The Relative Importance of CYP26A1 and CYP26B1 in Mediating Retinoid Homeostasis: Studies on the Formation, Elimination and Biological Activity of All-trans-Retinoic Acid Metabolites

Thesis (Ph.D.)--University of Washington, 2013All-trans-retinoic acid (atRA), the active metabolite of Vitamin A (retinol), is an essential nutrient during both fetal development and adult life. The concentration of atRA within a cell is tightly regulated by the synthesis and elimination of atRA. One of the major elimination pathways for atRA is oxidation, predominantly by the cytochrome P450s CYP26A1 and CYP26B1 that efficiently hydroxylate atRA. The role of both CYP26A1 and CYP26B1 is believed to be to inactivate atRA, and both are essential for correct fetal development. It is not known whether both enzymes play crucial roles during adult life. In this work, the differences in the catalytic efficiency of CYP26A1 and CYP26B1 and the metabolites formed from atRA by CYP26A1 and CYP26B1 were explored. The subsequent metabolism and the activity of atRA metabolites were also evaluated. CYP26A1 was determined to form 4-OH-RA from atRA more efficiently (Km = 50 nM, Clint = 190 µL/min/pmoles P450) than CYP26B1 (Km = 19 nM, Clint = 43 µL/min/pmoles P450). In addition, formation of 4-OH-RA by CYP26A1 was stereoselective resulting in formation of (4S)-OH-RA whereas no significant stereoselectivity in 4-OH-RA formation by CYP26B1was observed. The primary hydroxylated metabolites of atRA, (4S)-OH-RA, (4R)-OH-RA and 18-OH-RA, and the secondary metabolite, 4-oxo-RA, were all found to be substrates of CYP26A1 and CYP26B1. However, formation of 4-oxo-RA from 4-OH-RA was not P450 mediated in cells or in human fetal livers and was mediated by a yet to be identified enzyme. CYP26A1 mRNA was induced by atRA, 4-oxo-RA, (4S)-OH-RA, (4R)-OH-RA, and 18-OH-RA in HepG2 cells. Based on measured cell exposure to the retinoids, 4-oxo-RA is the most potent retinoid in terms of RAR activation. Since atRA is an important signaling molecule during pregnancy and fetal development, the metabolism of atRA in maternal liver during pregnancy was measured in a mouse model and the metabolism in human fetal liver was characterized. It was determined that during mouse pregnancy CYP26A1 activity was significantly increased in the liver, suggesting increased activation of RARs and increased atRA clearance during pregnancy, and decreased atRA concentration. The increased CYP26A1 activity in the maternal liver may function as a first-pass barrier to protect the developing fetus from excess atRA exposure. The human fetal liver also metabolized atRA, but the overall extraction ratio of atRA by the human fetal liver was predicted to be minimal (0.01-0.05), suggesting that CYP26 expression in other fetal tissues is critical for regulating atRA concentration. In conclusion the results of this study show that CYP26A1 and CYP26B1 are important enzymes regulating atRA clearance during pregnancy and adult life, and CYP26A1 and CYP26B1 have specific kinetic characteristics that likely explain their different roles in atRA clearance