20(S)-Hydroxyvitamin D3 (20(OH)D3) is an endogenous metabolite produced by the action of CYP11A1 on the side chain of vitamin D3 (D3). 20(OH)D3 can be further hydroxylated by CYP11A1, CYP27A1, CYP24A1 and/or CYP27B1 to several hydroxyderivatives. CYP11A1 also hydroxylates D3 to 22-monohydroxyvitamin D3 (22(OH)D3), which is detectable in the epidermis. 20-Hydroxy-7-dehydrocholesterol (20(OH)-7DHC) has been detected in the human epidermis and can be phototransformed into 20(OH)D3 following the absorption of ultraviolet B (UVB) energy by the B-ring. 20(OH)D3 and its hydroxyderivatives have anti-inflammatory, pro-differentiation and anti-proliferative effects, comparable to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Since cytochromes P450 with 20- or 25-hydroxylase activity are found in insects participating in ecdysone synthesis from 7-dehydrocholesterol (7DHC), we tested whether D3-hydroxyderivatives are present in honey, implying their production in bees. Honey was collected during summer in the Birmingham area of Alabama or purchased commercially and extracted and analyzed using LC-MS. We detected a clear peak of m/z = 423.324 [M + Na]+ for 20(OH)D3 corresponding to a concentration in honey of 256 ng/g. We also detected peaks of m/z = 383.331 [M + H − H2O]+ for 20(OH)-7DHC and 25(OH)D3 with retention times corresponding to the standards. We further detected species with m/z = 407.329 [M + Na]+ corresponding to the RT of 7DHC, D3 and lumisterol3 (L3). Similarly, peaks with m/z = 399.326 [M + H − H2O]+ were detected at the RT of 1,25(OH)2D3 and 1,20-dihydroxyvitamin D3 (1,20(OH)2D3). Species corresponding to 20-monohydroxylumisterol3 (20(OH)L3), 22-monohydroxyvitamin D3 (22(OH)D3), 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), 20,24/25/26-dihydroxyvitamin D3 (20,24/25/26(OH)2D3) and 1,20,23/24/25/26-trihydroxyvitamin D3 (1,20,23/24/25/26(OH)3D3) were not detectable above the background. In conclusion, the presence of 7DHC and D3 and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)2D3, 25(OH)D3 and 1,25(OH)2D3 in honey implies their production in bees, although the precise biochemistry and photochemistry of these processes remain to be defined.

No abstract

CYP11A1 and CYP27A1 hydroxylate tachysterol3, a photoproduct of previtamin D3, producing 20S‐hydroxytachysterol3 [20S(OH)T3] and 25(OH)T3, respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti‐oxidative genes in keratinocytes in a similar manner to 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3, and a minimal effect for their precursor, tachysterol3. Tachysterol3 hydroxyderivatives showed high‐affinity binding to the ligan‐binding domain (LBD) of the liver X receptor (LXR) α and β, and the peroxisome proliferator‐activated receptor γ (PPARγ) in LanthaScreen TR‐FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their natural ligands. The scores for the non‐genomic‐binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3.

Melatonin and serotonin, products of tryptophan metabolism, are endogenous neurotransmitters and hormones. We have identified and quantified these metabolites in natural honey from Australia, USA, and Poland using a Xevo G2 XS qTof LC− MS. To help ensure correct product identification, some samples were prepurified by RP-HPLC based on the retention times of standards, prior to LC−MS. The concentrations of the metabolites of interest depended on the source of the honey. For Australian honey, levels for melatonin and 2-hydroxymelatonin were 0.91 and 0.68 ng/g, respectively. Melatonin was detected in one brand of US commercial honey at 0.48 ng/g, while a second brand contained serotonin at 88.2 ng/g. In Polish natural honey, 20.6 ng/g of serotonin and 40.8 ng/g of N-acetylserotonin (NAS) were detected, while in Polish commercial honey 25.9 ng/g of serotonin and 7.30 ng/g of NAS were present. We suggest that addictive and health-related properties of honey may be in part dependent on the presence of serotonin, melatonin, and their metabolites, and that these compounds may play a role in the colony activities of bees.

We are commenting recent discoveries on the presence of L-DOPA, dopamine, 5-hydroxytryptophan, tryptamine, serotonin, N-acetylserotonin, melatonin, 2-hydroxymelatonin, AFMK and AMK in honey. Serotonin and melatonin, products of the tryptophan metabolism, are widely produced in nature, serving as hormones, neurotransmitters, biological regulators, neurotransmitters and antioxidants, in a context dependent fashion.  Dopamine and tryptamine are important neurotransmitters across different species. Honey is used as one of the most popular healthy food substances. Detection of above molecules in honey accompanied by detection of vitamin D3 and its hydroxyderivatives, is consistent with their detection in insects and plants. Their presence in honey enhances spectrum of its beneficial effects for human health and implicates that these molecules must play important role in social insects physiology, bees development and colony functions.

Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present at a concentration of 7.3±2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-GFP) from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Continuing our structure-activity studies on the vitamin D analogs with the altered intercyclic seco-B-ring fragment, we designed compounds possessing dienyne system conjugated with the benzene D ring. Analysis of the literature data and the docking experiments seemed to indicate that the target compounds could mimic the ligands with a good affinity to the vitamin D receptor (VDR). Multi-step synthesis of the C/D-ring building block of the tetralone structure was achieved and its enol triflate was coupled with the known A-ring fragments, possessing conjugated enyne moiety, using Sonogashira protocol. The structures of the final products were confirmed by NMR, UV and mass spectroscopy. Their binding affinities for the full-length human VDR were determined and it was established that compound substituted at C-2 with exomethylene group showed significant binding to the receptor. This analog was also able to induce monocytic differentiation of HL-60 cells.

No abstract