A markedly diminished pleiotropic response to phenobarbital and structurally-related xenobiotics in Zucker rats in comparison with F344/NCr or DA rats

doi:10.1016/0006-2952(92)90615-P

Biochemical Pharmacology

Volume 43, Issue 5, 3 March 1992, Pages 1079-1087

https://doi.org/10.1016/0006-2952(92)90615-P Get rights and content

Abstract

Phenobarbital (PB) and certain structurally-related compounds induce a variety of hepatic drug-metabolizing enzymes in many strains of rats. Thus, following administration of PB (300, 500 ppm), barbital (BB, 1500 ppm) or 5-ethyl-5-phenylhydantoin (EPH, 500 ppm), CYP2B1-mediated benzyloxyresorufin o-dealkylase activity and epoxide hydrolase activity were profoundly induced in female DA and F344/NCr rats. In contrast, outbred female lean and obese Zucker rats showed markedly reduced CYP2B1 responses (< 15% and < 5% of those observed in the female DA or F344/NCr rat) to PB (doses ≤ 300 ppm), BB (1500 ppm) or EPH (500 ppm). In parallel studies, profound increases in RNA levels coding for CYP2B1, glutathione S-transferases Ya/Yc (α subclass), or epoxide hydrolase were detected in the female F344/NCr rat following treatment with PB (300 ppm), BB (1500 ppm) or EPH (500 ppm). In contrast, lean Zucker rats showed a strong response only to the highest dose of PB (500 ppm), implying that the diminished response in the Zucker rats may occur at some pretranslational level. Similar studies with lower doses of PB, EPH or BB in male lean Zucker rats showed a decreased response, relative to that in male F344/NCr rats. However, this insensitivity was not as profound as that observed in the female Zucker rats. In fact, the response to PB-type inducers in male or female Zucker rats is probably most clearly explained as a shift of the dose-response curve sharply to the right (decreased responsiveness, compared to F344/NCr or DA rats of the same sex). This decreased responsiveness of female lean Zucker rats to induction of CYP2B1, relative to that of F344/NCr rats, was also observed with the structurally-diverse PB-type inducers clonazepam, clotrimazole and 2-hexanone. In contrast, the female Zucker rat (obese or lean) displayed a pronounced response to induction of CYP1A-mediated ethoxyresorufin O-deethylase activity by β-naphthoflavone, a prototype inducer of CYP1A1 and CYP1A2. The Zucker rat would thus appear to represent a potentially exploitable genetic model for examining the mechanism of enzyme induction by the myriad xenobiotics which induce a PB-type response.

References (55)

PE Thomas et al.
Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes
Arch Biochem Biophys
(1979)
LM Reik et al.
Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily
Arch Biochem Biophys
(1985)
CM Giachelli et al.
Regulation of cytochrome P-450b and P-450e mRNA expression in the developing rat: Hybridization to synthetic oligodeoxyribonucleotide probes
J Biol Chem
(1986)
M Atchison et al.
A cytochrome P-450 multigene family: Characterization of a gene activated by phenobarbital administration
J Biol Chem
(1983)
MD Burke et al.
Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: A series of substrates to distinguish between different induced cytochromes P-450
Biochem Pharmacol
(1985)
RA Lubet et al.
Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat
Arch Biochem Biophys
(1985)
AW Wood et al.
Regio- and stereoselective metabolism of two C₁₉ steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes
J Biol Chem
(1983)
M Christou et al.
Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues
Arch Biochem Biophys
(1987)
DL Simmons et al.
Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development
Arch Biochem Biophys
(1989)
PG Traber et al.
Expression of cytochrome P450b and P450e genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides
J Biol Chem
(1988)

RE Kouri et al.

Correlation of inducibility of aryl hydrocarbon hydroxylase with susceptibility to 3-methylcholanthrene-induced lung cancers

Cancer Lett

(1980)

CL Litterst

In vitro hepatic drug metabolism and microsomal enzyme induction in genetically obese rats

Biochem Pharmacol

(1980)

JA Koch et al.

Cytochrome P-450 induction in genetically obese rats

Biochem Biophys Res Commun

(1982)

P Böhlen et al.

Fluorometric assay of proteins in the nanogram range

Arch Biochem Biophys

(1973)

RA Lubet et al.

A pleiotropic response to phenobarbital-type inducers in the F344/NCr rat: Effects of chemicals of varied structure

Biochem Pharmacol

(1992)

PM Dansette et al.

Continuous fluorometric assay of epoxide hydrase activity

Anal Biochem

(1979)

AJ Sonderfan et al.

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

Arch Biochem Biophys

(1987)

CB Pickett et al.

Argenbright L and Lu AYH, Rat liver glutathione S-transferases: Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital

J Biol Chem

(1984)

JP Hardwick et al.

Transcriptional regulation of rat liver epoxide hydratase, NADPH-cytochrome P-450 oxidoreductase, and cytochrome P-450b genes by phenobarbital

J Biol Chem

(1983)

I Jansson et al.

A study of the interaction of a series of substituted barbituric acids with the hepatic microsomal monooxygenase

Arch Biochem Biophys

(1972)

T Cresteil et al.

In vivo administration of hydroxylated phenobarbital metabolites: Effect on rat hepatic cytochromes P-450, epoxide hydrolase and UDP-glucuronyltransferase

Biochem Pharmacol

(1980)

TJ Dunn et al.

Phenobarbital-inducible aldehyde dehydrogenase in the rat. cDNA sequence and regulation of the mRNA by phenobarbital in responsive rats

J Biol Chem

(1989)

AH Conney

Pharmacological implications of micro-somal enzyme induction

Pharmacol Rev

(1967)

DW Nebert et al.

P-450 genes: Structure, evolution and regulation

Annu Rev Biochem

(1987)

FJ Gonzalez

The molecular biology of cytochrome P450s

Pharmacol Rev

(1988)

H Kutt

Phenobarbital: Interactions with other drugs

CJ Omiecinski

Tissue-specific expression of rat mRN As homologous to cytochromes P-450b and P-450e

Nucleic Acids Res

(1986)

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^§: Present address: Department of Pathobiology, University of Tennessee, College of Veterinary Medicine, Knoxville, TN 37901-1071.

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