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Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androstane Receptor (CAR, NR1I3)

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Cytochrome P450

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

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Abstract

Constitutive androstane receptor (CAR, NR1I3) is among the orphan nuclear receptors important in altering drug elimination. In mechanisms similar to those ascribed to the pregnane X receptor, CAR governs the expression of many of the same drug metabolizing enzymes and transporters. Because of this important role, identifying new molecular entities (NMEs) that activate CAR has become a focus of investigation in the drug development arena. Most in vitro assays to identify NMEs that activate CAR are ligand binding in nature. However, there are limitations associated with these types of cell-based assays where only the ligand binding domain (LBD) of the receptors are employed. One major drawback is that activators of CAR can be either direct or indirect and using the truncated receptor will only provide the identification of direct activators. Described here is a medium to high throughput system to assess both types of stimulators, direct and indirect, by employing the full-length CAR1 and CAR3 variants in transiently transfected hepatomas. Methods are provided for culturing and transfecting hepatoma cells, freezing, and storing the transfectants, and thawing and seeding in 96-well plates. Furthermore, treatment of the CAR transfected cells with NMEs and simultaneous assessment of cytotoxicity and receptor activation are described.

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Abbreviations

ART:

Artemisinin

CAR:

Constitutive androstane receptor

CAR1+A:

Constitutive androstane receptor variant 1 + alanine

CAR3:

Constitutive androstane receptor variant 3

CITCO:

6-(4-Chlorophenyl)Imidazo[2,1-B][1,3]Thiazole-5-Carbaldehyde O-(3,4-Dichlorobenzyl)Oxime

CTF:

Cell-titer fluortm cell viability assay

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

FLU:

Fluorescent light units

NR:

Nuclear receptor

PBREM:

Phenobarbital-responsive enhancer module

PBS:

Phosphate buffered saline

RLU:

Relative light units

TCPOBOP:

1,4-Bis-[2-(3,5-Dichloropyridyloxy)]Benzene, 3,3′,5,5′-Tetrachloro-1,4-Bis(Pyridyloxy)Benzene

Wt:

Wild-type

XREM:

Xenobiotic responsive enhancer module

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Authors and Affiliations

  1. Puracyp Inc., Carlsbad, CA, USA

    Marija Pinne & Judy L. Raucy

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  1. Marija Pinne
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  2. Judy L. Raucy
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Correspondence to Marija Pinne .

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Editors and Affiliations

  1. Genentech Inc., South San Francisco, CA, USA

    Zhengyin Yan

  2. Blue Bell, PA, USA

    Gary W. Caldwell

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Pinne, M., Raucy, J.L. (2021). Cytochrome P450 Gene Regulation: Reporter Assays to Assess Direct and Indirect Activation of Constitutive Androstane Receptor (CAR, NR1I3). In: Yan, Z., Caldwell, G.W. (eds) Cytochrome P450. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1542-3_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1542-3_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1541-6

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