooxygenase activities by methylcholanthrene, phenobarbital and ethanol could be observed by the 5th day in MS cells/hepatocyte co-cultures. The fact that the results of our work show the suitability of MS cells, an epithelial-derived cell line, for improving the expression of biotransformation enzymes of cultured hepatocytes opens new possibilities of simplifying co-cultures for their use in drug-metabolism studies.Abbreviations AHH aryl hydrocarbon hydroxylase - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle's medium - ECOD 7-ethoxycoumarin O-deethylase - EDTA ethylenediamine tetraacetic acid - Et-OH ethanol - GSH reduced glutathione - GSH-t glutathione S-transferase - MC 3-methylcholanthrene - PB phenobarbital - UDP-Gt UDP-glucuronyltransferase     

The use of primary hepatocyte cultures for the evaluation of chemoprotective agents

Primary cultures of human and rat hepatocytes are widely used in pharmacotoxicological research. This model presents the advantages of retaining liver function for at least a few days, expressing both phase 1 and phase 2 enzymes, and responding to inducers. Recently we made use of primary hepatocytes to investigate the effects of chemoprotective agents on drug-metabolizing enzyme expression and activities. The treatment of rat and human hepatocytes with two chemoprotective agents, oltipraz (a synthetic derivative of 1,2-dithiole-3-thione) and sulforaphane (an isothiocyanate found in broccoli), clearly demonstrated that both of these compounds are inducers of glutathione transferases and transient inhibitors of cytochrome P450, suggesting that these two compounds could exert their chemoprotective effects by both reducing the formation of reactive metabolites of chemicals and enhancing their inactivation.