Chlorzoxazone metabolism by porcine cytochrome P450 enzymes and the effect of cytochrome b5.
Chlorzoxazone (CLZ) is a commonly used nontoxic in vivo and in vitro probe for the assessment of CYP2E1 activity. Human CYP1A1 and CYP3A4 have also been shown to contribute to CLZ metabolism. For pigs to be a potential model system for humans, it is necessary that human and pig cytochromes P450 (P450) have similar metabolizing capabilities. Therefore, CLZ metabolizing capabilities and specificities of porcine P450s were investigated. In this study, the complete coding regions of six porcine P450s were amplified from liver cDNA and cloned into pcDNA3.1/V5-His TOPO vector. Expression vectors for the individual P450s and microsomal cytochrome b(5) (CYB5A) were expressed in the human embryonic kidney HEK-293FT cell line to investigate their role in CLZ metabolism. As with the human enzymes, porcine CYP2E1 (K(m) = 290.3 microM and V(max) = 4980 pmol/h/mg total protein) and CYP1A1 (K(m) = 159.5 microM and V(max) = 1650 pmol/h/mg total protein) both contribute to CLZ metabolism. In addition, porcine CYP2A19 and CYP2C33v4 also metabolize the substrate, with K(m) = 212.1 microM and V(max) = 6680 pmol/h/mg total protein and K(m) = 126.3 microM and V(max) = 2100 pmol/h/mg total protein, respectively, whereas CYP3A does not. CYB5A augmented CYP2E1 and CYP2C33v4 activity in the pig, with a significant increase in activity of 85 and 73% compared with control, respectively. Thus, CLZ should be used with caution as a probe for CYP2E1 activity in the pig. However, further information regarding the abundance of different P450 isoforms is needed to fully understand their contribution in microsomal, hepatocyte, and in vivo systems in the pig.
Wiercinska
P
P
Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada.
Squires
E J
EJ
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002460:Cell Line; D002753:Chlorzoxazone / Q000031:analogs & derivatives / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism; D034421:Sus scrofa; D014162:Transfection
C000594834:CYB5A protein, human; C400322:cytochrome P-450 CYP2C subfamily; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C029009:steroid hormone 7-alpha-hydroxylase; D002753:Chlorzoxazone