Abstract
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 b5 (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 (Km = 290.3 μM and Vmax = 4980 pmol/h/mg total protein) and CYP1A1 (Km = 159.5 μM and Vmax = 1650 pmol/h/mg total protein) both contribute to CLZ metabolism. In addition, porcine CYP2A19 and CYP2C33v4 also metabolize the substrate, with Km = 212.1 μM and Vmax = 6680 pmol/h/mg total protein and Km = 126.3 μM and Vmax = 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.
Footnotes
This work was supported by the Natural Sciences and Engineering Research Council [Discovery Grant 400066]; and the Ontario Ministry of Agriculture and Food [Grant 026398] (to E.J.S.).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030528.
-
- P450
- cytochrome P450
- CLZ
- chlorzoxazone
- 6-OH-CLZ
- 6-hydroxy-chlorzoxazone
- HEK293FT
- human embryonic kidney cell line
- POR
- P450-oxidoreductase/P450 NADPH reductase
- CYB5R3
- cytochrome b5 reductase
- CYB5A
- microsomal cytochrome b5
- h
- human
- Cint
- intrinsic clearance.
- Received September 28, 2009.
- Accepted February 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|