Potential (co-)contamination of dairy milk with AFM1 and MC-LR and their synergistic interaction in inducing mitochondrial dysfunction in HepG2 cells.

Several toxic metabolites, such as aflatoxin M1 (AFM1), are known to contaminate dairy milk. However, as mentioned in an external EFSA report, there is a knowledge gap regarding the carry-over of certain emerging toxins such as microcystin-LR (MC-LR). Therefore, this work aimed to develop an LC-MS/MS method for MC-LR quantification in dairy milk. Also, the method included AFM1 as a common fungal metabolite and applied to analyze 113 dairy milk samples collected directly after the end of the summer peak. Both toxins were below their LODs, keeping the question on MC-LR carry-over still unanswered. Moreover, an in silico analysis, using a 3D molecular modeling was performed, pointing to a possible interaction between MC-LR and milk proteins, especially ß-lactoglobulin. Since AFM1 and MC-LR are hepatotoxic, their interaction in inducing mitochondrial dysfunction in HepG2 cells was investigated at low (subcytotoxic) concentrations. Live cell imaging-based assays showed an inhibition in cell viability, without involvement of caspase-3/7, and a hyperpolarization in the mitochondrial membrane potential after the exposure to a mixture of 100 ng mL-1 AFM1 and 1000 ng mL-1 MC-LR for 48h. Extracellular flux analysis revealed inhibitions of several key parameters of mitochondrial function (basal respiration, ATP-linked respiration, and spare respiratory capacity).

Quality Control and Safety Assessment of Online-Purchased Food Supplements Containing Red Yeast Rice (RYR).

Dietary supplements containing red yeast rice (RYR), a fermentation product of the fungus Monascus purpureus grown on white rice, remain popular in Europe as proclaimed cholesterol-lowering aids. The cholesterol-lowering effects are due to the occurrence of monacolin K, which is often present as a mixture of monacolin K lactone (MK) and as monacolin K hydroxy acid (MKA). MK is structurally similar to the cholesterol-lowering medicine lovastatin. Recently, due to safety concerns linked to the use of statins, the European Commission prohibited RYR supplements with a maximum serving exceeding 3 mg of total monacolins per day. Moreover, the amount of the mycotoxin citrinin, potentially produced by M. purpureus, was also reduced to 100 µg/kg. Evidently, manufacturers that offer their products on the European market, including the online market, must also be compliant with these limits in order to guarantee the safety of their products. Therefore, thirty-five different RYR supplements, purchased from an EU-bound e-commerce platform or from registered online pharmacies, were screened for their compliance to the European legislation for citrinin content and the amount of total monacolin K. This was conducted by means of a newly developed LC-MS/MS methodology that was validated according to ISO 17025. Moreover, these supplements were also screened for possible adulteration and any contamination by micro-organisms and/or mycotoxins. It was found that at least four of the thirty-five RYR supplements (≈11%) might have reason for concern for the safety of the consumer either due to high total monacolin K concentrations exceeding the European predefined limits for total monacolins or severe bacterial contamination. Moreover, three samples (≈9%) were likely adulterated, and the labeling of six of the seventeen samples (≈35%) originating from an EU-based e-commerce platform was not compliant, as either the mandatory warning was missing or incomplete or the total amount of monacolins was not mentioned.

Experimental accumulation and depuration kinetics and natural occurrence of microcystin-LR in basil (Ocimum basilicum L.).

Microcystin-LR (MC-LR) is a hepatotoxic metabolite that naturally occurs during some cyanobacterial blooms in eutrophic waterbodies, and irrigation of edible plants with MC-LR-contaminated water causes bioaccumulation of the toxin. However, sufficient information about accumulation and depuration mechanics in hydroculture-grown herb plants is still lacking. This work aimed at 1) investigating bioaccumulation and depuration of MC-LR in basil, 2) verifying the possible MC-LR detoxification mechanisms in the plant, and 3) detecting the natural occurrence of MC-LR in basil (n = 50) collected from the Belgian market. Basil plants grown in a hydroculture were exposed to MC-LR (5, 20, and 50 µg L-1) spiked in a Hoagland solution for seven days. MC-LR depuration was also studied by transferring the plants to a non-contaminated Hoagland solution after exposure to MC-LR for another seven days. MC-LR concentrations in Hoagland solution, basil leaves, and roots were quantified using a validated UHPLC-MS/MS method. In addition, ELISA and LC-HRMS (only basil leaves) were used for confirmation. The results showed an increase in the accumulated levels of MC-LR at higher exposure doses, with higher MC-LR levels in roots than in leaves for all the treatment conditions. For MC-LR depuration, significant reductions were observed in all the treatment conditions for roots only. No MC-LR conjugates, potentially related to metabolism, were detected by LC-HRMS. Finally, MC-LR was detected in one store-bought basil sample, representing the first occurrence of cyanotoxins in an edible crop from Belgium.

Development, validation, and application of a multimatrix UHPLC-MS/MS method for quantification of Datura-type alkaloids in food.

A quantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of tropane alkaloids (TAs), atropine and scopolamine, in a variety of food products. The sample preparation of cereal-based food, oilseeds, honey, and pulses consisted of a solid-liquid extraction with an acidified mixture of methanol and water, while an additional step of solid-phase extraction on a cation-exchange sorbent was introduced in the treatment of teas and herbal infusions, aromatic herbs, spices and food supplements. The limits of quantification of the method varied from 0.5 to 2.5 µg kg-1. Apparent recovery was in the range of 70-120%, and repeatability and intermediate precision were below 20%. The method was successfully applied in a proficiency testing exercise as well as in the analysis of various commercial foods. Only 26% of the analysed food samples contained one or both TAs. The mean concentrations for atropine and scopolamine amounted to 21.9 and 6.5 µg kg-1, respectively, while the maximum concentrations were 523.3 and 131.4 µg kg-1, respectively. Overall, the highest levels of TA sum were found in an herbal infusion of fennel and a spice mix containing fennel and anise seeds.

Analysis of Patulin in Apple Products Marketed in Belgium: Intra-Laboratory Validation Study and Occurrence.

Apple and apple derivatives (e.g., juices, puree) are the most important foodstuffs contaminated with patulin (PAT) in the human diet. To routinely monitor these foodstuffs and ensure that the PAT levels are below the maximum permitted levels, a method using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) has been developed. Afterwards, the method was successfully validated, reaching quantification limits of 1.2 µg/L for apple juice and cider, and 2.1 µg/kg for puree. Recovery experiments were performed with samples fortified with PAT in the range of 25-75 µg/L for juice/cider and 25-75 µg/kg for puree. The results show overall average recovery rates of 85% (RSDr = 13.1%) and 86% (RSDr = 2.6%) with maximum extended uncertainty (Umax, k = 2) of 34 and 35% for apple juice/cider and puree, respectively. Next, the validated method was applied to 103 juices, 42 purees and 10 ciders purchased on the Belgian market in 2021. PAT was not found in the cider samples, but it was present in 54.4% of the tested apple juices (up to 191.1 µg/L) and 7.1% of the puree samples (up to 35.9 µg/kg). When comparing the results to the maximum levels set by Regulation EC n° 1881/2006 (i.e., 50 µg/L for juices and 25 µg/kg for puree for adults, and 10 µg/kg for infants and young children), exceedances were observed in five apple juices and one puree sample, for infants and young children. Using these data, a potential risk assessment for consumers can be suggested, and it is found that the quality of apple juices and purees sold in Belgium needs further regular surveillance.

Validation of a Targeted LC-MS/MS Method for Cereulide and Application in Food and Faeces.

Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of 13C6-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg-1 for food and faeces, respectively. The linearity of the method was very good, with calculated R2 values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%-120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC-MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g-1 for food and between 6.32 and 773.37 ng g-1 for faecal samples.

LC-MS/MS Validation and Quantification of Cyanotoxins in Algal Food Supplements from the Belgium Market and Their Molecular Origins.

Food supplements are gaining popularity worldwide. However, harmful natural compounds can contaminate these products. In the case of algae-based products, the presence of toxin-producing cyanobacteria may cause health risks. However, data about the prevalence of algal food supplements on the Belgian market and possible contaminations with cyanotoxins are scarce. Therefore, we optimized and validated a method based on Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry to quantify eight microcystin congeners and nodularin in algal food supplements. Our analytical method was successfully validated and applied on 35 food supplement samples. Nine out of these samples contained microcystin congeners, of which three exceeded 1 µg g-1, a previously proposed guideline value. Additionally, the mcyE gene was amplified and sequenced in ten products to identify the taxon responsible for the toxin production. For seven out of these ten samples, the mcyE gene could be amplified and associated to Microcystis sp. EFSA and posology consumption data for algal-based food supplements were both combined with our toxin prevalence data to establish different toxin exposure scenarios to assess health risks and propose new guideline values.

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations.

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L-1) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L-1 to 2798.81 µg L-1 total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.

Organisation of Multi-Mycotoxin Proficiency Tests: Evaluation of the Performances of the Laboratories Using the Triple A Rating Approach.

In accordance with the International Standard Organization ISO 17043, two proficiency tests (PTs) for the simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2); deoxynivalenol; fumonisins FB1, FB2, and B3; ochratoxin A, the T-2 toxin; and the HT-2 toxin were conducted in 2019 and 2020 using cornflakes and rusk flours that were prepared in house. The homogeneity and the stability of these materials were verified according to the criteria laid down in ISO 13528 using randomly selected samples. Most of the targeted toxins were found to be homogenously distributed in both materials with no significant changes during the timescale of the PTs. Next, the materials were distributed to approximately 25 participating laboratories from Europe, Canada, and the United States. The obtained datasets were computed using robust statistics. The outliers were checked and removed, and the toxin concentrations were assigned as the consensus value of the results of the participants at Horwitz ratios <1.2. The z scores were generated for all mycotoxins, and the results were pooled to calculate the relative sum of squared z scores (SZ2) indexes and were clustered according to the triple A rating. Overall, at least 80% of the participating laboratories achieved good and acceptable performances. The most frequent categories assigned to good performances (SZ2 ≤ 2) were AAA (51%) and BAA (13%). Clusters of BBA + CBA (6%) included laboratories reporting acceptable z scores <90% of the total z scores for less than 90% or 50% of the mycotoxins targeted in the 2 matrices. The triple A rating seems to be appropriate in evaluating the performances of laboratories involved in multi-mycotoxin analyses. Accredited and non-accredited analytical methods achieved good and acceptable performances.

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market.

Following pending new legislation in the European Union setting a maximum of 20 ng g-1 for the total sum of ergot alkaloids in dry cereal-based baby food, a new UHPLC-MS/MS method was developed. It is suitable for the quantification of six ergot alkaloids: Ergocornine, ergocristine, ergometrine, ergosine, ergotamine, α-ergocryptine, and their corresponding epimers. The method is able to reliably detect individual ergot alkaloids at a level as low as 0.5 ng g-1. The method uses a modified QuEChERS extraction approach before UHPLC-MS/MS analysis. The method showed good sensitivity, accuracy, and precision. It has been applied to 49 samples from the Belgian market. In 26 samples, not a single ergot alkaloid was detected while in 23 out of 49 samples at least one ergot alkaloid was detected with 2 samples containing 12 ergot alkaloids. Ergometrine was the alkaloid most frequently detected i.e., 16 out of 49 samples. Only one sample, testing positive for all 12 ergot alkaloids, would be non-conforming to the newly proposed Maximum Residue Level (MRL).

N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition, but Not Fatty Acid Amide Hydrolase Inhibition, Prevents the Development of Experimental Autoimmune Encephalomyelitis in Mice.

N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.

Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Determination of ochratoxin A in edible pork offal: intra-laboratory validation study and estimation of the daily intake via kidney consumption in Belgium.

Pork-derived products can contribute to the overall ochratoxin A (OTA) intake via carry-over from contaminated feed or via mould spoilage of meat products (salami, dry-cured ham, sausage). An analytical method using liquid chromatography coupled with mass spectrometry (LC-MS/MS) was developed and validated in accordance with the specifications laid down by European Commission. It offered quantification limits of 0.2 for kidney, liver and 0.4 µg/kg for black sausage. Spiking experiments of blank samples at 5-10 µg/kg showed recoveries ranging from 88 to 101%, 89 to 97% and 80 to 85% for kidney, liver and black sausage, respectively. The respective intra-laboratory repeatabilities ranged between 9.8-11.1%, 9.4-14.4% and 9.7-14.2%, and extended measurement uncertainties MU(k = 2) were 33%, 35% and 43% for kidney, liver and black sausage. Next, the validated method was applied to kidney (110), liver (20) and black sausage (20) samples collected in Belgium in the period 2012-2019. Neither liver nor black sausage samples were contaminated with OTA. Kidney samples (37.3%) were OTA contaminated at the mean level of 0.22 ± 0.25 µg/kg (up to 1.91 µg/kg). These data combined with the offal consumption in the Belgian population revealed average daily OTA exposures ranged from 0.167 and 0.319 ng/kg bw for 3 age groups (3-9, 10-17 and 18-64 years). Taking into account, the OTA non-neoplastic and neoplastic effects, risk characterization assessed via the margin of exposure for reference endpoints revealed no potential health risk for the consumers. As the presence of low OTA content in foods together with other mycotoxins or derivatives may interactively potentiate its toxicity, monitoring of OTA and its metabolites in meat and meat by-products is advised.

Glycerophosphodiesterase 3 (GDE3) is a lysophosphatidylinositol-specific ectophospholipase C acting as an endocannabinoid signaling switch.

Endocannabinoid signaling plays a regulatory role in various (neuro)biological functions. 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and although its canonical biosynthetic pathway involving phosphoinositide-specific phospholipase C and diacylglycerol lipase α is known, alternative pathways remain unsettled. Here, we characterize a noncanonical pathway implicating glycerophosphodiesterase 3 (GDE3, from GDPD2 gene). Human GDE3 expressed in HEK293T cell membranes catalyzed the conversion of lysophosphatidylinositol (LPI) into monoacylglycerol and inositol-1-phosphate. The enzyme was equally active against 1-acyl and 2-acyl LPI. When using 2-acyl LPI, where arachidonic acid is the predominant fatty acid, LC-MS analysis identified 2-AG as the main product of LPI hydrolysis by GDE3. Furthermore, inositol-1-phosphate release into the medium occurred upon addition of LPI to intact cells, suggesting that GDE3 is actually an ecto-lysophospholipase C. In cells expressing G-protein-coupled receptor GPR55, GDE3 abolished 1-acyl LPI-induced signaling. In contrast, upon simultaneous ex-pression of GDE3 and cannabinoid receptor CB2, 2-acyl LPI evoked the same signal as that induced by 2-AG. These data strongly suggest that, in addition to degrading the GPR55 LPI ligand, GDE3 can act as a switch between GPR55 and CB2 signaling. Coincident with a major expression of both GDE3 and CB2 in the spleen, spleens from transgenic mice lacking GDE3 displayed doubling of LPI content compared with WT mice. Decreased production of 2-AG in whole spleen was also observed, supporting the in vivo relevance of our findings. These data thus open a new research avenue in the field of endocannabinoid generation and reinforce the view of GPR55 and LPI being genuine actors of the endocannabinoid system.

Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market.

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis® HLB SPE cartridges. Limits of quantification were in the range of 1.5-15 ng/g for herbs and 0.025-1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70-120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7-37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs.

Cancer-associated fibroblasts promote prostate cancer malignancy via metabolic rewiring and mitochondrial transfer.

Cancer-associated fibroblasts (CAFs) are the major cellular stromal component of many solid tumors. In prostate cancer (PCa), CAFs establish a metabolic symbiosis with PCa cells, contributing to cancer aggressiveness through lactate shuttle. In this study, we report that lactate uptake alters the NAD+/NADH ratio in the cancer cells, which culminates with SIRT1-dependent PGC-1α activation and subsequent enhancement of mitochondrial mass and activity. The high exploitation of mitochondria results in tricarboxylic acid cycle deregulation, accumulation of oncometabolites and in the altered expression of mitochondrial complexes, responsible for superoxide generation. Additionally, cancer cells hijack CAF-derived functional mitochondria through the formation of cellular bridges, a phenomenon that we observed in both in vitro and in vivo PCa models. Our work reveals a crucial function of tumor mitochondria as the energy sensors and transducers of CAF-dependent metabolic reprogramming and underscores the reliance of PCa cells on CAF catabolic activity and mitochondria trading.

The origin of neural stem cells impacts their interactions with targeted-lipid nanocapsules: Potential role of plasma membrane lipid composition and fluidity.

The adsorption of a peptide (NFL-TBS.40-63 peptide (NFL)) known to induce neural stem cells (NSC) differentiation in vitro, at the surface of lipid nanocapsules (LNC) provides a targeting drug delivery system (NFL-LNC) that penetrates subventricular zone-neural stem cells (SVZ-NSC) but not central canal-NSC (CC-NSC). We hypothesized preferential interactions could explaine, at least partially, the different properties of SVZ- and CC-NSC plasma membranes. The objective of this work was to compare SVZ- and CC-NSC plasma membrane lipid composition, fluidity and permeability. Plasma membranes of SVZ- and CC-NSC were isolated and analyzed by LC-MS for their lipid content. Membrane fluidity was evaluated by measuring the generalized polarization (GP) of Laurdan and membrane permeability by fluorescent dextran penetration. Liposomes with different lipid compositions and steady state fluidities were prepared. ΔGP was measured after incubation with NFL-LNC. A significantly higher proportion of cholesterol, ceramides, sphingomyelins, phosphatidylethanolamines and a lower proportion of phosphatidylcholines and sulfatides were observed in SVZ- compared to CC-NSC. Fluidity, probably more than lipid composition, drove NFL-LNC and NSC interactions, and SVZ-NSC were more sensitive to NFL permeabilization than CC-NSC. We demonstrated that NSC membrane lipid composition and fluidity depended of NSC origin and that these features could play a role in the specific interactions with NFL-LNC.

Lysophosphatidylinositols in inflammation and macrophage activation: Altered levels and anti-inflammatory effects.

Lysophosphatidylinositols (LPI) are bioactive lipids that are implicated in several pathophysiological processes such as cell proliferation, migration and tumorigenesis and were shown to play a role in obesity and metabolic disorders. Often, these effects of LPI were due to activation of the G protein-coupled receptor GPR55. However, the role of LPI and GPR55 in inflammation and macrophage activation remains unclear. Therefore, we thought to study the effect of macrophage activation and inflammation on LPI levels and metabolism. To do so, we used J774 and BV2 cells in culture activated with lipopolysaccharides (LPS, 100 ng/mL) as well as primary mouse alveolar and peritoneal macrophages. We also quantified LPI levels in the cerebellum, lung, liver, spleen and colon of mice with a systemic inflammation induced by LPS (300 µg/kg) and in the colon of mice with acute colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) and chronic DSS-induced colitis. Our data show that LPS-induced macrophage activation leads to altered LPI levels in both the cells and culture medium. We also show that cytosolic phospholipase A2α (cPLA2α) and α/ß­hydrolase domain 6 (ABHD6) are among the enzymes implicated in LPI metabolism in J774 macrophages. Indeed, ABHD6 and cPLA2α inhibition increased 20:4-LPI levels in LPS-activated macrophages. Furthermore, incubation of LPS-activated cells with LPI decreased J774 activation in a GPR55-dependent manner. In vivo, LPI levels were altered by inflammation in the liver, spleen and colon. These alterations are tissue dependent and could highlight a potential role for LPI in inflammatory processes.

Lysophosphatidylinositols, from Cell Membrane Constituents to GPR55 Ligands.

Lysophosphatidylinositols (LPIs) are membrane constituents that alter the properties of said membranes. However, recent data showing that the once orphan receptor, GPR55, can act as a receptor for LPIs has sparked a renewed interest in LPIs as bioactive lipids. As evidence supporting the importance of LPIs and/or GPR55 is continuously accumulating and because LPI levels are altered in a number of pathologies such as obesity and cancer, the coming years should bring new, exciting discoveries to this field. In this review, we discuss the recent work on LPIs and on their molecular target, the GPR55 receptor. First, we summarize the metabolism of LPIs before outlining the cellular pathways activated by GPR55. Then, we review the actions of LPIs and GPR55 that could have potential pharmacological or therapeutic applications in several pathophysiological settings, such as cancer, obesity, pain, and inflammation.