Critical evaluation of the potential of ICP-MS-based systems in toxicological studies of metallic nanoparticles.

The extensive application of metallic nanoparticles (NPs) in several fields has significantly impacted our daily lives. Nonetheless, uncertainties persist regarding the toxicity and potential risks associated with the vast number of NPs entering the environment and human bodies, so the performance of toxicological studies are highly demanded. While traditional assays focus primarily on the effects, the comprehension of the underlying processes requires innovative analytical approaches that can detect, characterize, and quantify NPs in complex biological matrices. Among the available alternatives to achieve this information, mass spectrometry, and more concretely, inductively coupled plasma mass spectrometry (ICP-MS), has emerged as an appealing option. This work critically reviews the valuable contribution of ICP-MS-based techniques to investigate NP toxicity and their transformations during in vitro and in vivo toxicological assays. Various ICP-MS modalities, such as total elemental analysis, single particle or single-cell modes, and coupling with separation techniques, as well as the potential of laser ablation as a spatially resolved sample introduction approach, are explored and discussed. Moreover, this review addresses limitations, novel trends, and perspectives in the field of nanotoxicology, particularly concerning NP internalization and pathways. These processes encompass cellular uptake and quantification, localization, translocation to other cell compartments, and biological transformations. By leveraging the capabilities of ICP-MS, researchers can gain deeper insights into the behaviour and effects of NPs, which can pave the way for safer and more responsible use of these materials.

Electrical asymmetric-flow field-flow fractionation with a multi-detector array platform for the characterization of metallic nanoparticles with different coatings.

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 Na2CO3 to achieve consistent µ values. However, ζ was one order of magnitude lower in EAF4-UV-vis-MALS than in DLS, probably due to different electric processes in the channel. From a practical point of view, EAF4 technique is still in its infancy and further studies are necessary for a robust implementation in the characterization of NPs.

Speciation of platinum nanoparticles in different cell culture media by HPLC-ICP-TQ-MS and complementary techniques: A contribution to toxicological assays.

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.

Screening-confirmation strategy for nanomaterials involving spectroscopic analytical techniques and its application to the control of silver nanoparticles in pastry samples.

The full characterization of nanomaterials (NMs), which requires a range of different and expensive equipment, is not always necessary to meet certain demands of information. Qualitative analytical methods are ideal alternatives when only a piece of information is required. In this work, a qualitative method for the screening of NMs has been developed and statistically validated for the first time, with silver nanoparticles (AgNPs) as a proof of concept. It is based on the generation of chemiluminescence of the luminol/Ag+ system in alkaline media in the presence of AgNPs. Measurements are obtained in a short time with a simple instrument. Probability of detection (POD) curves were constructed at three cut-off values next to the limit of detection of the chemiluminescent method. The unreliability region (UR) was from 0.50 to 1.82 µg L-1. Currently, no regulation on AgNPs exists, but the present method was successfully tested for a hypothetical threshold of 2.5 µg L-1 of 40 nm AgNPs. The method was applied to silver colored pearls, with silver (E-174) or aluminum (E-173) coating, used for decoration of pastry. Performance parameters such as false negative and positive rates as well as specificity, sensitivity and reliability rates were calculated for validation. The results of the screening method were confirmed by asymmetric flow field flow fractionation coupled to inductively coupled plasma mass spectrometry (AF4-ICP-MS). The proposed screening method is simple, fast, economic, and easy to transfer to routine laboratories in the field of food safety.

Temporal variability measurements of PM2.5 and its associated metals and microorganisms on a suburban atmosphere in the central Iberian Peninsula.

A novel and multidisciplinary observational analysis of atmospheric components in the Central Iberian Peninsula is presented here. PM2.5 concentrations and both populations of cultivable and non-cultivable microorganisms and concentrations of a wide range of trace elements associated have been simultaneously studied during multiple events along one year. The aim has been to characterize their potential relations and dependencies, and their seasonal, daily and hourly evolution. Tools that could explain the atmospheric mechanisms and sources from all these elements have been also evaluated. As it would be expected from a suburban environment, absolute levels obtained were not close to legislation limits. Anthropogenic and natural sources, such as heating home, soil resuspension, or Sahara dust intrusion; and atmospheric factors are responsible for higher PM2.5 and metals concentrations in months with both low and high temperatures. Daily and hourly evolution depends on University Campus activity, especially on traffic flow and resuspended dust due to human transit. No statistical significant differences on daily or seasonal scales between cultivable counts of fungi and bacteria were displayed. However, using the q-PCR technique, the bacterial population was lower in winter. Positive correlations between PM2.5 and relative humidity; and PM2.5 and cultivable microorganism have been established. It was also the case among 7 of the 11 trace elements, indicating then common natural or anthropogenic sources. In summary, this work illustrates the interest of a combined inspection of elements, interactions and dependencies when studying the unique and continuous atmospheric environment, which are typically analysed separately.

Screening and Preliminary Biochemical and Biological Studies of [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] in Breast Cancer Models.

Breast cancer is the second leading cause of cancer death worldwide. Despite progress in drug discovery, identification of the correct population is the limiting factor to develop new compounds in the clinical setting. Therefore, the aim of this study is to evaluate the effects of a new metallodrug, [RuCl(p-cymene)(N,N-bis(diphenylphosphino)-isopropylamine)][BF4] (pnpRu-14), as a lead pnp-Ru compound by screening and preliminary biochemical and biological studies in different breast cancer subtypes. The results show that complex pnpRu-14 is much more effective in promoting in vitro cytotoxic effects on HER2+ and RH+/HER2- breast cancer than the reference metallodrugs cisplatin, carboplatin, or RAPTA-C. It is important to highlight that pnpRu-14 shows an impressive cytotoxicity against BT474 cells. Caspase-dependent apoptosis is the mechanism of action for these compounds. In addition, treatment of SKBR3, BT474, T47D, and MCF7 cancer cells with pnpRu-14 caused an accumulation of cells in the G0/G1 phase cells. The human serum albumin, DNA, and H1 histones binding properties of the lead compound are reported. Pharmacokinetic and biodistribution studies show a quick absorption of pnpRu-14 in serum with no significant accumulation in any of the tested organs. This work provides evidence to support the preclinical and clinical development of pnpRu-14 in breast cancer.

Geno- and cytotoxicity induced on Cyprinus carpio by aluminum, iron, mercury and mixture thereof.

Metals such as Al, Fe and Hg are used in diverse anthropogenic activities. Their presence in water bodies is due mainly to domestic, agricultural and industrial wastewater discharges and constitutes a hazard for the organisms inhabiting these environments. The present study aimed to evaluate geno- and cytotoxicity induced by Al, Fe, Hg and the mixture of these metals on blood of the common carp Cyprinus carpio. Specimens were exposed to the permissible limits in water for human use and consumption according to the pertinent official Mexican norm [official Mexican norm NOM-127-SSA1-1994] Al (0.2mgL-1), Fe (0.3mgL-1), Hg (0.001mgL-1) and their mixture for 12, 24, 48, 72 and 96h. Biomarkers of genotoxicity (comet assay and micronucleus test) and cytotoxicity (caspase-3 activity and TUNEL assay) were evaluated. Significant increases relative to the control group (p<0.05) were observed in all biomarkers at all exposure times in all test systems; however, damage was greater when the metals were present as a mixture. Furthermore, correlations between metal concentrations and biomarkers of geno- and cytotoxicity were found only at certain exposure times. In conclusion, Al, Fe, Hg and the mixture of these metals induce geno- and cytotoxicity on blood of C. carpio.

Analysis of silica nanoparticles by capillary electrophoresis coupled to an evaporative light scattering detector.

A simple and rapid methodology has been developed to identify and separate silica nanoparticles (SiO2NPs) of different sizes in aqueous solution by capillary zone electrophoresis coupled to an evaporative light scattering detector (CE-ELSD). SiO2NPs were separated using 3 mM ammonium acetate buffer, containing 1% methanol at pH 6.9. SiO2NPs of 20, 50 and 100 nm were successfully separated under the optimum experimental conditions. CE coupled to ELSD has been proven to be an effective separation technique to determine particles with such small sizes, although the peaks are very close to each other, and it is a promising technique that may allow the separation of other types of nanoparticles. Confirmation by TEM and quantification of the SiO2 content was also carried out by inductively coupled plasma-mass spectrometry (ICP-MS). The new method was applied to the analysis of real samples, in order to assess its ability to avoid matrix effects in the determination of SiO2NPs in these kinds of samples.

Mercury exposure and mechanism of response in large game using the Almadén mercury mining area (Spain) as a case study.

Mercury (Hg) accumulation, transfer, defense mechanism and adverse effects were studied in red deer (Cervus elaphus) and wild boar (Sus scrofa) from the Almadén mining district (Spain), the largest (285,000 t of Hg) and the oldest (more than 2000 years) Hg mine/refining operation site in the world. Red deer (n=168) and wild boar (n=58) liver, kidney, bones (metacarpus), testis and muscle were analyzed for total Hg and selenium (Se) within a range of distances to the Almadén mining district. The highest Hg concentrations were found in kidney (0.092 and 0.103 µg/g d.w. for red deer and wild boar, respectively) followed by the levels in liver (0.013 and 0.023 µg/g d.w. for red deer and wild boar, respectively). A significant correlation (r=-0.609, p=0.007) was found between Hg concentrations and distance to the Almadén Hg mining district. However, both red deer and wild boar closest to the mining area still showed mercury concentrations well below the concentration associated with clinical signs of Hg poisoning. Highest Se concentrations were found in kidney (2.60 and 6.08 µg/g in red deer and wild boar, respectively) and testis (2.20 µg/g in red deer). For selenium, differences between red deer and wild boar were statistically significant (p<0.05) in all tissues, concentrations being higher in wild boar than in red deer. In the diagnosis of Se deficiency, the vast majority of the examined red deer livers were deficient. A significant correlation was found between Hg and Se in kidney (r=0.386, p>0.001 for red deer and r=0.567, p=0.005 for wild boar). Liver GSSG concentrations were negatively correlated to total mercury and Hg:Se molar ratio in male deer, which could indicate a hormetic response to Hg exposure. Moreover, a positive association was found between the antioxidant element Se and antioxidant vitamins in red deer tissues.

Biospeciation of tungsten in the serum of diabetic and healthy rats treated with the antidiabetic agent sodium tungstate.

It is known that oral administration of sodium tungstate preserves the pancreatic beta cell function in diabetic rats. Healthy and streptozotocin-induced diabetic rats were treated with sodium tungstate for one, three or six weeks, after which the species of W in serum, were analysed. An increase in serum W with treatment time was observed. After six weeks, the serum W concentration in diabetic rats (70 mg L(-1)) was about 4.6 times higher than in healthy specimens. This different behaviour was also observed for Cu accumulation, while the Zn pattern follows the contrary. The patterns observed in the retention of Cu and Zn may be attributable to a normalization of glycaemia. The speciation analysis of W was performed using 2D separations, including an immunoaffinity packing and a SEC (Size Exclusion Chromatography) column coupled to an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for elemental detection. Ultrafiltration data together with SEC-ICP-MS results proved that around 80% of serum W was bound to proteins, the diabetic rats registering a higher W content than their healthy counterparts. Most of the protein-bound W was due to a complex with albumin. An unknown protein with a molecular weight higher than 100 kDa was also found to bind a small amount of W (about 2%). MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) analysis of the desalted and concentrated chromatographic fractions confirmed albumin as the main protein bound to tungstate in rat serum, while no binding to transferrin (Tf) was detected. The interaction between glutathione and W was also evaluated using standard solutions; however, the formation of complexes was not observed. The stability of the complexes between W and proteins when subjected to more stringent procedures, like those used in proteomic methodologies (denaturing with urea or SDS, boiling, sonication, acid media, reduction with ß-mercaptoethanol (BME) or DTT (dithiotreitol) and alkylation with iodoacetamide (IAA), was also evaluated. Our results indicate that the stability of the complexes between W and proteins is not too high enough to remain unaltered during protein separation by SDS-PAGE in denaturing and reducing conditions. However, the procedures for in-solution tryptic digestion and for ESI-MS analysis in MeOH/H(2)O/with 0.1% formic acid could be used for protein identification without large loss of binding between W and proteins.

Study of tungstate-protein interaction in human serum by LC-ICP-MS and MALDI-TOF.

Oral administration of sodium tungstate is an effective treatment for type 1 and 2 diabetes in animal models; it does not incur significant side effects, and it may constitute an alternative to insulin. However, the mechanism by which tungstate exerts its observed metabolic effects in vivo is still not completely understood. In this work, serum-containing proteins which bind tungstate have been characterized. Size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a Phenomenex Bio-Sep-S 2000 column and 20 mM HEPES and 150 mM NaCl at pH 7.4 as the mobile phase was chosen as the most appropriate methodology to screen for tungsten-protein complexes. When human serum was incubated with tungstate, three analytical peaks were observed, one related to tungstate-albumin binding, one to free tungstate, and one to an unknown protein binding (MW higher than 300 kDa). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of the tungsten-containing fractions collected from SEC-ICP-MS chromatograms, after desalting and preconcentration processes, confirmed the association of tungstate with albumin and the other unknown protein. [figure: see text]