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114. Human Hepatocyte Culture.

Author

Phillips, Ian R., Shephard, Elizabeth A., Pichard, Lydiane, Raulet, Edith, Fabre, Gérard, Ferrini, Jean Bernard, Ourlin, Jean-Claude, and Maurel, Patrick

Abstract

Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocytes from the adult rat, this technique has been adapted to the ex vivo treatment of human liver from organ donors or from lobectomy resection for medical purposes. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short- and long-term cultures in which cells retain a differentiated phenotype for at least 1 mo. The various aspects emphasized here include the conditions for obtaining tissue, quality control of tissue for efficient perfusion, collagenase perfusion parameters, solutions for perfusion and culture media, cell substrate, cell plating, specific equipment, and safety conditions. [ABSTRACT FROM AUTHOR]

Published
2006
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116. The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants

Author

Zhang, Jiong, primary, Kuehl, Peter, additional, Green, Eric D., additional, Touchman, Jeffery W., additional, Watkins, Paul B., additional, Daly, Ann, additional, Hall, Stephen D., additional, Maurel, Patrick, additional, Relling, Mary, additional, Brimer, Cynthia, additional, Yasuda, Kazuto, additional, Wrighton, Steven A., additional, Hancock, Michael, additional, Kim, Richard B., additional, Strom, Stephen, additional, Thummel, Kenneth, additional, Russell, Christopher G., additional, Hudson, James R., additional, Schuetz, Erin G., additional, and Boguski, Mark S., additional

Published
2001
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117. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression

Author

Kuehl, Peter, primary, Zhang, Jiong, additional, Lin, Yvonne, additional, Lamba, Jatinder, additional, Assem, Mahfoud, additional, Schuetz, John, additional, Watkins, Paul B., additional, Daly, Ann, additional, Wrighton, Steven A., additional, Hall, Stephen D., additional, Maurel, Patrick, additional, Relling, Mary, additional, Brimer, Cynthia, additional, Yasuda, Kazuto, additional, Venkataramanan, Raman, additional, Strom, Stephen, additional, Thummel, Kenneth, additional, Boguski, Mark S., additional, and Schuetz, Erin, additional

Published
2001
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134. FK 506 renal toxicity and lack of detectable cytochrome P-450 3A in the liver graft of a patient undergoing liver transplantation

Author

Lemoine, Antoinette, primary, Azoulay, Daniel, additional, Dennison, Ashley, additional, Kiffel, Liliane, additional, Pichard, Lydiane, additional, Furlan, Valérie, additional, Bienvenu, Thierry, additional, Fredj, Gilles, additional, Debuire, Brigitte, additional, Maurel, Patrick, additional, Bismuth, Henri, additional, and Beaune, Philippe, additional

Published
1994
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141. The mycotoxin, patulin, increases the expression of PXR and AhR and their target cytochrome P450s in primary cultured human hepatocytes.

Author

Ayed-Boussema, Imen, Pascussi, Jean Marc, Rjiba, Karima, Maurel, Patrick, Bacha, Hassen, and Hassen, Wafa

Subjects
MYCOTOXINS, PATULIN, GENE expression, CYTOCHROME P-450, LIVER cells, MESSENGER RNA, GENETIC toxicology
Abstract

The mycotoxin, patulin (PAT), which is frequently found in apples, grapes, oranges, pear, peaches, and in apple juices, has previously been shown to be cytotoxic, genotoxic, and mutagenic. In this study, we have investigated the effect of PAT on mRNA level of pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and their corresponding target cytochrome P450s. Using primary cultures of adult human hepatocytes, we evaluated PAT cytotoxicity on hepatocytes after 24 hours of treatment. Real time reverse-transcriptase polymerase chain reaction procedure was employed to determine the effect of PAT on receptors (PXR, CAR, and AhR) and cytochrome (CYP3A4, 2B6, 3A5, 2C9, 1A1, and 1A2) genes. Our results showed that PAT reduced hepatocyte viability. At a noncytotoxic range of PAT concentrations, PAT induced an upregulation of the PXR gene in the three treated hepatocytes cultures, whereas CAR was overexpressed in only 1 treated liver. PXR gene induction was accompanied by the enhancement of CYP2B6, 3A5, 2C9, and 3A4 expression. PAT was also found to induce an overexpression of AhR and CYP1A1 and CYP1A2 mRNA expression. These findings suggested that PAT may activate PXR and/or CAR and AhR. However, further investigations are needed to confirm nuclear receptor activation by PAT and to elucidate the molecular mechanism of PAT action. [ABSTRACT FROM AUTHOR]

Published
2012
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142. Ochratoxin A induces CYP3A4, 2B6, 3A5, 2C9, 1A1, and CYP1A2 gene expression in primary cultured human hepatocytes: a possible activation of nuclear receptors.

Author

Ayed-Boussema, Imen, Pascussi, Jean Marc, Zaied, Chiraz, Maurel, Patrick, Bacha, Hassen, and Hassen, Wafa

Subjects
OCHRATOXINS, PENICILLIUM, ASPERGILLUS, IN vitro studies, LIVER cells, LABORATORY rats
Abstract

Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic, and immunotoxic to several species of animals and to cause kidney and liver tumors in mice and rats. Biotransformation of OTA has not been entirely elucidated. Several metabolites have been characterized in vitro and/or in vivo, whereas other metabolites remain to be characterized. At present, data available regarding OTA metabolism and cytochrome inductions concern only rodents or in vitro systems. The aim of the present study was to explore the effect of OTA on mRNA expression of some cytochromes known to be regulated by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR), using primary cultures of human hepatocytes. Our results showed that OTA reduced hepatocyte viability in a dose-dependent manner. Using quantitative real-time reverse-transcription polymerase chain reaction, our study showed that treatment of primary cultured human hepatocytes with noncytotoxic increasing concentrations of OTA for 24 hours caused a significant upregulation of CYP3A4, CYP2B6, and, to a lesser extent, CYP3A5 and CYP2C9. PXR mRNA expression increased in only 1 treated liver, whereas CAR mRNA expression was not affected. OTA was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that OTA could activate PXR and AhR; however, further investigations are needed to confirm nuclear-receptor activation by OTA. [ABSTRACT FROM AUTHOR]

Published
2012
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143. Effect of Aflatoxin B1 on Nuclear Receptors PXR, CAR, and AhR and Their Target Cytochromes P450 mRNA Expression in Primary Cultures of Human Hepatocytes.

Author

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

Subjects
AFLATOXINS, NUCLEAR receptors (Biochemistry), CYTOCHROME P-450, MYCOTOXINS, MESSENGER RNA, LIVER cells, GENE expression
Abstract

Aflatoxin B1 (AFB1), one of the most common mycotoxins found in human foods and animal feed, is principally hepatotoxic and hepatocarcinogenic. The aim of the present study was to explore the effect of AFB1 on messenger RNA (mRNA) expression of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and some of their target cytochromes using primary cultures of human hepatocytes. Our results showed that AFB1, at noncytotoxic increasing concentrations, caused a significant upregulation of cytochrome P 2B6 (CYP2B6), CYP3A5, and to a lesser extent CYP3A4 and CYP2C9. Pregnane X receptor and CAR mRNA expression increased in the 3 treated livers. Aflatoxin B1 was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that AFB1 could activate PXR, CAR, and AhR; however, further investigations are needed to confirm nuclear receptor activation by AFB1. [ABSTRACT FROM PUBLISHER]

Published
2012
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144. Lipid-Mediated Gene Transfer into Normal Adult Human Hepatocytes in Primary Culture.

Author

Walker, John M., Phillips, Ian R., Shephard, Elizabeth A., Ourlin, Jean-Claude, Vilarem, Marie José, Daujat, Martine, Harricane, Marie Cécile, and Maurel, Patrick

Abstract

Functional analysis of the 5′-flanking region of a gene has become a routme procedure for identifying the cis-acting DNA elements involved in the control of the transcriptional activity of the gene (1). This experimental approach requires the transfection of reporter plasmids, harboring various fragments of the 5′-flanking region of the gene, into appropriate cultured cells and the subsequent measurement of the activity of the reporter gene, either constitutively or in response to regulatory stimuli. Continuous cell lines derived from mahgnant tissues have been extensively used for this purpose because they can be easily cultured and transfected. However, these cell lines have two major drawbacks: they are dedifferentiated with respect to the normal tissue from which they originate; that is, the tissue-specific transcription factors are generally expressed at a low level, if at all; and their culture requires the use of serum, the chemical and hormonal composition of which is not fully defined. In fact, it would be preferable to transfect normal cells in a primary culture in which the phenotype of the tissue of origin is maintained. The problem with this approach is that the methods currently used to transfect cell lines, including electroporation and calcmm phosphate-or diethylaminoethyl (DEAE) dextran-DNA coprecipitation, are generally not convenient for primary cultures because of their toxicity (2-4). [ABSTRACT FROM AUTHOR]

Published
1998
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145. Human Hepatocyte Culture.

Author

Walker, John M., Phillips, Ian R., Shephard, Elizabeth A., Ferrini, Jean Bernard, Ourlin, Jean-Claude, Pichard, Lydiane, Fabre, Gérard, and Maurel, Patrick

Abstract

Primary culture of hepatocytes is an in vitro model widely used to investigate various aspects of liver physiology and pathology (1). In particular, such cultures have been extensively used for assessing the expression and function of drug-metabolizing enzymes including cytochromes P450 (CUP), drug metabolism, drug-drug interactions, and the mechanisms of cytotoxtcity and genotoxicity. Most of these studies have been carried out with rodent hepatocytes. However, because of species-specificity in both the regulation and activity of drug-metabolizing enzymes, extrapolation from animals to humans is not generally possible. For this reason, several groups have developed human hepatocyte culture systems (2-8). The technique used to isolate human hepatocytes is based on the two-step collagenase perfusion first mtroduced by Berry and Friend (9) and modified by Seglen (10). Originally performed in situ for obtaining hepatocytes from the adult rat, this technique of perfusion has been adapted to the ex vivo treatment of human liver tissue. The aim of this chapter is to describe the authors′ experience in the isolation of hepatocytes from human liver tissue and the preparation of short- and long-term cultures [ABSTRACT FROM AUTHOR]

Published
1998
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146. Induction, regulation and messenger half-life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes. CYP 1A1, 1A2 and 3A6 chromosome location in the rabbit and evidence that post-transcriptional control of gene IA2 does not involve mRNA stabilization

Author

DAUJAT, Martine, primary, CLAIR, Philippe, additional, ASTIER, Catherine, additional, FABRE, Isabelle, additional, PINEAU, Thierry, additional, YERLE, Martine, additional, GELLIN, Joel, additional, and MAUREL, Patrick, additional

Published
1991
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148. Constitutive androstane receptor–vitamin D receptor crosstalk: Consequence on CYP24 gene expression

Author

Moreau, Amélie, Maurel, Patrick, Vilarem, Marie-José, and Pascussi, Jean-Marc

Subjects
*CROSSTALK, *ELECTROACOUSTICS, *RADIO (Medium), *TELECOMMUNICATION
Abstract

Abstract: We previously reported that the pregnane X receptor (PXR) interferes with vitamin D receptor (VDR) target genes, notably CYP24, by targeting the same responsive elements. Since PXR and constitutive androstane receptor (CAR) share responsive elements in the promoter of their target genes, we wondered whether CAR also interferes with CYP24 expression. The current study shows that: (i) CAR-RXR heterodimer binds to and transactivates the proximal promoter of CYP24 (−1200/+22) and both VDRE-1 and VDRE-2 which control its expression in response to 1,25-dihydroxyvitamin D3, (ii) androstanol an inverse agonist of hCAR inhibits transactivation of VDREs by hCAR, (iii) mutations of either VDRE-1 or -2 half sites inhibit hCAR-mediated transactivation, and (iv) in primary human hepatocytes (n =11) CITCO, a specific hCAR agonist, is an inducer of CYP24 as well as of CYP2B6 and CYP3A4 mRNAs. In conclusion, CAR/PXR and VDR bind to and transactivate the same response elements in CYP24 promoter. [Copyright &y& Elsevier]

Published
2007
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