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Leaching experiments were carried out on fresh ash samples from Popocatépetl 2012, Etna 2011, and Etna 2012 eruptions, in order to investigate the release of compounds in both double-deionized and lake (Lake Ohrid, FYR of Macedonia) waters. The experiments were carried out using different grain sizes and variable stirring times (from 30 min to 7 days). Results were discussed in the light of changing pH and release of compounds for the different leachates. In particular, Etna samples induced alkalinization, and Popocatépetl samples induced acidification of the corresponding leachates. The release of different elements does not show correlation with the stirring time, with the measured maximum concentrations reached in the first hours of washing. General inverse correlation with grain size was observed only for Na+, K+, Cl-, Ca2+, Mg2+, SO2-4 , and Mn2+, while the other analysed elements show a complex, scattering relationship with grain size. Geochemical modelling highlights leachates' saturation only for F and Si, with Popocatépetl samples sometimes showing saturation in Fe. The analysed leachates are classified as undrinkable for humans on the basis of European laws, due to excess in F-, Mn2+, Fe, and SO2-4 (the latter only for Popocatépetl samples). Finally, the Etna 2012 and Popocatépetl leachates were used for toxicity experiments on living biota (Xenopus laevis). They are mildly toxic, and no significant differences exist between the toxic profiles of the two leachates. In particular, no significant embryo mortality was observed; while even at high dilutions, the leachates produced more than 20% of malformed larvae.

The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1–100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

The antibacterial properties of nano-metal oxides (ZnO, CuO) are based on the formation of reactive oxygen species (ROS). This work reveals that the antibacterial properties of these nano-metal oxides are strongly dependent on their crystalline structure. The antibacterial activity of the nanooxides was tested against four types of bacteria that commonly cause nosocomial infections. The sonochemical method was applied not only for synthesis of nanooxides but also to their coating on textiles. The antibacterial properties of textiles coated with commercial and sonochemically prepared nano-metal oxides were evaluated and compared. The toxicity was evaluated on human lung cells and amphibian embryos, as representative models for inhalation and aquatic toxicology. The sonochemically prepared metal nanooxides are better antimicrobials than commercially available metal oxides with the same particle size range. It was found that the crystallites which have more defects and less organized structure are more toxic. The formation of ROS was studied by electron spin resonance (ESR) measurements for both the sonochemically prepared and commercial samples of ZnO/CuO nanoparticles. A significant increase in the production of radical species was found in the more defective, sonochemically prepared samples, as compared to the commercial ones. Since modulation of the nanoparticle defects influenced their toxicity, the possibility of engineering safer nano-antibacterials is indicated.

The increased resistances to conventional antibiotics determine a strong need for new antibacterials, and specific syntheses at the nanoscale promise to be helpful in this field. A novel Zinc-doped Copper oxide nanocomposite (nZn-CuO) has been recently sonochemically synthesized and successfully tested also against multi-drug resistant bacteria. After synthesis and characterization of the physicochemical properties, the new nZn-CuO is here evaluated by the Frog Embyo Teratogenesis Assay-Xenopus test for its toxicological potential and this compared with that of nCuO and nZnO synthesized under the same conditions. No lethal effects are observed, while malformations and growth retardation slightly increase after nZn-CuO exposure. Nevertheless, these effects are smaller than those of nZnO. NP uptake by embryo tissues increase significantly with increasing NP concentrations, while no significant accumulation and adverse effects are seen after exposure to soluble Cu2+ and Zn2+ at the concentrations dissolved from the NPs. Key oxidative response genes are upregulated by nZn-CuO, as well as by nCuO and nZnO, suggesting the common mechanism of action. Considering the enhanced biocidal activity shown by the nanocomposite, together with the results presented in this study, we can affirm that the doping of the metal oxide nanoparticles should be considered a useful tool to engineer a safer nano-antibacterial.

Oxidative stress, pulmonary and systemic inflammation, endothelial cell dysfunction, atherosclerosis and cardiac autonomic dysfunction have been linked to urban particulate matter exposure. The chemical composition of airborne pollutants in Milano is similar to those of other European cities though with a higher PM2.5 fraction. Milano winter fine particles (PM2.5win) are characterized by the presence of nitrate, organic carbon fraction, with high amount of polycyclic aromatic hydrocarbons and elements such as Pb, Al, Zn, V, Fe, Cr and others, with a negligible endotoxin presence. In BALB/c mice, we examined, at biochemical and transcriptomic levels, the adverse effects of repeated Milano PM2.5win exposure in lung and heart. We found that ET-1, Hsp70, Cyp1A1, Cyp1B1 and Hsp-70, HO-1, MPO respectively increased within lung and heart of PM2.5win-treated mice. The PM2.5win exposure had a strong impact on global gene expression of heart tissue (181 upregulated and 178 down-regulated genes) but a lesser impact on lung tissue (14 up-regulated genes and 43 downregulated genes). Focusing on modulated genes, in lung we found two- to three-fold changes of those genes related to polycyclic aromatic hydrocarbons exposure and calcium signalling. Within heart the most striking aspect is the twofold to threefold increase in collagen and laminin related genes as well as in genes involved in calcium signaling. The current study extends our previous findings, showing that repeated instillations of PM2.5win trigger systemic adverse effects. PM2.5win thus likely poses an acute threat primarily to susceptible people, such as the elderly and those with unrecognized coronary artery or structural heart disease. The study of genomic responses will improve understanding of disease mechanisms and enable future clinical testing of interventions against the toxic effects of air pollutant

The developmental toxicity of nanostructured materials, as well as their impact on the biological barriers, represents a crucial aspect to be assessed in a nanosafety policy framework. Nanosized metal oxides have been demonstrated to affect Xenopus laevis embryonic development, with nZnO specifically targeting the digestive system. To study the mechanisms of the nZnO-induced intestinal lesions, we tested two different nominally sized ZnO nanoparticles (NPs) at effective concentrations. Advanced microscopy techniques and molecular marker analyses were applied in order to describe the NP-epithelial cell interactions and the mechanisms driving NP toxicity and translocation through the intestinal barrier. We attributed the toxicity to NP-induced cell oxidative damage, the small-sized NPs being the more effective. This outcome is sustained by a marked increase in anti-oxidant genes' expression and high lipid peroxidation level in the enterocytes, where disarrangement of the cytoskeleton and cell junctions' integrity were evidenced. These events led to diffuse necrotic changes in the intestinal barrier, and trans- and paracellular NP permeation through the mucosa. The uptake routes, leading NPs to cross the intestinal barrier and reach secondary target tissues, have been documented. nZnOs embryotoxicity was confirmed to be crucially mediated by the NPs' reactivity rather than their dissolved ions. The ZnO NPs' ability to overwhelm the intestinal barrier must be taken into high consideration for a future design of safer ZnO NPs. © 2014 Informa UK, Ltd.

The toxic effects of two differently sized ZnO nanopowders have been studied in Daphnia magna using advanced microscopy techniques. Five nanoZnO suspensions (0.1, 0.33, 1, 3.3 and 10mg/L) were tested. The results of the 48-h acute toxicity tests performed with ZnO<100nm (bZnO) and ZnO<50nm (sZnO) showed slight effects, with EC50 values of 3.1 and 1.9mg/L for bZnO and sZnO, respectively. Specimens exposed to 1 and 3.3mg/L have been microscopically analysed and nanoparticles (NPs) from both concentrations have been found into midgut cells: i) in the microvilli; ii) in endocytic vesicles near the upper cell surface; iii) in some endosomes, as well as in mitochondria, in multivesicular and multilamellar bodies; iv) into the enterocytes' nuclei; v) free in the cytoplasm; vi) in the paracellular space between adjacent cells; vii) into the folded basal plasma membrane, and viii) in the gut muscolaris, suggesting that not only both nanoZnOs are able to interact with the plasmatic membrane of D. magna enterocytes, but also that they are capable to cross epithelial barriers. The ultrastructural changes increased with increasing concentrations and the worst morphological fields came from samples exposed to 3.3mg/L of both nanoZnOs. Morphological effects were qualitatively similar between the two nanomaterials, but they appear to be much more frequent for sZnO NPs. Data from ICP-OES analyses demonstrated that the maximum Zn++ concentration in our tested suspensions was 0.137mg/L, which is well below the reported NOEC for the soluble Zinc. The corresponding Zn-salt exposures (0.1mg/L Zn++) gave 0% of immobilized daphnids for both NPs suggesting that in our test medium nanoZnO toxicity is not driven by their solubilized ions. The large presence of NPs inside midgut cells after only 48-h exposure to nanoZnOs and their effects on the intestinal cells highlighted the toxic potential of these nanomaterials, also suggesting that studies on chronic effects are needed. © 2014 Elsevier L

Exposure to engineered nanoparticles (NPs) and to ambient particles (PM) has increased significantly. During the last decades the application of nano-objects to daily-life goods and the emissions produced in highly urbanized cities have considerably augmented. As a consequence, the understanding of the possible effects of NPs and PM on human respiratory system and particularly on the air-blood barrier (ABB) has become of primary interest. The crosstalk between lung epithelial cells and underlying endothelial cells is indeed essential in determining the effects of inhaled particles. Here we report the effects of metal oxides NPs (CuO and TiO and of PM on an in vitro model of the ABB constituted by the type II epithelial cell line (NCI-H441) and the endothelial one (HPMEC-ST1.6R). The results demonstrate that apical exposure of alveolar cells induces significant modulation of proinflammatory proteins also in endothelial cells. © 2013 Rossella Bengalli et al.

Metal oxide NPs are abundantly produced in nanotech industries and are emitted in several combustion processes, suggesting the need to characterize their toxic impact on the human respiratory system. The acute toxicity and the morphological changes induced by copper oxide and titanium dioxide NPs (nCuO and nTiO2) on the human alveolar cell line A549 are here investigated. Cell viability and oxidative stress have been studied in parallel with NP internalization and cell ultrastructural modifications. TiO2 NPs were abundantly internalized by cells through the endocytic pathway, even they did not induce cell death and ultrastructural lesions. Only after 24h cells were affected by an abundant NP internalization presenting a consequent altered morphology. High cytotoxicity, oxidative stress and severe ultrastructural damages were produced by nCuO, since cell membrane and mitochondria resulted to be heavily affected, even at early exposure time. nCuO-induced toxicity has been interpreted as a consequence of both NPs reactivity and copper ions dissolution in lysosomal compartments, even the free NPs, scattered throughout all the cell compartments, might contribute to the toxicity. The antioxidant N-acetylcysteine was effective in recovering nCuO exposed cells viability and Bafilomycin A1 inhibited copper ions release in phagolysosomes and significantly rescued cells, suggesting a relevant cytotoxic mechanism relative to oxidative damages and authophagic cell death, together with NP internalization and dissolution. Our results support the previous data reporting CuO NPs are highly cytotoxic and genotoxic, and associate their toxic effects with their cell penetration and interaction with various compartments. In conclusion, the so-called "Trojan horse" mechanism and autophagy, are involved in nCuO-induced cell death, even a further research is needed to explain the events occurring at early exposure time. © 2013 Elsevier Ireland Ltd.

The advent of nanotechnology and the commercialization of several nanoparticle-containing products call to a serious assessment of the environmental and human risks derived from the exposure to these new materials. The emerging properties, the increasing world market and the several exposure scenarios are some of the main criticisms related to distribution of these products. Among nanomaterials metal oxides nanoparticles are considered at the same time an interesting and critical group; in particular titanium dioxide, zinc oxide and cupric oxide (nTiO2, nZnO, nCuO) have received particular attention due to their unique physical and chemical properties and their environmental and economical relevance. Previous studies focused on toxicity of these materials highlighted different adverse effects on the biological systems exposed but there is still a lack of information about the mechanisms involved. These research aims to evaluate the potential biological effects of these commercial transition metal nanoxides on two different experimental models: an in vivo model and an in vitro one. The in vivo model has been used to assess the aquatic toxicity, which has been evaluated by the use of the FETAX assay (Frog embryo teratogenesis assay - Xenopus), a sensitive test commonly used to investigate the embryotoxic effects induced by chemicals. After a preliminary physico-chemical characterization, nTiO2, nZnO, nCuO were administered to Xenopus embryos as suspensions in FETAX medium; at the end of the test the main end points of growth retardation, mortality, absolute and relative frequency of malformations have been analysed. In parallel, different microscopic techniques were used to better characterise the effects observed and to localise the particles inside the organisms. Our results revealed that even high NP concentrations (up to 500 mg/ml) were not lethal for such NPs with the exception of nCuO but all NPs induced relevant malformations. Exposure to titanium dioxide did p

PM10 was collected in a Milan urban site, representative of the city air quality, during winter and summer 2006. Mean daily PM10 concentration was 48 mug m(-3) during summer and 148 mug m(-3) during winter. Particles collected on Teflon filters were chemically characterized and the endotoxin content determined by the LAL test. PM10-induced cell toxicity, assessed with MTT and LDH methods, and proinflammatory potential, monitored by IL-6 and IL-8 cytokines release, were investigated on the human alveolar epithelial cell line A549 exposed to increasing doses of PM. Besides untreated cells, exposure to inert carbon particles (2-12 mum) was also used as additional control. Both cell toxicity and proinflammatory potency resulted to be higher for summer PM10 with respect of winter PM10, with IL-6 showing the highest dose-dependent release. The relevance of biogenic components adsorbed onto PM10 in eliciting the proinflammatory mediators release was investigated by inhibition experiments. Polymixin B (Poly) was used to inhibit particle-bind LPS while Toll-like receptor-2 antibody (a-TLR2) to specifically block the activation of this receptor. While cell viability was not modulated in cells coexposed to PM10 and Poly or a-TLR2 or both, inflammatory response did it, with IL-6 release being the most inhibited. In conclusion, Milan PM10-induced seasonal-dependent biological effects, with summer particles showing higher cytotoxic and proinflammatory potential. Cytotoxicity seemed to be unaffected by the PM biogenic components, while inflammation was significantly reduced after the inhibition of some biogenic activated pathways. Besides, the PM-associated biogenic activity does not entirely justify the PM-induced inflammatory effects. (c) 2010 Wiley Periodicals, Inc. Environ Toxicol 2010.

The impact of nano metal-oxides on human and environmental health is predicted to be increasing. Among metal oxides, nano ZnO (nZnO) is retained one of the most dangerous. Recently nZnO has invaded the market for its UV protective and antibacterial properties, that make it suitable for a wide range of application for functional coating formulations to protect wood, plastics, textiles from UV and microbial degradation. Previous data already showed that nZnO has a powerful embryotoxic potential on X. laevis and that it was able to mainly affect gut development. It was clearly demonstrated that nZnO produced severe lesions at the intestinal mucosa and potentially cross the gut barrier reaching the underlying tissues. In this work we used Xenopus laevis embryos to characterize the embryotoxic and teratogenic potential of nZnO according to the modulation of NP size and surface charge, as well as to the irradiation conditions. To optimize the stability of the NP suspensions and to achieve useful NP-surface functionalization, we worked in strict connection with a private nanotech company with both R&D and commercial activities. The purpose was to provide mechanistic data on nZnO ecotoxicology and to suggest criteria to design safer Zinc oxide NPs. We demonstrated that nZnO-induced embryotoxicity was mediated by NPs’ own reactivity rather than ion dissolution and that it is strongly associated with the modality of the biological interactions at the nano-level, which at last depend upon the physical and chemical NP surface properties. These properties are also at the base of the induced oxidative potential by nZNO, which is also very efficiently modulated by light irradiation. Finally NP dimension, and especially surface charge, played a crucial role in determining the embryotoxic potential and the intestinal translocation and lesions of nZnO. The present results showed how a comprehensive knowledge of the nZnO physical and chemical properties, affecting the interactions at

Lethal and teratogenic potentials of carbon nanoparticles (CNPs) in their amorphous form were investigated by the standardized Frog Embryo Teratogenesis Assay-Xenopus (FETAX), a 96-h in vitro whole-embryo toxicity test based on the amphibian Xenopus laevis. Embryos were acutely exposed to 1, 10, 100 and 500 mg/L CNP suspensions and evaluated for lethality, malformations and growth inhibition. Larvae were processed for histological and ultrastructural analyses to detect the main affected organs, to look for specific lesions at the subcellular level and to image and track CNPs into tissues. Only the highest CNP suspension resulted in being embryolethal for X. laevis larvae, while malformed larva percentages significantly differed from controls starting from 100 mg/L. The stomach and gut were the preferential CNP accumulation sites, on the contrary, the digestive epithelium remained intact. The analyses showed the presence of isolated nanoparticles and/or aggregates in different secondary target organs. CNPs were found in circulating erythrocytes. The research confirms the good tolerance of X. laevis towards pure elemental carbon in its nanoparticulate amorphous form, but highlights the possibility of CNP transfer toward all body areas

The results presented summarise the ones obtained in the coordinated research project TOSCA (Toxicity of Particulate Matter and Molecular Markers of Risk), which extensively analysed the impact of Milan urban PM on human health. The molecular markers of exposure and effects of seasonally and sizefractionated PMs (summer and winter PM10, PM2.5) were investigated in in vitro (human lung cell lines) and in vivo (mice) systems. The results obtained by the analyses of cytotoxic, pro-inflammatory and genotoxic parameters demonstrate that the biological responses are strongly dependent upon the PM samples seasonal and dimensional variability, which ultimately reflect their chemical composition and source. In fact, summer PM10, enriched in crustal elements and endotoxins, was the most cytotoxic and pro-inflammatory fraction, while fine winter PMs induced genotoxic effects and xenobiotic metabolizing enzymes (like CYP1B1) production, likely as a consequence of the higher content in combustion derived particles reach in PAHs and heavy toxic metals. These outcomes outline the need of a detailed knowledge of the PMs physicochemical composition on a local scale, coupled with the biological hazard directly associated to PM exposure. Apparently, this is the only way allowing scientists and policy-makers to establish the proper relationships between the respirable PM quantity/quality and the health outcomes described by clinicians and epidemiologists

The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO2 NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different. © 2012 Informa UK, Ltd.