Elsevier

Biochemical Pharmacology

Volume 50, Issue 11, 27 November 1995, Pages 1775-1782
Biochemical Pharmacology

Research paper
Validation of the (ω-l)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1

https://doi.org/10.1016/0006-2952(95)02040-3Get rights and content

Abstract

The (ω-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes. The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot (r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation (r = 0.73), 4-nitrophenol hydroxylation (r = 0.84), N-nitrosodimethylamine demethylation (r = 0.79) and n-butanol oxidation (r = 0.73). The (ω-1)-hydroxylation of lauric acid was inhibited by ethanol (Ki = 3.5 mM), acetone (IC50 = 10 mM), dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol Ki on the (ω-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (ω-1)-hydroxylation of lauric acid in human liver microsomes, and ω-hydroxylation is mediated by another enzyme. Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (ω-1)-hydroxylation of lauric acid.

References (38)

  • 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)
  • RT Okita et al.

    Effect of phenobarbital treatment and cytochrome P450 inhibition on the laurate ω and (ω-1) hydroxylase activities or rat liver microsomes

    Drug Metab Dispos

    (1980)
  • HAAM Dirven et al.

    Lauric acid hydroxylase activity and cytochrome P450 IV family proteins in humans liver microsomes

    Biochem Pharmacol

    (1991)
  • T Fukuda et al.

    Different mechanisms of regioselection of fatty acid hydroxylation by laurate (ω-1) hydroxylating P450s, P450 2C2 and P450 2E1

    J Biochem

    (1994)
  • SE Clarke et al.

    Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes

    Chem Res Toxicol

    (1994)
  • D Koop et al.

    Multiple mechanisms in the regulation of ethanol-inducible cytochrome P450IIE1

    BioEs-says

    (1990)
  • FP Guengerich et al.

    Role of human cytochrome P450 IIE1 in the oxidation of many low molecular weight cancer suspects

    Chem Res Toxicol

    (1991)
  • M Morimato et al.

    Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis

    Alcohol

    (1993)
  • F Berthou et al.

    Comparison of caffeine metabolism by slices, microsomes and hepatocyte cultures from adult human liver

    Xenobiotica

    (1989)
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