Induction of Drug-Metabolizing Enzymes by Phenobarbital in Layered Co-culture of a Human Liver Cell Line and Endothelial Cells
Advanced Medical Materials Group, Biomaterials Center, National Institute for Materials Science, MANA, Tsukuba, Ibaraki, Japan. Biological & Pharmaceutical Bulletin
(Impact Factor: 1.83).
06/2009; 32(5):813-7. DOI: 10.1248/bpb.32.813
Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various CYP genes were significantly increased compared with the monolayer cultured HepG2 cells. Here, we examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Moreover, the induction of hepatic drug transporters by phenobarbital was detected. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism.
Available from: Alexander S. Mosig
- "Co-culture approaches with NPCs have been shown to prevent hepatocyte dedifferentiation. Hepatocytes show improved urea production and a stable up-regulation of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 during long-term co-culture with ECs   . Similarly, co-culture of hepatocytes and HSCs was reported to increase albumin secretion and CYP2B1/2 expression . "
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ABSTRACT: Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.
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Biomaterials 08/2015; Biomaterials(71):119-131. DOI:10.1016/j.biomaterials.2015.08.043 · 8.56 Impact Factor
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ABSTRACT: We have developed two cell culture systems for use in pharmaceutical research using nano-biotechnology. First, we developed a double layered co-culture system using cell sheet technology, and showed that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various cytochrome P450 (CYP) genes were significantly increased compared to monolayer cultured HepG2 cells. In the layered HepG2 co-culture, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Next, we developed sensor cells. Living cells maintain homeostasis by responding quickly and with great sensitivity to changes in the external environment. Consequently, sensors using cells as active elements are thought to be able to perform analyses faster and with more sensitivity than previous methods. We have modified mammalian cells using genetic engineering techniques to develop next-generation cell sensors that can visually represent specific reactions. We successfully produced devices using sensor cells that can process a variety of specimens using Micro-Electro-Mechanical System (MEMS), Nano-Electro-Mechanical System (NEMS), and other nano/micro processing technologies. These systems may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of metabolism and cytotoxicity. In this review, we introduce our research and describe recent trends in this field.
Yakugaku zasshi journal of the Pharmaceutical Society of Japan 04/2010; 130(4):529-35. DOI:10.1248/yakushi.130.529 · 0.26 Impact Factor
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ABSTRACT: The efficacy of drugs acting within lymphocytes depends on their intracellular concentrations, which could be modulated by membrane efflux transporters including P-glycoprotein (P-gp), encoded by the MDR1 gene. In particular, P-gp induction may compromise the efficacy of its substrates. Rifampicin and phenobarbital have been shown to induce P-gp in hepatic and intestinal cells through the activation of the nuclear receptors PXR and CAR. However, controversial data exist in human lymphocytes. We investigated the effect of these drugs on P-gp activity and expression in lymphocytes in vitro and ex vivo. CCRF-CEM cells and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were incubated in the presence of rifampicin, phenobarbital, or without any drug. P-gp activity was measured by flow cytometry using DiOC(6) efflux. MDR1, PXR and CAR mRNA expression were measured by quantitative RT-PCR. Neither P-gp activity nor MDR1 mRNA expression were modified by rifampicin or phenobarbital both in CCRF-CEM cells and PBMCs. Moreover, P-gp protein expression at the membrane was neither detectable nor induced. The very weak PXR and CAR mRNA expression levels in these cells could partly explain these results. Therefore, P-gp induction by rifampicin and phenobarbital may play a negligible role in drug interactions occurring within lymphocytes.
International Journal of Pharmaceutics 08/2010; 395(1-2):98-103. DOI:10.1016/j.ijpharm.2010.05.016 · 3.65 Impact Factor
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