Search Results (21)

Background: Cytochromes P450 (CYPs) are heme-containing oxidoreductase enzymes with mono-oxygenase activity. Human CYPs catalyze the oxidation of a great variety of chemicals, including xenobiotics, steroid hormones, vitamins, bile acids, procarcinogens, and drugs. Findings: In our review article, we discuss recent data evidencing that the same CYP isoform can be involved in both bioactivation and detoxification reactions and convert the same substrate to different products. Conversely, different CYP isoforms can convert the same substrate, xenobiotic or procarcinogen, into either a more or less toxic product. These phenomena depend on the type of catalyzed reaction, substrate, tissue type, and biological species. Since the CYPs involved in bioactivation (CYP3A4, CYP1A1, CYP2D6, and CYP2C8) are primarily expressed in the liver, their metabolites can induce hepatotoxicity and hepatocarcinogenesis. Additionally, we discuss the role of drugs as CYP substrates, inducers, and inhibitors as well as the implication of nuclear receptors, efflux transporters, and drug–drug interactions in anticancer drug resistance. We highlight the molecular mechanisms underlying the development of hormone-sensitive cancers, including breast, ovarian, endometrial, and prostate cancers. Key players in these mechanisms are the 2,3- and 3,4-catechols of estrogens, which are formed by CYP1A1, CYP1A2, and CYP1B1. The catechols can also produce quinones, leading to the formation of toxic protein and DNA adducts that contribute to cancer progression. However, 2-hydroxy- and 4-hydroxy-estrogens and their O-methylated derivatives along with conjugated metabolites play cancer-protective roles. CYP17A1 and CYP11A1, which are involved in the biosynthesis of testosterone precursors, contribute to prostate cancer, whereas conversion of testosterone to 5α-dihydrotestosterone as well as sustained activation and mutation of the androgen receptor are implicated in metastatic castration-resistant prostate cancer (CRPC). CYP enzymatic activities are influenced by CYP gene polymorphisms, although a significant portion of them have no effects. However, CYP polymorphisms can determine poor, intermediate, rapid, and ultrarapid metabolizer genotypes, which can affect cancer and drug susceptibility. Despite limited statistically significant data, associations between CYP polymorphisms and cancer risk, tumor size, and metastatic status among various populations have been demonstrated. Conclusions: The metabolic diversity and dual character of biological effects of CYPs underlie their implications in, preliminarily, hormone-sensitive cancers. Variations in CYP activities and CYP gene polymorphisms are implicated in the interindividual variability in cancer and drug susceptibility. The development of CYP inhibitors provides options for personalized anticancer therapy. Full article

Despite the recent development of improved methods of treating melanoma such as targeted therapy, immunotherapy or combined treatment, the number of new cases worldwide is increasing. It is well known that active metabolites of vitamin D₃ and lumisterol (L₃) exert photoprotective and antiproliferative effects on the skin, while UV radiation is a major environmental risk factor for melanoma. Thus, many natural metabolites and synthetic analogs of steroidal and secosteroidal molecules have been tested on various cancer cells and in animal models. In this study, we tested the anti-melanoma properties of several natural derivatives of vitamin D₃ and L₃ in comparison to 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). A significant decrease in melanoma cell proliferation and cell mobility was observed for selected derivatives, with (25R)-27-hydroxyL₃ showing the highest potency (lowest IC50) in A375 cells but lower potency in SK-MEL-28 cells, whereas the parent L₃ failed to inhibit proliferation. The efficacy (% inhibition) by 1,24,25(OH)₃D₃ and 1,25(OH)₂D₃ were similar in both cell types. 1,25(OH)₂D₃ showed higher potency than 1,24,25(OH)₃D₃ in SK-MEL-28 cells, but lower potency in A375 cells for the inhibition of proliferation. As for 1,25(OH)₂D₃, but not the other derivatives tested, treatment of melanoma cells with 1,24,25(OH)₃D₃ markedly increased the expression of CYP24A1, enhanced translocation of the vitamin D receptor (VDR) from the cytoplasm to the nucleus and also decreased the expression of the proliferation marker Ki67. The effects of the other compounds tested were weaker and occurred only under certain conditions. Our data indicate that 1,24,25(OH)₃D₃, which has undergone the first step in 1,25(OH)₂D₃ inactivation by being hydroxylated at C24, still shows anti-melanoma properties, displaying higher potency than 1,25(OH)₂D₃ in SK-MEL-28 cells. Furthermore, hydroxylation increases the potency of some of the lumisterol hydroxy-derivatives, as in contrast to L₃, (25R)-27(OH)L₃ effectively inhibits proliferation and migration of the human malignant melanoma cell line A375. Full article

The active vitamin D metabolites, 25-hydroxyvitamin D₃ (25D₃) and 1,25-dihydroxyvitamin D₃ (1,25D₃), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D₃ is a major one. This study aims to investigate the structure–activity relationships of 4-hydroxy derivatives of 1,25D₃. Structural analysis indicates that 1,4α,25(OH)₃D₃ and 1,4β,25(OH)₃D₃ maintain the anchoring hydrogen bonds of 1,25D₃ and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D₃ and 1,4β,25D₃ are as potent as 1,25D₃ in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound. Full article

Background/Objectives: 25-hydroxy vitamin D (25-OH-D) is a fat-soluble compound that plays many essential functions, including bone formation, neuromuscular functions, and prevention of osteoporosis and inflammation. Recent data indicate that its metabolites are associated with rheumatoid arthritis (RA) progression and neuropathic pain in RA patients. We aimed to assess the effect of RA pharmacotherapy and seasonal variation on serum levels of 25-OH-D in RA patients who received treatment with methotrexate (MTX) or leflunomide (LEF) for at least one year. Methods: This study is a retrospective analysis of data collected from 101 patients with RA who received treatment for at least one year. All of them have supplemented 25-OH-D (2000 IU daily) for at least one year. Results: We observed a significant seasonal variation in 25-OH-D concentration (p = 0.004). Moreover, there were significant differences (p = 0.03) between LEF (50.63 ± 17.73 ng/mL) and MTX (34.73 ± 14.04 ng/mL) treatment groups, but only for the summer population. A correlation was observed between 25-OH-D and RA duration—once again, in the summer population (the whole group—r = −0.64; treatment subgroups—r = −0.82 for LEF and −0.61 for MTX). Deficiency of 25-OH-D (below 20 ng/mL) was confirmed in 28.7% of patients, while 18.8% had suboptimal 25-OH-D levels (20–30 ng/mL). Conclusions: Our results showed that both RA pharmacotherapy and seasonal variation affect the serum levels of 25-OH-D in patients with active RA. Full article

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH)₂D3, 1,20(OH)₂D3 and 1,20,23(OH)₃D3 were compared to those of classical 1,25(OH)₂D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR, RORs, or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)₂D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required. Full article

Background: Bipolar disorder is a chronic psychiatric disorder with depression and manic episodes. It is one of the leading causes of disease-related disability worldwide. Despite the presence of various alternative drug options for bipolar disorder, some patients do not adequately benefit from the treatment. Therefore, possible underlying mechanisms need to be clarified. Recently, studies on the relationship between bipolar disorder and vitamin D (Vit D) have attracted attention. Although many studies have found an association between depression and Vit D deficiency, little is known about the relationship between manic episodes and Vit D. The aim of this study was to compare Vit D and related metabolites of bipolar manic episodes prior to treatment, bipolar remission after treatment, and healthy control groups. Methods: This case–control study consisted of 34 bipolar manic episode patients and 34 healthy controls. Disease activity was evaluated with the Hamilton Depression Rating Scale (HAM-D) and Young Mania Rating Scale (YMRS). Firstly, serum 25-hydroxy vitamin D (25-OHD), calcium (Ca) and phosphorus (P) levels of patients in the bipolar manic episode were measured and compared with healthy control. Secondly, serum 25-OHD, Ca and P levels in the euthymic periods of the same patients were measured and compared with healthy control. Results: Bipolar manic episode Vit D levels were lower when compared to healthy controls; while there was no difference in terms of Ca and P levels. There was no significant difference between the bipolar euthymic period patients and the healthy control group in terms of 25-OHD, Ca and P levels. Conclusion: Our results demonstrated low serum Vit D concentrations in the acute manic episode of bipolar disorder. Decreased Vit D level may play a role in the onset of the manic episode, or malnutrition and insufficient sunlight during the manic episode may have caused Vit D deficiency. Future studies are needed to exclude potential confounding factors and to compare all mood episodes. Full article

Vitamin D₃ (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D₃ (6a) and 4β,25-dihydroxyvitamin D₃ (6b) are both produced from 25-hydroxyvitamin D₃ (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4β,24R,25-trihydroxyvitamin D₃ (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the β-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1. Full article

The hormonal form of vitamin D₃, 1,25(OH)₂D₃, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D₃ production in the skin, and continued UV exposure photoisomerizes pre-vitamin D₃ to produce “over-irradiation products” such as lumisterol₃ (L₃). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L₃ to produce three main derivatives: 24-hydroxy-L₃ [24(OH)L₃], 22-hydroxy-L₃ [22(OH)L₃], and 20,22-dihydroxy-L₃ [20,22(OH)L₃]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L₃, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L₃ or 1,25(OH)₂D₃ reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)₂D₃ was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L₃ provided photoprotection against UV damage similar to that of 1,25(OH)₂D₃. Full article

The role of vitamin D in atopic dermatitis (AD) is controversial. Conflicting data could be due to the use of inadequate markers for assessing vitamin D status. So far, directly measured free 25(OH)D concentrations have not been reported in AD patients. Ten adults with AD were treated with narrow band ultraviolet light B (NB-UVB) for 10–12 weeks. SCORing atopic dermatitis (SCORAD) and the visual analogue scale (VAS) were used to assess disease severity before and after NB-UVB therapy. Total and free 25(OH)D and 1,25(OH)₂D serum levels were analyzed before and after treatment. Free 25(OH)D concentrations were measured with a two-step immunosorbent assay (ELISA). The majority of patients had sufficient levels of 25(OH)D before treatment (mean 76.4 nmol/L). Mean free 25(OH)D was 11.9 pmol/L and mean 1,25(OH)₂D was 108.9 pmol/L. Median SCORAD decreased from 37.1 to 19.8 and VAS improved significantly after phototherapy. Total and free 25(OH)D increased in all subjects. No correlations between disease severity and vitamin D levels were found. There was no correlation between total and free 25(OH)D levels. Larger studies are needed to test the applicability of the free hormone hypothesis in AD pathogenesis. Full article

Vitamin D deficiency is associated with a higher risk of SARS-CoV-2 infection and poor outcomes of the COVID-19 disease. However, a satisfactory mechanism explaining the vitamin D protective effects is missing. Based on the anti-inflammatory and anti-oxidative properties of classical and novel (CYP11A1-derived) vitamin D and lumisterol hydroxymetabolites, we have proposed that they would attenuate the self-amplifying damage in lungs and other organs through mechanisms initiated by interactions with corresponding nuclear receptors. These include the VDR mediated inhibition of NFκβ, inverse agonism on RORγ and the inhibition of ROS through activation of NRF2-dependent pathways. In addition, the non-receptor mediated actions of vitamin D and related lumisterol hydroxymetabolites would include interactions with the active sites of SARS-CoV-2 transcription machinery enzymes (M^pro;main protease and RdRp;RNA dependent RNA polymerase). Furthermore, these metabolites could interfere with the binding of SARS-CoV-2 RBD with ACE2 by interacting with ACE2 and TMPRSS2. These interactions can cause the conformational and dynamical motion changes in TMPRSS2, which would affect TMPRSS2 to prime SARS-CoV-2 spike proteins. Therefore, novel, CYP11A1-derived, active forms of vitamin D and lumisterol can restrain COVID-19 through both nuclear receptor-dependent and independent mechanisms, which identify them as excellent candidates for antiviral drug research and for the educated use of their precursors as nutrients or supplements in the prevention and attenuation of the COVID-19 disease. Full article

β-Hydroxy-β-methylbutyrate (HMB), a leucine metabolite, can increase skeletal muscle size and function. However, HMB may be less effective at improving muscle function in people with insufficient Vitamin D₃ (25-OH-D < 30 ng/mL) which is common in middle-aged and older adults. Therefore, we tested the hypothesis that combining HMB plus Vitamin D₃ (HMB + D) supplementation would improve skeletal muscle size, composition, and function in middle-aged women. In a double-blinded fashion, women (53 ± 1 yrs, 26 ± 1 kg/m², n = 43) were randomized to take placebo or HMB + D (3 g Calcium HMB + 2000 IU D per day) during 12 weeks of sedentary behavior (SED) or resistance exercise training (RET). On average, participants entered the study Vitamin D₃ insufficient while HMB + D increased 25-OH-D to sufficient levels after 8 and 12 weeks. In SED, HMB + D prevented the loss of arm lean mass observed with placebo. HMB + D increased muscle volume and decreased intermuscular adipose tissue (IMAT) volume in the thigh compared to placebo but did not change muscle function. In RET, 12-weeks of HMB + D decreased IMAT compared to placebo but did not influence the increase in skeletal muscle volume or function. In summary, HMB + D decreased IMAT independent of exercise status and may prevent the loss or increase muscle size in a small cohort of sedentary middle-aged women. These results lend support to conduct a longer duration study with greater sample size to determine the validity of the observed positive effects of HMB + D on IMAT and skeletal muscle in a small cohort of middle-aged women. Full article

In humans, most free tryptophan is degraded via kynurenine pathways into kynurenines. Kynurenines modulate the immune system, central nervous system, and skeletal muscle bioenergetics. Consequently, kynurenine pathway metabolites (KPMs) have been studied in the context of exercise. However, the effect of vitamin D supplementation on exercise-induced changes in KPMs has not been investigated. Here, we analyzed the effect of a single high-dose vitamin D supplementation on KPMs and tryptophan levels in runners after an ultramarathon. In the study, 35 amateur runners were assigned into two groups: vitamin D supplementation group, administered 150,000 IU vitamin D in vegetable oil 24 h before the run (n = 16); and control (placebo) group (n = 19). Blood was collected for analysis 24 h before, immediately after, and 24 h after the run. Kynurenic, xanthurenic, quinolinic, and picolinic acids levels were significantly increased after the run in the control group, but the effect was blunted by vitamin D supplementation. Conversely, the decrease in serum tryptophan, tyrosine, and phenylalanine levels immediately after the run was more pronounced in the supplemented group than in the control. The 3-hydroxy-l-kynurenine levels were significantly increased in both groups after the run. We conclude that vitamin D supplementation affects ultramarathon-induced changes in tryptophan metabolism. Full article

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages. Full article

Introduction. Chronic low-grade inflammation is a characteristic of women with polycystic ovary syndrome (PCOS), although this may be obesity-driven rather than an intrinsic facet of PCOS; furthermore, vitamin D deficiency, another common feature of PCOS, is reported to have an association with increased inflammation. Therefore, circulating inflammatory protein levels and circulating levels of vitamin D may be linked in PCOS, though it is unclear which vitamin D metabolites may be important. Methods. We measured plasma levels of 24 inflammatory proteins and 12 matrix metalloproteinases (proteins modulated by the inflammatory process) by slow off-rate modified aptamer (SOMA)-scan plasma protein measurement in weight and aged-matched non-obese non-insulin resistant PCOS (n = 24) and control (n = 24) women. Inflammatory proteins and matrix metalloproteinases were correlated to 25-hydroxy vitamin D₃ (25(OH)D₃), its epimer 25-hydroxy-3epi-vitamin D (3epi25(OH)D) and the active 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) as measured by gold standard isotope-dilution liquid chromatography tandem mass spectrometry. Results. PCOS women had both an elevated free androgen index and circulating anti-mullerian hormone, though insulin resistance was comparable to controls. C-reactive protein, as a standard circulatory marker of inflammation, was comparable between cohorts. Levels of circulating inflammatory proteins and matrix metalloproteinases were not different between the PCOS and control women, with no correlation of 25(OH)D_3, 1,25(OH)₂D₃ or 3epi25(OH)D with any of the inflammatory proteins. Conclusion. In a non-obese PCOS population matched for age and insulin resistance, circulating inflammatory proteins and matrix metalloproteinases were not elevated and did not correlate with 25(OH)D_3, its epimer 3epi25(OH)D or 1,25(OH)₂D₃ in either control or PCOS women, indicating that the inflammatory response is absent and the vitamin D-metabolite independent in non-obese women with PCOS. Full article

In the life sciences, automation solutions are primarily established in the field of drug discovery. However, there is also an increasing need for automated solutions in the field of medical diagnostics, e.g., for the determination of vitamins, medication or drug abuse. While the actual metrological determination is highly automated today, the necessary sample preparation processes are still mainly carried out manually. In the laboratory, flexible solutions are required that can be used to determine different target substances in different matrices. A suitable system based on an automated liquid handler was implemented. It has been tested and validated for the determination of three cannabinoid metabolites in blood, urine and saliva. To extract Δ9-tetrahydrocannabinol-D₃ (Δ9-THC-D₃), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) from serum, urine and saliva both rapidly and cost-effectively, three sample preparation methods automated with a liquid handling robot are presented in this article, the basic framework of which is an identical SPE method so that they can be quickly exchanged against each other when the matrix is changed. If necessary, the three matrices could also be prepared in parallel. For the sensitive detection of analytes, protein precipitation is used when preparing serum before SPE and basic hydrolysis is used for urine to cleave the glucuronide conjugate. Recoveries of developed methods are >77%. Coefficients of variation are <4%. LODs are below 1 ng/mL and a comparison with the manual process shows a significant cost reduction. Full article