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During the twenty-first century, engineered nanomaterials (ENMs) have attracted rising interest, globally revolutionizing all industrial sectors. The expanding world population and the implementation of new global policies are increasingly pushing society toward a bioeconomy, focused on fostering the adoption of bio-based nanomaterials that are functional, cost-effective, and potentially secure to be implied in different areas, the medical field included. This research was focused on silica nanoparticles (SiO2-NPs) of bio-based and synthetic origin. SiO2-NPs are composed of silicon dioxide, the most abundant compound on Earth. Due to their characteristics and biocompatibility, they are widely used in many applications, including the food industry, synthetic processes, medical diagnosis, and drug delivery. Using zebrafish embryos as in vivo models, we evaluated the effects of amorphous silica bio-based NPs from rice husk (SiO2-RHSK NPs) compared to commercial hydrophilic fumed silica NPs (SiO2-Aerosil200). We evaluated the outcomes of embryo exposure to both nanoparticles (NPs) at the histochemical and molecular levels to assess their safety profile, including developmental toxicity, neurotoxicity, and pro-inflammatory potential. The results showed differences between the two silica NPs, highlighting that bio-based SiO2-RHSK NPs do not significantly affect neutrophils, macrophages, or other innate immune system cells.

Questo lavoro fa parte del progetto Europeo H2020 ASINA, il quale mira a promuovere delle soluzioni Safe-by-design (SbD) per nuovi nanomateriali (NMs) lungo tutto il loro ciclo vitale. L’adozione di innovativi approcci SbD è di grande importanza nel quadro del Green Deal della Commissione Europea. Negli ultimi decenni, la produzione e l’uso di NMs ha subito una sostanziale crescita, sollevando preoccupazioni a proposito del potenziale rischio associato al loro rilascio e all’esposizione della popolazione. Le nanoparticelle (NPs) di argento (Ag) e di biossido di titanio (TiO2) sono tra i NMs più ampiamente utilizzati e le loro applicazioni in vari prodotti, a causa delle proprietà antibatteriche delle prime e dell’attività fotocatalitica delle seconde, stanno aumentando. Lo scopo di questo lavoro è stato di identificare il pericolo posto da NMs metallici di nuova sintesi, disegnati secondo un approccio SbD, verso la salute umana durante la loro produzione e il loro utilizzo. Il primo passo ha previsto il test delle nuove NPs di Ag e TiO2 con diversi rivestimenti per confrontare i loro effetti tossicologici con NPs di riferimento senza alcun rivestimento. Gli endpoint sono stati selezionati secondo un approccio Adverse Outcome Pathway (AOP), il quale ha consentito l’identificazione dei principali eventi connessi alla tossicità di queste NPs. La vitalità cellulare, la risposta infiammatoria, i livelli di specie reattive dell’ossigeno, il danno ossidativo al DNA, e le interazioni tra cellule e NPs sono state analizzate nel modello alveolare in vitro A549. In seguito, un New Approach Methodology (NAM) è stato sviluppato per identificare e valutare il rischio delle NPs identificate come più sicure nella prima parte dello studio considerando la potenziale esposizione umana durante la loro produzione. Lo scopo è stato quello di definire meglio il pericolo di queste NPs. La selezione di dosi di esposizione appropriate per la definizione del pericolo ha un’importanz, This work is part of the H2020 European project ASINA which aims to promote Safe-by-Design (SbD) solutions for new nanomaterials (NMs) across all life cycle stages. The adoption of SbD novel approaches for new NMs is of pivotal importance in the framework of the European Commission’s Green Deal. Over the past decades, the production and use of NMs have seen substantial growth, raising concerns regarding the potential risk associated with their release and population exposure. Silver (Ag) and titania (TiO2) nanoparticles (NPs) are among the most widely used NMs and their applications in various products, due to the antibacterial properties of the first and photocatalytic activity of the latter, are increasing. The aim of this work is to identify the potential hazard posed by newly developed metal based NMs, designed according to a SbD approach, toward human health during their production and use. The initial step involved testing the newly developed Ag and TiO2 NPs coated with different agents to compare their toxicological effects to reference uncoated NPs. The endpoints were selected according to an Adverse Outcome Pathway (AOP) approach that enabled the identification of the main events linked to the toxicity of these NPs. Cell viability, inflammatory response, reactive oxygen species levels, oxidative DNA damage, and cell-NPs interactions were analyzed in the alveolar in vitro model, A549. Furthermore, a New Approach Methodology (NAM) was developed to identify and evaluate the hazard of the safer NPs (based on the results of the first part of this study) considering the potential human exposure during NMs production. The goal was to improve their hazard and risk definition. The selection of appropriate exposure doses for hazard assessment has gained increasing importance in the risk assessment framework. To this end, starting from monitoring campaign data and applying the MPPD model to determine the lung retained dose of NPs, it was possible to estimate the doses

Microglial cells play a key role in protecting the central nervous system from pathogens and toxic compounds and are involved in the pathogenesis of different neurodegenerative diseases. Cadmium is a widespread toxic heavy metal, released into the environment at a rate of 30,000 tons/year by anthropogenic activities; it is easily uptaken by the human body through diet and cigarette smoke, as well as by occupational exposure. Once inside the body, cadmium enters the cells and substitutes to zinc and other divalent cations altering many biological functions. Its extremely long half-life makes it a serious health threat. Recent data suggest a role for heavy metals in many neurodegenerative diseases; however, the role of cadmium is still to be elucidated. In this work we report the investigation of cadmium toxicity towards murine BV2 microglial cells, a widely used model for the study of neurodegeneration. Results show that increasing cadmium concentrations increase oxidative stress, a proposed mechanism of neurodegeneration, but also that BV2 cells can keep oxidative stress under control by increasing glutathione reduction. Moreover, cadmium induces alterations of cell morphology and metabolism leading to mitochondrial impairment, without switching the cells to Warburg effect. Finally cadmium induces the release of proinflammatory cytokines, but does not markedly switch BV2 cells to M1 phenotype.

The growing demand for effective antimicrobials determined a significant increase of new nanoformulated materials to fight against bacteria and viruses, to be used in many industrial sectors including textiles, cosmetics, pharmaceuticals, and air and water filtering. Nano silver (Ag)-based materials, well-known for their antibacterial properties, are among the most exploited ones. However, the toxicity mechanisms of Ag nanoparticles (NPs) are still debated, requiring more evidence to support a safe-by-design (SbD) strategy for these materials. This study used zebrafish (D. rerio) to assess and compare the toxic and adverse effects (AEs) of commercial Ag-NPs (naked and PVP-coated) and newly synthesized hydroxyethylcellulose-coated Ag-NPs in solution (Ag-HECs) or the powder (Ag-HECp) form. Statistical correlation analysis between AEs and NP physico-chemical (p-chem) properties, such as size, surface charge, and solubility, was also performed. The results ranked the materials as follows in terms of acute lethality (LC50) and malformation (EC50) effects: Ag-HECp > Ag-HECs > Ag-PVP > Ag-NKD. Notable AEs included axial defects, pericardial edema, and reduced lipid yolk consumption, impacting embryo growth and hatching time. Correlation analyses showed that the stabilizing agent HEC, though a safe polymer, played a significant role in modulating Ag-NPs' reactivity toward embryonic structures. The study discusses the biological mode of action and potential molecular events underlying the observed effects. These findings contribute to understanding the biological targets and AEs modulated by tuning Ag-NPs' properties and will additionally feed the SbD frameworks under development for implementing safe and sustainable Ag-based nano-enabled antimicrobial materials.

COVID-19 pandemic had a significant impact on global public health. The spread of the disease was related to the high transmissibility of SARS-CoV-2 virus but incidence and mortality rate suggested a possible relationship with environmental factors. Air pollution has been hypothesized to play a role in the transmission of the virus and the resulting severity of the disease. Here we report a plausible in vitro toxicological mode of action by which fine particulate matter (PM2.5) could promote a higher infection rate of SARS-CoV-2 and severity of COVID-19 disease. PM2.5 promotes a 1.5 fold over-expression of the angiotensin 2 converting enzyme (ACE2) which is exploited by viral particles to enter human lung alveolar cells (1.5 fold increase in RAB5 protein) and increases their inflammatory state (IL-8 and NF-kB protein expression). Our results provide a basis for further exploring the possible synergy between biological threats and air pollutants and ask for a deeper understanding of how air quality could influence new pandemics in the future.

Lung toxicity of carbon nanotubes (CNTs) is matter of concern since very long time. However, their mechanism of toxicity is still not yet well defined. In this work, the role of structural defects as organic stressors of CNTs able to trigger their potential toxicity is investigated. Four commercial CNTs, with different carbon purity grade, are morphologically characterized by transmission electron microscopy (TEM) and the relative amount of structural defects are estimated through Raman spectroscopy, by measuring the intensity ratio D/G (ID/IG). The oxidative potential of CNTs is evaluated with cytochrome-C assay and reactive oxygen species (ROS) detection. Data show that CNTs with larger amounts of structural defects (higher ID/IG ratio) induce an increased ROS generation and consequent cytotoxicity and cellular damage, shown by TEM images of CNTs-cells interaction. Raman analyses of cells exposed to CNTs point out that the spectra of the CNTs inside the cells show no differences with respect of the signal recorded for cell-free CNTs, evidencing their biopersistence in lung cells. Raman spectra cannot provide direct indication of the existence of metals as impurity. It follows that the intensity ratio ID/IG can be taken as a predictive marker of the toxicity of a given CNT.

The assessment of the environmental footprint of an organization or product is based on methods published by the European Union Joint Research Centre, which take 16 impact areas into account. Among the listed categories are human and freshwater ecosystem toxicities. Standard protocols utilize just chemical parameters as input data, hindering the determination of the full impact of complex mixes, such as pollutants released into the environment. Biological assays enable us to overcome this gap: in the present work, assays were employed to determine both baseline and specific toxicity to aquatic species (green algae, luminescent bacteria, and crustacean cladocera) as well as specific toxicity (mutagenicity and carcinogenicity). Ecological footprint was estimated with regard to the impact categories “freshwater toxicity” and “human cancer toxicity” following the standard methodology. In parallel, the impact on the above categories was estimated using the results of biological assays as input. Standard and bioassay-based results are not always congruent, and conventional methods generally underestimate the effects. Likewise, the choice of reference substance (metals or organics) influences the quantification of impact. Appropriate batteries of biological assays could therefore be utilized to complement LCA (Life Cycle Assessment) techniques in order to make them more sensitive when considering toxicity in mid-term impact categories.

Classical dynamins (DNMs) are GTPase proteins engaged in endocytosis, a fundamental process for cargo internalization from the plasma membrane. In mammals, three DNM genes are present with different expression patterns. DNM1 is expressed at high levels in neurons, where it takes place in the recycling of synaptic vesicles; DNM2 is ubiquitously expressed, while DNM3 is found in the brain and in the testis. Due to the conservation of genes in comparison to mammals, we took advantage of a zebrafish model for functional characterization of dnm1a, ortholog of mammalian DNM1. Our data strongly demonstrated that dnm1a has a nervous tissue-specific expression pattern and plays a role in the formation of both axon and synapse. This is the first in vivo study that collects evidence about the effects of dnm1a loss of function in zebrafish, thus providing a new excellent model to be used in different scientific fields.

Airborne pathogens represents a topic of great scientific relevance, especially considering the recent COVID-19 pandemic. Air pollutant such as Particulate Matter (PM) can in fact be associated with an increased incidence of respiratory viral diseases.1 To provide useful insights into the mechanisms by which PM could be involved into infection, we exposed human lung cells to fine PM (PM2.5) and SARS-CoV-2, in order to evaluate different particles cytotoxicological properties and the molecular pathways activated after 72 hours of treatment. We also explored the combined effects of PM2.5 and SARS-CoV-2, to verify the possibility that PM2.5 may play a role in facilitating SARS-CoV-2 infection. Results demonstrated that inflammation is the key process involved in the cell response to PM2.5 and viral particles, albeit with differences. Interestingly, the harmful effects are increased when SARS-CoV-2 and PM2.5 are combined. We also found that PM2.5 induces an over-expression of the angiotensin 2 converting enzyme (ACE2), a preferential entry for the viral particles into respiratory host cells.2 We then verified the possibility that a sub-chronic exposure to PM could induce a reduction of cellular defenses, thus making people more susceptible to infection. Cells were treated with PM2.5 for 72 hours, afterward SARS-CoV-2 was added for additional 2 and 24 hours. Data showed that pre-treatment with PM2.5 facilitates virus entry within the endosomal route already after 2 hours of exposure to SARS-CoV-2. We also demonstrated that the cells exposed to PM and successively treated with virus for 24 hours, respond with an intensified inflammatory state. Taken together, these results showed that PM2.5 appears to facilitate the virus entry in the lung, besides worsening the potency of the inflammatory response to viral infection. The times we are living in, solicit the urgency to carry out a more in-depth investigation, to provide useful elements for understanding the different mecha

Several harmful modifications in different tissues-organs, leading to relevant diseases (e.g., liver and lung diseases, neurodegeneration) are reported after exposure to cadmium (Cd), a wide environmental contaminant. This arises the question whether any common molecular signatures and/or Cd-induced modifications might represent the building block in initiating or contributing to address the cells towards different pathological conditions. To unravel possible mechanisms of Cd tissue-specificity, we have analyzed transcriptomics data from cell models representative of three major Cd targets: pulmonary (A549), hepatic (HepG2), and neuronal (SH-SY-5Y) cells. Further, we compared common features to identify any non-specific molecular signatures. The functional analysis of dysregulated genes (gene ontology and KEGG) shows GO terms related to metabolic processes significantly enriched only in HepG2 cells. GO terms in common in the three cell models are related to metal ions stress response and detoxification processes. Results from KEGG analysis show that only one specific pathway is dysregulated in a significant way in all cell models: the mineral absorption pathway. Our data clearly indicate how the molecular mimicry of Cd and its ability to cause a general metal ions dyshomeostasis represent the initial common feature leading to different molecular signatures and alterations, possibly responsible for different pathological conditions.

The hypothesis that particulate matter (PM) may promote SARS-CoV-2 infection is of great interest. PM has been suggested to act either as a “carrier” of SARS-CoV-2 or by reducing the cellular defenses and making people more susceptible to infections. PM ability to induce a state of chronic lung inflammation, characterized by the over-expression of the angiotensin 2 converting enzyme (ACE2), for which SARS-CoV-2 has a high affinity, is currently under evaluation1,2. Thus, understanding how ACE2-related pathways in pulmonary epithelial cells could be perturbed by PM and exploited by SARS-CoV-2 infection can help in suggesting new strategies to prevent/mitigate the effects of COVID-19 pandemic. This project proposes to evaluate the in vitro interactions between SARS-CoV-2 and PM2.5, using human alveolar epithelial cells (A549) to define if and how PM facilitates virus entry and to identify biological markers of exposure and risk. PM2.5 was collected in Milan during winter 2021. Cells were firstly treated for 72h with PM2.5 in combination or not with SARS-CoV-2 inactivated form. Successively cells were exposed only to PM for 72h, afterward SARS-CoV-2 was added for additional 24h to mimic a sub-chronic exposure to air pollution. The activation of the inflammatory response was studied at biochemical and molecular level. The endosomal pathway was also explored as parameter for the internalization of viral particles. Results demonstrated that exposure to PM2.5 induced an ACE2 over-expression in A549 cells, even in absence of SARS-CoV-2. Moreover, PM/SARS-CoV-2 co-exposure promoted an increase of the pro-inflammatory cytokine IL-6 release. Preliminary data suggested that co-exposure might facilitate the virus entry within the endosomal route. Our data support the hypothesis that the mechanism by which PM exposure can contribute to enhancing SARS-CoV-2 infection and consequent COVID-19 severity is related to an increase in the basal level of the ACE2 receptor in lung epitheli

In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand the early metabolic deregulation and events in cadmium-induced carcinogenesis.

In three different biological models, the recombinant protein expressed in E. coli, the neuronal cells SH-SY5Y and the nematode Caenorhabditis elegans, cadmium inhibits SOD1 activity without affecting its expression level.

Epidemiological data have related cadmium exposure to neurotoxicity. The combination of multi-level and complementary approaches provides a comprehensive view of mechanistic processes in neurotoxicity.

L'inquinamento atmosferico rimane un problema globale per il 21 °secolo, in quanto causa di malattie cardiovascolari e respiratorie. È urgente stabilire il reale impatto del particolato (PM) sulla salute umana includendo l'analisi delle fonti, la distribuzione delle dimensioni, la composizione fisico-chimica (P-chem) e i meccanismi tossicologici. L'aumento dei dati in vitro per stabilire i meccanismi di tossicità utilizzando linee cellulari umane esposte a specifici inquinanti atmosferici da fonti di emissione rigorosamente caratterizzate, potrebbe aiutare a validare approcci scientifici nella caratterizzazione dei rischi per la salute, che alla fine possono determinare azioni normative potenzialmente più efficaci per proteggere la salute pubblica. Lo scopo di questa tesi è stato quello di studiare gli effetti in vitro delle particelle emesse da diverse fonti di combustione utilizzando cellule polmonari umane, concentrandosi sulla relazione tra le caratteristiche P-chem del PM e i processi cellulari e molecolari che inducono la tossicità. Sono stati utilizzati modelli in vitro rappresentativi del sistema respiratorio umano per studiare la bio-interazione e gli effetti tossicologici delle particelle. Sono state confrontate diverse particelle derivate da combustione, con particolare attenzione alle particelle fini e ultrafini (UFPs), ovvero le particelle emesse allo scarico di veicoli di vecchia e nuova generazione diesel (DEP) e alle particelle emesse dalla combustione di biomassa solida per il riscaldamento residenziale. Le emissioni dei veicoli e delle stufe a biomasse, così come la raccolta del PM, sono state eseguite in collaborazione con INNOVHUB SSI (Area Combustibili), mentre la caratterizzazione P-chem e gli studi tossicologici sono stati effettuati presso il Dipartimento di Scienze della Terra e dell'Ambiente (DISAT) - Centro di ricerca POLARIS (Polveri in Ambiente e Rischio per la Salute) e presso l'Unità Consumer Products Safety del Centro Comune di, Due to cardiovascular and respiratory diseases, air pollution remains a global issue for the 21st century. There is an urgent need to establish the real impact of Particulate Matter (PM) on human health by including the analysis of sources, size distribution, physico-chemical (P-chem) composition, and toxicological mechanisms. Increasing in vitro data for establishing pathways of toxicity in human cell lines exposed to specific air pollutants from rigorously characterized emission sources, could help to improve scientifically sound approaches in health risk characterizations, which finally may result in regulatory actions potentially more effective to protect public health. The aim of this thesis was to study the in vitro effects of particles emitted from different combustion sources using human lung cells, focusing on the relationship between the PM P-chem attributes and the cellular and molecular pathways that drive the toxicity. In vitro models, representative of the human respiratory system, were used to study the bio-interaction and toxicological effects of particles. Different exhaust particles were compared, with special emphasis on fine and ultrafine Particles (UFPs), namely Diesel exhaust particles (DEPs) from old and new generation vehicles and on particles emitted from the combustion of solid biomass fuels for residential heating. The emissions from vehicles and biomass-propelled stoves, as well as the PM collection, were performed in collaboration with INNOVHUB SSI (Fuels Department), while the P-chem characterization and toxicological studies were carried out in the Department of Earth and Environmental Sciences (DISAT)- POLARIS research centre (Polveri in Ambiente e Rischio per la Salute) and at the Consumer Products Safety Unit of the European Commission's Joint Research Centre. The results of this work show that testing different material sampled in realistic conditions allows evaluating how the toxicity of particles may vary in relation to the sourc

Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 mu m and <50 mu m (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.

Cadmium is a widespread pollutant, which easily accumulates inside the human body with an estimated half-life of 25–30 years. Many data strongly suggest that it may play a role in the pathogenesis of neurodegenerative diseases. In this paper we investigated cadmium effect on human SH-SY5Y neuroblastoma cells metabolism. Results showed that, although SH-SY5Y cells already showed hyperactivated glycolysis, cadmium further increased basal glycolytic rate. Both glycolytic capacity and reserve were also increased following cadmium administration, endowing the cells with a higher compensatory glycolysis when oxidative phosphorylation was inhibited. Cadmium administration also led to an increase in glycolytic ATP production rate, paralleled by a decrease in ATP production by oxidative phosphorylation, due to an impairment of mitochondrial respiration. Moreover, following cadmium administration, mitochondria increased their dependency on glutamine, while decreasing lipids oxidation. On the whole, our data show that cadmium exacerbates the Warburg effect and promotes the use of glutamine as a substrate for lipid biosynthesis. Although increased glutamine consumption leads to an increase in glutathione level, this cannot efficiently counteract cadmium-induced oxidative stress, leading to membrane lipid peroxidation. Oxidative stress represents a serious threat for neuronal cells and our data confirm glutathione as a key defense mechanism.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder of the motor system. The etiology is still unknown and the pathogenesis remains unclear. ALS is familial in the 10% of cases with a Mendelian pattern of inheritance. In the remaining sporadic cases, a multifactorial origin is supposed in which several predisposing genes interact with environmental factors. The etiological role of environmental factors, such as pesticides, exposure to electromagnetic fields, and metals has been frequently investigated, with controversial findings. Studies in the past two decades have highlighted possible roles of metals, and ionic homeostasis dysregulation has been proposed as the main trigger to motor-neuron degeneration. This study aims at evaluating the possible role of environmental factors in etiopathogenesis of ALS, with a particular attention on metal contamination, focusing on the industrial Briga area in the province of Novara (Piedmont region, North Italy), characterized by: i) a higher incidence of sporadic ALS (sALS) in comparison with the entire province, and ii) the reported environmental pollution. Environmental data from surface, ground and discharge waters, and from soils were collected and specifically analyzed for metal content. Considering the significance of genetic mechanisms in ALS, a characterization for the main ALS genes has been performed to evaluate the genetic contribution for the sALS patients living in the area of study. The main findings of this study are the demonstration that in the Briga area the most common metal contaminants are Cu, Zn, Cr, Ni (widely used in tip-plating processes), that are above law limits in surface waters, discharge waters, and soil. In addition, other metals and metalloids, such as Cd, Pb, Mn, and As show a severe contamination in the same area. Results of genetic analyses show that sALS patients in the Briga area do not carry recurrent mutations or an excess of mutations in the four main ALS caus

Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryo gut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification.

L'inquinamento atmosferico è una considerevole minaccia, sia per l’ambiente che per la salute. È stato classificato come cancerogeno per l'uomo (gruppo 1) e contribuisce notevolmente all’aumento di morbilità, mortalità e all'esacerbazione delle malattie respiratorie e cardiovascolari. L'inquinamento atmosferico può essere generato sia da fonti naturali che antropogeniche. Tra le fonti antropogeniche i principali contribuenti sono le particelle emesse dalla combustione di biomassa e motori a diesel. Numerosi studi sperimentali ed epidemiologici sono stati condotti su queste particelle, considerate tra le principali responsabili degli effetti avversi sulla salute dopo l'esposizione al particolato (PM). Questi studi descrivono gli effetti tossicologici delle particelle riportando risposte diverse in funzione di specifici endpoint biologici studiati in sistemi in vitro e in vivo. Le proprietà del PM possono infatti variare in base a diversi parametri, tra cui le fonti di emissione. Infatti, in base alle fonti di emissione le particelle possono mostrare diverse proprietà fisico-chimiche, come dimensioni, forma, superficie, solubilità e composizione chimica. Tutte queste proprietà possono influenzare in maniera significativa la riposta biologica e quindi l'impatto finale sulla salute umana. Tuttavia, la nostra conoscenza delle diverse risposte biologiche e dei meccanismi molecolari innescati da particelle con proprietà fisico-chimiche variabili è ancora scarsa. Nella presente tesi sono state studiate le proprietà citotossiche, genotossiche e cancerogene delle particelle raccolte durante la combustione di diverse fonti di biomassa e diesel (CDP): - particelle di biomassa raccolte dall'emissione di un sistema di riscaldamento funzionante rispettivamente con pellet, carbone o legno; - particelle ultrafini di diesel (DEP) campionate direttamente da un motore leggero Euro IV senza filtro antiparticolato. Nella prima parte del lavoro, è stata prestata attenzione al, Airborne pollution is a noteworthy environmental and health threat. It has been classified as carcinogenic to humans (Group 1) and greatly contributes to increased morbidity, mortality and respiratory and cardiovascular diseases exacerbation. Air pollution can be generated both by natural and anthropogenic sources. Among anthropogenic sources the main contributors are diesel exhaust and biomass-derived particles. Several experimental and epidemiological studies have been performed on traffic-related and biomass burning-derived particles as chief contributors to the adverse health effects reported after exposure to particulate matter (PM). These studies describe the particle toxicological effects reporting different responses on the biological endpoints investigated on in vitro and in vivo systems. PM properties indeed may vary depending on several parameters, including emission sources. According to the different emission sources, particles can show different physico-chemical properties, such as size, shape, surface area, solubility and chemical composition. All these properties might seriously affect the biological reactivity and thus the final impact on human health. Nevertheless, our knowledge on the different biological responses and molecular mechanisms triggered by particles with variable physico-chemical properties is still poor. In the present thesis, the cytotoxic, genotoxic and pro-carcinogenic properties of particles collected during combustion of different biomass and diesel sources (CDPs) have been investigated: - biomass particles collected from the emission of a heating system operating with pellet, charcoal or wood respectively; - diesel exhaust ultrafine particles (DEP) directly sampled from a Euro IV vehicle run over a chassis dyno. In the first part of the work, attention was given to particles collected from biomass combustion. The role possibly played by biomass particles collected during the combustion of different fuels under identical conditi

The ecotoxicological tools reveal to be profitably employable within the assessment of the so-called environmental footprint, which is commonly based on the results of a chemical monitoring. Due to the heterogeneity of biological endpoints and the possibility to explore several exposure frames, as well as to consider higher levels of organization (from cells to organisms and mesocosms), the definition of a protocol is desirable.

As glyphosate-based herbicides, sold under the commercial name Roundup®, represent the most used herbicides in the world, contamination of the freshwater environment by glyphosate has become a widespread issue. In Italy, glyphosate was detected in half of the surface waters monitoring sites and its concentrations were higher than environmental quality standards in 24.5% of them. It can last from days to months in water, leading to exposure for aquatic organisms and specifically to amphibians’ larvae that develop in shallow water bodies with proven effects to development and behaviour. In this study, we tested the effects of a 96 h exposure during embryonic development of marsh frog's tadpoles to three ecologically relevant Roundup® Power 2.0 concentrations. As expected, given the low concentrations tested, no mortality was observed. Morphological measurements highlighted a reduction in the total length in tadpoles exposed to 7.6 mg a.e./L, while an increase was observed at lower concentrations of 0.7 and 3.1 mg a.e./L compared to control group. Tadpoles raised in 7.6 mg a.e./L also showed a smaller tail membrane than those raised in the control solution. Regarding behaviour, we tested tadpoles in two different sessions (Gosner stages 25 and 28/29) for lateralization, antipredator response and basal activity. Lower intensity of lateralization was detected in tadpoles raised at the highest Roundup® concentration in the first session of observation, while no significant difference among treatments was observed in the second one. In both sessions, effects of Roundup® Power 2.0 embryonic exposure on antipredator response, measured as the proportional change in activity after the injection of tadpole-fed predator (Anax imperator) cue, were not detected. Tadpoles exposed during embryonic development to Roundup® exhibited lower basal activity than the control group, with the strongest reduction for the 7.6 mg a.e./L treatment. Our results reinforce the concern of Roundup® c

Aim: To demonstrate that cobalt nanoparticles doses are safe for use in humans and to understand the consequences of the particulate effects, which may persist inside the cells. Materials & methods: Human adipose stem cells were used. We evaluated cell recovery by viability test, morphology and ultrastructure using electronic and optical microscopy, while gene expression was assessed utilizing real-time PCR. Results: After exposure, most stem cells recovered their normal function. Co3O4-nanoparticles remained inside the cell for the entirety of the considered time. A slight modification of gene expression was observed in the exposed cells. Conclusion: After exposure to 100 M cobalt nanoparticles, most cells returned to normal function. Nanoparticle toxicity was due to ions released by dissolution as well as from the nanoparticles themselves.

Metal oxide (MeO) nanoparticles (NPs), such as copper (CuO) and zinc (ZnO) oxides, thanks to their antibacterial properties, are among the most eligible nanomaterials for the coating of textiles. Nevertheless, there is a general concern about their safety for human health. Within the EU funded H2020 project “PROTECT”, the skin toxicity of textiles coated with antibacterial CuO and ZnO nanoparticles was assessed by different in vitro approaches. CuO and ZnO NPs were produced by sonochemical process and characterized by TEM and DLS. Textiles, coated with ZnO and CuO NPs, were subjected to extraction procedure (ISO 10993-12), including 72 hours incubation in artificial sweat solution (AS, pH 4.7 and 6.3) at 37°C. The stability of the NPs at the different AS pHs, so as the release of NPs and/or ions from the textiles after the extraction procedure in AS, was evaluated through CPS and ICP-OES techniques. For the Skin Corrosion test, Epiderm™ 3D in vitro skin models (Mattek) were exposed to different concentration of CuO and ZnO in water, according to the OECD TG.431 protocol, while for the Skin Irritation test, the 3D models were exposed to the textile extracts, according to ISO/TC 194/WG 8 for Medical Devices. Balb/3T3 fibroblasts were also used to understand the mechanisms of action of the cytotoxicity trigged by CuO and ZnO NPs. Cell viability, inflammatory mediators release, morphological changes and wound healing process (scratch assay) were investigated. Data from the Corrosion test showed that CuO and ZnO NPs resulted non-corrosive up to 1000 ppm in water, according to the Globally Harmonized System (GHS) adopted by the OECD. For the Skin Irritation test, a significant reduction of tissue cells viability was induced by both metal oxide NPs extracted from textiles in AS pH 4.7, likely due to the high content of free Cu and Zn ions released in these conditions and detected by ICP-OES. At higher pH, the effects were observed at lower extent, due to the less solubilit

Among nano metal oxides, Zn-doped CuO nanocomposite (nZn-CuO) showed antibacterial activity more effective than nZnO or CuO nanoparticles, due to higher ROS production. Hence, nZn-CuO is considered a promising nanotool for coating different materials, including reverse osmosis membranes for water purification, to confer antibacterial properties. However, the effects of nZn-CuO on water purification membranes to the aquatic ecosystem health, when released into the aquatic environment, are still unknown. Consequently, this work aim is to define: 1) the eventual release of nZn-CuO in water and 2) the toxicological potential of nZn-CuO functionalized membranes on aquatic organisms. Zebrafish (Danio rerio) is widely accepted as a model for ecotoxicological studies. Organisation for Economic Co-operation and Development (OECD) recommends the Fish Embryo Acute Toxicity (FET) test (OECD n. 236) to determine toxicity of a wide variety of chemicals on zebrafish embryonic stages. Fertilized eggs are exposed to chemicals for 96 hours and different morphological indicators of lethality are recorded every 24 hours. At the end of the exposure, acute toxicity is determined based on a positive outcome in any of the observations recorded. During FET test, nZn-CuO affects zebrafish hatching and this effect likely would have an impact on development at later phases. Therefore, FET test is considered a powerful tool to evaluate Zn-doped CuO nanocomposite and reverse osmosis leachate toxicological potential in aquatic environment. Further investigations are necessary to better comprehend the real potential of nZn-CuO as an antibacterial coating for reverse osmosis membrane. Moreover, biomarkers of sublethal effect will be evaluated.

Introduzione La gestione delle opere afferenti al ciclo idrico integrato può avvalersi di strumenti consolidati e, spesso, standardizzati nell’ambito dei sistemi di gestione della qualità, al fine di perseguire obiettivi di efficienza e sostenibilità (tecnico-economica e ambientale). Tuttavia, l’individuazione della soluzione ottimale implica talvolta la necessità di confrontare e valutare innumerevoli fattori, spesso tra loro contrastanti: per esempio, nel caso della depurazione delle acque reflue, l’obiettivo di rimozione degli inquinanti dalle emissioni liquide potrebbe pesare negativamente sui consumi energetici. Sarebbe utile disporre di uno strumento che consenta sia di definire gli obiettivi, sia di misurare idonei parametri che ne descrivano il raggiungimento, e, infine, di confrontare tra loro il valore di tali parametri, avvalendosi di criteri per l’attribuzione di un peso a questi ultimi. Scopo del progetto è lo sviluppo di protocolli e strumenti integrati per valutare possibili effetti di un’opera (segnatamente, impianti di depurazione delle acque reflue) sulle matrici ambientali e sulla salute umana, fornendo criteri utili al processo decisionale. Materiali e metodi Influenti ed effluenti di due impianti di depurazione di acque reflue urbane sono stati analizzati mediante una batteria di saggi biologici in organismi posti a diversi livelli trofici (batteri, alghe unicellulari, crostacei cladoceri, macrofite, cellule murine e umane) e differenti endpoint (tossicità aspecifica, genotossicità, interferenza endocrina, cancerogenicità). Sulla base della valenza tossicologica dei diversi saggi è stato definito un protocollo di valutazione. Risultati Dai diversi test è emerso un quadro assai complesso. Le acque in entrata agli impianti sono dotate di molta tossicità, che in generale diminuisce in uscita. Relativamente agli interferenti endocrini il trattamento non sembra ridurne la concentrazione. Per quanto riguarda le sostanze che agiscono dannegg

Glyphosate is the active ingredient in broad-spectrum herbicide formulations used in agriculture, domestic area and aquatic weed control worldwide. Its market is growing steadily concurrently with the cultivation of glyphosate-tolerant transgenic crops and emergence of weeds less sensitive to glyphosate. Ephemeral and lentic waters near to agricultural lands, representing favorite habitats for amphibian reproduction and early life-stage development, may thus be contaminated by glyphosate based herbicides (GBHs) residues. Previous studies on larval anuran species highlighted increased mortality and growth effects after exposure to different GBHs in comparison to glyphosate itself, mainly because of the surfactants such as polyethoxylated tallow amine present in the formulations. Nevertheless, these conclusions are not completely fulfilled when the early development, characterized by primary organogenesis events, is considered. In this study, we compare the embryotoxicity of Roundup® Power 2.0, a new GBH formulation currently authorized in Italy, with that of technical grade glyphosate using the Frog Embryo Teratogenesis Assay–Xenopus (FETAX). Our results evidenced that glyphosate was not embryolethal and only at the highest concentration (50 mg a.e./L) caused edemas. Conversely, Roundup® Power 2.0 exhibited a 96 h LC50 of 24.78 mg a.e./L and a 96 h EC50 of 7.8 mg a.e./L. A Teratogenic Index of 3.4 was derived, pointing out the high teratogenic potential of the Roundup® Power 2.0. Specific concentration-dependent abnormal phenotypes, such as craniofacial alterations, microphthalmia, narrow eyes and forebrain regionalization defects were evidenced by gross malformation screening and histopathological analysis. These phenotypes are coherent with those evidenced in Xenopus laevis embryos injected with glyphosate, allowing us to hypothesize that the teratogenicity observed for Roundup® Power 2.0 may be related to the improved efficacy in delivering glyphosate to cells, gu

Negli ultimi decenni abbiamo assistito all’esplosione delle nanotecnologie, sviluppatesi con lo scopo di manipolare la materia a livello nanometrico. Secondo l’International Standard Organization (ISO) per Nanoparticelle si intendono nano-oggetti con dimensioni comprese tra 1 e 100 nm (ISO TS27687/2008). Questi innovative e sconosciuti nanomateriali (NMs) sono stati portati continuamente sul mercato, causando un’esposizione massiva per l’Uomo e l’Ambiente. La principale via di esposizione per l’uomo è l’inalazione. Le NPs, caratterizzate dale loro piccolo dimensioni (meno di 100 nm) sono in grado di raggiungere gli alveoli, ultima parte del nostro tratto respiratorio. La capacità dei NMs di depositarsi a livello alveolare è la ragione per la quale è d’obbligo considerare prioritari gli studi tossicologici a livello polmonare. Per quanto riguarda l’esposizione ambientale, le NPs vengono considerate come una classe di nuovi contaminanti soprattutto per gli habitat acquatici. Alla luce di ciò, studi nanotossicologici diventano fondamentale per comprendere gli effetti avversi delle NPs sugli organismi acquatici. L’utilizzo di vari modelli con diversi livelli di complessità permette di ottenere una caratterizzazione del comportamento delle NPs sempre più complete e dettagliato. Tre diversi moelli in vitro con un crescente livello di complessità sono proposti in questa tesi: da una semplice monocoltura di cellule alveolari, a un innovative modello 3D alveolare di tetracoltura e un organismo completo in via di sviluppo (Xenopus laevis). Le monocolture sono I sistemi più comunemente usati per uno screening ppeliminare degli effetti di composti chimici e particelle. In particolare, la linea cellular A549 (Human Alveolar Adenocarcinoma Cell Line) è un modello largamente utilizzato in esperimenti tossicologici. Negli ultimi anni, numeri modelli 3D in vitro sono stati proposti per studi predittivi di tossicologia. Questi modelli, più o meno complessi, mimano l’organo, During the last decades, we are witnessing the explosion of the nanotechnologies, which have developed with the aim of manipulating matter in the nanoscale dimension. According to The International Standard Organization (ISO) “Nanoparticles indicate nano-objects with the three external dimensions ranging from 1 to 100 nm” (ISO TS27687/2008). The main route of human exposure is represented by inhalation. NPs characterized by their small size (less than 100 nm) are able to reach the alveoli site, the latest part of our air way tract. The capability of NMs to deposit into alveoli, is the reason why considerer human lung toxicological studies mandatory. About the environmental exposure, NPs are considered as a new class of pollutants especially for the aquatic habitats always considered as a sink for wastes. In the light of this, nanotoxicology studies have become fundamental for assessing the adverse effects of NPs on the aquatic organisms. The main physico-chemical properties that characterize NMs are ascribable to their size, shape, surface charge and coating agents. NM size and shape contribute significantly to their interaction with cells, however, functional groups on the NP surface are the primary dictators of many important nanomaterial properties, such as solubility and macromolecule and cell surface interactions. The knowledge of how cells interact with NPs and how their physico-chemical properties are relevant to toxic effects is of great importance in the perspective of proposing and supporting the development a Safe-by-Design (SbD) approach for the production of safer NPs. The use of several models with different level of complexity allows to obtain a more complete and well-described characterization of NP behavior. Different in vitro models with increasing complexity have been proposed in this thesis for a complete assessment: a simple alveolar monoculture, a novel 3D tetraculture alveolar model and a whole developing organism, the Xenopus laevis early emb

Air pollution, mainly with airborne particulate matter (PM), is one of the leading global risks to human health. The toxicity of airborne PM is linked to particles’ dimension and chemical composition and is greatly affected by regional and seasonal variations, as well as by the local emission sources. Fine (FP) and ultrafine (UFP) particles are nowadays considered the most deleterious fractions, because they penetrate in the profound lung, where can hit the respiratory barrier and translocate to secondary organs. In this presentation, the cytotoxic and genotoxic effects of PM2.5 collected in Milan (Italy) and Giza (Egypt) on in vitro lung cells will be displayed. The variations in PM2.5 chemical composition, especially polycyclic aromatic hydrocarbons (PAHs) and heavy metals, will also be discussed in relation to the geographical and seasonal variations. Since diesel and biomass combustion-derived ultrafine particles (UFPs) largely contribute to the PM2.5 pollution, additional toxicity studies have been performed by testing combustion particles from different diesel and biomass sources. The results clearly indicate the variability in the cytotoxic effects exerted by UFPs derived from different sources and burning conditions. The chemical and toxicity data presented will improve our knowledge on the health hazards associated with PM2.5 and UFP exposure and may be considered in the planning of future mitigation strategies. Acknowledgments: Italian Ministry of Foreign Affairs and International Cooperation (proj. ID PGR00786); Egyptian Science and Technology Development Fund (STDF) (proj. ID 26001); Fondazione Cariplo (proj. ID 2013-1038)