Sonia D'Ilio

Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

Arsenic content of various types of rice as determined by plasma- based techniques

Abstract The determination of total As in rice is of special importance because rice forms a major dietary staple for many populations and contains a relatively high amount of this element as compared to other foodstuffs. Arsenic is in fact abundant in many minerals and its compounds are frequently employed in the chemical industry for producing pharmaceuticals, agricultural chemicals (herbicides, fungicides, wood preservatives, insecticides, rodenticides and sheep-dips) or as pig and poultry growth-promoters. Ingestion of As with food is one major route for this element to enter the human organism. Moreover, As in general, and its chemical forms in particular, are well known to cause acute and chronic toxicity, which may range from minor disorders to cancer and even death. In consideration of the above, the Community Reference Laboratory for Residues at the Istituto Superiore di Sanita (ISS-CRL) has undertaken a study aimed at quantifying the total content of As in eight different types of rice produced in northern Italy, seven of which belong to the variety Japonica and one to the variety Indica. Prior to analysis the rice samples were subjected to grinding and mineralized by acid-assisted microwave digestion. Determinations were performed by means of inductively coupled plasma atomic emission spectrometry (ICP-AES), quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) and magnetic sector high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). The As concentration intervals found for the eight types of rice investigated are as follows (in ng g−1): 205±16 (Arborio, Japonica, very fine); 80.7±1.0 (Baldo, Japonica, very fine); 136±4 (Carnaroli, Japonica, very fine); 283±9 (Ribe, Japonica, fine); 121±4 (Ribe Parboiled, Japonica, fine); 223±12 (Vialone Nano, Japonica, semi-fine); 187±1 (Originario, Japonica, common) and 133±4 (Thaibonnet Parboiled, Indica). Other trace elements were also quantified, the concentrations of which spanned the ranges (in ng g−1): Cd, 15.6–228; Co, 4.40–30.9; Cr, 2.1–4.7; Cu, 1958–3165; Fe, 2378–5014; Mn, 4656–11847; Pb, 4.57–17.3; V, 2.48–5.16 and Zn, 7510–20529. Grinding of rice prior to digestion caused no detectable effects on the actual values of As and of the other trace elements.

Occupational exposure of goldsmith workers of the area of Rome to potentially toxic metals as monitored through hair analysis

In continuation of an investigation recently carried out to monitor through hair analysis the occupational exposure of goldsmith workers to potentially toxic elements, another study was performed to extend the same methodological approach to the goldsmiths of Rome. This research was part of the project P.R.O.Art. undertaken by the Italian National Research Council in cooperation with the Ministry of Industry and the National Craftsmens Federation with the purpose of supporting goldsmith activities and trade. Sampling of hair, washing and sample digestion followed well-established procedures. Silver, Au, Cd, Co, Cr, In, Ni, Pb and Pt were determined by means of inductively coupled plasma mass spectrometry (ICP-MS), whereas Hg was analyzed using the flow injection mercury system (FIMS). On the other hand, the expected relatively high concentrations of Cu and Zn in hair allowed for the use of inductively coupled plasma atomic emission spectrometry (ICP-AES). Data obtained were statistically treated by applying the non-parametric Kruskal-Wallis test. A significant difference, at the level of P < 0.05, between exposed and unexposed subjects in the Rome area was observed only for Au.

A pilot study for the production of a certified reference material for trace elements in honey

In the context of the general mandate of the European Union Community Reference Laboratory (CRL) for residues in living animals and their products established at the Istituto Superiore di Sanita, a pilot study was undertaken to assess the possibility of producing a new certified reference material (CRM) for trace elements in a matrix of honey. The elements considered were As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Sn, V and Zn. Their determination was performed by inductively coupled plasma (ICP)-based techniques. Data obtained with different ICP techniques were generally in good agreement. In light of these results, the next step was the effective production of a candidate CRM in a honey matrix. In the preliminary phase, two different types of honey, i.e., Eucalyptus (solid and sticky) and Robinia (viscous and sticky), were pretreated at the Institute for Reference Materials and Measurements, Joint Research Centre, European Commission (EC-JRC-IRMM) in order to produce the materials candidate for the certification process. Approximately 600 ampoules were thus produced for the Robinia honey and 450 ampoules for the Eucalyptus honey, each ampoule containing 5 g of an aqueous solution of honey (with 20% and 30% high purity water, respectively) and sealed under inert gas (Ar). A ring test to determine the levels of the chemical elements and a long-term study to evaluate the stability of the samples is in progress. Tentative figures for the analytes of interest are (in ng g -1 ): Robinia, As, 1.28 ± 0.09; Cd, 0.59 ± 0.08; Cr, 2.36 ± 0.21; Cu, 57.6 ± 3.2; Fe, 209 ± 9; Mn, 90.8 ± 3; Ni, 18.1 ± 0.6; Pb, 23 ± 1.5; Sn, 8.10 ± 0.35; V. 1.19 ± 0.37; and Zn, 178 ± 4; Eucalyptus, As, 3.18 ± 0.21; Cd, 0.70 ± 0.08; Cr, 2.73 ± 0.22; Cu, 141 ± 6; Fe, 926 ± 16; Mn, 1905 ± 81; Ni, 7.77 ± 0.4; Pb, 138 ± 4; Sn, 7.97 ± 0.16; V, 3.47 ± 0.15; and Zn, 405 ± 9.

Determination of Cd and Pb in Honey by SF‐ICP‐MS: Validation Figures and Uncertainty of Results

Abstract A method based on microwave‐assisted acid digestion of honey and quantification of Cd and Pb by Sector Field Inductively Coupled Plasma Mass Spectrometry was in‐house validated and the combined uncertainty was estimated according to the Eurachem/Citac Guide. Limits of detection and quantification were 0.07 and 0.20 ng g−1 for Cd, and 0.70 and 2.10 ng g−1 for Pb; recovery was 103.9% for Cd and 98.5% for Pb; repeatability was 10.7% for Cd and 18.5% for Pb; within‐laboratory reproducibility was 15.2% for Cd and 21.4% for Pb. Relative combined uncertainty in honey was 15% for Cd and 22% for Pb, with the main contribution coming from the within‐laboratory reproducibility. The method showed robustness when subjected to different working conditions and when applied to various Italian honeys. Cadmium content ranged 0.2–1.37 ng g−1 and Pb 4.6–30.5 ng g−1 in flower honeys, while the highest concentrations were presented by honeydew honeys.

Method validation for the quantification of As, Cd, Hg and Pb in blood by ICP-MS for monitoring purposes

Medical laboratories dedicated to the quantification of trace and ultra trace elements in human biological fluids should be accredited according to the ISO standards 15189 : 2007. The use of validated methods for elements determination in blood becomes crucial for laboratories involved in medical analysis. The toxicological importance of As, Cd, Hg and Pb is well known and internationally recognized. These trace elements may be accumulated in human body through the exposure to pollutants in the environment as a result of uncontrolled anthropogenic activities. The internal levels of these elements can be estimated by the collection of blood samples from groups of selected population (biomonitoring). A fast and simple method to allow routine analysis of hundreds of blood samples was developed and fully validated. Arsenic, cadmium, mercury and lead were quantified in blood by quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS). A high throughput open digestion procedure was applied. Trueness, repeatability, within-laboratory reproducibility and standard measurement uncertainty were evaluated as method characteristics. The limits of quantification of the method were: 2.3 (As) μg l−1, 0.10 (Cd) μg l−1, 0.66 (Hg) μg l−1 and 0.56 (Pb) μg l−1. The method can be applied in the following ranges of concentration: 5–120 (As) μg l−1, 0.5–32 (Cd) μg l−1, 2.5–160 (Hg) μg l−1, 10–640 (Pb) μg l−1.

Application of routine estimation of Pb isotopic ratios by inductively coupled plasma mass spectrometry for studying the Pb origin in hair of children living in polluted areas. A pilot study

The analysis of 204 Pb, 206 Pb, 207 Pb and 208 Pb isotope ratios for environmental Pb markers (leaded gasoline, air-borne particulate matter, house window dust) and hair of children was undertaken by the routine quadrupole inductively coupled argon plasma mass spectrometry (Q-ICP-MS). Hair samples collected from 10-year-old children living in Krakow in 1995 and 35 randomly selected children, aged 11, both sexes were included in the current study. Air-borne particles were collected by PM-2.5 (an aerodynamic diameter dac < 2.5 μm) and PM-10 (fraction with an dac < 10 μm) samplers in the proximity of steel factories situated near Krakow. Imprecision of routine estimations was approximately 0.6-0.7% or less, and was the lowest for the 207 Pb/ 208 Pb ratio (approx. 0.2%). These conditions were sufficient to distinguish Pb from air-borne dust from that in gasoline or window dust. No differences were found between Pb in PM-2.5 and PM-10 particles. The associations of the Pb in the these environmental sources and that in hair of children were discussed. The relationships of the Pb isotope ratios and other parameters related to environmental pollution were also analyzed. The analysis of distribution of the 207 Pb/ 208 Pb ratio in the hair of children, provided some evidence of the fact that hair lead of approximately 20% of the investigated population could arise from gasoline, while the Pb from air-borne dust and remaining sources can be attributed to approximately 80% of the population.

Hair analysis and the early detection of imbalances in trace elements for members of expeditions in Antarctica

Abstract In the framework of the Human Biology and Medicine Project of the National Program for Research in Antarctica (Programma Nazionale di Ricerche in Antartide, PNRA) a study was undertaken to explore the suitability of trace element determination in human hair as a tool for the early detection of the impact of extreme environmental conditions on the health of members of the Italian and French expeditions. This study included three campaigns, namely, the 1995–1996 expedition to the Italian base of Terra Nova Bay, as well as the 1997–1998 and 1998–1999 expeditions to both the Italian base of Terra Nova Bay and the Italian–French base of Dome C. Sampling of hair of participants in the expeditions was carried out both prior to departure and at the end of the period spent at the bases. As regards storage, pre-treatment and analysis of hair samples they were performed in accordance with previously established procedures. Acid-assisted microwave digestion was adopted as the best approach for preparing solutions for subsequent analysis by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. In the first part of this study, only the elements Ca, Cu, Fe, Mg and Zn were selected, while at a later stage it was decided to also include Co, Cr, Mn, Mo and Ni. Experimental results were subjected to basic statistical analysis to detect possible significant differences in element concentrations that occurred during the sojourn at the bases, with a view to identify possible remedial action to minimize adverse consequences.

Determination of depleted uranium in human hair by quadrupole inductively coupled plasma mass spectrometry: method development and validation

The presence of depleted uranium (DU) in the environment and its possible transfer to humans has been a matter of concern for the potential risks to human health. Depleted uranium has been used for military purposes in the last few years of conflict due to its great availability and low cost. Uranium content has been mainly determined in biological fluids, such as urine and blood. Human hair can be considered as a good alternative indicator of exposure to DU thanks to its ability of accumulating chemical elements over a long period of time. Determining the isotopic composition of elements always requires using a sensitive and precise analytical technique. In this regard, isotope ratio quadrupole inductively coupled plasma mass spectrometry was considered as a qualified analytical technique and consequently employed in the present study in combination with a desolvating sample introduction system. The method was in-house validated at three different levels of concentration of depleted uranium (50, 500 and 1000 ng/l) according to common standards and guidelines. The limit of quantification of the method in hair was 7.21 μg/kg with a within-laboratory reproducibility, in terms of variation coefficient, of 16.8, 6.1 and 5.5% for the three levels, respectively. A novel analytical approach was applied to the specificity studies for hair contaminated by DU with different background levels of NU.

Trace elements in bovine muscle: An ongoing project for a new certified reference material

Abstract In the framework of the activities of the Community Reference Laboratory (CRL) for residues at the Istituto Superiore di Sanita (ISS) of Rome, a number of proficiency tests were performed in order to assess and improve, wherever necessary, the quality of regulatory residue analysis within the EU. In this context, a pilot study was undertaken with the purpose of ascertaining the feasibility of a project for the certification of a new reference material for trace elements in bovine muscle. On behalf of the ISS, approximately 70 kg of bovine muscle, collected at a slaughterhouse in Geel (Belgium), were freeze-dried, homogenized, stabilized and bottled under argon atmosphere by the Institute for Reference Materials and Measurements, Joint Research Centre, European Commission (IRMM-JRC-EC). Preliminary analyses were carried out with Quadrupole (Q) and High Resolution (HR) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to evaluate the content of As, Cd, Cu and Pb, whereas the Flow Injection Mercury System (FIMS) was used to quantify Hg. Digestion of the matrix was achieved by acid-assisted microwave irradiation. The mean values of experimentally obtained data were the following (in nanograms per gram): As, 24.2±1.2; Cd, 5.48±0.29; Cu, 4765±95; Hg, 3.37±0.73; and Pb, 184±7. Subsequently, 16 National Reference Laboratories (NRLs) for residues in the Member States plus the Norwegian one and eight Italian public laboratories were accepted to participate in the project and received one bottle of the freeze-dried bovine muscle. The laboratories were requested to perform three different analytical runs (in three different days), each consisting of three individual measurements. Preliminary results showed good agreement and fair evidence for homogeneity of the entire mass, thus setting the stage for the future certification of this candidate CRM.