Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel

Goodwin, Bryan; Liddle, Christopher; Murray, Michael T.; Tapner, Michael; Rooney, T. P.; Farrell, Geoffrey C.

doi:10.1016/0006-2952(96)00179-7

Cited by 9 publications

(4 citation statements)

References 35 publications

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“…Efforts to improve the quality of hepatocyte cultures for drug metabolism studies have been performed by altering cell culture medium composition (69), using extracellular matrices (70)(71)(72), and utilizing cocultures (47,48) to optimize culture conditions. Although hepatocyte cultures allow reproduction of in vivo pattern of biotransforamtion of xenobiotics, it must be realized that these cultures cannot be used to predict in vivo clearance of drugs.…”

Section: In Vitro Hepatic Drug Metabolism and Toxicology Systemsmentioning

confidence: 99%

Predictive Value of in Vitro Model Systems in Toxicology

Dávila¹,

Rodriguez

Melchert

et al. 1998

Annu. Rev. Pharmacol. Toxicol.

120

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The application of in vitro model systems to evaluate the toxicity of xenobiotics has significantly enhanced our understanding of drug- and chemical-induced target toxicity. From a scientific perspective, there are several reasons for the popularity of in vitro model systems. From the public perspective, in vitro model systems enjoy increasing popularity because their application may allow a reduction in the number of live animals employed in toxicity testing. In this review, we present an overview of the use of in vitro model systems to investigate target organ toxicity of drugs and chemicals, and provide selective examples of these model systems to better understand cutaneous and ocular toxicity and the role of drug metabolism in the hepatotoxicity of selected agents. We conclude by examining the value and use of in vitro model systems in industrial development of new pharmaceutical agents.

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Section: In Vitro Hepatic Drug Metabolism and Toxicology Systemsmentioning

confidence: 99%

Predictive Value of in Vitro Model Systems in Toxicology

Dávila¹,

Rodriguez

Melchert

et al. 1998

Annu. Rev. Pharmacol. Toxicol.

120

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“…Blots were incubated in Cell Culture-All cell lines were cultured under standard conditions at 37°C, 5% CO 2 in a humidified incubator. Hepatocytes were isolated from 230 -270 g of anesthetized male Wistar rats by in situ two-stage collagenase perfusion of the liver, as described elsewhere (12). The liver was excised and dispersed in modified Waymouth medium containing sodium bicarbonate (24 mM), penicillin (100 units/ml), HEPES (18 mM), and insulin (25 milliunits/ml).…”

Section: Methodsmentioning

confidence: 99%

Soluble Low Density Lipoprotein Receptor-related Protein (LRP) Circulates in Human Plasma

Quinn¹,

Grimsley²,

Dai³

et al. 1997

Journal of Biological Chemistry

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120

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Our studies have identified a soluble molecule in normal human plasma and serum with the characteristics of the ␣-chain of the low density lipoprotein receptorrelated protein (LRP). LRP is a large multifunctional receptor mediating the clearance of diverse ligands, including selected lipoproteins, various protease inhibitor complexes, and thrombospondin. A soluble molecule (sLRP) has been isolated from plasma using an affinity matrix coupled with methylamine-activated ␣ 2 -macroglobulin, the ligand uniquely recognized by LRP, and eluted with EDTA. This eluate contains a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with authentic human placental LRP ␣-chain, is recognized by anti-LRP ␣-chain monoclonal antibodies, and binds the 39-kDa receptor-associated protein (RAP) and tissue plasminogen activator-inhibitor complexes. A similar RAP-binding molecule was detected in medium conditioned for 24 h by primary cultures of rat hepatocytes, suggesting that the liver may be the in vivo source of sLRP. In contrast, immunoprecipitation experiments failed to detect the production of sLRP by cultured HepG2 hepatoma and primary human fibroblast cells. Addition of a soluble form of LRP to cultured HepG2 cells resulted in a significant inhibition of capacity of these cells to degrade tPA, a process that has been demonstrated to be mediated by cell surface LRP. Preliminary data indicate that the concentration of sLRP is altered in the plasma of patients with liver disease. Increased levels of sLRP may antagonize the clearance of ligands by cell bound LRP perturbing diverse processes including lipid metabolism, cell migration and extracellular proteinase activity.The low density lipoprotein receptor-related protein (LRP) 1 has been previously identified as a membrane-bound endocytic receptor (1, 2). Studies have demonstrated that LRP mediates the internalization of multiple, structurally unrelated ligands, including selected lipoproteins, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (reviewed in Refs. 3 and 4). The binding of all ligands to LRP is inhibited by the receptor-associated protein (RAP), a protein that was copurified with LRP (2, 5). The range of ligands recognized by LRP suggests that it plays a role in diverse processes including lipid metabolism, cell growth, migration, and tissue invasion. LRP expression is widespread; however, it is most highly expressed in the liver, brain, and placenta. The remarkable degree of cross-species identity conserved in the LRP amino acid sequence (3) and the embryonic lethal phenotype obtained after targeted disruption of the LRP gene in the mouse (6) underscore the biological importance of this molecule.Here we report the identification of a soluble form of LRP circulating in human plasma. The characterization of this molecule, which maintains the ligand binding characteristics of cell surface LRP, introduces a new dimension to the biology of LRP. Accumulation of soluble LRP in plasma may antagonize the clearance of ligands by cell-...

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“…Microsomal proteins were quantitated by immunoblotting as previously described (26). The primary antibodies used were as follows.…”

Section: Immunoquantitation Of P450 Proteinsmentioning

confidence: 99%

Separate and Interactive Regulation of Cytochrome P450 3A4 by Triiodothyronine, Dexamethasone, and Growth Hormone in Cultured Hepatocytes¹

Liddle

Goodwin

George

et al. 1998

The Journal of Clinical Endocrinology & Metabolism

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CYP3A4, the predominant cytochrome P450 expressed in human liver, is responsible for the metabolism of endogenous steroids and many drugs. On the basis of pharmacokinetic studies in patients with hormonal derangements and the effects of replacement therapy, it has been suggested that iodothyronines decrease CYP3A4-mediated drug metabolism, whereas glucocorticoids and GH enhance CYP3A4 activity. The aim of the present study, using well differentiated human hepatocytes in primary culture, was to examine directly whether hormonal factors regulate CYP3A4 gene expression. Addition of T3 to primary hepatocytes resulted in a marked reduction of CYP3A4-catalyzed testosterone 6 beta-hydroxylase activity and corresponding levels of CYP3A4 protein and messenger ribonucleic acid compared to those in untreated cells. Conversely, both dexamethasone and GH treatment substantially increased CYP3A4 gene expression. None of the hormones studied consistently altered the expression of other human cytochrome P450 genes. We conclude that iodothyronines, glucocorticoids, and GH act directly on human hepatocytes to regulate the expression of CYP3A4, and these effects appear to be exerted at a pretranslational level. Altered regulation of hepatic CYP3A4 is, therefore, likely to account for previous observations concerning the effects of endocrine diseases and hormonal treatments on human cytochrome P450-mediated drug and steroid metabolism.

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Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel

Cited by 9 publications

References 35 publications

Predictive Value of in Vitro Model Systems in Toxicology

Predictive Value of in Vitro Model Systems in Toxicology

Soluble Low Density Lipoprotein Receptor-related Protein (LRP) Circulates in Human Plasma

Separate and Interactive Regulation of Cytochrome P450 3A4 by Triiodothyronine, Dexamethasone, and Growth Hormone in Cultured Hepatocytes¹

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Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel

Cited by 9 publications

References 35 publications

Predictive Value of in Vitro Model Systems in Toxicology

Predictive Value of in Vitro Model Systems in Toxicology

Soluble Low Density Lipoprotein Receptor-related Protein (LRP) Circulates in Human Plasma

Separate and Interactive Regulation of Cytochrome P450 3A4 by Triiodothyronine, Dexamethasone, and Growth Hormone in Cultured Hepatocytes1

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Separate and Interactive Regulation of Cytochrome P450 3A4 by Triiodothyronine, Dexamethasone, and Growth Hormone in Cultured Hepatocytes¹