Toluene metabolism by cDNA-Expressed human hepatic cytochrome P450

doi:10.1016/S0006-2952(96)00652-1

Biochemical Pharmacology

Volume 53, Issue 3, 7 February 1997, Pages 271-277

https://doi.org/10.1016/S0006-2952(96)00652-1 Get rights and content

Abstract

The metabolism of toluene in human liver microsomes and by cDNA-expressed human cytochrome P450s (CYPs) was investigated. Toluene was metabolized mainly to benzyl alcohol and slightly to o- and p-cresol by human liver microsomes. Formation of o-cresol was elevated in microsomes from human livers derived from cigarette smokers, but the induced CYP isoforms were not clear. Of the eleven human CYP forms studied, CYP2E1 was the most active in forming benzyl alcohol, followed by CYP2B6, CYP2C8, CYP1A2, and CYP1A1, in that order. The activities of CYP2A6, CYP2C9, CYP2D6, CYP3A3, CYP3A4, and CYP3A5 were negligible. In addition, CYP2B6 and CYP2E1 catalyzed the formation of p-cresol (11–12% of total metabolites), and CYP1A2 catalyzed the formation of both o-(22%) and p-cresol (35%). The relationship between the amino acid sequence of rat CYP2B1 cDNA and the activity for toluene metabolism was investigated using variants, because of great differences in the forming of toluene ring products between CYP2B1 and CYP2B6. These results suggest that the structure of CYP2B1 at the site of Leu 58 rather than Ile-114 and Glu-282 plays an important role in the formation of toluene ring products, whereas in CYP2B1 Ile-114 plays an important role in the formation of benzyl alcohol. These results may explain, in part, the lower activity of CYP2B6, which has Phe at position 58 of the protein, for toluene ring oxidations than that of CYP2B1.

References (28)

T Nakajima et al.
Monoclonal antibody-directed characterization of cytochrome P450 isozymes responsible for toluene metabolism in rat liver
Biochem Pharmacol
(1991)
FJ Gonzalez et al.
Expression of mammalian cytochrome P450 using vaccinia virus
Methods Enzymol
(1991)
T Aoyama et al.
Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver
J Biol Chem
(1989)
T Aoyama et al.
Sequence requirement for cytochrome P450 IIB1 catalytic activity: Alteration of the stereospecificity and regioselectivity of steroid hydroxylation by a simultaneous change of two hydrophobic amino acid residues to phenylalanine
J Biol Chem
(1989)
OH Lowry et al.
Protein measurement with the Folin phenol reagent
J Biol Chem
(1951)
T Omura et al.
The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature
J Biol Chem
(1964)
SJ Duthie et al.
Status of reduced glutathione in the human hepatoma cell line, HEP G2
Biochem Pharmocol
(1988)
International Agency for Research on Cancer
Toluene
IARC Monogr Eval Carcinog Risks Chem Hum
(1989)
DR Nelson et al.
The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature
DNA Cell Biol
(1993)
T Nakajima et al.
Immunochemical assessment of the influence of nutritional, physiological and environmental factors on the metabolism of toluene
Int Arch Occup Environ Health
(1993)

DJ Waxman et al.

Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: Purification, characterization, and interactions with metyrapone and cytochrome b₅

Biochemistry

(1983)

G Nise

Urinary excretion of o-cresol and hippuric acid after toluene exposure in rotogravure printing

Int Arch Occup Environ Health

(1992)

M Dossing et al.

Urinary hippuric acid and orthocresol excretion in man during experimental exposure to toluene

Br J Ind Med

(1983)

T Nakajima et al.

A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver

Biochem Pharmacol

(1992)

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Better insight into these interactions can be obtained by visualizing the scheme for toluene metabolism. The main metabolic pathway of toluene consists of its conversion to benzyl alcohol (catalyzed mainly by CYP2E1 enzymes), further to benzoic acid (catalyzed by alcohol and aldehyde dehydrogenases), and to the final product hippuric acid (Angerer et al., 1998; Nakajima and Wang, 1994; Nakajima et al., 1997). In fact, about 75–80% of inhaled toluene that is absorbed can be detected as its principal metabolite, hippuric acid, in urine (Löf et al., 1993; Tardif et al., 1998).
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Another recent report in petrochemical workers (Piccoli et al., 2010) showed no significant differences in the 6-OH-CHZ/CHZ ratio between participants exposed to very low levels of benzene (one or two orders of magnitude below the current TLV) and controls. Benzene, however, has shown different properties on CYP2E1 activity as compared to toluene (Nakajima et al., 1997; González-Jasso et al., 2003). Our data showing a positive correlation between BMI and the 6-OH-CHZ/CHZ ratio in the E group suggest a possible synergism on CYP2E1 activity between exposure to solvents and physiological phenomena (Fig. 5) These results confirm those from a cluster of reports that have shown obesity as a crucial factor for CYP2E1 activity regulation (Lucas et al., 1999; Chtioui et al. 2007) and relate CYP2E1 activity with fatty acids metabolism (Lieber, 1997; Niwa et al., 2009).
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^☆: This work was supported, in part, by a Grant-in-Aid for General Scientific Research (No 03454202) from the Japan Ministry of Education, Science, Sports and Culture of japan. The critical comments of M. Ohara are also gratefully acknowledged.

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Research paperToluene metabolism by cDNA-Expressed human hepatic cytochrome P450☆

Abstract

Biochem Pharmacol

Methods Enzymol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Pharmocol

Toluene

IARC Monogr Eval Carcinog Risks Chem Hum

The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature

DNA Cell Biol

Immunochemical assessment of the influence of nutritional, physiological and environmental factors on the metabolism of toluene

Int Arch Occup Environ Health

Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: Purification, characterization, and interactions with metyrapone and cytochrome b5

Biochemistry

Urinary excretion of o-cresol and hippuric acid after toluene exposure in rotogravure printing

Int Arch Occup Environ Health

Urinary hippuric acid and orthocresol excretion in man during experimental exposure to toluene

Br J Ind Med

A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver

Biochem Pharmacol

Research paper
Toluene metabolism by cDNA-Expressed human hepatic cytochrome P450☆

Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: Purification, characterization, and interactions with metyrapone and cytochrome b₅