Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques

doi:10.1016/0006-2952(93)90639-E

Biochemical Pharmacology

Volume 46, Issue 11, 3 December 1993, Pages 1975-1981

https://doi.org/10.1016/0006-2952(93)90639-E Get rights and content

Abstract

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetic; the respective apparent K_m values were 30 ± 7 and 21 μM. Mutual competetive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent K_m and K_i values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (r_s ⩾ 0.75, P < 0. 005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (α-naphthoflovone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.

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Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques

Abstract

FEBS Lett

Toxicol Appl Pharmacol

Br J Anaesth

Biochem Biophys Res Commun

Trends Pharmacol Sci

Biochem Pharmacol

Biochem Pharmacol

Biochim Biophys Acta

Cancer Lett

J Chromatogr

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

FEBS Lett

Arch Biochem Biophys

J Biol Chem

Toxicol Appl Pharmacol

Arch Biochem Biophys

Arch Biochem Biophys

Arch Biochem Biophys

Life Sci

Methods Enzymol

P450 genes: structure, evolution, and regulation

Annu Rev Biochem

Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes

Chem Res Toxicol

Role of human cytochrome P-450IIE1 in the oxidation of many low molecular weight cancer suspects

Chem Res Toxicol