Dietary squalene supplementation decreases triglyceride species and modifies phospholipid lipidomic profile in the liver of a porcine model of non-alcoholic steatohepatitis.

Squalene is a key minor component of virgin olive oil, the main source of fat in the Mediterranean diet, and had shown to improve the liver metabolism in rabbits and mice. The present research was carried out to find out whether this effect was conserved in a porcine model of hepatic steatohepatitis and to search for the lipidomic changes involved. The current study revealed that a 0.5% squalene supplementation to a steatotic diet for a month led to hepatic accumulation of squalene and decreased triglyceride content as well as area of hepatic lipid droplets without influencing cholesterol content or fiber areas. However, ballooning score was increased and associated with the hepatic squalene content. Of forty hepatic transcripts related to lipid metabolism and hepatic steatosis, only citrate synthase and a non-coding RNA showed decreased expressions. The hepatic lipidome, assessed by liquid chromatography-mass spectrometry in a platform able to analyze 467 lipids, revealed that squalene supplementation increased ceramide, Cer(36:2), and phosphatidylcholine (PC[32:0], PC[33:0] and PC[34:0]) species and decreased cardiolipin, CL(69:5), and triglyceride (TG[54:2], TG[55:0] and TG[55:2]) species. Plasma levels of interleukin 12p40 increased in pigs receiving the squalene diet. The latter also modified plasma lipidome by increasing TG(58:12) and decreasing non-esterified fatty acid (FA 14:0, FA 16:1 and FA 18:0) species without changes in total NEFA levels. Together this shows that squalene-induced changes in hepatic and plasma lipidomic profiles, non-coding RNA and anti-inflammatory interleukin are suggestive of an alleviation of the disease despite the increase in the ballooning score.

Squalene through Its Post-Squalene Metabolites Is a Modulator of Hepatic Transcriptome in Rabbits.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

Squalene Loaded Nanoparticles Effectively Protect Hepatic AML12 Cell Lines against Oxidative and Endoplasmic Reticulum Stress in a TXNDC5-Dependent Way.

Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5.

Occurrence of Pesticide Residues in Spanish Honey Measured by QuEChERS Method Followed by Liquid and Gas Chromatography-Tandem Mass Spectrometry.

In the current study, the QuEChERS extraction method with slight modifications, followed by liquid and gas chromatography-tandem mass spectrometry, was applied for the determination of 399 pesticide residues in 91 raw honey samples from northeastern Spain. The quality control procedure established in Document No. SANTE/12682/2019 was successfully followed: the responses in reagent blank and blank honey samples were below 30% of the reporting limit (0.01 mg kg-1) for all analysed compounds, the correlation coefficients (R2) were higher than 0.99 in most calibration curves, the deviation of back-calculated concentration from the true concentration was below ±20% (using the standard of 50 µg L-1 concentration), and the recoveries of spiked samples on matrix were within the range of 70-120% for almost all analytes. Only chlorfenvinphos (2-7.8 ng/g) and coumaphos (8.8-37 ng/g) were detected in 13 samples, and neither were observed to exceed their maximum residue limits (MRLs). Dietary risk assessment for pesticide residues in honey above their lowest calibrated level (LCL) was performed, and two different age groups, adults and infants, were considered as populations at risk. The contribution of honey lay far below the acceptable daily intake (ADI) for both pesticide residues. Therefore, according to our results, honey is unlikely to pose concerns for consumer health in terms of its contribution to dietary long-term exposure. However, to maintain the level of compliance, pesticide residues in honey should be continuously monitored.

Chitosan hydrogel synthesis to remove arsenic and fluoride ions from groundwater.

Groundwater samples from eight deep drinking water wells that cover three aquifers in Chihuahua City, northern Mexico, were fully characterized. Water is naturally contaminated with arsenic, fluoride, and uranium, according to the Environmental Protection Agency (EPA) and local standards. The results from the Geochemist's Workbench (GWB) program revealed that the minerals in equilibrium with the groundwater were calcite and dolomite, while others, such as fluoride, schoepite, rutherfordite and K(UO2)(AsO4), were also dissolved. The hydrogeochemical characterization of water samples indicates that they were sodium bicarbonate-type water samples at neutral to slightly alkaline pH (7.6-8.3). A batch equilibrium sorption procedure was implemented using natural groundwater, a synthesized chitosan network (net-CS) and a chitosan binary network grafted with N-vinylcaprolactam/N-N-dimethylacrylamide (net-CS)-g-NVCL/DMAAm hydrogels. Isotherms and kinetics sorption tests were evaluated. The adsorption capacity of net-CS hydrogels for As ions was 0.0022 mg/g and F ions 0.15 mg/g after 50 h. Scanning electron microscopy coupled with energy-dispersive spectroscopy (SEM-EDS) was used to investigate the hydrogel surface before and after the sorption process, and TGA was used to evaluate the stability of the adsorbents. Freundlich adsorption isotherm constants for As and F ions indicate heterogeneous sorption and the mechanism of retention by physisorption.