The relative importance of CYP26A1 in hepatic clearance of all-trans retinoic acid
Graphical abstract
Introduction
All-trans retinoic acid (RA) is the biologically active metabolite of Vitamin A (Retinol). RA serves as a critical signaling molecule, regulating gene transcription, cell division and apoptosis during development and adult life [1], [2], [3]. Both too much and too little RA have been linked with detrimental effects, including increased risk of infection leading to death [4], decreased fertility [5], Alzheimer's disease [6] and developmental defects [7]. Therefore, concentrations of RA must be tightly regulated.
Cellular exposure to RA is regulated by controlled synthesis and metabolism. The liver plays a role in maintaining RA homeostasis by contributing to RA synthesis and clearance, serving as the major storage organ for retinyl esters and RA, and by providing a first pass barrier for dietary intake of RA [8]. The liver is also likely to be the main organ clearing therapeutically administered RA. The exact metabolic pathway by which RA is cleared from the body is still unclear, but the primary oxidative pathway for RA clearance is believed to be via P450 mediated formation of 4-OH RA [9], [10], [11]. There is an abundance of data showing the oxidation of RA to 4-OH RA and other metabolites, including a subsequent oxidation to 4-oxo RA, in in vitro systems such as COS-1, V79-4, and HeLa cells; liver, lung, kidney, and testis microsomes and recombinant P450 expression systems [9], [10], [11], [12], [13], [14], [15], [16]. In all of these studies, 4-OH RA was detected as the primary oxidation product formed from RA. Despite the in vitro data, the identity of in vivo metabolites of RA is still mainly qualitative and the only metabolite detected in plasma is the 4-oxo RA. 4-oxo RA has been detected in rabbits administered RA or retinyl palmitate [17] and in human plasma [18]. At present the identity of the enzyme forming 4-oxo RA is unknown. However, since 4-OH RA is a necessary primary metabolite leading to 4-oxo RA, the presence of 4-oxo RA in vivo demonstrates that 4-OH RA is also formed in vivo.
Several P450s have been demonstrated to oxidize RA in vitro, including CYP2C8, CYP2C9, CYP2C18, CYP3A4, CYP3A5, CYP3A7, and the newer family of P450s, CYP26. [9], [10], [11], [19] The general consensus of the previously published studies evaluating the importance of P450 isoforms in the metabolism of RA is that CYP2C8 and members of the CYP3A subfamily are the primary P450s responsible for RA clearance. However, the proposed relative importance of these enzymes varies and published data suggests that other enzymes may be involved in RA metabolism as well. Quinidine, a classic selective inhibitor of CYP2D6, caused a 50% reduction in 4-OH RA formation in human liver microsomes despite the fact that recombinant CYP2D6 did not form 4-OH RA from RA [11]. In another study, ketoconazole (20 μM) inhibited 50% of the 4-hydroxylation activity indicating that CYP3A4 contribution to RA hydroxylation does not exceed 50% [10]. These findings suggest that enzymes other than CYP3A4 and CYP2C8 may be important for RA hydroxylation in human liver. A possible additional enzyme would be a member of the CYP26 family, as the contribution of CYP26 enzymes to hepatic RA oxidation was not analyzed in these previous studies [20].
CYP26A1 was first identified in 1996 and has been demonstrated to be inducible by RA and to metabolize RA [13]. The enzymes of the CYP26 family, including CYP26A1 as well as CYP26B1 [21] and CYP26C1 [22], are believed to be the primary CYP enzymes responsible for clearing RA based on the fact that these enzymes metabolize RA efficiently in vitro and are inducible by RA [23]. Cyp26a1−/− and Cyp26b1−/− mice are not viable [24], [25] and show developmental defects similar to the teratogenic effects associated with excess RA [24]. Studies examining mRNA expression have identified CYP26A1 mRNA in the human liver [26], [27] whereas CYP26B1 and CYP26C1 mRNA were present in only a fraction of livers and at a lower level of expression than CYP26A1. Based on mRNA data CYP26A1 was suggested to be the major liver CYP26 isoform [27].
The goal of this study was to determine the role of CYP26A1 in hepatic clearance of RA. We hypothesized that if CYP26A1 is expressed in the liver, it will be the major contributor to RA clearance in this organ, due to the efficiency with which CYP26A1 metabolizes RA [19]. We also hypothesized that due to the low Km of RA towards CYP26A1, with increasing exposure to RA during therapeutic treatment with RA, CYP26A1 will be saturated and the role of other P450 isoforms in RA clearance will increase. To test this hypothesis, RA hydroxylation kinetics was determined for individual P450 isoforms and the relative importance of these enzymes to RA clearance was predicted. Using human liver microsomes (HLMs) from multiple donors, the expression of CYP26A1 protein in the liver was quantified, and RA hydroxylation measured in multiple donors. The obtained data was used in simulating the contribution of CYP26A1 to RA clearance for the purpose of establishing the relative importance of CYP26A1 between individuals and over a variety of RA concentrations.
Section snippets
Reagents
RA, acitretin, cholate, imidazole and NADPH were purchased from Sigma–Aldrich (St. Louis, MO). 4-OH RA was synthesized in-house as previously described [19]. Supersomes® were purchased from Gentest (BD Biosciences, Woburn, MA.) All supersomes were co-expressed with reductase and, with the exception of CYP4A11, CYP1A1, CYP1A2, CYP2C18 and CYP2D6, with cytochrome b5. Purified CYP1A1 protein was purchased from Panvera (Madison, WI). The remaining purified proteins were obtained as gifts from the
Quantification of CYP26 in human liver microsomes
To determine the relative importance of CYP26A1 in RA clearance, the amount of CYP26A1 in a panel of livers was quantified. The specificity and sensitivity of the commercial and in-house antibodies was validated by evaluating them against two CYP26 insect cell membrane preparations and a panel of supersomes of ten P450s abundant in human liver. Cross reactivity with specific P450 isoforms was further confirmed using seven purified CYP proteins. Supplemental Figure 1 shows the ability of the
Discussion
The enzymes in the CYP26 family have been suggested to be the main RA hydroxylases in mammals and other chordates. Based on previously published RNA work, CYP26A1 appears to be the main liver CYP26 isoform [26]. CYP26B1 mRNA was not quantifiable in 46% of livers in the donor bank used in this study, and when detected, the amounts were significantly lower than CYP26A1 mRNA [27]. CYP26A1 has been previously demonstrated to be very efficient in metabolizing RA and has a high affinity to RA [19].
Acknowledgments
This work was supported in part by NIH grants T32 GM007750, P01 GM32165, and R01 GM081569. While the cytochrome P450 concentrations for CYP2C8, CYP3A4, and CYP3A5 used in the predictions for individual HLMs have been previously reported [32], [33], we thank Dr. Rheem Totah, University of Washington, Department of Medicinal Chemistry and Dr. Yvonne Lin, University of Washington, Department of Pharmaceutics for providing us a matched list of the concentrations to the specific HLM donor number so
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