Identification of ethanol-inducible P450 isozyme 3a (P450IIE1) as a benzene and phenol hydroxylase

doi:10.1016/0041-008X(89)90233-0

Toxicology and Applied Pharmacology

Volume 98, Issue 2, April 1989, Pages 278-288

https://doi.org/10.1016/0041-008X(89)90233-0 Get rights and content

Abstract

In this report, the identity of the cytochrome P450 isozyme(s) catalyzing the hydroxylation of benzene and the major hydroxylated metabolite of benzene, phenol, was investigated using rabbit hepatic microsomes and six purified isozymes of hepatic P450. Microsomes from acetone-treated rabbits showed about a 5-fold induction of benzene hydroxylation to phenol and hydroquinone. This increase correlated with the increase in form 3a determined immunochemically (about 7-fold). Antibody to isozyme 3a inhibited greater than 90% of the benzene and phenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits. At high benzene concentrations (2 mm) in the presence of cytochrome b₅, form 3a was 1.3 times more active than form 2 and 7- to 10-fold more active than forms 3b, 3c, 4, and 6. At lower benzene concentrations (about 0.3 mm) form 3a was 5-fold more active than form 2. Furthermore, form 3a was the only isozyme to produce significant quantities of hydroquinone as did microsomes from acetone-treated rabbits. When phenol was used as the substrate, hydroquinone was the only product detected, and acetone treatment induced its formation 4- to 5-fold. Purified form 3a was 20- to 30-fold more active than the next most active isozyme, form 6, depending on the presence or absence of cytochrome b₅. These results suggest that isozyme 3a (P450IIE1) is a low-K_m benzene hydroxylase and the principal phenol hydroxylase in rabbit hepatic microsomes. As a result, the induction of isozyme 3a could potentiate the toxicity of benzene by catalyzing an increase in the formation of both phenol and hydroquinone.

References (49)

L.S. Andrews et al.
³H-Benzene metabolism in rabbit bone marrow
Life Sci.
(1979)
K.A. Baarson et al.
The hematotoxic effects of inhaled benzene on peripheral blood, bone marrow, and spleen cells are increased by ingested ethanol
Toxicol. Appl. Pharmacol.
(1982)
M.J. Coon et al.
Two forms of liver microsomal cytochrome P-450, P-450_LM2 and P-450_LM4 (rabbit liver)
B.J. Dean
Recent findings on the genetic toxicology of benzene, toluene, xylenes, and phenols
Mutat. Res.
(1985)
D.R. Dutton et al.
Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: Evidence against cytochrome P-450 isozyme:isozyme interactions
Arch. Biochem. Biophys.
(1987)
D.A. Eastmond et al.
An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure
Toxicol. Appl. Pharmacol.
(1987)
J.S. French et al.
Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes
Arch. Biochem. Biophys.
(1979)
M.M. Gad-El-Karim et al.
Modifications in the myeloclastogenic effect of benzene in mice with toluene, phenobarbital, 3-methylcholanthrene, Aroclor 1254, and SKF-525A
Mutat. Res.
(1984)
D.P. Gill et al.
Modifications of benzene myelotoxicity and metabolism by phenobarbital, SKF-525A and 3-methylcholanthrene
Life Sci.
(1979)
L.M. Gonasun et al.
Benzene metabolism in mouse liver microsomes
Toxicol. Appl. Pharmacol.
(1973)

W.F. Greenlee et al.

A novel method for the separation and quantification of benzene metabolites using high-pressure liquid chromatography

Anal. Biochem.

(1981)

I. Johansson et al.

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

J. Biol. Chem.

(1983)

I. Johansson et al.

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

FEBS Lett.

(1985)

D.R. Koop et al.

Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes from ethanol-treated rabbits

J. Biol. Chem.

(1982)

D.R. Koop et al.

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Arch. Biochem. Biophys.

(1984)

D.R. Koop et al.

Properties of electrophoretically homogeneous constitutive forms of liver microsomal cytochrome P-450

J. Biol. Chem.

(1981)

W. Levin et al.

N-Demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome p-450: Isozyme specificity and role of cytochrome b₅

Arch. Biochem. Biophys.

(1986)

O.H. Lowry et al.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

(1951)

T. Nakajima et al.

Effects of ethanol and phenobarbital administration on the metabolism and toxicity of benzene

Chem.-Biol. Interact.

(1985)

D.E. Ryan et al.

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Arch. Biochem. Biophys.

(1986)

P.J. Sabourin et al.

Differences in the metabolism and disposition of inhaled [³H]-benzene by $F344 >N$ rats and B6C3F1 mice

Toxicol. Appl. Pharmacol.

(1988)

P.J. Sabourin et al.

Effect of dose on the absorption and excretion of [¹⁴C]-benzene administered orally or by inhalation in rats and mice

Toxicol. Appl. Pharmacol.

(1987)

M. Adesnik et al.

Genes for cytochrome P-450 and their regulation

CRC Crit. Rev. Biochem.

(1986)

M. Askoy

Benzene as a leukemogenic and carcinogenic agent

Amer. J. Ind. Med.

(1985)

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^☆: This work was supported in part by Grants P30 CA-43703 to the Ireland Cancer Center and AA-07219 to D.R.K.

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Identification of ethanol-inducible P450 isozyme 3a (P450IIE1) as a benzene and phenol hydroxylase☆

Abstract

Life Sci.

Toxicol. Appl. Pharmacol.

Mutat. Res.

Arch. Biochem. Biophys.

Toxicol. Appl. Pharmacol.

Arch. Biochem. Biophys.

Mutat. Res.

Life Sci.

Toxicol. Appl. Pharmacol.

Anal. Biochem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Chem.-Biol. Interact.

Arch. Biochem. Biophys.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Genes for cytochrome P-450 and their regulation

CRC Crit. Rev. Biochem.

Benzene as a leukemogenic and carcinogenic agent

Amer. J. Ind. Med.