Validation of the (omega-1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1.
The (omega-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 (omega-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 (omega-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 (omega-1)-hydroxylation of lauric acid in human liver microsomes, and omega-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 (omega-1)-hydroxylation of lauric acid.
Amet
Y
Y
Equipe d'Accueil DGRT EA 948, Laboratoire de Biochimie-Nutrition Faculté de Médecine, Brest, France.
Berthou
F
F
Baird
S
S
Dreano
Y
Y
Bail
J P
JP
Menez
J F
JF
D019392:Cytochrome P-450 CYP2E1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D007850:Lauric Acids; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating