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Domeneghetti, Stefania (2011) Valutazione di ceppi mutanti di S. TYPHIMURIUM e di
M. GALLOPROVINCIALIS come sistemi per la caratterizzazione tossicologica di nanoparticelle di ampio utilizzo.
[Tesi di dottorato]

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Abstract (inglese)

A variety of nanomaterials are currently in use or under development in our society. Small size, particular shape, large surface area and activity make them attractive to different application fields, ranging from medical products to industrial processes.
Owing to the unique chemico-physical properties, the most common nanoparticles lack of adequate toxicological characterization in different biological systems. To date, first-level assays for the detection of possible cytotoxic and genotoxic effects have not yet identified and validated. The European Centre for the Sustainable Impact of Nanotechnology (ECSIN) was created to ascertain the biological risks related to the use of nanoparticles (NPs) by in vitro and in vivo studies on prokaryotic and eukaryotic cells (only fragmentary or conflicting data were available for bacterial cells at the start of this study).
Aiming to evaluate the suitability of protocols based on rfa mutant Salmonella thyphimurium cells, Ludox® silica NPs were tested on the strains TA 98, TA 100 (plate and fluctuation test) and TA 1535 pSK1002 (SOS umu test). Following incubation with log-phase TA 100 cells at 37 °C in PBS, flow cytometry and fluorimetric measures were carried out to estimate the intracellular uptake of Qtracker®800 and fluorescein-loaded silica NPs. The incubation with these NPs slightly increased the fluorescence associated to bacterial cells after 3-6 h and 3-11 h of contact, respectively. Possible internalization of electron-dense quantum dots and γFe2O3 was also studied by TEM imaging. Although partially cytotoxic, the silica NPs did not induce mutagenic or genotoxic effects, nor the γFe2O3 did it. The inability of the tested particles to enter the mutant bacterial cells can explain the overall results, as confirmed by the TEM images. NP-related oxidative stress with indirect alteration of cellular functions cannot be excluded.
Hence protocols based on prokaryotes are not suitable for routine NP testing, however they could be rationally used to characterize the combined effect of NPs and genotoxic components occurring together in complex environmental mixtures. For instance, in our hands, γFe2O3 significantly attenuated the mutagenic effects directly induced by hexavalent (K2Cr2O7) on TA 100 cells.
As alternative experimental model, we considered haemocytes and other cell types of the marine invertebrate Mytilus galloprovincialis, already used as sentinel organism in environmental studies.
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Preliminary data obtained with the Single-Cell gel Electrophoresis (Comet Assay) and Micronucleus test support the use of both assays as a first-screening tools for assessing the DNA damage possibly induced by industrial NPs.

Abstract (italiano)

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Nonostante il rapido sviluppo e il molteplice utilizzo di una varietà di nanoparticelle e nanomateriali, a tutt‟oggi mancano regolamentazione adeguata e protocolli standard per la loro caratterizzazione tossicologica. In questi anni, il centro di ricerca ECSIN (The European Centre for the Sustainable Impact of Nanotechnology) si è fatto promotore di studi in vitro e in vivo volti ad accertare i rischi biologici associabili a NP di ampio uso industriale, definendo tra gli altri obiettivi la valutazione di specifiche NP in cellule procariotiche ed eucariotiche.
Benchè i batteri siano incapaci di endocitosi, alcuni dati conflittuali e la frammentarietà delle informazioni disponibili all‟inizio di questo studio hanno indotto a considerare come potenziale sistema bersaglio cellule derivate da S. thyphimurium LT2, mutanti rfa caratterizzati da sintesi difettiva delle catene polisaccaridiche dell‟LPS, lipopolisaccaride di membrana.
Le Ludox® sono NP di silice di ampio utilizzo e di interesse prioritario in ECSIN. Esse vengono usate nella chiarificazione delle bevande e come additivi in polimeri, vernici, rivestimenti e detergenti per la loro capacità di migliorare le proprietà di resistenza, brillantezza e adesione delle superfici e recentemente in prodotti cosmetici. Ludox® AS 30 e SM 30, caratterizzate per diametro dinamico e potenziale zeta, sono state incubate con cellule TA 98 e TA 100 per 30 minuti in soluzione salina tamponata a 37°C. Rispetto alle NP come tali, quelle sottoposte ad ultrafiltrazione sono risultate meno citotossiche. Saggiate per attività mutagena sugli stessi ceppi (plate incorporation test) le Ludox® sono risultate innocue, indipendentemente dalla presenza della frazione microsomiale S9. Per una valutazione più esauriente, le NP AS 30 e SM 30 sono state saggiate anche in micropiastra (Ames fluctuation test) e nell‟SOS/ umu test, saggi che hanno confermato l‟incapacità di indurre, rispettivamente, mutazioni o danno al DNA.
Per accertare se anche le cellule LPS-difettive escludessero le NP sono state applicate altre tecniche sperimentali. NP elettrondense quali Qtracker®800 e maghemite (γ Fe203) sono state incubate con le cellule TA 100 per 30 minuti e per 6 ore procedendo quindi a visualizzarle al TEM, analisi che hanno dimostrato l‟esclusione di entrambe le NP.
Dati ottenuti in citometria a flusso incubando NP di silice marcate con fluoresceina oppure Qtracker®800 con cellule TA 100 in PBS a 37°C per 0.5-24 ore hanno rivelato una percentuale molto modesta di cellule fluorescenti.
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A compendio del lavoro svolto, e per suggerire un uso alternativo dei test sopra illustrati, è stata valutata l‟interazione tra NP (γ Fe203) e ioni cromato (K2Cr2O7).
L‟incubazione in PBS di maghemite e bicromato di potassio in dosi combinate ha dimostrato che le nanoparticelle causano una significativa attenuazione dell‟attività mutagena del cromo esavalente.
Come approccio sperimentale alternativo, è stata messa a punto l‟analisi di danno al DNA in emociti di Mytilus galloprovincialis, un invertebrato marino già ampiamente usato come organismo sentinella dell‟ambiente costiero e che sta emergendo come modello anche per lo studio dell‟immunità innata. I primi risultati ottenuti con le particelle Ludox® indicano un aumento di frammentazione del DNA negli emociti trattati rispetto al controllo, più accentuato per le SM 30 rispetto alle AS 30. L‟individuazione di danno genetico in cellule di mitilo è stato consolidato ottimizzando l‟analisi della frequenza di cellule micronucleate come indice di danno cromosomico.

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Tipo di EPrint:Tesi di dottorato
Relatore:Celotti, Lucia - Venier, Paola
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > TERRITORIO, AMBIENTE, RISORSE E SALUTE > MEDICINA AMBIENTALE, NUTRIZIONE E INQUINAMENTO
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:31 Gennaio 2011
Parole chiave (italiano / inglese):Nanoparticelle, cellule procariotiche, test di mutagenesi, Mytilus galloprovincialis, test della cometa. Nanoparticles, prokaryotic cells, mutagenicity test, Mytilus galloprovincialis, comet assay
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Struttura di riferimento:Dipartimenti > Dipartimento Territorio e Sistemi Agro-Forestali
Codice ID:4022
Depositato il:20 Lug 2011 09:50
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