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Vitamin A toxicity possibly mediated by stereoisomer of metabolite 4-hydroxyretinoic acid
I found an inconsistency while comparing gene expression in liver biopsies from nonalcoholic fatty liver disease (NAFLD) patients by two groups, (Wruck and Adjaye 2017) (Group A) and (Hoang, Oseini et al. 2019) (Group B). Cytochrome P450 Family 26 Subfamily A Member 1 (CYP26A1) and CYP26 Subfamily B Member 1 (CYP26B1) were found to be oppositely regulated, as shown in Table 1. In Group A, CYP26A1 was downregulated (-0.93 log2 fold change) and CYP26B1 was unaffected (-0.003 log2 FC), whereas in Group B CYP26A1 was unaffected (0.066 log2 FC) and CYP26B1 was downregulated (-0.638 log2 FC).
CYP26A1 catalyzes the formation of (4R)-hydroxyretinoic acid ((4R)-OH-RA) from all-trans-RA (atRA) and the formation of 4-oxo-RA from (4S)-OH-RA, whereas CYP26B1 catalyzes the formation of both stereoisomers of 4-OH-RA from atRA. The disease progression of Group B, in which 80% of patients had progressed to NASH, was confirmed to be more advanced than that of Group A, which consisted of multiple data sets, due to modified expression of NASH biomarkers as described by (Suppli, Rigbolt et al. 2019) and shown in Table 2.
This data raises the possibility that a change in retinoic acid metabolism causes disease progression from NAFLD to NASH. The observed changes in CYP enzyme expression in NASH would lead to an accumulation of 4-oxo-RA whereas in NAFLD, (4S)-OH-RA would accumulate, and be conjugated by UGT enzymes to facilitate elimination. This is supported by the expression of adipocyte Protein 2 (FABP4), an intracellular retinoic acid-binding protein, which is highly overexpressed in NAFLD but even more in NASH (1.778 log2 FC in Group A vs 2.391 in Group B). There is also the possibility of 4-oxo-RA being preferentially transported by FABP5, which is 60% more expressed in NASH than in NAFLD (1.487 log2 FC in Group B vs. 0.812 in Group A). FABP4 was found to migrate to the nucleus in response to PPARγ ligands (which can be induced by interaction of extracellular retinol with STRA6) whereas FABP5 was found to migrate to the nucleus in response to PPARδ ligands (Schug, Berry et al. 2007).
It should be considered that (4R)-OH-RA and (4S)-OH-RA may have evolved to have opposite biological effects, since the cholesterol precursor (S)-2,3-epoxysqualene is a substrate for lanosterol synthase whereas (R)-2,3-epoxysqualene is an inhibitor of lanosterol synthase. Some proposed metabolic pathways for atRA are shown in Figure 1, adapted from (Foti 2016) and (Shimshoni, Roberts et al. 2012).
Even though 4-oxo-RA is less lipophilic than (4R)-OH-RA, its TPSA is lower with 54.53 Å2 compared to 57.53 Å2. 4-oxo-RA was reported to be a highly active modulator of positional specification (Pijnappel, Hendriks et al. 1993) and a potent in vivo inducer of proliferation (Gaemers, van Pelt et al. 1996). The β-glucuronide of (4S)-OH-RA was predicted to interact with 9 targets with 11% probability, notably GLI1 but not RARG and HSD11B2, whereas retinoyl-β-glucuronide was predicted to interact with 11 targets with 12% probability, notably RARG and HSD11B2 but not GLI1. GLI1 is a downstream transcription factor in the sonic hedgehog signaling pathway that is inhibited by citral, and overexpressed in NAFLD.
Possible therapeutics would therefore include selective CYP26B1 inhibitors, which would attenuate (4R)-OH-RA formation and promote atRA elimination. Adding more support to the theory, the selective CYP26B1 inhibitor DX314 was recently characterized by (Veit, De Glas et al. 2021), who note that they “unexpectedly discovered that DX314, but not all-trans-RA or previous RA metabolism blocking agents, appears to protect epidermal barrier integrity. In addition, DX314-induced keratinization and epidermal proliferation effects are observed in a rhino mice model.”
Bibliography
Foti, R. (2016). Characterization of xenobiotic substrates and inhibitors of CYP26A1, CYP26B1 and CYP26C1 using computational modeling and in vitro analyses.
Gaemers, I. C., A. M. van Pelt, P. T. van der Saag and D. G. de Rooij (1996). "All-trans-4-oxo-retinoic acid: a potent inducer of in vivo proliferation of growth-arrested A spermatogonia in the vitamin A-deficient mouse testis." Endocrinology 137(2): 479-485.
Hoang, S. A., A. Oseini, R. E. Feaver, B. K. Cole, A. Asgharpour, R. Vincent, M. Siddiqui, M. J. Lawson, N. C. Day, J. M. Taylor, B. R. Wamhoff, F. Mirshahi, M. J. Contos, M. Idowu and A. J. Sanyal (2019). "Gene Expression Predicts Histological Severity and Reveals Distinct Molecular Profiles of Nonalcoholic Fatty Liver Disease." Scientific Reports 9(1): 12541.
Pijnappel, W. W., H. F. Hendriks, G. E. Folkers, C. E. van den Brink, E. J. Dekker, C. Edelenbosch, P. T. van der Saag and A. J. Durston (1993). "The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification." Nature 366(6453): 340-344.
Schug, T. T., D. C. Berry, N. S. Shaw, S. N. Travis and N. Noy (2007). "Opposing Effects of Retinoic Acid on Cell Growth Result from Alternate Activation of Two Different Nuclear Receptors." Cell 129(4): 723-733.
Shimshoni, J. A., A. G. Roberts, M. Scian, A. R. Topletz, S. A. Blankert, J. R. Halpert, W. L. Nelson and N. Isoherranen (2012). "Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes." The Journal of biological chemistry 287(50): 42223-42232.
Suppli, M. P., K. T. G. Rigbolt, S. S. Veidal, S. Heebøll, P. L. Eriksen, M. Demant, J. I. Bagger, J. C. Nielsen, D. Oró, S. W. Thrane, A. Lund, C. Strandberg, M. J. Kønig, T. Vilsbøll, N. Vrang, K. L. Thomsen, H. Grønbæk, J. Jelsing, H. H. Hansen and F. K. Knop (2019). "Hepatic transcriptome signatures in patients with varying degrees of nonalcoholic fatty liver disease compared with healthy normal-weight individuals." American Journal of Physiology-Gastrointestinal and Liver Physiology 316(4): G462-G472.
Veit, J. G. S., V. De Glas, B. Balau, H. Liu, F. Bourlond, A. S. Paller, Y. Poumay and P. Diaz (2021). "Characterization of CYP26B1-Selective Inhibitor, DX314, as a Potential Therapeutic for Keratinization Disorders." Journal of Investigative Dermatology 141(1): 72-83.e76.
Wruck, W. and J. Adjaye (2017). "Meta-analysis reveals up-regulation of cholesterol processes in non-alcoholic and down-regulation in alcoholic fatty liver disease." World J Hepatol 9(8): 443-454.
As usual, my interpretation was a bit oversimplified. 4-oxo-RA seems to be equally potent or more potent than atRA, but the pathway from atRA to 4-oxo-RA has not yet been discovered. What is known is that
- 4-oxo-RA slowly gets reduced back to racemic 4-OH-RA in human liver microsomes, but the enzyme catalyzing this reaction is unknown (maybe AKR1B10?)
- (4S)-OH-RA is rapidly metabolized to 4-oxo-RA by human liver microsomes, whereas metabolization of (4R)-OH-RA was about 50% slower and resulted in 50% less formation of 4-oxo-RA (Topletz, Tripathy et al. 2015)
- Three metabolites of 18-OH-RA by CYP26A1 and CYP26B1 have been detected but not yet been identified, a dihydroxyretinoic acid, another novel substance and a ketone, possibly 18-oxo-4-OH-RA (Topletz, Thatcher et al. 2012)
- One explanation could be (assuming 18-OH-RA is metabolized like 4-OH-RA) that the orange spike identified as 4,18-(OH)2-RA is really (4R),18-(OH)2-RA, and the red spike next to it is (4S),18-(OH)2-RA, which would leave 16,18-(OH)2-RA for the remaining spike, but that is pure speculation
- A third metabolite of 4-oxo-RA apart from 4-oxo-16-OH-RA and 4-oxo-18-OH-RA has been detected but not identified
- CYP26C1, which is normally regulated in NAFLD (no data for NASH), was found to preferentially form (4R)-OH-RA from atRA (Zhong, Ortiz et al. 2018)
- It seems to me that CYP26B1 is more involved in embryogenesis, since CYP26B1 null male mice did not form sperm, and expression of CYP26B1 decreases after gestation. In adults, CYP26A1 possibly prevents RA elimination due to preferential formation of (4S)-OH-RA whereas CYP26C1 possibly facilitates RA elimination through preferential formation of (4R)-OH-RA, even though the pathway is yet to be discovered. Also interesting is that CYP26C1 shows higher affinity and clearance for 9-cis-RA (which is more similar to the endogenous RXRB ligand 9-cis-13,14-dihydroretinoic acid) than for atRA.
- UGT2B7 shows a higher preference for glucuronidation of 4-OH-RA, 5,6-epoxy-RA (probably not a metabolic product of atRA, it is more likely formed during the degradation of cell membranes by atRA) and atRA than for glucuronidation of 4-oxo-RA (Samokyszyn, Gall et al. 2000)
- Either (4R),16-dihydroxy-RA or (4S),16-dihydroxy-RA may get metabolized by CYP19A1 via formation of a benzene ring, which would produce a molecule similar to the synthetic retinoid acitretin, but this has not been tested or even suggested in literature
- Even though (4R) and not (4S)-OH-RA seems to be responsible for RA clearance, CYP26B1 inhibitors could still have some benefits due to decreased production of (4S)-OH-RA. Additionally, CYP26A1 expression is induced directly by retinoic acid, and inhibition of CYP26B1 by DX314 led to a 222x increase in CYP26A1 mRNA expression (Veit, De Glas et al. 2021).
I’ve updated the metabolic pathway graphic below (if it’s too small to read try downloading it). All in all the current state of research is pretty unsatisfying and I probably won’t be spending much more time on the CYP26 enzymes because there just isn’t enough data available.
Bibliography
Samokyszyn, V. M., W. E. Gall, G. Zawada, M. A. Freyaldenhoven, G. Chen, P. I. Mackenzie, T. R. Tephly and A. Radominska-Pandya (2000). "4-Hydroxyretinoic Acid, a Novel Substrate for Human Liver Microsomal UDP-glucuronosyltransferase(s) and Recombinant UGT2B7 *." Journal of Biological Chemistry 275(10): 6908-6914.
Topletz, A. R., J. E. Thatcher, A. Zelter, J. D. Lutz, S. Tay, W. L. Nelson and N. Isoherranen (2012). "Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases." Biochemical pharmacology 83(1): 149-163.
Topletz, A. R., S. Tripathy, R. S. Foti, J. A. Shimshoni, W. L. Nelson and N. Isoherranen (2015). "Induction of CYP26A1 by metabolites of retinoic acid: evidence that CYP26A1 is an important enzyme in the elimination of active retinoids." Molecular pharmacology 87(3): 430-441.
Veit, J. G. S., V. De Glas, B. Balau, H. Liu, F. Bourlond, A. S. Paller, Y. Poumay and P. Diaz (2021). "Characterization of CYP26B1-Selective Inhibitor, DX314, as a Potential Therapeutic for Keratinization Disorders." Journal of Investigative Dermatology 141(1): 72-83.e76.
Zhong, G., D. Ortiz, A. Zelter, A. Nath and N. Isoherranen (2018). "CYP26C1 Is a Hydroxylase of Multiple Active Retinoids and Interacts with Cellular Retinoic Acid Binding Proteins." Molecular Pharmacology 93(5): 489-503.