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Electrical asymmetric-flow field-flow fractionation (EAF4) is a new and interesting analytical technique recently proposed for the characterization of metallic nanoparticles (NPs). It has the potential to simultaneously provide relevant information about size and electrical parameters, such as electrophoretic mobility (μ) and zeta-potential (ζ), of individual NP populations in an online instrumental setup with an array of detectors. However, several chemical and instrumental conditions involved in this technique are definitely influential, and only few applications have been proposed until now. In the present work, an EAF4 system has been used with different detectors, ultraviolet-visible (UV-vis), multi-angle light scattering (MALS), and inductively coupled plasma with triple quadrupole mass spectrometry (ICP-TQ-MS) for the characterization of gold, silver, and platinum NPs with both citrate and phosphate coatings. The behavior of NPs has been studied in terms of retention time and signal intensity under both positive and negative current with results depending on the coating. Carrier composition, particularly ionic strength, was found to be critical to achieve satisfactory recoveries and a reliable measurement of electrical parameters. Dynamic light scattering (DLS) has been used as a comparative technique for these parameters. The NovaChem surfactant mix (0.01%) showed a quantitative recovery (93 ± 1%) of the membrane, but the carrier had to be modified by increasing the ionic strength with 200 μM of Na

A rapid and simple methodology based on single particle-ICP-MS (SP-ICP-MS) has been developed for the study of gold nanoparticles (AuNPs) of different sizes in a cell culture medium commonly used in toxicological studies (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics as it is used in cell culture). Key instrumental parameters for the signal acquisition in single particle mode (e.g. dwell time and acquisition time) have been optimized. This new approach accomplishes the characterization and quantification of AuNPs in only one minute of analysis at concentrations in the ng L−1 range with a minimum sample preparation because only dilution was required. Precise determinations of particle size and concentration (%RSD around 1%) were reported with 22.4 nm and 2.1 × 105 particles per L as limit of detection for particle size (LODsize) and for particle-number concentration (LODNP), respectively. AuNPs were incubated with DMEM at 37 °C at different times up to 96 h to study the possible transformations undergone in this medium. The incubation time had an effect on the concentrations (number- and mass-based), even though different trends for AuNP concentrations over time were observed depending on the AuNP size. However, no differences were observed regarding the particle size.

A simple method based on the use of inductively coupled plasma mass spectrometry in single particle mode (SP-ICP-MS) has been proposed, for the first time, for the study of platinum nanoparticles (PtNPs) in complex clinical matrices such as human urine and blood serum. Critical parameters for signal acquisition were optimized to achieve a correct and simultaneous sizing and counting (particle-based in particles L−1 and mass-based in ng L−1) of 50 and 70 nm PtNPs. Different reagents, as tetramethylammonium hydroxide (TMAH) and/or Triton X-100, and concentrations have been tested to ensure an adequate stabilization and extraction of PtNPs. Finally, TMAH at 1% is demonstrated to be the best reagent to extract the NPs guaranteeing their integrity. No heating or any additional treatment was required, which allows sample preparation, and the overall process, to be simple and fast. Good precisions for size (2% RSD) and particle number and mass concentrations (

Toxicological studies of nanoparticles (NPs) are highly demanded nowadays but they are very challenging. In the in vitro assays, the understanding of the role of cell culture media is crucial to derive a proper interpretation of the toxicological results and to do so, new analytical tools are necessary. In this context, an analytical strategy based on reversed-phase liquid chromatography hyphenated to inductively coupled plasma-triple quadrupole mass spectrometry (HPLC-ICP-TQ-MS) has been developed for the first time for the detection and characterization of both 5 and 30 nm PtNPs, as well as ionic platinum species, in commonly used cell culture media. For this purpose, Dulbecco's Modified Eagle Medium, DMEM-high glucose, DMEM-F12, DMEM 31053–028, and Roswell Park Memorial Institute, RPMI-1640 (supplemented with 10% fetal bovine serum (FBS) and antibiotics) at several incubation times (24, 48, and 96 h at 37 °C) were tested. After a careful optimization and analytical performance, the developed method allows to simultaneously study the oxidation process, leading to the release of ionic species, and the increase in the hydrodynamic volume of PtNPs, probably related to the formation of new biological entities (protein corona). The magnitude of both processes was found to be dependent on the tested cell culture media and incubation times. Dynamic light scattering (DLS) and high-resolution scanning electron microscopy (HR-SEM) were used as complementary techniques to study the important process of both soft and hard protein corona formation. The feasibility of the HPLC-ICP-TQ-MS to get relevant information for toxicological studies has been demonstrated and in light of our results, the influence of the cell culture media on the behavior of PtNPs should not be underestimated.

The Tablas de Daimiel National Park (TDNP) is a floodplain ecosystem in central Spain with a potential risk of heavy metal and metalloid pollution. The objective of this study was to know the accumulation of arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), and selenium (Se) in muscle of six species of freshwater fish from the floodplain of TDNP. We obtained muscle samples of Cyprinus carpio (n = 89); Squalius pyrenaicus (n = 16); Ameiurus melas (n = 9); Lepomis gibbosus (n = 8); Micropterus salmoides (n = 6) and Carassius auratus (n = 5). A. melas, which is a predatory species, had significantly higher Hg concentrations than omnivorous or herbivorous species (i.e. C. carpio). On the contrary, A. melas showed lower concentrations of As, Pb and Se than omnivorous species (i.e. S. pyrenaicus and L. gibbosus ). The concentration of Hg was positively associated with fish size in C. carpio and A. melas. Some individuals of C. carpio (5.7%) and S. pyrenaicus (12.5%) showed Pb muscle concentrations above the maximum residue levels established by the European legislation for human consumers. The observed muscle Se concentrations can be associated with adverse effects on fish such as blood changes, reduced growth, mortality of juveniles and reproductive failure. The accumulation of Se in this floodplain located in a seleniferous area and the contamination produced by spent Pb shot pellets used for hunting in the past are discussed as potential sources of the elevated levels of these two elements in fish from this floodplain wetland., This study was supported by grant of research in National Parks (OAPN 755/2012) from the Ministry of Agriculture, Food and Environment Spain. Jhon J. López-Perea was supported by a doctoral FPU grant (AP-2012-04966) from the Spanish Ministry of Education. Sergio Fernández-Trujillo also thanks University of Castilla-La Mancha for the research starting scholarship (BIN1611), and Junta de Comunidades de Castilla-La Mancha for his pre-doctoral contract, SBPLY/16/180501/000356.

Human exposure to mercury is a serious problem of public health in Amazon. As in other vulnerable populations throughout the world, Amazonian riverine populations are chronically exposed to this metal and some symptoms of mercury intoxication were already detected in these populations. However, studies on the genetic susceptibility to mercury toxicity in the Amazon are scarce, and they tested a limited number of individuals. In this context, apolipoprotein E gene (APOE) is a key element with a well-established association among their alleles and the neurodegenerative consequences of mercury intoxication. However, no studies have addressed APOE genotyping in Amazonian exposed populations. Additionally, epidemiological studies with APOE genotyping in Amazon have been restricted to indigenous populations. Therefore, this work analyzed for the first time the genotypic and allelic profiles of APOE in Amazonian riverine populations chronically exposed to mercury. Eight hundred and twenty three individuals were enrolled in our study donating blood (794) and/or hair (757). APOE genotyping was analyzed by real-time PCR. Total mercury and mercury species were quantified by ICP-MS and GC-pyro-AFS, respectively. Genomic ancestry markers were evaluated by multiplex-PCR reaction, separated by capillary electrophoresis on the ABI 3130 Genetic Analyzer instrument and analyzed on GeneMapper ID v3.2. The 3 and 3/3 were the most frequent allele and genotype, respectively, followed by 4 allele and 3/4 genotype. Only 2/2 genotype was not found, suggesting that the absence of this genotype is a generalized phenomenon in Amazon. Also, our data supported an association between the presence of APOE4 and the Amerindian origin in these populations. Fifty-nine individuals were identified at maximum risk with levels of mercury above 10 μg/g and the presence of APOE4. Interestingly, among individuals with high mercury content, APOE4-carriers had high mercury levels than APOE2-carriers, pointing to a different heavy metal accumulation according to the APOE allele. These data suggest that APOE4, in addition to a possible pharmacodynamic effect, may influence pharmacokinetically the mercury exposure causing its higher accumulation and leading to worse deleterious consequences. Our results may aid in the development of prevention strategies and health policy decision-making regarding these at-risk vulnerable populations. A exposição humana ao mercúrio é um grave problema de saúde pública na Amazônia. Assim como em outras populações vulneráveis ​​em todo o mundo, populações ribeirinhas amazônicas estão cronicamente expostas a esse metal e alguns sintomas de intoxicação por mercúrio já foram detectados nessas populações. No entanto, estudos sobre a suscetibilidade genética à toxicidade do mercúrio na Amazônia são escassos e testaram um número limitado de indivíduos. Nesse contexto, o gene da apolipoproteína E (APOE) é um elemento chave com uma associação bem estabelecida entre seus alelos e as consequências neurodegenerativas da intoxicação por mercúrio. No entanto, nenhum estudo abordou a genotipagem da APOE em populações expostas na Amazônia. Além disso, estudos epidemiológicos com genotipagem APOE na Amazônia têm sido restritos a populações indígenas. Portanto, este trabalho analisou pela primeira vez os perfis genotípicos e alélicos de APOE em populações ribeirinhas amazônicas expostas cronicamente ao mercúrio. Oitocentos e vinte e três indivíduos foram incluídos em nosso estudo doando sangue (794) e/ou cabelo (757). A genotipagem da APOE foi analisada por PCR em tempo real. O mercúrio total e as espécies de mercúrio foram quantificados por ICP-MS e GC-piro-AFS, respectivamente. Os marcadores de ancestralidade genômica foram avaliados por reação multiplex-PCR, separados por eletroforese capilar no instrumento ABI 3130 Genetic Analyzer e analisados ​​no GeneMapper ID v3.2. O 3 e 3/3 foram os alelos e genótipos mais frequentes, respetivamente, seguidos do alelo 4 e do genótipo 3/4. Apenas o genótipo 2/2 não foi encontrado, sugerindo que a ausência desse genótipo é um fenômeno generalizado na Amazônia. Além disso, nossos dados apoiaram uma associação entre a presença de APOE4 e a origem ameríndia nessas populações. Cinquenta e nove indivíduos foram identificados em risco máximo com níveis de mercúrio acima de 10 μg/ge presença de APOE4. Curiosamente, entre os indivíduos com alto teor de mercúrio, os portadores de APOE4 apresentaram níveis de mercúrio mais elevados do que os portadores de APOE2, apontando para um acúmulo diferente de metais pesados ​​de acordo com o alelo APOE. Esses dados sugerem que a APOE4, além de um possível efeito farmacodinâmico, pode influenciar farmacocinéticamente a exposição ao mercúrio causando seu maior acúmulo e levando a piores consequências deletérias. Nossos resultados podem auxiliar no desenvolvimento de estratégias de prevenção e tomada de decisão de políticas de saúde em relação a essas populações vulneráveis ​​em risco.