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Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by the hypoxia-inducible factor (HIF)-1α inhibition demands for a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of the aryl hydrocarbon receptor (AhR), a versatile environmental sensor highly expressed in the lungs where it plays a homeostatic role. We resorted to murine models of Aspergillus fumigatus infection in vivo and to human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential crosstalk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR: ARNT/HIF-1β heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent the hypoxia-driven inflammation, restore immune homeostasis and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of the hypoxia:xenobiotic receptor cross-talk for anti-inflammatory therapy in CF.

Sample manipulation for storage and storage itself, interfere with the stability of labile lipids in human plasma, including vitamin E (α-tocopherol), polyunsaturated fatty acids (PUFAs), and their enzymatic and free radical-derived oxidation metabolites. This remains a main limit of lipidomics studies that often lack of sufficient standardization and validation at the pre-analytical level. In order to characterize the stability of these lipids in human plasma and to develop a standardized pre-analytical protocol for lipidomics methods, the oxidation metabolites of α-tocopherol, the free form of ω3 and ω6 PUFAs, and some arachidonic acid (AA)-derived eicosanoids were investigated in human plasma during storage at different freezing temperatures. The effect of a protection/defense cocktail of antioxidants and lipoxygenase inhibitors (PD solution) on these lipid parameters was also evaluated. The temperature of storage markedly affected the formation of α-tocopheryl quinone (α-TQ), the main lipoperoxyl radical-derived oxidation metabolite of vitamin E, with the lowest production rate observed in samples stored at −80 °C or in liquid nitrogen. A similar effect of the storage temperature was observed for the free form of the ω-3 species eicosapentaenoic and docosahexaenoic acid, and for the ω-6 AA. Freezing samples at −20 °C resulted in a time-dependent formation of the pro-inflammatory eicosanoid LTB4. The PD solution prevents non-specific alterations of these lipid parameters in samples that are processed for direct analysis and protects from the temperature-dependent modifications of free PUFAs. Combining PD solution and preservation at −80 °C or in liquid nitrogen, resulted in levels of α-TQ and PUFAs that remained stable over 1 month and up to 8 months of storage, respectively. This method paper provides indications for the optimal processing and storage of human plasma utilized in lipidomics studies.

Recently, several health benefits associated with the consumption of foods rich in chlorogenic acid (5-CQA) have been reported. However, an important issue is its low stability during extraction and food processing, resulting in isomerization to neochlorogenic and cryptochlorogenic acids and the formation of further degradation products. This work describes the evaluation of 5-CQA reactivity in commercial waters after microwave treatment. An optimized HPLC-UV method was used to monitor 5-CQA conversion to its main isomers, while LC-HRMS/MS was performed for the elucidation of transformation products. Results revealed different degrees of isomerization in 5-CQA depending on the water sample, and the formation of oxidation derivatives of CQA isomers. This study highlights the importance of analytical monitoring of food compounds, during microwave treatment for example. In the case of 5-CQA, understanding of the degradation process would allow better preservation of bioactive constituent in foods and beverages and health promoting effects.

The metabolic syndrome (MetSyn) is a disorder characterized by a cluster of diseases where the regulation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor endowed with ligand- and context-dependent activities, can have a major therapeutic relevance. We have recently discovered a tryptophan metabolite of microbial origin, indole-3-carboxaldehyde (3-IAld), able to regulate intestinal mucosal homeostasis by acting as a ligand of AhR. This makes 3-IAld a potential candidate to treat MetSyn related ailments. Herein, we provide a proof of concept on the efficacy and safety of 3-IAld encapsulated in enteric microparticles (MP) in vivo in a MetSyn murine model. In particular, we showed that 3-IAld: i) is released from MPs in the intestine and potentially metabolized; ii) is able to activate AhR locally; iii) prevents the metabolic complications and the intestinal epithelial barrier dysfunction; iv) is not associated with toxic events. This study does not only extend the biological activity of 3-IAld in vivo, but also provides hints on the therapeutic potential of 3-IAld delivered by enteric MP in MetSyn related diseases.

An analytical method for the quantification of thirty-three perfluoroalkyl and polyfluoroalkyl substances (PFASs) in animal liver was developed applying the isotopic dilution methodology with twenty-one labelled isotopologues of native compounds. The proposed protocol involved the determination of short and long aliphatic chain PFASs (C4C18) extracting liver with acetonitrile followed by two clean-up steps. The instrumental analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. The acquisition method combined full MS/dd-MS2, t-SIM/dd-MS2 and SIM experiments with variable resolution in order to maximize in one chromatographic run accuracy, sensitivity and selectivity. An eight-level validation study was performed evaluating linearity, trueness, precision, quantification and detection limits. Trueness was from 94 to 126% with intra-laboratory reproducibility lower than 20%. Limits of quantification were in the range 2-100 pg g-1, except for 2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)-propanoic acid, HFPO-DA (500 pg g-1). The analysis of a certified reference material (IRMM-427) and participation in a proficiency test scheme (FAPAS - 0687) confirmed these satisfactory performances. Finally, the application of the developed procedure to detect PFASs in sixteen liver samples of farm animals revealed that chicken was the less contaminated species.

A multiclass method for the determination of antimicrobial substances in eggs has been developed and validated, covering sixty-three antibiotics belonging to ten families. After extraction with acidified acetonitrile and EDTA, the analytes were injected into a liquid-chromatography high-resolution mass-spectrometry system operating in positive ionization mode. The procedure was successfully validated as confirmatory method evaluating selectivity, linearity, precision, recovery, decision limit, detection capability, limits of detection and quantitation. The here developed is the first method including all the antibiotics with Maximum Residue Limit (MRL) in eggs, except very polar drugs (aminoglycosides and colistins), which require specific protocols. Finally, a wide survey was carried out analyzing 100 commercial eggs samples from local markets. Only 3% contained residues of authorized substances at concentrations lower than the relevant MRLs.

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.

The role of vitamin E in both enzymatic and free radical-dependent metabolism of polyunsaturated fatty acids (PUFAs) has been well demonstrated. This study proposed a new LC-MS/MS method to quantify the main vitamin E forms, their metabolites and main PUFA species in human blood, since, at present, there are not procedures able to simultaneously determine these two classes of compounds. After the optimization of sample treatment and reverse-phase separation conditions, tandem mass spectrometry detection was evaluated experimenting both positive and negative electrospray ionisation modes. The procedure was also preliminarily adapted to assess five arachidonic acid-derived eicosanoids that could be under the influence of vitamin E function, such as LTB4 (leukotriene B4), 20-HETE (20-hydroxyeicosatetraenoic acid) and their ω-oxidation metabolites. After the validation study, the performance characteristics were confirmed analysing a certified reference material (SRM® 1950 - frozen human plasma by NIST). Finally, an application of the method in the analysis of lipid abnormalities of chronic kidney disease patients was shown.

A multiclass method has been developed to screen and confirm a wide range of anti-microbial residues in muscle and milk, and validated using liquid-chromatography coupled to (low-resolution, LR) tandem mass spectrometry (LC-QqQ). Over sixty antibiotics, belonging to ten distinct families, were included in the method scope. The development process was rapidly concluded as a result of two previously implemented methods. This consisted of identical sample treatments, followed by liquid chromatography, and coupled with high-resolution (HR) mass spectrometry (LC-Q-Orbitrap). The validation study was performed in the range between 10&ndash, 1500 &mu, g&middot, kg&minus, 1 for muscles and 2&ndash, 333 &mu, 1 for milk. The main performance characteristics were estimated and, then, compared to those previously obtained with HR technique. The validity of the method transfer was ascertained also through inter-laboratory studies.

A multi-group screening method to detect residues of veterinary drugs in meat and environmental contaminants in wheat flour has been developed using liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS). The procedure was tested for over 300 representative compounds (173 veterinary drugs, 122 pesticides and 9 mycotoxins) analysing in parallel negative and positive (spiked) samples according to European validation rules. The Screening Target Concentrations (STCs) were chosen conservatively with respect to the method purposes. Interpretation of results was based on retention time, mass accuracy of precursor and MS2 spectral library. Evaluating the percentage of false negative results, 280 out of the 304 analytes were detectable at the STCs (false compliant rate ≤ 5%). In wheat flours, incurred levels of mycotoxins, deoxynivalenol and 3-acetyldeoxynivalenol, higher than STCs, were frequently found, whereas in meat, the most detected veterinary drugs were antibiotics generally at negligible concentrations (

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.