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Apical-out enteroids mimic the in vivoenvironment well due to their accessible apical surface and mucus layer, making them an ideal model for studying the impact of (bioactive) food compounds. Generated human ileal apical-out enteroids showed a fucose-containing mucus layer surrounding the apical brush border on their exposure side, indicating their physiological relevance. Effects on the mucosal epithelium of antibacterial prenylated phenolics (glabridin, licochalcone A, and glycycoumarin) from licorice roots were investigated for cytotoxicity, cell viability, barrier integrity, and biotransformation. At concentrations up to 500 μg mL–1, licochalcone A and glycycoumarin did not significantly affect apical-out enteroids, with cytotoxicities of −6 ± 2 and −2 ± 2% and cell viabilities of 77 ± 22 and 77 ± 13%, respectively (p> 0.05). Conversely, 500 μg mL–1glabridin induced significant cytotoxicity (31 ± 25%, p< 0.05) and reduced cell viability (21 ± 14%, p< 0.01). Apical-out enteroids revealed differential sensitivities to prenylated phenolics not observed in apical-in enteroids and Caco-2 cells. Both enteroid models showed phase II biotransformation but differed in the extent of glucuronide conversion. The apical mucus layer of apical-out enteroids likely contributed to these differential interactions, potentially due to differences in electrostatic repulsion. This study underscores the relevance of 3D apical-out enteroid models and highlights the promise of prenylated phenolics for antimicrobial applications.

Cachexia‐anorexia syndrome is a complex metabolic condition characterized by skeletal muscle wasting, reduced food intake and prominent involvement of systemic and central inflammation. Here, the gut barrier function was investigated in pancreatic cancer‐induced cachexia mouse models by relating intestinal permeability to the degree of cachexia. We further investigated the involvement of the gut–brain axis and the crosstalk between tumour, gut and hypothalamus in vitro. Two distinct mouse models of pancreatic cancer cachexia (KPC and 4662) were used. Intestinal inflammation and permeability were assessed through fluorescein isothiocyanate dextran (FITC‐dextran) and lipopolysaccharide (LPS), and hypothalamic and systemic inflammation through mRNA expression and plasma cytokines, respectively. To simulate the tumour–gut–brain crosstalk, hypothalamic (HypoE‐N46) cells were incubated with cachexia‐inducing tumour secretomes and LPS. A synthetic mimic of C26 secretome was produced based on its secreted inflammatory mediators. Each component of the mimic was systematically omitted to narrow down the key mediator(s) with an amplifying inflammation. To substantiate its contribution, cyclooxygenase‐2 (COX‐2) inhibitor was used. In vivo experiments showed FITC‐dextran was enhanced in the KPC group (362.3 vs. sham 111.4 ng/mL, P< 0.001). LPS was increased to 140.9 ng/mL in the KPC group, compared with sham and 4662 groups (115.8 and 115.8 ng/mL, P< 0.05). Hypothalamic inflammatory gene expression of Ccl2was up‐regulated in the KPC group (6.3 vs. sham 1, P< 0.0001, 4662 1.3, P< 0.001), which significantly correlated with LPS concentration (r= 0.4948, P= 0.0226). These data suggest that intestinal permeability is positively related to the cachexic degree. Prostaglandin E2 (PGE2) was confirmed to be present in the plasma and PGE2 concentration (log10) in the KPC group was much higher than in 4662 group (1.85 and 0.56 ng/mL, P< 0.001), indicating a role for PGE2 in pancreatic cancer‐induced cachexia. Parallel to in vivo findings, in vitro experiments revealed that the cachexia‐inducing tumour secretomes (C26, LLC, KPC and 4662) amplified LPS‐induced hypothalamic IL‐6 secretion (419%, 321%, 294%, 160%). COX‐2 inhibitor to the tumour cells reduced PGE2 content (from 105to 102pg/mL) in the secretomes and eliminated the amplified hypothalamic IL‐6 production. Moreover, results could be reproduced by addition of PGE2 alone, indicating that the increased hypothalamic inflammation is directly related to the PGE2 from tumour. PGE2 secreted by the tumour may play a role in amplifying the effects of bacteria‐derived LPS on the inflammatory hypothalamic response. The cachexia‐inducing potential of tumour mice models parallels the loss of intestinal barrier function. Tumour‐derived PGE2 might play a key role in cancer‐related cachexia‐anorexia syndrome via tumour–gut–brain crosstalk.

Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.

Gamma-aminobutyric acid (GABA) is mainly known as an endogenously produced neurotransmitter. However, GABA intake from dietary sources like tomatoes and fermented foods can be considerable. Studies in rodent models have shown beneficial effects of oral GABA supplementation on glucose homeostasis and cardiovascular health. Still, it is currently unknown whether oral GABA supplementation produces cardiometabolic benefits in humans.

17α-Boldenone (17α-BOL) and/or 17β-boldenone (17β-BOL) appear occasionally in fecal matter of cattle. In addition to 17α-BOL, a whole array of boldenone related substances can be found in the same samples. In vitro experiments with microsomal liver preparations and isolated hepatocytes combined with the excretion profiles found in urine and feces samples of in vivo experiments made it possible to identify several metabolites of 17β-BOL in 17β-BOL positive feces samples. In one animal treated with 17β-BOL, no 17β-BOL or its metabolites were present before treatment and most of these compounds disappeared gradually in time after the treatment was stopped. It is not clear what the origin is of 17α-BOL and boldenone metabolites in samples screened routinely for the abuse of anabolic steroids and considered to be ‘negative’ because of the absence of 17β-BOL since other workers showed some evidence that 17α-BOL can be of endogenous origin. However, in our hands, most of these 17α-BOL positive samples, obtained during routinely performed screenings of cattle, contained large amounts of Δ4-androstene-3,17-dione (AED), which normally is absent from routinely screened negative samples. Furthermore, AED was absent in all samples obtained from the animals treated with 17β-BOL. We have no direct evidence that 17α-BOL or 17β-BOL is of endogenous origin.

The purpose of this review is to illustrate the expanding view of the endocannabinoid system (ECS) in relation to its roles in inflammation.

The selective synthesis of unsaturated N-acylethanolamines 1b-6b by lipase-catalyzed direct condensation between unsaturated fatty acids 1a-6a and ethanolamine is reported. Reactions were carried out in hexane at 40 °C, in the presence of Candida antarctica Lipase B as the catalyst, to give the corresponding amides 1b-6b with yields ranging from 80 to 88%.

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 μg mL–1lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.

β2-Adrenoceptor agonists (β-agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional β-agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a β2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 ± 100 fmol mg⁻¹ of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different β-agonists, with IC₅₀ values ranging from 2.1 ± 0.76 × 10⁻⁷ M for salmeterol to 1.1 ± 0.62 × 10⁻⁵ M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of β2-adrenoceptors are a convenient source of active receptor material. Solubilised β2-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

Screening for the presence of anabolic growth promoters in urine samples from cattle grown for meat production can be performed by (semi)quantitative methods such as immuno-, receptor- or cell-based assays or by quantitative methods with mass spectrometric detection which can also include confirmation of compounds. In this study conventional immunoassays used at two different institutes [Veterinary Sciences Division (VSD) in Northern Ireland and TNO Nutrition and Food Research Institute (TNO) in The Netherlands] were compared with the oestrogen radioreceptor assay (ORRA), with GC-MS as the reference method. Urine samples were generated by treating calves (n = 2 per group) intramuscularly with ethynyloestradiol (EE2), diethylstilbestrol (DES) or α-zearalanol (zeranol, ZER). Urine samples were collected up to 21 d after administration of the oestrogenic compounds. Samples were screened by enzyme immunoassay or radioimmunoassay and by the ORRA and also by GC-MS. Values found by VSD were lower by a factor of 1–20 than those measured by TNO. These differences could be explained by differences in sample clean-up (immunoaffinity chromatography versus solid-phase extraction) and by differences in cross-reactivities between the antisera used. The ORRA and GC-MS showed similar results for EE2 and DES, but produced lower results (by a factor of ca. 3) for ZER owing to the relatively low affinity of ZER for the oestrogen receptor. The most important finding was that the withdrawal period for calves treated with EE2, DES or ZER was similar for each of the screening methods used. Therefore, it is concluded that the choice of screening method does not affect the probability of finding a positive sample.

Proinflammatory cytokines play an important role in the depression of cytochrome P450 (CYP450)-dependent drug metabolism in mammals during inflammation and infection. Although much has been learned concerning the effects and mechanisms of cytokine-mediated suppression of CYP450, there is limited knowledge about how cytokines affect UDP glucuronosyl transferases (UDPGT). The aim of the present study was to investigate the effects and dose dependency of recombinant human proinflammatory cytokines on both CYP450- and UDPGT-dependent enzyme activities in primary cultures of pig hepatocytes. A possible role of nitric oxide in cytokine-induced suppression of enzyme activities was studied by incubating hepatocytes in the presence ofNG-nitro-L-arginine (L-NAME), a competitive inhibitor of nitric oxide (NO) biosynthesis. Incubation of hepatocytes with interleukin-1α (IL-1α), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) decreased both oxidation and glucuronidation activities dose dependently, in which the effects on glucuronidation activities were even more pronounced. IL-6 differed from IL-1α and TNF-α by inhibiting CYP450 and UDPGT more effectively after 24 hr of incubation, whereas the inhibition by IL-1α and TNF-α was more pronounced after 12 hr. Only at a concentration of 500 U/ml did interferon-γ (IFN-γ) inhibit CYP450 and UDPGT. The inhibition of CYP450 enzyme activities by cytokines was probably not due to the production of NO, because L-NAME totally blocked NO production but had no effect on the cytokine-induced suppression of CYP450 enzyme activities. However, there might be a role for NO in the decrease of glucuronidation by cytokines, as L-NAME slightly though significantly prevented the inhibition of glucuronidation.

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Introduction: Studies suggest a role of vitamin D in the progression and symptomatology of Alzheimer's disease (AD), with few in vitro studies pointing to effects on serotonergic and amyloidogenic turnover. However, limited data exist in AD patients on the potential association with cognition and behavioral and psychological signs and symptoms of dementia (BPSD). In this retrospective cross-sectional study, we, therefore, explored potential correlations of serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations, indicative of vitamin D status, with serum serotonin (5-hydroxytryptamine, 5-HT) levels, cognitive/BPSD scorings, and cerebrospinal fluid (CSF) biomarker levels., Methods: Frozen serum samples of 25 well-characterized AD subjects as part of a previous BPSD cohort were analyzed, of which 15 had a neuropathologically confirmed diagnosis. Serum 25(OH)D3 levels were analyzed by means of LC-MS/MS, whereas 5-HT concentrations were quantified by competitive ELISA., Results: Among AD patients, vitamin D deficiency was highly prevalent, defined as levels below 50 nmol/L. Regression analyses, adjusted for age, gender, and psychotropic medications, revealed that serum 25(OH)D3 and 5-HT levels were positively associated (p = 0.012). Furthermore, serum 25(OH)D3 concentrations correlated inversely with CSF amyloid-beta (Aβ1-42) levels (p = 0.006), and serum 5-HT levels correlated positively with aggressiveness (p = 0.001), frontal behavior (p = 0.001), depression (p = 0.004), and partly with cognitive performance (p &lt; 0.005). Lastly, AD patients on cholinesterase inhibitors had higher serum 25(OH)D3 (p = 0.030) and lower serum 5-HT (p = 0.012) levels., Conclusions: The molecular associations between low vitamin D status, serum 5-HT, and CSF Aβ1-42 levels are highly remarkable, warranting further mechanistic and intervention studies to disclose potential involvement in the clinico-biobehavioral pathophysiology of AD., (© 2023 The Author(s). Published by S. Karger AG, Basel.)