Se and Hg in processed fish-derived products and their fish raw materials: Occurrence, Se:Hg molar ratio, HBVSe index, bioaccessibility and Caco-2 cells toxicity.

Processing conditions applied during food production could affect food component contents and bioaccessibility. Here, possible changes in Hg and Se total and species contents and bioaccessibility have been tracked in each stage of the production chain of processed fish-derived products. Therefore, Se:Hg molar ratio and Selenium Health Benefit Value (HBVSe) were calculated for final products and raw materials, resulting favorable in all cases, suggesting the safety of surimi-based products regarding mercury. Speciation studies revealed the presence of SeMeSeCys and SeMet in all samples. Thus, the integrity of the selenium species seems to be maintained. Moreover, in vitro gastrointestinal digestion model evidenced that Se bioaccessibility ranged between 20-39 % for all samples, while in case of Hg was between 8-37 %. Additionaly, SeMeSeCys and SeMet were also identified in the gastrointestinal extracts. Finally, no cytotoxicity was observed after exposure of Caco-2 cells to the gastrointestinal extracts.

Development of a fast and low-cost aqueous based-extraction protocol for the simultaneous extraction and characterization of SiO2 and TiO2 (nano)particles in confectionary products.

BACKGROUND: The determination of (nano)particulate content from food additives has been a long-standing concern for authorities since it is of vital importance for ensuring food safety, regulatory adherence, and transparent consumer information. Nonetheless, a critical step in these determinations is the refinement of a careful and quantitative extraction process for particles that may be found within complex matrices such as confectionery products. The development of new technologies and analysis methods for nanoparticles is ongoing. Whereas new technologies and analysis methods for nanoparticles are being developed, the extraction of (nano)particles of different nature has not been adequately addressed in the literature. RESULTS: A simple aqueous extraction procedure was found to be suitable for the simultaneous extraction of TiO2 and SiO2 (nano)particles from five confectionery products. Neither the extraction agents (water, lipase, pancreatin and Tris-HCl solutions) nor the methods (manual shaking, ultrasonic bath, ultrasonic probe and ultrafiltration) altered the size, morphology, or aggregation state of either type of particle, as revealed by the micrographs obtained by Transmission Electron Microscopy (TEM). Single-particle ICP-MS (spICP-MS) determined that the optimal conditions for extracting both types of particles involve manual shaking using water as the solvent. Furthermore, the use of enzymes seemed to hinder the determination of both types of particles by spICP-MS. (Nano)particles of TiO2 and SiO2 were detected in all the confectionaries, even though the E171 additive was only labeled in one of them. The average percentage of nanoparticulate TiO2 material in the evaluated products was 30 %, while no nanometer-sized particles of SiO2 were detected. SIGNIFICANCE: Ensuring food safety, regulatory compliance and transparent consumer information relies on getting reliable results that connect with the application of sample treatment procedures for detecting unaltered nanoparticles in food products. The presented research introduces an economical, swift, user-friendly, environmentally responsible, and harmonious extraction method for the concurrent analysis of TiO2 and SiO2 particles in confectionery samples. Furthermore, particles from additives not included in the labeling have been detected, characterized, and quantified in the confectionary products.

Single-cell ICP-MS for evaluating the Se-protective effect against MeHg+-induced neurotoxicity in human neuroblastoma cell line (SH-SY5Y).

The protective effect of selenium (Se) against Hg-induced neurotoxicity has been widely investigated; however, the mechanisms behind this interaction have not been fully elucidated yet. In the current work, the role of Se against MeHg+-induced cytotoxicity in the human neuroblastoma cell line (SH-SY5Y) is reported for the first time by tracking Hg uptake and accumulation at the single-cell level by inductively coupled plasma-mass spectrometry in single-cell mode (SC-ICP-MS). The influence of different Se species (SeMet, SeMeSeCys, citrate-SeNPs, and chitosan-SeNPs) on MeHg+ cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. SeMet and SeMeSeCys exhibited protective effects against MeHg+-induced cell death, particularly at high MeHg+ concentrations (LC50). In addition, chitosan-SeNPs showed greater protection compared to citrate-SeNPs when co-exposed with MeHg+. Interestingly, SC-ICP-MS unveiled the heterogeneous distribution of Hg uptake by SH-SY5Y cells. Co-exposure of SeMet and SeMeSeCys with MeHg+ led to a reduction of the amount of Hg accumulated per individual cell, which decreased the maximum level of Hg per cell by half (from 60 fg Hg cell-1 to 30 fg Hg cell-1) when SeMet was present, along with a decrease in the percentage of cells that accumulated the highest quantity of MeHg+. All these data corroborate the protective role of Se against Hg toxicity at the cellular level.

Fish acute toxicity of nine nanomaterials: Need of pre-tests to ensure comparability and reuse of data.

Fish acute toxicity tests are commonly used in aquatic environmental risk assessments, being required in different international substances regulations. A general trend in the toxicity testing of nanomaterials (NMs) has been to use standardized aquatic toxicity tests. However, as these tests were primarily developed for soluble chemical, issues regarding particle dissolution, agglomeration or sedimentation during the time of exposure are not considered when reporting the toxicity of NMs. The aim of this study was to characterize the NM behaviour throughout the fish acute test and to provide criteria to assay the toxicity of nine NMs based on TiO2, ZnO, SiO2, BaSO4, bentonite, and carbon nanotubes, on rainbow trout following OECD Test Guideline (TG) nº203. Our results showed the importance of conducting a preliminary test (without fish) when working with NMs. They provide valuable information on, sample monitoring, agglomeration, sedimentation, dissolution, actual concentrations of NMs, needed to design the test. Among the NMs tested, only bentonite nanoparticles were stable during the 96-h pre-test and test in aquarium water. In contrast, the remaining NMs exhibited considerable loss and sedimentation within the first 24 h. The high sedimentation observed for almost all NMs highlights the need of consistently measuring the concentrations throughout the entire duration of the fish acute toxicity test to make reliable concentration-response relationships. Notable differences emerged in LC50 values when using actual concentrations as nominal concentrations overestimated concentrations by up to 85.6%. Among all NMs tested, only ZnO NMs were toxic to rainbow trout. A flow chart was specifically developed for OECD TG 203, aiding users in making informed decisions regarding the selection of test systems and necessary modifications to ensure accurate, reliable, and reusable toxicity data. Our findings might contribute to the harmonization of TG 203 improving result reproducibility and interpretability and supporting the development of read-across and QSAR models.

Cytotoxicity, uptake and accumulation of selenium nanoparticles and other selenium species in neuroblastoma cell lines related to Alzheimer's disease by using cytotoxicity assays, TEM and single cell-ICP-MS.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, representing 80% of the total dementia cases. The "amyloid cascade hypothesis" stablishes that the aggregation of the beta-amyloid protein (Aß42) is the first event that subsequently triggers AD development. Selenium nanoparticles stabilized with chitosan (Ch-SeNPs) have demonstrated excellent anti-amyloidogenic properties in previous works, leading to an improvement of AD aetiology. Here, the in vitro effect of selenium species in AD model cell line has been study to obtain a better assessment of their effects in AD treatment. For this purpose, mouse neuroblastoma (Neuro-2a) and human neuroblastoma (SH-SY5Y) cell lines were used. Cytotoxicity of selenium species, such as selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys) and Ch-SeNPs, has been determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry methods. Intracellular localisation of Ch-SeNPs, and their pathway through SH-SY5Y cell line, have been evaluated by transmission electron microscopy (TEM). The uptake and accumulation of selenium species by both neuroblastoma cell lines have been quantified at single cell level by single cell- Inductively Coupled Plasma with Mass Spectrometry detection (SC-ICP-MS), with a previous optimisation of transport efficiency using gold nanoparticles (AuNPs) ((69 ± 3) %) and 2.5 mm calibration beads ((92 ± 8) %). Results showed that Ch-SeNPs would be more readily accumulated by both cell lines than organic species being accumulation ranges between 1.2 and 89.5 fg Se cell-1 for Neuro-2a and 3.1-129.8 fg Se cell-1 for SH-SY5Y exposed to 250 µM Ch-SeNPs. Data obtained were statistically treated using chemometric tools. These results provide an important insight into the interaction of Ch-SeNPs with neuronal cells, which could support their potential use in AD treatment.

Characterization of AgNPs and AuNPs in sewage sludge by single particle inductively coupled plasma-mass spectrometry.

This study develops for the first time an analytical method for the characterization of silver and gold nanoparticles in sewage sludge. The evaluation of the effect of temperature, extracting agent and centrifugation speed and time on the extraction yield was carried out through a multifactorial analysis of variance which allows us to select 289 g, 5 min and 20 mM sodium pyrophosphate tetrabasic as optimal extraction conditions. Under these conditions, the analysis of the extract by single particle inductively coupled plasma-mass spectrometry provided recovery percentages of 70 ± 2% and 56 ± 1% for silver and gold nanoparticles, respectively. Moreover, the complementary results obtained upon analysis of these extracts by transmission electron microscopy and single particle inductively coupled plasma-mass spectrometry showed that the developed method did not modify the original size and shape of these nanoparticles during the extraction procedure. Size detection limits of 23 nm and 16 nm as well as number concentration limits of 3.12 × 109 particles kg-1 and 1.38 × 109 particles kg-1 were obtained for silver and gold nanoparticles, respectively. Moreover, a stability study of silver and gold nanoparticles in sewage sludge for 12 months showed differences between the two nanoparticle types. Although the sizes were not affected during the 12 months, silver nanoparticles underwent an oxidation process from 6 months onwards, which was reflected in an increase in the percentage of ionic silver from 14 ± 1% at 6 months to 24 ± 2% at 12 months. The developed methodology represents a simple, reliable and fast tool for detecting, quantifying and assessing the stability of nanoparticles in an important environmental sample such as sewage sludge.

Analysis of Se and Hg biomolecules distribution and Se speciation in poorly studied protein fractions of muscle tissues of highly consumed fishes by SEC-UV-ICP-MS and HPLC-ESI-MS/MS.

Distribution of Se and Hg in sarcoplasmic, myofibrillar and alkali-soluble protein fractions extracted from muscle tissue of tuna, swordfish and salmon (wild vs. farmed) is investigated for the first time. SEC-UV-ICP-MS analyses revealed that Se and Hg are mostly bound to proteins of 2-12 kDa and up to 574 kDa, respectively. Moreover, Se and Hg appeared associated to proteins of same molecular weight, evidencing that Se-Hg interaction may occur at the level of the fish tissue evaluated. Important differences were found between farmed and wild salmon, suggesting the effect of the type of feed and growing conditions on Se and Hg content and their distribution through protein fractions. Finally, Se speciation studies performed by HPLC-ICP-MS and confirmed by HPLC-ESI-MS/MS showed SeMeSeCys as the only Se specie found in soluble, sarcoplasmic, myofibrillar and alkali-soluble proteins of all fishes analysed, except in soluble proteins extracted from tuna, where SeMet was also identified.

Combined single cell and single particle ICP-TQ-MS analysis to quantitatively evaluate the uptake and biotransformation of tellurium nanoparticles in bacteria.

Assessing the impact of nanoparticles in living systems implies a proper evaluation of their behaviour at single-cell level. Due to the small size of nanoparticles, their accumulation, transformation and location within single cells is challenging. In this work, the combination of single cell/single particle triple quadrupole inductively coupled plasma mass spectrometry (SC/SP-ICP-TQ-MS) analysis along with X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements has been applied to go deeper into the uptake and biotransformation of tellurium nanoparticles (TeNPs) in two bacterial model organisms, S. aureus and E. coli. The use of SC-ICP-TQ-MS enabled the individual introduction of bacterial cells where tellurium and phosphorous (as constitutive element) were monitored and detected at concentration levels down to femtogram (fg) per cell. S.aureus uptake of TeNPs was 0.5-1.9 fg Te cell-1 and 7-30 fg Te cell-1 in presence of 0.5 and 15 mg Te L-1 of TeNPs, respectively, whereas for E. coli, the amount of Te ranged from 0.08 to 0.88 fg Te cell-1 and from 2 to 36 fg Te cell-1 in presence of 0.5 and 15 mg Te L-1 of TeNPs, respectively. TEM and XRD analysis confirmed the occurrence of TeNPs biotransformation (from nanospheres to nanorods) as the nanoparticles were incorporated into both bacterial strains. Finally, SP-ICP-MS analysis after cell lysis was applied to determine the number of particles/rods per bacteria cell and to perform the dimensional characterization of the rod-shaped TeNPs. The results obtained clearly confirmed high cell-to-cell variability in terms of Te nanorods dimensions and TeNPs uptake. To the best of our knowledge, this is the first time that SC/SP-ICP-TQ-MS along with TEM and XRD analysis have been applied to investigate, quantitatively, nanoparticle uptake in bacterial cells and to estimate the dimensions of biogenic Te nanorods.

Fate and effect of in-house synthesized tellurium based nanoparticles on bacterial biofilm biomass and architecture. Challenges for nanoparticles characterization in living systems.

The unexpected impact of nanoparticles on environment and human health remains as a matter of concern. In this sense, understanding the interaction between nanoparticles and biological indicators such as microorganism may help to understand their fate and effect in environmental systems. However, the adverse effect of nanoparticles greatly depends on their properties and, therefore, a precise evaluation of nanoparticles physicochemical characteristics is mandatory as the first step in accurately elucidating their behaviour in different ecosystems. Here in this work, in house-synthesized tellurium-based nanoparticles have been fully characterized for first time by means of a multi-method approach. Once characterized, the effect of these nanoparticles on Staphylococcus aureus and Escherichia coli biofilm biomass and structure was explored and quantified for first time. Moreover, the morphological transformations of tellurium based nanoparticles within the confines of a biofilm are also highlighted. Architectural metric calculations evidenced that nanoparticles were able to reduce the biovolume of the biofilm produced for both bacteria. Interestingly, the interaction between nanoparticles and bacterial communities led to the transformation of tellurium nanoparticles from sphere to rod-shaped nanoparticles. These findings open new insights into the behaviour of a type of uncommon nanoparticles such as tellurium-based nanoparticles on microbial communities.

Using single-particle ICP-MS for unravelling the effect of type of food on the physicochemical properties and gastrointestinal stability of ZnONPs released from packaging materials.

This article discusses the application of Single-Particle Inductively Coupled Plasma Mass Spectrometry (SP-ICP-MS) to study the effect of different types of food (orange juice and chicken breast) on the fate of zinc oxide nanoparticles (ZnONPs) migrated from two widely employed food packaging materials (polyethylene terephthalate (PET) and low density polyethylene (LDPE)). The gastrointestinal stability of ZnONPs was also evaluated. The idea behind this study is to track for first time the transformations underwent of nanoparticles in the different steps of their route from packaging to the consumer. The presence of high amount of dissolved zinc in the samples notably influenced size detection limit and the accuracy of SP-ICP-MS measurements. The diameter limits of detection (LODd) were 26 nm, 95 nm, 108 nm and 129 nm for aqueous solution, chicken breast extract and for oral and intestinal extracts, respectively. ZnONPs characterization in juice was not possible with SP-ICP-MS due to nanoparticles size was below LODd. Besides difficulties, SP-ICP-MS after extraction with Tris-HCl allowed us to determine that a 72% of the ZnONPs that migrated to chicken breast were smaller than 95 nm. Complementary to SP-ICP-MS, transmission electron microscopy (TEM) enabled to detect small nanoparticles (<3 nm). The combination of TEM and SP-ICP-MS measurements indicated that nanoparticles in chicken reach the intestine wall as small particles (<10 nm), as aggregates (>200 nm) and as ionic zinc whereas in case of juice only small nanoparticles (<3 nm) and ionic zinc were detected in the intestinal step.

Unravelling mechanisms of bacterial quorum sensing disruption by metal-based nanoparticles.

Nanoparticles are released in the environment causing a negative impact in several ecosystems such as microbial communities. To adapt to environmental changes some bacteria use a collective behaviour ruled by a cell-to-cell communication process called quorum sensing (QS). In this study, the impact of some of the most employed metal-based nanoparticles, such as zinc oxide nanoparticles (ZnONPs), titanium dioxide nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs) on bacterial QS has been assessed by using two different strains of the model organism Chromobacterium violaceum and by employing different experimental conditions. TiO2NPs were tested with and without applying a previous step of UV-irradiation while the effect of AgNPs of two diameter sizes (40 and 60 nm) and two different coating agents (PVP and citrate) was evaluated. Results evidenced that all nanoparticles produced a significant effect on violacein production and therefore, in the QS system. ZnONPs mainly disrupted the QS steps related to signal perception and response whereas TiO2NPs and AgNPs affected the autoinducer biosynthesis. AgNPs with the smallest size and citrate as capping agent produced the most deleterious effect while the impact of TiO2NPs was not affected by UV irradiation. The present study provides new insights into the mechanisms by which these commonly employed metal-based nanoparticles disturb bacterial QS-based communication and clearly evidences the potential risk of releasing nanoparticles to the environment, especially for microbial communities which play a key role in many environmental and technological processes.

Selenium and tellurium-based nanoparticles as interfering factors in quorum sensing-regulated processes: violacein production and bacterial biofilm formation.

A cell-to-cell communication system called quorum sensing (QS) promotes the transcription of certain target genes in bacterial cells leading to the activation of different cellular processes, some of them related to bacterial biofilm formation. The formation of bacterial biofilms favours antibiotic resistance, which is nowadays a significant public-health problem. In this study, the effect of selenium (SeNPs) and tellurium (TeNPs) nanoparticles was examined in two bacterial processes mediated by QS: violacein production by Chromobacterium violaceum and biofilm formation by Pseudomonas aeruginosa. For this purpose, quantification of the pigment production in the presence of these nanoparticles was monitored using the C. violaceum strain. Additionally, a combination of different microscopical imaging techniques was applied to examine the changes in the 3D biofilm structure of P. aeruginosa, which were quantified through performing architectural metric calculations (substratum area, cell area coverage and biovolume). SeNPs produce an 80% inhibition in the violacein production by C. violaceum and a significant effect on the P. aeruginosa biofilm architecture (a reduction of 80% in the biovolume of the bacterial biofilm was obtained). TeNPs similarly affect violacein production and the P. aeruginosa biofilm structure but at lower concentration levels. The results obtained suggest an important disruption of the QS signalling system by SeNPs and TeNPs, supporting nanotechnology as a promising tool to fight against the emerging problem of bacterial resistance related to bacterial biofilm formation.

Silac-based quantitative proteomic analysis of Lactobacillus reuteri CRL 1101 response to the presence of selenite and selenium nanoparticles.

Stable isotope labeling in cell culture (SILAC) was applied for the first time on a lactic acid bacterium strain (L. reuteri CRL1101) for analyzing differential protein expression associated to selenite(Na2SeO3) and selenium nanoparticles (SeNPs) exposure. 57 and 47 proteins were found de-regulated by >1,5 fold in presence of selenite and SeNPs, respectively. Only 16 out of 104 proteins differentially expressed were commonly altered by selenite and SeNPs. The use of a clustered heat map allows us to visualize relations between the de-regulated proteins and exposure conditions. We identified a number of proteins involved in diverse functions and biological processes such as metabolism of carbohydrates, selenium and lipids; folding, sorting and degradation; environmental information and processing. In presence of both, selenite and SeNPs, proteins related to selenium metabolism such as cystathione beta-lyase and oxidoreductases (thioredoxine reductase and NAD/FAD oxidoreductase) were over expressed. Interestingly, the over expression of thioredoxin reductase could protect the host from oxidizing compounds. An over expression of phage proteins and chaperones with selenite was observed; this result and the fact that a lower cell count was detected when selenite was added could indicate that this latter Se species has a more deleterious effect than the nanoparticles.