[Extracellular Matrix Stiffness Induces Mitochondrial Morphological Heterogeneity via AMPK Activation]. / 细胞外基质硬度通过AMPK调控干细胞线粒体的形态异质性.

Objective: To investigate the mechanical responses of mitochondrial morphology to extracellular matrix stiffness in human mesenchymal stem cells (hMSCs) and the role of AMP-activated protein kinase (AMPK) in the regulation of mitochondrial mechanoresponses. Methods: Two polyacrylamide (PAAm) hydrogels, a soft one with a Young's modulus of 1 kPa and a stiff one of 20 kPa, were prepared by changing the monomer concentrations of acrylamide and bis-acrylamide. Then, hMSCs were cultured on the soft and stiff PAAm hydrogels and changes in mitochondrial morphology were observed using a laser confocal microscope. Western blot was performed to determine the expression and activation of AMPK, a protein associated with mitochondrial homeostasis. Furthermore, the activation of AMPK was regulated on the soft and stiff matrixes by AMPK activator A-769662 and the inhibitor Compound C, respectively, to observe the morphological changes of mitochondria. Results: The morphology of the mitochondria in hMSCs showed heterogeneity when there was a change in gel stiffness. On the 1 kPa soft matrix, 74% mitochondria exhibited a dense, elongated filamentous network structure, while on the 20 kPa stiff matrix, up to 63.3% mitochondria were fragmented or punctate and were sparsely distributed. Western blot results revealed that the phosphorylated AMPK (p-AMPK)/AMPK ratio on the stiff matrix was 1.6 times as high as that on the soft one. Immunofluorescence assay results revealed that the expression of p-AMPK was elevated on the hard matrix and showed nuclear localization, which indicated that the activation of intracellular AMPK increased continuously along with the increase in extracellular matrix stiffness. When the hMSCs on the soft matrix were treated with A-769662, an AMPK activator, the mitochondria transitioned from a filamentous network morphology to a fragmented morphology, with the ratio of filamentous network decreasing from 74% to 9.5%. Additionally, AMPK inhibition with Compound C promoted mitochondrial fusion on the stiff matrix and significantly reduced the generation of punctate mitochondria. Conclusion: Extracellular matrix stiffness regulates mitochondrial morphology in hMSCs through the activation of AMPK. Stiff matrix promotes the AMPK activation, resulting in mitochondrial fission and the subsequent fragmentation of mitochondria. The impact of matrix stiffness on mitochondrial morphology can be reversed by altering the level of AMPK phosphorylation.

Risk/Benefit Evaluation of Chia Seeds as a New Ingredient in Cereal-Based Foods.

Chia seed (Salvia hispanica L.) is a food rich in protein, fiber, polyunsaturated fatty acids and antioxidants. Consequently, its incorporation in food formulations may be desirable from a nutritional and healthy point of view. However, there is concern regarding the formation of process contaminants when they are subjected to thermal processing. The objective of this study was to incorporate different amounts of ground chia seeds in a biscuit model to evaluate the effect on the antioxidant capacity and formation of acrylamide and furfurals. Seven standard "Maria-type" biscuit formulations were prepared, replacing wheat flour with different amounts of ground chia seeds (defatted and non-defatted), from 0% (control biscuit) to 15% (respect to total solids in the recipe). Samples were baked at 180 °C for 22 min. Compared with the control biscuit, chia formulations increased the content of nutrients, antioxidant capacity (ABTS) and phenolic compounds (Folin-Ciocalteau method) but also doubled acrylamide levels and even raised more than 10 times furanic compound concentrations. Results indicate that the use of chia seeds as ingredients in new cereal-based formulations would improve the nutritional profile but also increase the occurrence of chemical process contaminants. This paradox should be carefully considered in the context of risk/benefit analysis.

Processing contaminants in wheat-based foods - a systematic review, meta-analysis and bibliometric analysis.

Wheat is one of the main cereals grown around the world and is the basis for several foods such as bread, cakes and pasta. The consumption of these foods raises a concern with food safety, as toxic substances such as acrylamide, 5-hydroxymethylfurfural and polycyclic aromatic hydrocarbons are formed during their processing. To assess the occurrence of processing contaminants in wheat-based foods, a systematic search was carried out in four databases: PubMed, Embase, Web of Science and Scopus. Of the 1479 results, 28 were included for a meta-analysis. Most studies (69.7%) evaluated acrylamide in bread, cookies, and pasta, while PAHs (26.2%) were determined mainly in wheat grains and pasta. HMF was the least determined contaminant (4.1%), with only four studies on cookies included in the meta-analysis. The highest concentration was for acrylamide (136.29 µg·kg-1) followed by HMF (70.59 µg·kg-1) and PAHs (0.11 µg·kg-1). Acrylamide is the main processing contaminant researched, and no studies on the subject have been found in commercial samples in some regions of the world. This result shows a gap in the dates available about process contaminants in wheat-based foods and how the levels can change depending on the process parameters and the ingredients used.

Quantification of Osimertinib and Metabolite-Protein Modification Reveals Its High Potency and Long Duration of Effects on Target Organs.

Covalent drugs are newly developed and proved to be successful therapies in past decades. However, the pharmacokinetics (PK) and pharmacodynamic (PD) studies of covalent drugs now ignore the drug and metabolite-protein modification. The low abundance of modified proteins also prevents its investigation. Herein, a simple, selective, and sensitive liquid chromatography-mass spectrometry (LC-MS)/MS quantitative method was established based on the mechanism of a drug and its metabolite-protein adducts using osimertinib as an example. Five metabolites with covalent modification potential were identified. The drug and its metabolite-cysteine adducts released from modified proteins by a mixed hydrolysis method were developed to characterize the level of the modified proteins. This turned the quantitative objects from proteins or peptides to small molecules, which increased the sensitivity and throughput of the quantitative approach. Accumulation of protein adducts formed by osimertinib and its metabolites in target organs was observed in vivo and long-lasting modifications were noted. These results interpreted the long duration of the covalent drugs' effect from the perspective of both parent and the metabolites. In addition, the established method could also be applied in blood testing as noninvasive monitoring. This newly developed approach showed great feasibility for PK and PD studies of covalent drugs.

LC-MS/MS Estimation of Rociletinib Levels in Human Liver Microsomes: Application to Metabolic Stability Estimation.

BACKGROUND: Rociletinib (CO-1686; RLC) is a new, small molecule that is orally administered to inhibit mutant-selective covalent inhibitor of most epidermal growth factor receptor (EGFR)-mutated forms, including T790M, L858R, and exon 19 deletions, but not exon 20 insertions. Non-small-cell lung cancer (NSCLC) with a gene mutation that encodes EGFR is sensitive to approved EGFR inhibitors, but usually resistance develops, which is frequently mediated by T790M EGFR mutation. RLC is an EGFR inhibitor found to be active in preclinical models of EGFR-mutated NSCLC with or without T790M. METHODS: In silico drug metabolism prediction of RLC was executed with the aid of the WhichP450 module (StarDrop software package) to verify its metabolic liability. Second, a fast, accurate, and competent LC-MS/MS assay was developed for RLC quantification to determine its metabolic stability. RLC and bosutinib (BOS) (internal standard; IS) were separated using an isocratic elution system with a C18 column (reversed stationary phase). RESULTS: The developed LC-MS/MS analytical method showed linearity of 5-500 ng/mL with r2 ≥ 0.9998 in the human liver microsomes (HLMs) matrix. A limit of quantification of 4.6 ng/mL revealed the sensitivity of the analytical method, while the acquired inter- and intra-day accuracy and precision values below 4.63% inferred the method reproducibility. RLC metabolic stability estimation was calculated using intrinsic clearance (20.15 µL/min/mg) and in vitro half-life (34.39 min) values. CONCLUSION: RLC exhibited a moderate extraction ratio indicative of good bioavailability. The developed analytical method herein is the first LC-MS/MS assay for RLC metabolic stability.

Optimization, extraction, and purification of three bioactive compounds from Entada phaseoloides by high-speed countercurrent chromatography.

The objective of this paper was to develop a preparative method for the separation and purification of phaseoloidin, entadamide A, and entadamide A-ß-D-glucopyranoside from the crude extract of Entada phaseoloides by high-speed countercurrent chromatography (HSCCC) for the first time. Optimized by orthogonal experiments, the extraction conditions were extraction temperature of 65°C, solid-to-liquid ratio of 1:15 (g/mL), ethanol concentration of 40%, and extraction time of 2.5 h. Using n-butanol-acetic acid-water (4:1:5, v/v/v) as the two-phase solvent system, 38.79 mg phaseoloidin (the purity was 99.3% with a recovery of 98.1%), 34.85 mg entadamide A (the purity was 96.4% with a recovery of 98.5%), and 33.97 mg entadamide A-ß-D-glucopyranoside (the purity was 98.6% with a recovery of 97.7%) were obtained from 500 mg crude extract by HSCCC in head-to-tail elution mode. The retention ratio of stationary phase was 51.0%. According to the antioxidant activity assays, phaseoloidin, entadamide A, and entadamide A-ß-D-glucopyranoside had certain scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl free radicals and hydroxyl free radicals.

Determination of intracellular anlotinib, osimertinib, afatinib and gefitinib accumulations in human brain microvascular endothelial cells by liquid chromatography/tandem mass spectrometry.

RATIONALE: Brain metastases are a common complication in patients with non-small-cell lung cancer (NSCLC). Anlotinib hydrochloride is a novel multi-target tyrosine kinase inhibitor (TKI) exhibiting a superior overall response rate for brain metastases from NSCLC. The penetrability of anlotinib and three generations of epidermal growth factor receptor (EGFR) TKIs (osimertinib, afatinib and gefitinib) into brain microvascular endothelial cells (HBMECs) was compared. METHODS: A sensitive quantification method for the four TKIs was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Anlotinib and the three EGFR TKIs were separated on an ACQUITY BEH C18 column after a direct protein precipitation, and then analyzed using electrospray ionization in positive ion mode. The linearity, accuracy, precision, limit of quantification, specificity and stability were assessed. RESULTS: The four analytes could be efficiently quantified in a single run of 3.8 min. The validation parameters of all analytes satisfy the acceptance criteria of bioanalytical method guidelines. The calibration range was 0.2-200 ng mL-1 for anlotinib and gefitinib, 1-500 ng mL-1 for osimertinib and 1-200 ng mL-1 for afatinib. The penetration of anlotinib across HBMECs was comparable with that of afatinib and gefitinib but less than that of osimertinib. CONCLUSIONS: A sensitive LC/MS/MS method to simultaneously measure anlotinib, osimertinib, afatinib and gefitinib in cell extracts was successfully validated and applied to determine their uptake inside HBMECs, which could pave the way for future research on the role of anlotinib in NSCLC brain metastases.

Dietary risk evaluation of acrylamide intake with bread in Poland, determined by two comparable cleanup procedures.

Acrylamide is regarded as a food chemical contaminant. The aims of this work included: (i) to develop sample cleanup procedures applicable for determination of AA in soft bread samples; (ii) to determine AA levels in soft bread available in retail trade in Poland and to compare them with currently standing benchmark levels; (iii) to determine dietary risk related to AA in soft bread. The procedure based on ion-exchange solid phase extraction was more suitable to obtain LOQs corresponding to AA concentrations in soft bread samples. AA levels found in bread samples were in 3.6-163 µg kg-1 range. AA levels varied greatly from sample to sample, which suggests that both food composition and manufacturing processes play a crucial role in AA generation. When considering reference point for neoplastic effects, Margin of Exposure calculated for AA taken with soft bread ranged between 543 and 3,035.

Carcinogenic risk associated with popular Korean dishes: An approach of combined risk assessments using Oral Slope Factor and BMDL10 values.

Cooking-related carcinogens are formed during the heating or processing of foods. To date, numerous studies analyzing carcinogens present in cooking ingredients or formed through different cooking methods have been conducted. However, combined risk assessment is important for practical reasons. The purpose of this study was to conduct a combined risk assessment of five cooking-related genotoxic carcinogens encompassing 25 chemicals: heterocyclic amines, acrylamide, furan, polycyclic aromatic hydrocarbons, and nitrosamines. Oral Slope Factor (OSF) and benchmark dose lower-bound confidence limit 10% (BMDL10) of the compounds were obtained from public databases, and the values for the compounds that did not have published reference values were approximated using related toxicity values. The high-risk contributing food items and cooking methods for each carcinogen were selected for the study based on the Korean Total Diet Study (TDS) and Korea National Health and Nutrition Examination Survey (KNHANES). Exposure to the carcinogens from selected dishes per serving was estimated based on concentrations determined in TDS and consumption data gathered from 24-h recalls in the 2014 to 2016 KNHANES. The combined cancer risks were obtained by summing the risks of individual compounds in a dish, which were calculated by multiplying the OSF values by the concentrations of carcinogens per serving. The combined risks were used to compare the risk of different dishes, not to calculate the lifetime risk from the individual dishes. The risks of the dishes prepared with potatoes were found to be high, whereas namul (vegetable dish) had the lowest risk. Soup or stew dishes exhibited relatively high risks. Estimated combined risks based on BMDL10 showed similar trends, except for fried potatoes and roasted or fried meat dishes. Combined risks of cooking-related carcinogens may vary based on the major contributors of individual carcinogens. The results of this study could provide an insightful guideline for selecting menus for consumers.

Toxicity of crude organic extracts from the zoanthid Palythoa caribaeorum: A biogeography approach.

Marine isolates such as palytoxin (PTX) are of concern within the Caribbean region due to their toxicity. PTX for example has been described as a one of the most known potent marine toxins, known to prevent predation from larger species (e.g. vertebrates) as well as the prevention of being overgrown from other coral species. PTX is a polyhydroxylated polyether toxin with a very large and complex chemical structure that possesses both hydrophilic and lipophilic properties. Previous acute toxicity tests using brine shrimp (Artemia salina) and PTX extract had shown it to be moderately toxic. In humans, PTX has been credited to be responsible for extreme symptoms such anaphylactic shock, rapid cardiac failure and eventual death occurring within minutes. Extrapolation for human dose ranges has therefore been suggested to be between 2.3 and 31.5 µg. This study isolates a potentially PTX-enriched extract from Palythoa caribaeorum and examines its organic extract toxicity from a biogeography perspective from a within-colony to a variety of reef sites around Trinidad and Tobago that are popular for marine visitors. This research represents an acute study with a high level of crude organic extract toxicity on A. salina whilst postulating potential factors which may contribute to its extreme toxicity and the risk posed to users of these environments.

Determination of osimertinib in human plasma, urine and cerebrospinal fluid.

Aim: Osimertinib (Tagrisso, AZD9291) has been approved for the treatment of patients with metastatic EGFRm T790M non-small-cell lung cancer. Results: Rapid and sensitive LC-MC/MS methods were developed for osimertinib and its metabolites, AZ13597550 and AZ13575104, in human plasma (low- and high-range), urine and cerebrospinal fluid. We discuss the challenges of these multi-analyte and multiple matrix assays. The methods have been successfully validated and used for the analysis of over 20,000 clinical samples, with successful incurred sample reproducibility. Conclusion: The assays have been shown to be selective, accurate and robust, providing high-throughput analysis during the clinical development of osimertinib.

Integrated Analytical Tools for Accessing Acridones and Unrelated Phenylacrylamides from Swinglea glutinosa.

In natural product studies, the purification of metabolites is an important challenge. To accelerate this step, alternatives such as integrated analytical tools should be employed. Based on this, the chemical study of Swinglea glutinosa (Rutaceae) was performed using two rapid dereplication strategies: Target Analysis (Bruker Daltonics®, Bremen, Germany) MS data analysis combined with MS/MS data obtained from the GNPS platform. Through UHPLC-HRMS data, the first approach allowed, from crude fractions, a quick and visual identification of compounds already reported in the Swinglea genus. Aside from this, by grouping compounds according to their fragmentation patterns, the second approach enabled the detection of eight molecular families, which presented matches for acridonic alkaloids, phenylacrylamides, and flavonoids. Unrelated compounds for S. glutinosa have been isolated and characterized by NMR experiments, Lansamide I, Lansiumamide B, Lansiumamide C, and N-(2-phenylethyl)cinnamamide.

A Sensitive LC-MS/MS Method for Palytoxin Using Lithium Cationization.

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]3+. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg-1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg-1 in mussel, cockle, and ensis, and at 60 µg PlTX kg-1 in oyster.

A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels.

The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10-11 M (32.2 pg/mL) and 2.8 × 10-11 M (75.0 pg/mL), respectively, with good accuracy (bias = 2.5%) and repeatability (15% and 9% interday and intraday relative standard deviation of repeatability (RSDr), respectively). Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation.

Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives.

Biochemically modified proteins have attracted significant attention due to their widespread applications as biomaterials. For instance, chemically modified gelatin derivatives have been widely explored to develop hydrogels for tissue engineering and regenerative medicine applications. Among the reported methods, modification of gelatin with methacrylic anhydride (MA) stands out as a convenient and efficient strategy to introduce functional groups and form hydrogels via photopolymerization. Combining light-activation of modified gelatin with soft lithography has enabled the materialization of microfabricated hydrogels. So far, this gelatin derivative has been referred to in the literature as gelatin methacrylate, gelatin methacrylamide, or gelatin methacryloyl, with the same abbreviation of GelMA. Considering the complex composition of gelatin and the presence of different functional groups on the amino acid residues, both hydroxyl groups and amine groups can possibly react with methacrylic anhydride during functionalization of the protein. This can also apply to the modification of other proteins, such as recombinant human tropoelastin to form MA-modified tropoelastin (MeTro). Here, we employed analytical methods to quantitatively determine the amounts of methacrylate and methacrylamide groups in MA-modified gelatin and tropoelastin to better understand the reaction mechanism. By combining two chemical assays with instrumental techniques, such as proton nuclear magnetic resonance (1H NMR) and liquid chromatography tandem-mass spectrometry (LC-MS/MS), our results indicated that while amine groups had higher reactivity than hydroxyl groups and resulted in a majority of methacrylamide groups, modification of proteins by MA could lead to the formation of both methacrylamide and methacrylate groups. It is therefore suggested that the standard terms for GelMA and MeTro should be defined as gelatin methacryloyl and methacryloyl-substituted tropoelastin, respectively, to remain consistent with the widespread abbreviations used in literature.

First Identification of Palytoxin-Like Molecules in the Atlantic Coral Species Palythoa canariensis.

Palytoxin (PLTX) is a complex marine toxin produced by Zoanthids (Palyhtoa), dinoflagellates (Ostreopsis), and cyanobacteria (Trichodesmium). Contact with PLTX-like compounds present in aerosols or marine organisms has been associated with adverse effects on humans. The worldwide distribution of producer species and seafood contaminated with PLTX-like molecules illustrates the global threat to human health. The identification of species capable of palytoxin production is critical for human safety. We studied the presence of PLTX analogues in Palythoa canariensis, a coral species collected in the Atlantic Ocean never described as a PLTX-producer before. Two methodologies were used for the detection of these toxins: a microsphere-based immunoassay that offered an estimation of the content of PLTX-like molecules in a Palythoa canariensis extract and an ultrahigh-pressure liquid chromatography coupled to an ion trap with a time-of-flight mass spectrometer (UPLC-IT-TOF-MS) that allowed the characterization of the toxin profile. The results demonstrated the presence of PLTX, hydroxy-PLTX and, at least, two additional compounds with PLTX-like profile in the Palythoa canariensis sample. The PLTX content was estimated in 0.27 mg/g of lyophilized coral using UPLC-IT-TOF-MS. Therefore, this work demonstrates that Palythoa canariensis produces a mixture of PLTX-like molecules. This is of special relevance to safeguard human health considering Palythoa species are commonly used for decoration by aquarium hobbyists.

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin.

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d-1 for O. cf. ovata, 0.35 ± 0.01 d-1 for C. monotis and 0.33 ± 0.04 d-1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed.

A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels.

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 µg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.

Toxicological Perspective on Climate Change: Aquatic Toxins.

In recent years, our group and several others have been describing the presence of new, not previously reported, toxins of high toxicity in vectors that may reach the human food chain. These include tetrodotoxin in gastropods in the South of Europe, ciguatoxin in fish in the South of Spain, palytoxin in mussels in the Mediterranean Sea, pinnatoxin all over Europe, and okadaic acid in the south of the U.S. There seem to be new marine toxins appearing in areas that are heavy producers of seafood, and this is a cause of concern as most of these new toxins are not included in current legislation and monitoring programs. Along with the new toxins, new chemical analogues are being reported. The same phenomenom is being recorded in freshwater toxins, such as the wide appearance of cylindrospermopsin and the large worldwide increase of microcystin. The problem that this phenomenon, which may be linked to climate warming, poses for toxicologists is very important not only because there is a lack of chronic studies and an incomplete comprehension of the mechanism driving the production of these toxins but also because the lack of a legal framework for them allows many of these toxins to reach the market. In some cases, it is very difficult to control these toxins because there are not enough standards available, they are not always certified, and there is an insufficient understanding of the toxic equivalency factors of the different analogues in each group. All of these factors have been revealed and grouped through the massive increase in the use of LC-MS as a monitoring tool, legally demanded, creating more toxicological problems.

Acrylamide elution from roasted barley grains into mugicha and its formation during roasting.

This paper investigated acrylamide elution from roasted barley grain into mugicha and its formation during roasting of the grain. Mugicha is an infusion of roasted barley grains. Highly water-soluble acrylamide was easily extracted to mugicha from milled roasted barley grains in teabags. On the other hand, the acrylamide concentration in mugicha prepared from loose grain increased with longer simmering and steeping times. During roasting in a drum roaster, the acrylamide concentration of the grain increased as the surface temperature rose, reaching a maximum at 180-240°C. Above this temperature, the acrylamide concentration decreased with continued roasting, exhibiting an inverted 'U'-shaped curve. For most of the samples, the acrylamide concentration showed good correlation with the value of the colour space parameter L*. The dark-coloured roasted barley grains with lower L* values contained lower amounts of acrylamide as a result of deep roasting. The level of asparagine in barley grains was found to be a significant factor related to acrylamide formation in roasted barley products. The data are an important contribution to the mitigation of acrylamide intake from mugicha.