Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes: Analysis with a novel reverse transcriptase–polymerase chain reaction assay

doi:10.1016/S0006-2952(00)00454-8

Biochemical Pharmacology

Volume 60, Issue 10, 15 November 2000, Pages 1509-1518

https://doi.org/10.1016/S0006-2952(00)00454-8 Get rights and content

Abstract

The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzymes for drug metabolism, can be elevated by chemical inducers. In the present study, we investigated in cultured rat hepatocytes the induction of all four currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vivo. To this end, CYP3A mRNA levels were quantified with a novel, radioactive reverse transcriptase–polymerase chain reaction (RT–PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT–PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucleotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT–PCR assay, it was found that the expression of all CYP3A isoforms in rat hepatocytes, cultured on a collagen matrix, was decreased by 80–90% within one day of cultivation. After addition of dexamethasone, at one day after isolation, CYP3A1 mRNA levels were elevated to levels comparable to those in freshly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and CYP3A18 mRNA levels were not affected by dexamethasone treatment, and were hardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addition of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after dexamethasone administration. However, in contrast to the situation in cultured hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser extent (4- and 7-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interference by any of the other CYP3A isoenzymes.

Section snippets

Materials

Collagenase type IV, BSA fraction V, insulin, dexamethasone, testosterone, corticosterone, glucose 6-phosphate, NADP, DEPC, and yeast tRNA were obtained from Sigma. Amylym (Lintner’s Starch) was from J.T. Baker. [α-³²P]dCTP was purchased from Amersham. 6β-OH-testosterone was from Steraloids. Guanidinium isothiocyanate was purchased from Promega. Collagen-S type I and glucose-6-phosphate dehydrogenase were from Boehringer Mannheim. DMEM, fetal bovine serum, and penicillin/streptomycin were

Development of a sensitive and quantitative RT–PCR assay

To detect changes in cytochrome P450 levels in small amounts of cells or tissue, a sensitive RT–PCR assay was developed. The expression of the different CYP3A isoenzymes was quantified by relating their expression to that of the housekeeping protein GAPDH. For this purpose, cDNA was synthesized from total cellular RNA by reverse transcriptase in the presence of oligo(dT) primers. Thereafter, a multiplex PCR reaction was performed on the reverse-transcribed RNA with two sets of primers, one

Discussion

In the present study, the mRNA levels of all four currently identified CYP3A isoenzymes were determined in isolated hepatocytes and in rat liver using a novel, quantitative RT–PCR assay. RT–PCR assays have several advantages over conventional assays for RNA quantification such as Northern blotting and nuclease protection assays. Both the sensitivity and specificity of the RT–PCR assay are higher 27, 28. We further improved the sensitivity and accuracy of the RT–PCR assay by including

References (39)

V. Rogiers et al.
Rat hepatocyte cultures and co-cultures in biotransformation studies of xenobiotics
Toxicology
(1993)
F.J. Gonzalez et al.
Complete cDNA and protein sequence of a pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450. A representative of a new gene family
J Biol Chem
(1985)
H. Wang et al.
cDNA cloning of a novel CYP3A from rat brain
Biochem Biophys Res Commun
(1996)
A. Mahnke et al.
Expression and inducibility of cytochrome P450 3A9 (CYP3A9) and other members of the CYP3A subfamily in rat liver
Arch Biochem Biophys
(1997)
D. Strotkamp et al.
A novel CYP3 gene from female rats
Biochim Biophys Acta
(1995)
S. Kirita et al.
cDNA cloning and characterization of a novel member of steroid-induced cytochrome P450 3A in rats
Arch Biochem Biophys
(1993)
K. Nagata et al.
Structure and expression of the rat CYP3A1 geneIsolation of the gene (P450/6betaB) and characterization of the recombinant protein
Arch Biochem Biophys
(1999)
D.L. Morris et al.
Analysis of rat cytochrome P450 isoenzyme expression using semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR)
Biochem Pharmacol
(1996)
K.O. Cooper et al.
Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats
Arch Biochem Biophys
(1993)
P.O. Seglen
Preparation of isolated rat liver cells
Methods Cell Biol
(1976)

H.M. Wortelboer et al.

The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers

Biochem Pharmacol

(1990)

K. Dukas et al.

Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction

Anal Biochem

(1993)

A.P. Li et al.

Primary hepatocyte culture as an experimental model for the evaluation of interactions between xenobiotics and drug-metabolizing enzymes

Chem Biol Interact

(1997)

M. Ogino et al.

Hepatocyte nuclear factor 4-mediated activation of rat CYP3A1 gene and its modes of modulation by apolipoprotein AI regulatory protein I and v-ErbA-related protein 3

Arch Biochem Biophys

(1999)

J.M. Huss et al.

Nuclear receptor involvement in the regulation of rat cytochrome P450 3A23 expression

J Biol Chem

(1998)

L.C. Quattrochi et al.

Characterization of DNA-binding proteins required for glucocorticoid induction of CYP3A23

Arch Biochem Biophys

(1998)

M. Miyata et al.

Transcriptional elements directing a liver-specific expression of P450/6 beta A (CYP3A2) gene-encoding testosterone 6 beta-hydroxylase

Arch Biochem Biophys

(1995)

S.A. Kliewer et al.

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway

Cell

(1998)

J.M. Huss et al.

Differential glucocorticoid responses of CYP3A23 and CYP3A2 are mediated by selective binding of orphan nuclear receptors

Arch Biochem Biophys

(1999)

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^§: Abbreviations: CYP, cytochrome P450; RT–PCR, reverse transcriptase–polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DEPC, diethylpyrocarbonate; DMEM, Dulbecco’s modified Eagle’s medium; HNF, hepatocyte nuclear factor; DexRE, dexamethasone response element; DR-3, direct repeat-3; RXR, retinoid X receptor; PXR, pregnenolone X receptor; and COUP-TF, chicken ovalbumin upstream promoter-transcription factor.

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Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes: Analysis with a novel reverse transcriptase–polymerase chain reaction assay§

Abstract

Section snippets

Materials

Development of a sensitive and quantitative RT–PCR assay

Discussion

Toxicology

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Arch Biochem Biophys

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