UNIVERSITÀ DEGLI STUDI DI TRIESTE

XXVIII CICLO DEL DOTTORATO DI RICERCA IN

NANOTECNOLOGIA

Effects of nanomaterials on biological barriers, fetal

and post-natal, and evaluation of epigenetic toxicity

Settore scientifico-disciplinare: BIO/ 14

DOTTORANDA

Francesca Cammisuli

COORDINATORE

Prof.ssa Lucia Pasquato

SUPERVISORE DI TESI

Dott.ssa Lorella Pascolo

ANNO ACCADEMICO 2014 / 2015

TABLE OF CONTENTS

ABSTRACT (En).............................................................................................................6

ABSTRACT (It)...............................................................................................................8

ACKNOWLEDGEMENTS..........................................................................................11

CHAPTER 1 – GENERAL INTRODUCTION..........................................................13

1.1 Nanotoxicology: the dark side of Nanotechnology................................................13

1.1.1 The impact of physico-chemical properties of nanomaterials on biological

system.......................................................................................................................15

1.1.2 The predominant routes of nanomaterial exposure.........................................17

1.1.2.1 Pleura and placenta barriers.....................................................................18

1.1.3 The impact of nanotoxicity on more susceptible populations.........................22

1.2 Pharmacological and Toxicological studies of nanomaterial exposure.................24

1.3 Asbestos and Carbon nanotubes: a comparison.....................................................25

1.3.1 Mesothelioma: do carbon nanotubes determine the same health risk of

asbestos?..................................................................................................................30

1.3.2 Disorders of iron metabolism in nanomaterial toxicity..................................31

1.3.3 New approaches for Nanotoxicology: imaging and elemental analysis by

Synchrotron-based X-ray Fluorescence...................................................................32

1.3.4 Asbestos as inductor of carcinogenesis microenvironment: Do carbon

nanotubes behave like asbestos?.............................................................................34

1.4 Role of genetic predisposition in cancer development during nanomaterial

exposure: BAP1, a promising candidate gene in susceptibility to mesothelioma.......37

1.5 Conclusions...........................................................................................................41

1.6 Aims of the research..............................................................................................42

References...................................................................................................................43

CHAPTER 2 – Biochemical and microscopic methods in the study of intracellular

iron alteration after nanomaterial exposure..............................................................61

2.1 Material and methods............................................................................................61

2.1.1 Carbon nanotubes...........................................................................................61

2.1.2 Crocidolite......................................................................................................62

2.1.3 Nanofibre suspensions....................................................................................62

2.1.4 Iron sulphate (FeSO4).....................................................................................62

2.1.5 Cell culture and treatments.............................................................................62

2.1.6 Evaluation of citotoxicity...............................................................................63

2.1.7 Ferritin assay...................................................................................................63

2.1.8 Synchrotron-based X-ray analysis..................................................................64

2.1.9 Atomic force microscope analysis..................................................................65

2.2 RESULTS I: Iron related toxicity of single-walled carbon nanotubes and

crocidolite in mesothelial cells (MeT5A) investigated by synchrotron XRF

microscopy..................................................................................................................66

2.2.1 State of the art in in vitro tests of mesothelial cell line..................................66

2.2.2 Results of viability test...................................................................................67

2.2.3 Results of elemental mapping with XRF microscopy....................................68

2.2.4 Results of ferritin assay..................................................................................78

2.3 RESULTS II: Toxicity effects of single-walled carbon nanotubes and crocidolite

in human placental cells (BeWo) investigated by Synchrotron XRF microscopy and

AFM microscope.........................................................................................................80

2.3.1 State of the art in in vitro tests of placental cell line......................................80

2.3.2 Results of viability test...................................................................................82

2.3.3 Results of elemental mapping with XRF and AFM microscopy....................83

2.3.4 Results of ferritin assay..................................................................................90

2.4 Conclusions and comparison between pleura and placenta cell responses...........91

References...................................................................................................................92

CHAPTER 3 - A “nanotoxicogenomic” study: the role of BAP1 in mesothelioma

development...................................................................................................................96

3.1 Material and Methods............................................................................................98

3.1.1 Patients and tissue samples.............................................................................98

3.1.2 Nucleic acid extraction and PCR amplification..............................................99

3.1.3 Sanger sequencing and mutation data handling...........................................100

3.1.4 Multiplex ligation-dependent probe amplification.......................................101

3.2 Results.................................................................................................................102

3.3 Discussion and conclusions.................................................................................104

References.................................................................................................................107

CHAPTER 4 - UV resonant Raman spectroscopy for investigation of oxidative

DNA damage induced by carbon nanotubes............................................................111

4.1 Material and Methods..........................................................................................112

4.1.1 UV Resonant Raman spectroscopy..............................................................112

4.1.2 Computational simulations: Gaussian-03 software......................................114

4.1.3 Oxidative DNA damage...............................................................................114

4.2 Results and discussion.........................................................................................115

4.2.1 dNTPs Raman analysis before Fenton’s reaction.........................................115

4.2.2 dNTPs Raman analysis after Fenton’s reaction............................................119

4.2.3 Plasmid DNA Raman analysis......................................................................123

4.3 Conclusions..........................................................................................................125

References.................................................................................................................126

CHAPTER 5 – GENERAL CONCLUSION AND OUTLOOK............................129

Appendix A..................................................................................................................132

Appendix B..................................................................................................................134

Abbreviation list..........................................................................................................138

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ABSTRACT (En)

Beyond the merits and the substantial opportunities offered by nanotechnology research,

there is a great need of understanding the possible harmful effects of general exposure

to nanomaterials.

My PhD project aimed to contribute to this topic by focusing on the interaction and the

induction of possible toxic effects of fibrous nanomaterials at two critical internal

biological barriers: the pleura and the placenta. Different physico-chemical properties

may determine the toxicity of nanomaterials, and among them this work was mainly

intended to investigate the impact of iron presence in old and new nanofibres: asbestos

and carbon nanotubes. It was worth noting that it has been recently demonstrated that

the fibrous structure of nanotubes might cause asbestos-like pathology.

The work was carried out by using advanced, synchrotron-based X-ray microscopy and

fluorescence (µXRM and XRF), together with more standard imaging techniques such

as SEM and AFM, conventional molecular analysis (PCR and Sanger sequencing) and

advanced spectroscopic measurements (UV-Raman).

We conducted biochemical studies by using XRM and XRF techniques, operated at two

synchrotron facilities, the ID21 and TwinMic beamlines, respectively at the European

Synchrotron Radiation Facility (ESRF, located in Grenoble, France) and Elettra

Synchrotron (Trieste, Italy). The aim was to reveal mechanisms of toxicity in human

mesothelial (MeT5A) and placental (BeWo) cell lines exposed to carbon nanotubes

(raw-SWCNTs, purified- and highly purified-SWCNTs) or asbestos (crocidolite fibres).

Other state-of-the-art imaging techniques microscopes were performed in some

experiments, to gather complementary data about the morphology and the cell-nanofiber

interactions.

The results obtained with the combination of these microscopic techniques allowed to

understand toxic mechanisms in the two internal barriers. The cells treated with raw-

SWCNTs and crocidolite fibres compared to the control showed a severe alteration of

iron metabolism, which was maximal in the pleural cells and was clearly related to the

presence of iron into the fibre. This study also found that highly purified nanotubes did

not altered iron metabolism. X-ray microscopy images (absorption and phase contrast

imaging) confirmed that the toxicity of nanomaterials was characterized by membrane

damage with vesicle secretion and filipodia formation.

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While X-ray fluorescence analysis clearly revealed a homogeneous intracellular iron

increase in pleural cells after fibre exposure, the same technique showed that the iron

changes inside placental cells were restricted to small intracellular regions, thought to

be just in contact with the exogenous fibre.

In relation to this complex, and still unknown toxic mechanism, we evaluated the

presence of intracellular ferritin in treated cells. The results demonstrated that

crocidolite and “raw” carbon nanotubes increased the amount of intracellular ferritin in

both cell models, while highly purified carbon nanotubes gave values comparable to

control. The stimulation was clearly lower in placental cells and clearly linked to a

different uptake of fibres in these cells, suggesting that this barrier is less vulnerable

than the pleura.

In my thesis, I was also interested in investigating genetic effects to toxicity of

nanomaterials (nanotoxicogenomic). Since we have to learn from asbestos, one study

investigated the possible genetic predisposition to develop mesothelioma after asbestos

exposure by looking for BAP1 gene mutations in 29 cases of mesothelioma. BAP1 is

located on the short arm of chromosome 3 (3p21) and belongs to the ubiquitin C-

terminal hydrolase subfamily of deubiquitinating enzymes, involved in the removal of

ubiquitin from proteins. In addition, BAP1 is also involved in regulation of

transcription, regulation of cell cycle and growth, response to DNA damage and

chromatin dynamics (epigenetic mechanisms).

Sanger sequencing of BAP1 gene in the 29 patients identified one non-synonymous

variant and two intronic variants: patient 9 carried in heterozygous a missense variant

(c.T1028C; p.L343P) at exon 11; patient 4 carried in heterozygous a known intronic

variation 8 bases downstream of the exon 13 (c.1729+8T>C); in patient 15 carried a

heterozygous intronic variation 8 bases upstream of the exon 10 (c.784-8G>A). Sanger

sequencing of cDNA revealed no alternative splicing due to the nucleotide change for

each mutations. In silico mutation analysis was performed in a predicted protein

structure of BAP1 protein without any significant possible effect of the amino acid

change about exonic mutations of patient 9. Finally, MLPA (Multiplex ligation-

dependent probe amplification) analysis revealed no significant copy number variations

at exonic level in all samples.

The last aim of molecular studies was to test the feasibility of UV-Raman (IUVS

beamline, Elettra Synchrotron of Trieste) spectroscopy to reveal epigenetic changes at

DNA level after nanomaterial exposure. An oxidative environment was created in vitro

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by using carbon nanotubes (raw-SWCNT), which contain some impurity in metal traces

(iron), and free radicals OH• (derived from H2O2). In this condition the elements of

DNA, in particular the nucleotides (dATP, dCTP, dGTP and dTTP), were resulted in

increased susceptibility to oxidative damage. The results demonstrated that UV-Raman

spectroscopy is useful to reveal the chemical changes that affect the nitrogenous bases

after nanomaterials exposure, providing a “fingerprint” of the oxidative DNA damage.

ABSTRACT (It)

Oltre ai meriti e alle notevoli opportunità offerte dalla ricerca per le nanotecnologie, c’è

un maggiore bisogno di studi volti a capire il possibile effetto dannoso della generale

esposizione ai nanomateriali.

Il mio progetto di dottorato si è proposto di essere parte di questa valutazione,

focalizzando l’attenzione sull’interazione e l’induzione di possibili effetti tossici dei

nanomateriali fibrosi a livello di due cruciali barriere biologiche interne: la pleura e la

placenta. Le diverse proprietà fisico-chimiche potrebbero determinare la tossicità dei

nanomateriali, e tra queste il progetto è stato maggiormente interessato ad investigare

l’impatto della presenza di ferro in un vecchio e nuovo nanomateriale: l’amianto e i

nanotubi di carbonio, rispettivamente. E’ interessante notare che è stato recentemente

dimostrato che la struttura fibrosa dei nanotubi di carbonio potrebbe causare un

meccanismo di patogenicità simile a quello dell’amianto.

Il lavoro è stato svolto usando avanzate microscopie basate sulla radiazione di

sincrotrone (µXRM and XRF) ed altre microscopie (SEM e AFM), oltre ad analisi

molecolari convenzionali (PCR e sequenziamento Sanger) e analisi spettroscopiche

avanzate (UV-Raman).

Gli studi biochimici hanno previsto l’uso di microscopia avanzata e fluorescenza a raggi

X (µXRM and XRF), operata in due diversi sincrotroni - Beamline ID21, Sincrotrone

Europeo in Francia (ESRF), e Beamline TwinMic, Sincrotrone di Trieste in Italia

(ELETTRA) - al fine di rilevare meccanismi di tossicità in cellule umane di mesotelio

pleurico (MeT5A) e di placenta (BeWo), esposte a nanotubi di carbonio (raw-SWCNT,

purificati- e altamente purificati-SWCNT) ed amianto (fibre di crocidolite). Altre

microscopie (AFM, microscopio a forza atomica e SEM, microscopio a scansione

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elettronica) sono state aggiunte in qualche esperimento, per meglio investigare sulla

morfologia della cellula e sulla sua interazione con i nanomateriali.

I risultati ottenuti con la combinazione di queste tecniche microscopiche hanno

permesso di rivelare la presenza di simili e diversi meccanismi di tossicità nelle due

barriere biologiche interne. Le cellule trattate con i raw-SWCNT e le fibre di crocidolite

rispetto al controllo hanno mostrano una severa alterazione del metabolismo del ferro,

che era massimale nella cellule del mesotelio pleurico ed era chiaramente legato alla

presenza di ferro nelle fibre. Infatti, i nanotubi di carbonio altamente purificati non

alteravano il metabolismo del ferro. Le immagini ottenute con la microscopia a raggi X

(in assorbimento e contrasto di fase) hanno confermato che la tossicità dei nanomateriali

è caratterizzata anche dal danno di membrana con la secrezione di vescicole e la

formazione di filipodi.

La barriera placentare ha mostrato una differenza nella risposta cellulare e

nell’alterazione del metabolismo del ferro. Infatti, mentre nelle cellule di mesotelio

pleurico le analisi ottenute dalla fluorescenza a raggi X hanno rivelato un aumento del

ferro intracellulare omogeneo dopo l’esposizione ai nanomateriali, la stessa tecnica ha

mostrato che nelle cellule di placenta i cambiamenti del ferro sono ristretti a piccole

regioni intracellulari laddove c’è stata un’interazione con le nanofibre o i nanotubi.

In relazione a questo meccanismo abbastanza complesso e ancora sconosciuto abbiamo

valutato la presenza di ferritina intracellulare. I risultati hanno dimostrato che la

crocidolite e i nanotubi di carbonio “raw” aumentavano la quantità di ferritina

intracellulare in entrambi i modelli cellulari, mentre i nanotubi altamente purificati

davano valori paragonabili a quelli controllo. La stimolazione era chiaramente più bassa

nelle cellule di placenta, e chiaramente legata a un differente o comunque più basso up-

take delle fibre in queste cellule, suggerendo che la barriera placentare è meno

venerabile di quella pleurica.

Nella mia tesi, mi sono interessata dello studio degli effetti genetici in relazione alla

tossicità dei nanomateriali (nanotossicogenomica). Poiché abbiamo da imparare

dall’amianto, lo studio ha indagato sulla possibile predisposizione genetica a sviluppare

il mesotelioma dopo esposizione all’amianto, osservando mutazioni nel gene BAP1 in

29 casi di mesotelioma. BAP1 è localizzato sul cromosoma 3 (3p21.1) ed appartiene

alla sottofamiglia degli enzimi ubiquitina C-terminale idrolasi che sono coinvolti nella

rimozione di ubiquitina dalle proteine. Inoltre, BAP1 è anche coinvolto nella

regolazione della trascrizione, regolazione del ciclo e della crescita cellulare, nella

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risposta al danno al DNA e quindi alle dinamiche della cromatina (meccanismi

epigenetici). Il sequenziamento Sanger dell’intero gene BAP1 nei 29 soggetti esposti ha

permesso di identificare una mutazione non senso e due mutazioni introniche in tre

diversi pazienti: il paziente 9 portava una mutazione non senso in eterozigosi sull’esone

11 (c.T1028C; p.L343P), il paziente 4 portava una mutazione intronica in eterozigosi ad

otto basi a valle dall’esone 13 (c.1729+8T>C), il paziente 15 portava un’altra mutazione

intronica in eterozigosi ad otto basi a monte dall’esone 10 (c.784-8G>A). Per quanto

riguarda queste mutazioni introniche è stato studiato lo splicing alternativo, eseguendo

il sequenziamento Sanger del cDNA: i risultati hanno dimostrato che sono assenti forme

alternative di splicing. Per valutare se la mutazione esonica del paziente 9 avesse effetti

sulla struttura proteica è stata fatta un’analisi in silico: i risultati hanno predetto che non

si ha un significativo effetto sul cambiamento amminoacidico e quindi sulla struttura

della proteina. Infine, l’analisi molecolare MLPA (Multiplex ligation-dependent probe

amplification) per ricercare delezioni-duplicazioni (copy number variations) non ha

rivelato a livello esonico variazioni significative in tutti i campioni.

L’ultimo obiettivo dello studio molecolare è stato di testare la fattibilità della

spettroscopia UV-Raman (Beamline IUVS, Sincrotrone di Trieste) per valutare possibili

cambiamenti epigenetici a livello del DNA dopo trattamento con nanomateriali. E’ stato

ricreato in vitro un ambiente ossidativo in presenza nanotubi di carbonio “raw”, che

contengono delle impurità in metalli tra cui anche ferro, e radicali liberi OH•(derivati da

H2O2). In questa condizione i componenti del DNA, in particolare i nucleotidi (dATP,

dCTP, dGTP e dTTP), sono risultati essere suscettibili al danno ossidativo. I risultati

hanno dimostrato che la spettroscopia UV-Raman è utile per studiare i cambiamenti

chimici che interessano le basi azotate del DNA in seguito all’esposizione ai

nanomateriali, ottenendo rapidamente un “fingerprint” del danno ossidativo al DNA.

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ACKNOWLEDGMENTS

This PhD work has been supported by “Institute for Maternal and

Child Health - IRCCS Burlo Garofolo, Trieste” and

“Istituto nazionale per l'assicurazione contro gli infortuni sul

lavoro - INAIL; provincial seat in Trieste”.

Before anything else, I would like to thank my supervisor, Lorella Pascolo, for allowing

me to continue my academic experience. During these three years of doctoral she was

an excellent guide in my research activity, giving me the opportunity to develop my

knowledge and enriching my professional and human growth.

I would like to acknowledge Professor Mauro Melato (Institute of Child Health IRCCS

Burlo Garofolo, Trieste), Dr Clara Rizzardi (University of Trieste) and Dr Vincenzo

Canzonieri (Oncological Referral Center, Aviano) for their helpful teaching and

research support.

My gratitude goes to Giammaria Severini, Bortot Barbara and De Martino Eleonora for

the way they accepted me in their laboratory at the IRCCS Burlo Garofolo Institute

during three years of doctoral.

This research work was carried out in collaboration with several institutions and I would

like to acknowledge all participants which supported me.

Thanks to Alessandra Gianoncelli, Matteo Altissimo and Diana Bedolla, which

supporting me in the TwinMic beamtimes at Elettra-Sincrotrone (Trieste) carried out

during this PhD project. And particularly gratitude goes to Alessandra Gianoncelli and

Matteo Altissimo which helped me and my supervisor during the beamtimes at ESRF

(European Synchrotron Radiation Facility in Grenoble, France). I would like to thank

all scientists which supported me at ESRF: Salome Murielle and Marine Cotte (ID21

beamline), Yohan Fuchs and Fabio Comin (AFM Service). Thanks to Damiano Cassese

(TASC-INFM National Laboratory, Trieste) and Francesca Vita (University of Trieste)

for contributing to obtain AFM and SEM images shown in Appendix A. All these

participants allowed the biochemical study discussed in Chapter two of the PhD thesis.

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Thanks to Emmanouil Athanasakis (Institute of Child Health IRCCS Burlo Garofolo),

Simeone Dal Monego and Danilo Licastro (Cluster in Biomedicine, Trieste) which

helped me in the BAP1 study discussed in Chapter three of the PhD thesis.

I would like to thank Francesco D’amico, Claudio Masciovecchio and Alessandro

Gessini (IUVS beamline at Elettra-Sincrotrone, Trieste) which supported the molecular

study already published and discussed in Chapter four of the PhD thesis.

Moreover, thanks to Silvia Giordani (Istituto Italiano di Tecnologia, Genova) who

provided me carbon nanotubes used in this PhD work.

To conclude, I would like to thank my family and friends.

Grazie mamma, grazie papà, senza il vostro amore tutto questo non sarebbe stato

possibile. Non posso che ringraziarvi esprimendo l’ amore incondizionato che nutro per

voi sin dal primo respiro. Grazie anche al mio fratellone ed alla sua famiglia, facendomi

dono di due fantastici nipotini che anche se lontani riempiono di gioia la mia vita. Un

grazie speciale al mio ragazzo che ha la capacità di farmi sorridere anche quando sono

triste, e grazie anche alla sua famiglia meravigliosa che mi ha accolto come una figlia.

Grazie alla mia adorata e preziosa amica Giorgia che trovo sempre dalla mia parte anche

a distanza di anni e di chilometri.

Grazie alle amiche ed anche colleghe, Eleonora, Alessandra e Vanessa, che mi hanno

accompagnato in questi tre anni di dottorato.

Infinite thanks to all of you!

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CHAPTER 1 – GENERAL INTRODUCTION

1.1 Nanotoxicology: the dark side of Nanotechnology

Nanotechnology is often described as a set of new technologies, but is not a newly field;

it is only recently that discoveries have advanced so far as to assure their impact upon

the world revolutionizing our lives to common problems [1].

The concept was formulated for the first time by Richard Feynman, 1965 Nobel Prize

Laureate in physics. During the 1959 American Physical Society meeting at Caltech, he

presented a lecture titled “There’s Plenty of Room at the Bottom”, introducing the

concept of manipulating matter at the atomic and molecular level to build devices of

various type. This novel idea demonstrated new ways of thinking so much that

Feynman is considered the father of modern nanotechnology [2].

Fifteen years after Feynman’s lecture, Norio Taniguchi, a Japanese scientist, was the

first to use the term “nanotechnology” to describe semiconductor processes that

occurred on the scale order of a nanometer. He suggested that nanotechnology could be

considered as the processing, separating, consolidating, and deforming materials atom

by atom or molecule by molecule [3].

The golden age of nanotechnology began in the 1980s when Kim Eric Drexler used

term nanotechnology in his 1986 book titled, “Engines of Creation: The Coming Era of

Nanotechnology”, delineating the possible consequences of an emerging field called

“molecular nanotechnology” [2].

The term has been defined by various players over the decades and, despite of the desire

for the adoption of a single definition for nanotechnology, to date, there are several

accepted ones. For example, the Royal Society and Royal Academy of Engineering

(UK) gives the following definition: “Nanotechnologies are the design,

characterisation, production and application of structures, devices and systems by

controlling shape and size at nanometre scale”. In USA, the National Nanotechnology

Initiative (NNI) defines nanotechnology as: “... the understanding and control of matter

at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel

applications... At this level, the physical, chemical, and biological properties of

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materials differ in fundamental and valuable ways from the properties of individual

atoms and molecules or bulk matter” [4].

Nanotechnology is not a unique technology, but the term encloses several other

technologies as indicated by the definitions above. Some of the most well-known

technologies and methods include lithography, chemical vapor deposition, atomic force

microscopy and scanning probe- and tunnelling microscopy but according to a recent

standard on the terminology for nanofabrication and nanomaterials (NMs) from the

British Standard Institute (BSI) the number of methods, processes and techniques easily

exceeds 30 [5].

Tipically, nanotechnology is classified into two main approaches: (a) the “top-down”

approach, in which a bulk materials are reduced to the nanoscale size while maintaining

their original properties and (b) the “bottom-up” approach, in which materials from the

nanoscopic scale, such as molecules and atoms are engineered to form larger structures

through a process of assembly or self-assembly [6].

In nanotechnology fabrication, the physical chemical characteristics of solid material

processed into small pieces can be very different from those of the same material in

bulk form [7].

Nanotechnology is one of the leading scientific fields today combining knowledge from

Physics, Chemistry, Biology, Medicine, Informatics, and Engineering. Today and in the

near future, the application and use of nanostructure materials in electronic and

mechanical devices, in optical and magnetic components, in quantum computing, in

tissue engineering, and other biotechnologies, will constitute the most important

contribution of the nanotechnology in the marketplace [8-9].

Nanomaterials are actually being used in an extremely large number of applications

(medical imaging, drug delivery, cosmetic, electronic, etc.) and contain various NMs,

including nanotubes, metal oxides, and quantum dots [10].

It is interesting to note that, because of extensive human exposure to nanomaterials,

there is a significant and growing concern about the potential adverse health and

environmental risks associated with the use of nanostructured materials, their disposal

and their dispersion in the environment. These concerns led to the emergence of a new

scientific discipline: nanotoxicology. Its field of application is the study of potential

adverse health effects of nanomaterials investigating on the relationships between their

toxicity in relation to their dose levels and physicochemical properties (i.e., size, shape,

reactivity and material composition) [11].

15

Many of these concerns have been raised about the potential adverse health risks of

associated with nanotechnology and nanomaterials derive from the analogies with

ambient ultrafine particles in general, and asbestos in particular. It has been shown that

they produce a number of adverse health reactions after inhalation, including

inflammation, genotoxicity, and carcinogenesis [12-13].

Given these considerations, this PhD work focused on the study of the possible similar

toxic effects induced by two types of nanomaterials, asbestos and carbon nanotubes,

during fetal and post natal life.

1.1.1 The impact of physico-chemical properties of nanomaterials

on biological system

The advances in nanotoxicology research have shown that the interactions between

nanomaterials and cells, animals, humans, and the environment are very complex; this

complexity derives from ability of nanomaterials to bind and interact with biological

matter and then modify their surface characteristics [14]. The physical, chemical, and

biological properties of nanomaterials often differ from individual atoms, molecules,

and from bulk matter, making them attractive for commercial development and

application. However, these properties of nanomaterials may exert negative effects into

the body, such as an increased rate of pulmonary deposition, the ability to penetrate in

systemic circulation, and a high inflammatory potential [14].

Nanomaterial toxicity seems to originate from the nanomaterial’s size and surface area,

composition, and shape [15].

Nanomaterials come in varied shapes and can be divided into main groups:

nanoparticles, (i.e., rings, spheres), and nanofibres (i.e., tubes, rod). The wide variety of

nanomaterial’s shapes influences the in vivo membrane wrapping processes, either

during endocytosis or phagocytosis. Non-spherical nanomaterials are more prone to

flow through the capillaries inducing inflammatory response and DNA damage [15-16].

For instance, it has been shown that rod shaped single-walled carbon nanotubes

(SWCNTs) can block potassium (K+) ion channels more efficiently than spherical

carbon fullerenes [15-17].

Nanomaterials can be also varied in the chemical composition, from completely

inorganic, such as metals (iron, nickel, zinc, titanium, gold, silver, palladium, iridium,

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and platinum), and metal oxides (titanium oxide, zinc oxide, silica, iron oxide, etc.), to

entirely organic, suche as fullerenes, CNT, nanopolymers, and biomolecules [18].

Particle size and surface area play a major role in interaction of materials with

biological system: the small size may lead to the high surface to volume ratio, making

the nanomaterial’s surface more biologically reactive [15]. At the same time, it has been

reported that nanoparticles of lower dimensions are less toxic than bigger [19].

Moreover, it has been established that various biological mechanisms (i.e., endocytosis,

cellular uptake) also depend on size of the material [20].

The aspect ratio is an important feature of nanomaterials that establishes the surface to

volume ratio. Various types of nanomaterials (i.e., carbon nanotubes or asbestos fibres)

show a higher aspect ratio determining their toxic effects. For example, in the case of

asbestos induced toxicity, it has been revealed that the asbestos fibres longer than 10

microns cause lung carcinoma, while fibres >5 microns cause mesothelioma and fibres

>2 microns cause asbestosis [21]. Longer fibres will not be effectively cleared from the

respiratory tract since the macrophages are unable to fully phagocytize them [21-22]. At

the same way, long-aspect ratio of SWCNTs produce significantly higher pulmonary

toxicity respect to the spherical particles. It has also been reported that long multi-

walled carbon nanotubes (MWCNTs) cause inflammation responses after intra-

abdominal instillation, while no inflammatory response are observed in case of short

MWCNTs [22-23].

Surface charge is important to nanomaterial toxicity, affecting their interactions with

the biological systems. The plasma membrane is negatively charged, thus positively

charged nanomaterials show significant cellular uptake compared to negatively charged

or neutral ones. Additionally, DNA is negatively charged, thus cationic NMs are more

reactive with the genetic material [24]. Besides, many nanomaterials are functionalized

on the surface to increase the circulation time of them in blood and their

biocompatibility, such as for targeted therapy. Although the functionalization seems to

be very promising in many applications, functional groups added to the surface can

potentially interact with biological components, altering some biological functions and

allowing the passage of nanomaterials in certain cells. Nanomaterial purity is another

important aspect in determining their toxicity. Some nanomaterials contain

contaminants, such as residual metals catalysts (i.e. iron, cobalt, and nickel) that may be

responsible for toxicological responses and the quantity of which depends upon

particular synthetic route. However, where metals are present as impurities, iron is one

17

of the primary sources of biological damage as it induces oxidative stress through

Fenton or Haber-Weiss reactions [25].

1.1.2 The predominant routes of nanomaterial exposure

The number of humans exposed to nanomaterials, generated by both natural processes

and industrial activities, will increase over the next years. The main factors determining

the toxic effects of NMs in the body are the characteristics of the exposure (i.e.,

penetration route, duration, and concentration) and of the exposed organism (i.e.,

individual susceptibility, activity at time of exposure, and the particular route the NMs

follow in the body), and the intrinsic toxicity of NMs (i.e., catalytic activity,

composition, electronic structure, capacity to bind or coat surface species, surface area)

[14]. Nanomaterials have a small size similar to DNA (about 2.5 nm) and proteins (i.e.,

albumin 7.2 nm), which allows the migration of the nanomaterials through capillaries,

cell membranes and cell substructures.

The predominant routes of human exposure to NMs intended for industrial or

environmental applications include inhalation exposure, dermal uptake, and oral

ingestion with possible entering into systemic circulation by absorption or directly into

the brain (Figure 1) [26-27].

Figure 1. The predominant routes of NMs exposure and uptake, and potential routes of their

translocation.

18

Three anatomical primary barriers separate the deep tissues of the body from external

environment and are represented by the epithelia of the skin, the gastrointestinal tract

and the respiratory system.

The inhalation route has been of biggest concern since it is the most common route of

exposure to airborne particles, mainly in the workplace [28].

Particle uptake through the skin results from diffusion through its three constituting

layers: the epidermis, dermis and hypodermis. For example, prolonged dermal

application of microfine titania sunscreen seems to lead to its penetration into the

epidermis and dermis [29].

To date, little is known about oral and ocular exposure. Regarding to the oral exposure

nanomaterials seem to penetrate only in regions where the integrity of gastric epithelial

is disrupted [30]. While the ocular exposure may occur from cornea into the inner eye,

when accidently using cosmetics or transferring NMs from the hands to eyes [31].

When nanomaterials overcome the primary barriers (epithelia of the skin, the

gastrointestinal tract and the respiratory system), there are further biological barriers

protecting specific organs of human body. The secondary or internal barriers are

represented by the blood–brain barrier (BBB), protecting the neural tissue, the blood–

testis barrier (BTB), protecting the germ cells, and the placenta, protecting the fetus

[32]. In addition, pleura can be considered an additional internal barrier, involved in the

regulation of the passage of some substrates from lung to circulation (see 1.3 section).

1.1.2.1 Pleura and placenta barriers

My PhD work involved investigating two main biological barriers: the pleura and the

placenta internal barriers.

The choice was directed to two biological barriers because, particularly in accidental or

environmental exposure, the lung is considered to be the most important portal of entry

for nanomaterials into the human body, while little is known on biological interaction

and biodistribution of nanomaterials across the placenta.

The respiratory system acts as a barrier between the “outside” and the “inside”. The

respiratory tract consists of three structurally and functionally distinct areas, which are

depicted in Figure 2. The extrathoracic region which consists of the nasal cavity, the

mouth, the pharynx and the larynx. At the end of the extrathoracic zone, there is the

tracheobronchiolar region which includes the trachea, the main bronchi, the bronchi, the

19

bronchioles and the terminal bronchioles. The proximal part of the alveolar-interstitial

region is composed of the respiratory bronchioles with only a few adjacent alveoli [33].

Inhaled nanomaterials can be deposited in different parts of the respiratory system (i.e.,

nasal, tracheabronchial, and alveolar regions) in a manner dependent in large part on

particle size. They can enter into the systemic circulation crossing the epithelia of the

lung, especially the alveolar epithelium with its very large surface area and thin barrier

thickness [34].

Figure 2. The human respiratory tract.

However, the respiratory tract is protected from a series of structural and functional

defences [35]. A thin film of surfactant and an aqueous surface-lining layer with the

mucociliary escalator are the primary defence of respiratory system. The next lung

defence is composed of macrophages (professional phagocytes), the epithelial cellular

layer with tight junctions as well as adherens junctions between the cells, and a network

of dendritic cells inside and underneath the epithelium [36-37].

But, another important defences are the pleural cells that trigger cellular responses to

expel the harmful substances that after penetrating the lung parenchyma arrive at the

pleural space [38-39]. The pleura is a double monolayer of mesothelial cells that

surrounds the lung. The outer cell layer is called the visceral pleura, and the inner layer

is called the parietal pleura. In between of the two pleural layers there is the pleura

space with a serous liquid (Figure 2). A range of in vitro models have been developed to

20

investigate the mechanisms of cellular interaction and clearance of toxic substances

(i.e., MeT5A and H-MESO-1 human cell lines).

Nanomaterials are first inhaled and caught on mucus on the walls of the trachea,

bronchi, and larger bronchioles, which are lined with ciliated epithelial cells covered

with a thin layer of mucous. The mucous traps foreign material, while the ciliated

bodies act to move it towards the throat where it can be swallowed or expelled from the

body [40].

The immune defences in the upper respiratory system (nasal cavity, the mouth, the

pharynx and the larynx) cannot remove all nanomaterials and some of them can

penetrate in the deepest parts of the lung (terminal bronchioles and alveoli). The

terminal bronchioles and alveoli contain specialized cells called macrophages, which

find, digest, and assist in expelling foreign matter from deep in the lungs. Since

macrophages range in diameter from about 10 to 20 μm, shorter NMs (i.e. nanoparticles,

nanofibers and nanotubes) are more likely to be completely phagocytized by alveolar

macrophages than longer ones. This causes the incomplete or “frustrated” phagocytosis

of fibres longer than 20 μm, which is characterized by prolonged production of reactive

oxygen species (ROS); oxidative stress leads to inflammation with release of

inflammatory cytokines and cell death [41].

Nanomaterials that crossed the alveolar-capillary barrier after inhalation (or the gastro-

intestinal barrier after ingestion) end into circulation and may interact further with

internal barriers through passive diffusion (simple or facilitated), active transport and

endocytosis [32]. Endocytosis is an active cellular transport of large, polar molecules

that cannot pass through the cell’s hydrophobic membrane ending with invagination of

the hydrophobic membrane around the molecules deposited on the surface and their

inclusion into the cytoplasm. In passive diffusion, the crossing occurs through a

concentration gradient across the barrier. In the case of passive facilitated diffusion, the

passage of substances is always regulated by a concentration gradient without any

energy expenditure, while active transport is mediated by transporters, occurs against a

concentration gradient and involves an expenditure of energy [42]. While the

intervention of real transporter is unlikely, endocytosis and passive diffusion are the

most observed mechanisms for fibre uptake. Fibre passage through the pleural barrier

will be described in section 1.3.

The placenta is a fundamental barrier between the mother and the fetus to prevent or

greatly limit drug and toxic agent passage into fetal circulation. The placenta is an organ

21

of fetal origin, but shared with his mother, who is called multifunction because it

performs functions that in adult life are made by the liver, kidney, intestines, endocrine

and immune systems. At the level of the chorionic villi, the placenta has an important

barrier function, finely adjusting the bidirectional passage of gases and solutes, ions,

nutrients and metabolites and drugs. The functional unit through which vectorial

transport take place is syncytiotrophoblast, a highly polarized epithelial structure where

the fetal blood is in contact with the basolateral membrane, while the maternal blood

wets the apical side. The apical and the basolateral membranes not only are structurally

distinct, but also differ for the presence of specific transporters, enzymes and hormone

receptors. The concerted action of hundreds of maternal-fetal and vice versa transport

mechanisms ensure the effective removal of metabolites and toxic substances from

fetus, as well as proper procurement of nutrients and active substances.

It is the great selectivity of membrane transport systems that determines the

effectiveness of the placental barrier, which prevents the risk of fetal exposure to

elements and toxic agents. However, similarly to what happens in other body districts,

also the placenta seems to be partially permeable to nanomaterials, either accidentally

through exposure to these materials, or intentionally in the case of potential

nanomedical applications (therapeutic, diagnostic and imaging) [43].

To date, a variety of in vitro models have been developed to clarify the mechanisms of

cellular interaction and transport across the human placenta (i.e., BeWo and JEG-3 cell

lines) [44-45].

Figure 3. Structure of the placenta barrier in the first trimester and at term. At term, the placental barrier

is much thinner due to thinning of the syncytiotrophoblast (ST) and the spreading of the cytotrophoblastic

layer (CT). SOURCE: Buerki-Thurnherr T et al., Swiss Med Wkly (2012) [43].

22

The syncytiotrophoblast layer is thick in early pregnancy and becomes thinner during

gestation. This reduction in thickness and the increase of vascularization fetal enhances

the efficiency of maternal-fetal exchange during fetal growth. Consequently, the

possibility to cross the placenta is higher during the third trimester of pregnancy (Figure

3) [43].

1.1.3 The impact of nanotoxicity on susceptible populations

Although there are numerous benefits of nanotechnology applications (i.e. drug

delivery, all the modern electronics) it increases the need to define the risk assessment

of susceptible populations. In fact, very little is known on the health effects of

nanomaterials in susceptible populations. Recently, nanotoxicological studies are

mainly interesting on pregnant women, neonate, diseased and elderly population. The

susceptible populations are more sensitive to toxic mechanisms of nanomaterial (i.e.,

oxidative stress and inflammation), having alterations in physiological structures and

functions [46-47].

The susceptibility of pregnant women to nanomaterial exposure may depend mainly on

physiological changes in the neuroendocrine network and the placental uptake or

transfer of NMs. The alterations in the neuroendocrine system are important to initiating

and maintaining the pregnancy, fetal development and in the end parturition [48]. Some

research demonstrated that NMs can cross the placenta and enter the fetus [49].

Whereas the fetus is missing of protective mechanisms, it is more sensitive to foreign

substances that are taken up by mother both during and in some cases before gestation.

For example, a study has demonstrated that a single intranasal administration of

titanium dioxide nanoparticles causes a acute inflammation in pregnant BALB/c mice

[46,50]. In another study has found that intravenous injection of silica (70nm) and

titanium dioxide nanoparticles (35nm) in pregnant BALB/c mice decrease the gestional

success rate [46,51]. Moreover, nanomaterial exposure can cause structural and

functional abnormalities in the placenta and can lead to placental dysfunction (i.e.

oxidative stress). In addition, and in some cases, nanomaterials can enter the fetus

through placenta and yolk sac. For example, in pregnant mice, a single intravenous

injection of platinum nanoparticles and PEG-coated quantum dots can translocate after

exposure [52]. Also, in pregnant CD-1 mice, silver NPs (average diameter 50 nm) are

found in extra-embryonic tissues including the visceral yolk sac and endometrium after

23

intravenously injection [53]. In another study, after intravenous injection, silica (70 nm)

and titanium dioxide nanoparticles (35 nm) are found in the placenta, fetal liver, and

fetal brain [51].

Although the fetus is protected by the placenta barrier, these studies confirmed that the

fetus is more sensitive to nanomaterial exposure. Therefore, this uncontrolled exposure

may affect the health of the fetus, potentially leading to abnormal fetal development and

malfunctions (i.e. reproductive toxicity and neurotoxicity). Furthermore, maternal

exposure to NMs can induce the production of inflammatory cytokines that may enter

the fetus and induce alterations in gene expression and cause DNA damage [54].

In diseased population there is a higher risk of toxicity after nanomaterial exposure,

since most of the physiological functions are compromised. In more recent studies, the

toxicity of nanomaterials is investigated more thoroughly in subjects with

cardiovascular and respiratory diseases.

Studies have suggested that nanoparticle exposure causes vascular dysfunctions and

progression of atherosclerosis by enhancing oxidative stress, inflammation,

mitochondrial DNA damage in aortas, and damage to vessel endothelial cells

[55,56,57].

Subjects with chronic respiratory diseases, especially chronic obstructive pulmonary

diseases (COPD) and asthma, are more susceptible to allergens, such as airborne

particles. Asthmatic subjects show higher particulate matter deposition in the lung

compared with healthy ones [58]. A study in humans confirmed that inhaled ultrafine

carbon particles are retained for longer time period in the human lung of asthmatic

patients compared to healthy subjects [59]. Further, NMs deposited in respiratory tract

system of these subjects aggravate the pre-existing inflammation enhancing

hypersensitivity [46,60].

The elderly population is more sensitive to the potential effects of nanomaterials due to

their less effective physiological functions (i.e. protein degradation, regenerative

capability, and immune defences) than the young or adult population. Recent studies

using elderly animal models showed that animals are more susceptible to the harmful

effects of nanoparticles compared with young or adults. For example, the exposure to

SiO2 nanoparticles (24.1 mg/m3; 40 min/day) by inhalation for four weeks in 20-month-

old rats (equivalent to 60–80 years old in humans) causes cardiovascular dysfunctions

[46,61].

24

Therefore the future research in nanotoxicity field should include susceptible

populations in order to provide a comprehensive understanding of transport and

adsorption of NMs compared to healthy populations.

1.2 Pharmacological and Toxicological studies of nanomaterial exposure

Although a large number of research papers are published in recent years to better

understand the cellular uptake of nanomaterials, the underlying mechanisms are still

poorly understood both in vitro and in vivo. It is well recognized that physical and

chemical properties of materials can varied dramatically at nanoscale size, and the

increasing use of nanotechnologies requires further investigations to unravel unexpected

toxicities and biological interactions.

In general, three approaches can be used to study the effects of nanomaterial toxicity: in

vivo experiments on animals, ex vivo studies on biopsies and in vitro experiments using

more or less complex cell culture systems. Cells used for in vitro experiments can stem

either from a continuous cell line (secondary cultures) or freshly isolated tissues

(primary cultures). Mostly, of in vitro nanotoxicology studies are performed using

immortalized cell lines for their practicality.

The most widely used and best characterised in vitro models of the human alveolar and

bronchial epithelia are A549 and BEAS-2B cell lines, respectively. Senlin Lu et al.

[62] investigated on cellular toxicity of A549 cells induced by the different engineered

metal oxide NPs (ZnO, NiO, and CeO2) after exposure and tried to determine

differences in cytotoxicity induced by ambient ultrafine particles (UFPs). The study

demonstrated that metal nanoparticles oxides and UFPs at low concentration induce cell

damage. Interestingly, man-made metal oxide nanoparticles showed a lower toxicity as

compared to UFPs. Kim IS et al. [63] assessed and compared the toxicity of four

different oxide nanoparticles (Al2O3, CeO2, TiO2 and ZnO) to human lung epithelial

cells (A549), demonstrating that ZnO exhibit the highest cytotoxicity in terms of cell

proliferation, cell viability, membrane integrity and colony formation; however, TiO2

induce oxidative stress in a concentration- and time-dependent manner, CeO2 cause

membrane damage and inhibit colony formation in the long-term, and Al2O3 seem to be

less toxic than the other nanoparticles even after long time exposure.

Gurr JR et al. [64] showed that the exposure to ultrafine titanium dioxide particles

induce in BEAS-2B cells oxidative DNA damage, lipid peroxidation, increased H2O2

25

and nitric oxide production, decrease cell growth, and increase micronuclei formation

(indicating genetic toxicity). Davoren M. et al. [65] described the in vitro cytotoxicity

assessment of single-walled carbon nanotubes (SWCNTs) on A549 cells. The exposure

of A549 cells to a wide dose range of SWCNTs (1.56–800 µg/ml) for 24 h revealed that

the SWCNTs have low acute toxicity. TEM (Transmission Electron Microscopy)

studies confirmed that there is an increased number of surfactants storing lamellar

bodies as defensive response of these lung cells to SWCNT exposure. Jia G. et al. [66]

demonstrated the cytotoxicity of single-walled carbon nanotubes (SWCNTs), multi-wall

nanotubes (with diameters ranging from 10 to 20 nm, MWCNT10), and fullerene (C60)

in alveolar macrophage (AM), after a 6 h exposure in vitro. No significant toxicity are

observed for C60 at high dose. SWCNTs significantly impaired phagocytosis of AM at

the low dose. Whereas MWNT10 and C60 induce injury at the high dose and the

macrophages exposed show typical features of necrosis and degeneration.

In vivo studies using animal models have shown that pulmonary exposure to CNT can

led to an immediate macrophage-mediated inflammatory response characterized by a

rapid macrophage influx in the bronchoalveolar fluid and an internalization of the CNT

by the resident or attracted macrophages [67-68].

For pregnant women and their unborn children there are yet concerns about their

nanomaterial exposure. So far there is little coverage in the published literature about

placental uptake or transfer of NMs. A range of in vitro models are used to study

transplacental transfer of several drugs and compounds. Particularly, the human BeWo

cell line, representing a choriocarcinoma-derived placental cell line, is strongly similar

to the cytotrophoblastic cells.

The first study that confirmed the ability of nanosized materials to cross the placental

barrier comes from an in vivo study in pregnant rats where the gold NPs are transferred

to the embryos after intravenous administration [69]. In humans, it has been reported

that polystyrene nanoparticles can cross the placenta in a size-dependent manner [49],

while gold nanoparticles are shown to be retained inside the cells of trophoblastic layer

[70].

1.3 Asbestos and Carbon nanotubes: a comparison

Asbestos is the generic term of a variety of mineral silicates that in the 20th century

became widely used in industrial products, automotive parts, buildings, fabrics, and in a

26

huge array of other domestic and industrial products as a result of its fibrous nature. By

the last few decades of the 20th century asbestos is recognised as potentially harmful,

since it turned out that the exposure to asbestos caused pulmonary diseases (i.e.,

asbestosis, pleural effusions, and pleural plaques) and cancer (i.e., lung cancer and

mesothelioma) [71-72].

Figure 4. Two groups of asbestos: serpentines and amphiboles. Chrysotile is the only member of the

serpentine group. Actinolite, amosite, anthophyllite, crocidolite, and tremolite belong to the amphibole

group. SOURCE: http://www.nationaldryout.com.

Asbestos fibres can be divided into two groups: serpentines and amphiboles. Chrysolite

is the only member of the serpentine group. Actinolite, amosite, anthophyllite,

crocidolite, and tremolite belong to the amphibole group. All asbestos types are

hazardous to human health, but crocidolite is considered most dangerous (Figure 4).

After many years of study, the “fibre pathogenicity paradigm” (FPP) has been proposed

by Donaldson K. [73] to define the toxic characteristics of asbestos and other fibres.

The FPP recognises the geometry of fibres as their most important toxicological

characteristic, superior to the chemical composition. In particular, the paradigm argues

that the pathogenicity of fibres depends on a specific combination of length, thickness

and biopersistence (Figure 5). Fibres that cannot be cleared by physiological processes

are considered to be biopersistent. For instance, to be high hazardous in the body

following inhalation, a fibre must be thinner than 3 µm, longer than 10 to 20 µm, and

non-degradable [73].

27

Figure 5. Diagram illustrating the “pathogenicity fibre paradigm” of Donaldson K. The fibre are

pathogenic if they are thin, biopersisten and long.

The high aspect-ratio makes carbon nanotubes (CNTs) a useful technological material.

In fact, there is a growing development and use of CNTs in a wide range of applications

(industrial, electronics, medicine, etc.). But, their strong similarities to asbestos fibres

(i.e., shape, size, fibrous morphology and pathogenic potential) have raised concerns

about potential toxicity [74-75].

CNTs are manufactured into two main forms, SWCNT and MWCNT. Single-walled

carbon nanotubes are a single layer graphene sheet rolled up in a cylindrical shape with

a diameter of 0.4-2 nm and a length of 0.2-5 µm, while multi-walled carbon nanotubes

contain several layers of grapheme, are 2-100 nm in diameter of and tens of nanometers

to several microns in length (Figure 6) [76].

Figure 6. Carbon nanotubes: single-walled carbon nanotubes (SWCNTs) and multiwalled carbon

nanotubes (MWCNTs). SOURCE: S. Iijima,Physica B: Condensed Matter (2002).

The main risk for adverse effects is associated to the inhalation route both for asbestos

fibres and carbon nanotubes, because they may be deposited and retained in the lung.

The effects of carbon nanotubes are studied following in vivo exposure of rodents, and

in vitro cell culture models highlighting their pulmonary toxicity [77-78].

28

Poland et al. [79] reported that long fibres, either they are asbestos fibres or carbon

nanotubes, are able to evade complete phagocytosis by macrophages, leading to

frustrated phagocytosis and chronic inflammation (Figure 7).

Figure 7. The phagocytosis of short and long fibres. The macrophages can enclose and eliminate short

fibres, while they cannot enclose long fibres, resulting in incomplete or frustrated phagocytosis and

inflammation.

After deposition in the lung, the fibres may translocate to the pleura where they can be

retained depending on their length and biopersistence, leading to inflammation and

oxidative stress. In addition to fibre dimension, the chemical composition of asbestos

and CNTs can increase their stability and render them more biopersistence in the

respiratory system [80]. Highly biopersistent fibres may induce inflammation and

carcinogenesis [81-82].

Surface properties of fibres showed particular biological importance for their uptake by

the cell and cytotoxicity. When asbestos fibres reach the lung lining fluid or alveolar

macrophages can adsorb endogenous proteins, such as ferritin [83], leading to the

formation asbestos bodies [84]. The presence of endogenous molecules may determine

more easily interactions with biological systems.

Research studies showed that surface properties are also crucial to cellular uptake of

carbon nanotubes. Unfunctionalized carbon nanotubes are hydrophobic, but the wide

variety of surface functionalizations can increase their the potential to adsorb a wide

range of small molecules and macromolecules in biological environments. Therefore,

29

the type of functional groups may determine the hydrophilic/hydrophobic properties of

nanotubes, modifying the translocation, distribution and excretion of carbon nanotubes

from the organisms [85].

Considerable evidences suggested that reactive oxygen species (ROS) such as hydrogen

peroxide (H2O2), superoxide anion (O2ˉ) and the hydroxyl radical (HO·), and the

reactive nitrogen species (RNS) can be generated directly by the fibres themselves or

indirectly through interactions with inflammatory cells [86].

The presence of transition metals on the fibres and their ability to attract them is a

central process to explain carcinogenic effects of asbestos. The presence of both ferrous

(Fe2+

) and ferric (Fe3+

) forms, is considered to be responsible for the genotoxic and

cytotoxic responses after deposition of asbestos fibres [87]. Among commercially used

asbestos fibres, crocidolite and amosite asbestos are contain 20-30% iron by weight and

are considered the most carcinogenic. It has been reported that in cell-free systems the

iron mobilization can contribute to redox-cycling reactions leading to production of

hydroxyl radicals, DNA breaks, and formation of premutagenic DNA adduct such as 8-

OHdG (8-hydroxy-2-deoxyguanosine) [88].

Typically, metals (i.e., iron, nickel, cobalt and molybdenum) are used as catalyst in the

synthesis of CNTs to promote the CNTs growth. However, the purification is not

efficient to remove all of the metal contaminants, and often the purified samples are not

completely metal-free.

The presence of metals is known as a mediator of fibre toxicity and carcinogenicity in

diverse carbon nanotubes [89]. Similarly to iron toxic effect, it has been shown that

nickel may cause acute inflammation and pulmonary injury in rat models after

intrapleural injection [90]. Nickel toxicity is carried out by epigenetic mechanisms in

target cells. Alterated DNA methylation or protein acetylation may result in

transcriptional silencing of tumor suppressor genes [91]. Iron related toxicity could be

also epigenetic, and direct demonstration need to be provided.

A second mechanism by which fibres can activate oxidative stress was by the

recruitment of inflammatory cells to the site of fibre deposition. If fibres exceed 20 µm

in length, the macrophages are unable to remove them, leading to persistent macrophage

activation (frustrated phagocytosis) and chronic inflammation [92-93]. It has been

reported that macrophages recognize both asbestos and carbon nanotubes fibres through

the similar mechanism, i.e. via the class A scavenger receptor, and their uptake activates

the NLRP3 inflammasome [94].

30

1.3.1 Mesothelioma: do carbon nanotubes determine the same health

risk of asbestos?

Asbestos exposure can lead to a wide range of pleural pathologies including pleural

effusion which is an excessive build-up of fluid in the pleural space, pleural fibrosis and

pleural mesothelioma. The latter is a cancer of the lining of the pleural cavity caused by

the neoplastic transformation of mesothelial cells. It is aggressive and uniformly fatal

tumor and is seen as a hallmark disease of asbestos exposure. A report of the

International Agency for Research on Cancer (IARC) classified all types of asbestos as

carcinogenic to humans with sufficient evidence for different types of cancers [95].

Recently, a specific multi-walled carbon nanotube (MWCNT-7) has been classified as

possibly carcinogenic to humans [96].

In the years, the aim was to understand if carbon nanotubes may present a hazard to the

pleural district. The first observation derives from the study of Kane AB and co-

workers, in which demonstrated the failure to eliminate long fibres in peritoneal and

pleural cavity [97]. Viallat JR and co-workers [98] provided same hypothesis about

asbestos removal from the parietal pleura and the mesothelioma development.

Poland et al. [99] showed that introperitoneal injection of MWCNTs in mice induces

inflammation and granuloma formation on the mesothelial surface of the peritoneum.

These results demonstrated that only long samples of MWCNTs and amosite produce

inflammation and granuloma as compared with short fibres. Histological analyses

revealed the presence of frustrated phagocytosis by macrophages. Another study

performed by Takagi et al. [100] reported that mice with a deficiency in the p53 tumor

suppressor develop mesothelioma formation in the abdominal cavity after injection of

MWCNTs and crocidolite fibres used as positive control. Murphy et al. [101]

demonstrated that long carbon nanotubes are retained in the pleural space initiating

sustained mesothelial inflammation. The ability of carbon nanotubes to move into the

pleural space after inhalation has also been shown by Mercer et co-workers [102].

A more recent study demonstrated how MWCNT exposure can promote the growth and

neoplastic progression in B6C3F1 mice (a strain used by the National Toxicology

Program to evaluate chemicals for potential carcinogenicity) associated with the use of

a tumor initiator methylcholanthrene (MCA) [103].

31

All these findings suggested that some MWCNTs are likely to pose a carcinogenic risk

similar to asbestos fibres and lead to develop of mesothelioma.

There is significant correlation between the in vivo and in vitro pulmonary responses

studies after CNTs exposure. Some of these studies demonstrated that CNTs induce

ROS generation in mesothelial cells leading to activation of several signalling

pathways, such as AKT, mitogen-activated protein kinase (MAPK), nuclear factor-

kappa B (NF-κB), AP-1 and p53 [104-105]. These results may suggest that the

oxidative stress induced by CNTs is an important aspect of pulmonary toxicity of CNTs

[106]. In vitro studies of MWCNT and SWCNT treatments have revealed genotoxic

effects, such as DNA strand breakage, DNA base oxidation, chromosomal aberrations

and gene mutations [107-108].

1.3.2 Disorders of iron metabolism in nanomaterial toxicity

Iron is a trace element and has a crucial role in living cells, participating in a wide

variety of metabolic processes, such as oxygen transport, DNA synthesis and electron

transport. The disposition of iron in the human body is regulated by a complex

mechanism to maintain homeostasis [109]. Disorders of iron metabolism are among the

most prominent diseases of humans, such as Alzheimer’s and Parkinson’s diseases,

metabolic syndrome, diabetes, atherosclerosis and even cancer [110].

In general, fibre toxicity is also related to the chemical composition. Asbestos toxicity is

ascribed to its particular physico-chemical characteristics, and one of them is the

presence and ability to adsorb iron (Fe3+

and Fe2+

ions), which may cause an alteration

of iron homeostasis in the tissue [111-112]. When the asbestos fibres are deposited in

the lower tract of respiratory system, iron complexation by the fibre surface can cause

an accumulation of metal. But the coordination sites of metal can be incomplete

allowing its participation in metal-catalyzed oxidative stress [113].

Oxidative stress is one of the main epigenetic mechanisms that contributes to

development of carcinogenesis [114]: free radicals (H2O2, ·O2ˉ,

·HO) are capable of

causing DNA damage, protein oxidation and lipid peroxidation [115-116].

The lung defence is performed by alveolar macrophages, which are a type of

macrophages that protect lung tissue against oxidative damage, having the ability to

scavenge iron [111-117]. In fact, superoxide generated by these recruited inflammatory

cells reduces Fe3+

to Fe2+

, allowing metal carrier proteins (i.e., DMT1) to transport

32

metal across the cell membrane to intracellular sites where it can be detoxified [118-

119]. A second mechanism limits the capacity of iron to generate free radicals and is

played by ferritin the iron storage protein [120].

The presence of contaminating transition metals, catalysts used in the synthesis of

carbon nanotubes, is considered an important determinant of their toxicity [121]. Some

studies have reported that CNTs, like asbestos, are capable of inducing ROS generation

and oxidative stress [71,122,123]. For example, unpurified single-walled carbon

nanotubes with 30% of iron impurities showed to cause severe oxidative stress in

human keratinocytes and bronchial epithelial cells [71,124,125].

1.3.3 New approaches for Nanotoxicology: imaging and elemental

analysis by Synchrotron-based X-ray Fluorescence

Biologists and life scientists are enjoying a number of very interesting experimental

developments revolving around X-ray based probes [126]. This has been happening

largely due to third generation synchrotron light sources becoming operational, coupled

with advancements in the fabrication of X-ray optics [127,128,129,130]. Their

combination allows imaging of biological samples at a resolution that is reaching the 10

nm limit.

The scattering of X-rays by solid matter is governed by several interactions, which are

in turn used to gain information about the sample topography (in the case of imaging),

elemental composition (in the case of X-Ray Fluorescence, XRF), or chemical status (in

the case of X-ray Absorption Near Edge Spectroscopy, XANES).

In the course of this work, part of the synchrotron-based experiments involved imaging

and XRF analysis of tissues and cells. The two techniques are often present in the same

end-station, and experiments are often conducted in parallel. An X-ray beam, produced

by a suitable device in the storage ring of a third generation synchrotron light source, is

steered and monochromatized to the working energy by a set of conditioning optical

elements. A lens working on the basis of Fresnel diffraction (called Zone Plate, ZP)

then focuses the beam onto the sample, which is raster-scanned under the beam. A set of

further optical elements, namely a central stop and an Order Sorting Aperture (OSA),

reduce the contribution of unwanted diffraction orders, thereby increasing the signal-to-

noise ratio. A detector downstream the sample collects the transmitted photons,

allowing for a micro-radiography to be constructed onto a computer screen.

33

The primary beam impinging on the sample also excites the characteristics X-ray

fluorescence of the atoms constituting the specimen. When collected and analyzed,

these spectra allow determining the elemental composition of the area that has been

scanned (Figure 8).

Figure 8. Schematics of a typical X-ray fluorescence microscopy end-station.

This is a very valuable tool for the biologist, since it can reveal the variation of the

atomic composition of specimens treated in various ways.

Pascolo L. et al. [87] demonstrated the potential of the advanced synchrotron-based X-

ray imaging and microspectroscopy techniques for studying the response of the lung

tissue to the presence of asbestos fibres. The X-ray absorption and phase contrast

images and the simultaneously monitored XRF maps of tissue samples allowed to

reveal the location, distribution and elemental composition of asbestos bodies and

associated nanometric structures. Another work of Pascolo L. et al. [111] showed iron

mobilization features during asbestos permanence in lung tissue by combination of

advanced synchrotron-based X-ray imaging and micro-spectroscopic techniques. The

XRF elemental mapping allowed clear identification of asbestos fibre and asbestos body

shape, determined from silicon and iron distributions respectively, while phosphorus

and sulphur signals are used to recognise cell morphologies in unstained histological

sections.

34

Marmorato P. et al. [131] investigated the distribution of CoFe2O4 NPs in mouse

Balb/3T3 fibroblasts, exploring also possible chemical changes of the NPs once

penetrated inside the cells. The study demonstrated that the sensitivity of synchrotron-

based X-ray fluorescence is useful to reveal not only the spatial distribution of the NPs

in Balb/3T3 cells exposed to different NPs concentrations but also the possible changes

in the NPs composition as result of intracellular interactions.

1.3.4 Asbestos as inductor of carcinogenesis microenvironment: Do

carbon nanotubes behave like asbestos?

The mechanisms of nanotubes toxicity are not fully understood, but the points of

similarity with asbestos fibres are shown, as described above.

Both asbestos and carbon nanotubes have attracted a great deal of research to evaluate

their carcinogenic potential in human health. In fact, fibrous materials may have the

potential to transform normal cells into cancer cells by causing chromosomal

aberrations and/or gene mutations that can lead to invasion and destruction of the

surrounding tissue.

Figure 9. Long fibres interact with epithelial and mesothelial cells and trigger macrophage activation in

tissues, with chronic inflammation. Epithelial and mesothelial cells with fibres inside can become cancer

cells causing lung cancer and mesothelioma, respectively.

A study reported that fibrous material induces carcinogenesis in mesothelial cells or

epithelial cells, while macrophages are not affected [132]. After fibre exposure, some of

35

these cells undergo apoptosis or programmed necrosis. The cells with the long fibres

inside which are able to evade the killing mechanisms and can become cancer cells

(Figure 9).

Fibre carcinogenicity associated with development of lung cancer and mesothelioma is

well documented for asbestos fibres. Direct mechanisms of asbestos fiber

carcinogenesis include genotoxic and non-genotoxic (mitogenic and citotoxic) pathways

(Table 1) [133]. Asbestos fibres can promote carcinogenicity through iron-dependent

generation of reactive metabolites, such as superoxide, hydrogen peroxide, hydroxyl

radical, and nitric oxide [134]. ROS produced by asbestos can damage cellular

environment through lipid peroxidation and oxidative DNA damage (i.e., oxidative

DNA adducts, DNA single- and double-strand breaks, mutations and chromosomal

aberrations) [135]. Carbon nanomaterials may cause generation of ROS both directly, in

the presence of redox-active transition metals, and indirectly, as a result of ROS

production by target cells [71-124]. Preliminary evidence demonstrated that carbon

nanotubes may also be genotoxic [107-136].

The fibre interaction with the mitotic spindle can cause chromosomal abnormalities

inducing the genetic instability (i.e., micronuclei and chromosomal imbalances,

aneuploidy or polyploidy). Direct interference with the mitotic apparatus could lead to

aneuploidy or polyploidy, and these chromosomal alterations are reported in human

mesotheliomas [133,137,138]. It has been reported that carbon nanomaterials interfere

with mitosis and induce aneuploidy in hamster lung fibroblasts [107] and in a human

lung epithelial cell line [136].

It has also been showed that asbestos fibres induce the activation of downstream

mitogen-activated protein kinase (MAPK) signaling leading to chronic cellular

proliferation [139]. Recently, it has been demonstrated that carbon nanomaterials, like

asbestos fibers, may activate some of the same stress-response pathways; for example,

SWCNT-induced oxidative stress is associated with NF-κB activation via MAPK

signalling in human keratinocytes as well as in normal and malignant mesothelial cells

[140]. In human fibroblasts, exposure to MWCNTs activates genes involved in

p38/MAPK stress-signalling cascade [141].

36

Table 1. Direct and indirect mechanisms of fibre carcinogenesis. SOURCE: Bernstein et al. (2005) [142].

The mechanisms mentioned above are the direct mechanisms of fibre carcinogenesis.

Multiple indirect mechanisms may contribute to a synergistic interaction between fibres

and carcinogenesis, as shown in Table 1 [142]. Asbestos fibres may also exert their

carcinogenic effects by indirect mechanisms. Epidemiological studies suggested that

tobacco smoking can alter mucociliary functions and cause a reduced mucociliary

clearance from the bronchi and alveoli increasing the risk for lung cancer after asbestos

exposure [143-144]. The high surface area of asbestos fibres may facilitate adsorption

of polycyclic aromatic hydrocarbons (PAHs), transport them into the lungs, and

facilitate metabolic activation [133-145]. PAHs and asbestos fibres can be synergistic in

causing squamous metaplasia in vitro [134-146].

The combined effects of asbestos fibres and tobacco smoke on development of lung

cancer may be explained at a molecular level. Asbestos exposure can increase K-ras and

p53 gene mutations, enhancing chromosomal instability [147]. Some smokers may be

genetically predisposed to lung cancer as a result of mutations in DNA repair pathways

[128-142]. Alternatively, acquired mutations or deletions in key genes involved in DNA

repair may facilitate accumulation of additional genetic mutations induced by tobacco-

smoke carcinogens during early stages of development of lung cancer [133-149].

Epigenetic mechanisms (such as DNA methylation or histone modifications) play a role

in carcinogenesis and cellular functions, including the regulation of inflammatory gene

expression, DNA repair, and cell proliferation. For example, chronic inflammation and

37

generation of ROS are also associated with epigenetic gene silencing induced by

hypermethylation [150]. Epigenetic silencing of tumor suppressor genes are described

in human lung cancers [151] and in human malignant mesotheliomas [152].

SV40 (polyomavirus) may act as a cofactor in the pathogenesis of malignant

mesothelioma [153]. However, a role for SV40 as a carcinogen or co-carcinogen is

demonstrated in cellular and animal models on basis of molecular mechanisms of action

[154]. Human mesotheliomas containing SV40 viral sequences showed a significantly

higher index of gene methylation [133]. One of the most frequently methylated genes,

RASSF1A, is progressively methylated during passage of SV40-infected mesothelial

cells in vitro [155].

Persistent inflammation in response to biopersistent asbestos fibers may lead to

secondary genotoxicity caused by release of reactive oxygen and reactive nitrogen

species from activated macrophages [156]. It has been reported that ROS contribute to

alter DNA methylation [157]. Activated macrophages, recruited in the lungs, produce

chemokines, cytokines, proteases, and growth factors causing cell proliferation [158], as

well as adducts, oxidized bases, single- and double-strand breaks [145]. These lesions

can culminate in mutagenic events if they are not repaired. It has been demonstrated that

asbestos-induced TNF-α release is associated with upregulation of TNF-α receptor on

mesothelial cells, as well as NF-κB activation, suggesting that activation of prosurvival

pathways by inflammatory cytokines may prolong the exposure of mesothelial cells to

the genotoxic effects of asbestos fibers [159]. A recent work showed that mice deficient

in Vitamin E, a potent antioxidant, have an enhanced inflammatory and fibrotic

response after exposure to SWCNTs. These events are accompanied by increased

recruitment of inflammatory cells and enhanced production of proinflammatory (TNF-α

and interleukin-6) and profibrotic (TGF -β) cytokines [160].

1.4 Role of genetic predisposition in cancer development during

nanomaterial exposure: BAP1, a promising candidate gene in susceptibility

to mesothelioma.

Recent epidemiological studies have shown a strong correlation between NMs,

respiratory and cardiovascular diseases, various cancers, and mortality. The potential

adverse effects of nanomaterials on human health also involve individual factors, such

as genetics and existing disease. For example, diseases associated with inhaled

38

nanoparticles are asthma, bronchitis, emphysema, lung cancer, and neurodegenerative

diseases, such as Parkinson’s and Alzheimer’s diseases [161]. Nanoparticles that enter

the circulatory system are related to occurrence of arteriosclerosis, and blood clots,

arrhythmia, heart diseases, and ultimately cardiac death [161]. Exposure to some

nanoparticles is associated to the occurrence of autoimmune diseases, such as: systemic

lupus erythematosus, scleroderma, and rheumatoid arthritis [161].

Genetic polymorphisms, genotoxic changes, epigenetic profiles and host factors may

play an important role in the response of humans and their sensitivity to NMs exposure.

Recently, there is growing interest in the potential of epigenetic processes can

significantly modulate cellular behaviour and potentially complex disease risk including

cancer, especially in response to NMs exposure. In vitro animal and human studies have

allowed to identify air pollutants (particulate matter, black carbon, and benzene) which

are able to modify epigenetic processes [162,163]. Epigenetic mechanisms cause

heritable changes in gene expression without changes in DNA sequence. Several

epigenetic mechanisms (i.e., DNA methylation, histone tails modifications, and non-

coding RNA expression) can be modified by exogenous influence, including

nanomaterials, altering genome functions [164-165].

On the contrary, the role of genetic variants in genes, as determinants of susceptibility

to nanomaterials, has not been widely explored. Therefore, this topic merits further

investigations in order to define candidate genes useful as potential biomarkers of NMs

susceptibility. A new approach to understand the link between genetic variation and

susceptibility to nanomaterials is the “toxicogenomics”. It has become apparent that the

effects of exposures vary greatly depending on the genotype and genetic predisposition

of an individual and may result in significant differences from no effects to severe

effects upon exposure to same dose [166].

Asbestos is the principal risk factor in the etiology of malignant mesothelioma (MM).

Malignant mesothelioma is a rare, but aggressive neoplasm, which arises from the

mesothelial lining of the pleura, peritoneum, pericardium, and tunica vaginalis.

However, on average less than 10% of exposed subjects develop mesothelioma,

suggesting the possible involvement of other risk factors. One of these risk factor is

genetic predispositions [167].

Genetic susceptibility to mesothelioma was observed in the Cappadocian villages of

Tuzkoy, Karain, and “Old” Sarihidir [168]. Although mineralogical studies showed that

all the houses appear to contain similar amounts of erionite, mesothelioma is prevalent

39

in certain families but not in others. Pedigree studies showed that tumor seems to be

inherited in an autosomal dominant pattern. The results of mineralogical studies and

pedigree analysis indicate that the MM epidemic in Cappadocia is caused by fibres

exposure in genetically predisposed individuals [169]. Genetically predisposed family

members born and raised outside the MM villages do not seem to develop cancer,

supporting the observation that the combination of genetics and erionite exposure (gene

and environment) is involved in causing MM in these villages [169].

BRCA1-associated protein 1 (BAP1) is located on chromosome 3p21, a region, which

is frequently deleted in MM and other cancers, such as cutaneous and uveal melanoma

and cancers of the lung and breast. But, it has also been reported that BAP 1 germline

mutations predispose to the development of several tumours, such as lung

adenocarcinoma and meningioma [170], uveal and cutaneous melanoma as well as

atypical melanocytic tumours, which they call “melanocytic BAP1-mutated atypical

intradermal tumours” (MBAITs) [171]. Testa et al. [167] demonstrated that BAP1

germline mutations are associated with uveal melanoma and malignant mesothelioma.

This study suggested that individuals with uveal melanoma who carry germline BAP1

mutations have a high risk of developing mesothelioma when exposed to asbestos.

Figure 10. Schematic representation of BAP1 domains and predictive protein folding: UCH, nuclear

ubiquitin carboxy-terminal hydrolase domain; Ba, BARD1 binding domain; H, HCF-1 binding domain;

Br, BRCA1 binding domain; NLS, nuclear localization signal. Possible mechanisms of BAP1 function:

I) BAP1 had deubiquitinase activity, II) BAP1 regulates gene transcription via association with other

protein partners; III) BAP1 performs the epigenetic regulation with other members of the polycomb-

group proteins (PcG) of highly conserved transcriptional repressors.

40

BAP1 is a 729 residue, nuclear-localized deubiquitinating enzyme with an ubiquitin

carboxy-terminal hydrolase function (Figure 10) [172]. But, a nuclear protein BAP1

performs other several functions, including transcriptional regulation, chromatin

regulation, and forming part of multiprotein complexes that regulate cellular

differentiation, gluconeogenesis, cell cycle checkpoints, transcription and apoptosis

[173]. It has been suggested to be a tumour suppressor gene with a role in cell

proliferation and growth inhibition [174].

Only a few mutations common in malignant mesothelioma have been identified so far.

The recent findings of a study on peritoneal mesothelioma discovered two genes which

are associated with a shorter survival: CDKN2A and NF2 [175]. Molecular genetic

study has revealed frequent inactivation in CDKN2A and NF2 genes. The most

common genetic alterations are homozygous 9p21 deletions centered on CDKN2A

found in up to 72% of tumours and 80 % of MPM cell lines [176] and NF2, located on

chromosome 22q12, mutated in 50% of pleural mesotheliomas with corresponding loss

of the wild-type allele by deletion of either 22q or all of chromosome 22. Hemizygous

loss of NF2 is associated with increased mesothelioma proliferation, invasiveness,

spreading, and migration [177].

Carbone M. of the University of Hawaii Cancer Center and other researchers

demonstrated that mesothelioma patients with the BAP1 mutation live at least four

years longer than those without the genetic mutation: the five-year survival rate was to

about 47% for those with the mutation, respect to the relative survival rate of just 6.7%.

Therefore, it seems BAP1 increases the risk of developing mesothelioma, but it also

improves chances of long-term survival [178]. Based on these results, the BAP1 gene

could be used as a prognostic tumour factor. Moreover, the discovery of a genetic

predisposition could be useful to determine the high-risk versus low-risk groups of

asbestos-exposed individuals by using a novel non invasive biomarkers [179].

1.5 General conclusions

Nanotechnology is a fast growing field that will allow for development of materials

with new properties. The number of subjects exposed to NMs will increase over the

next few years, in a context in which the impact of NMs on occupational health and

safety is already difficult to predict. The main factors that determine the toxicological

effects of NMs in the body are the characteristics of the exposure (e.g. penetration route,

41

duration, and concentration), of the exposed organism (e.g. individual susceptibility,

activity at time of exposure, and the particular route the NMs follow in the body), and

the intrinsic toxicity of NMs (e.g. catalytic activity, composition, electronic structure,

capacity to bind or coat surface species, surface area). During the last decade, there

were many studies into the effects of nanomaterials on the respiratory tract and, via

systemic distribution, on the internal biological barriers (blood-brain, blood-testis and

the placenta barriers). It is therefore critically important to understand the nature of the

biological barriers and how NMs may cross them, or interact with their cellular

components.

Carbon nanotubes are promising product in industry and medicine, but there are several

concerns for human pulmonary exposure due to their similarities with asbestos, which

caused a pandemic disease in the 20th

century that continues into present. A growing

body of literature assesses the potential health risk to workers and others subjects (i.e.,

consumers and/or patients). Therefore, the evaluation through in vitro studies to

investigate inflammatory and genotoxicity effects of CNTs is becoming urgent.

In general, the penetration and deposition depth of inhaled fibres is determined by their

length, thickness and biopersistence, in accordance with the “fibre pathogenicity

paradigm”. However, in addition to size and geometry of fibers, the composition and

surface properties seem to play a major role in biological activities. Particularly, the

presence of transition metals (iron) onto the fiber surface is thought to be responsible

for the genotoxic and cytotoxic effects of asbestos fibres by generating oxygen reactive

species. Similarly the presence of metals as trace contaminants during CNT preparation,

in particular iron (Fe) impurities, plays an important role in cellular response. In fact,

iron content in CNTs is one of the primary sources of damage, with increased oxidative

stress and inflammatory responses.

A combination of advanced techniques could be useful in order to unravel the toxic

effects of nanomaterials. In our case, X-ray imaging and X-ray fluorescence microscopy

synchrotron-based techniques allowed to monitor and investigate at high spatial

resolution the presence of iron and other biological elements in cells exposed to

nanomaterials without artefacts (such as the pre-treatment of the sample in conventional

microscopies), and with correlated morphological information.

Although DNA is a stable and well-protected molecule, ROS generated by fibres can

interact with DNA and cause several types of chemical changes. Their main effects

include the oxidation of DNA nucleobases, de-purination and DNA strand breaks.

42

Raman spectroscopy is extensively used in biology field to characterize the chemical

structure of DNA and to reveal specific changes such as the formation of oxidative

products and chain breaks. Moreover, conventional molecular techniques are

indispensable to evaluate the genetic factors which are involved in susceptibility to

nanomaterial exposure.

Therefore, the goal of nanotoxicology should be to recognize the potential harmful risk

of nanomaterials. The multidisciplinary approach will help shedding light on the

mechanisms underlying NMs toxicity in cellular models.

1.6 Aims of the research

My Ph.D. work aimed to contribute in the field of nanotoxicology by unravelling the

possible toxic effects and the epigenetic changes induced by two types of nanomaterials

(NMs), namely asbestos and carbon nanotubes (CNTs), in two internal biological

barriers: the pleura and the placenta.

Appropriate cell line models allowed investigating the chemical properties and

reactivity of these nanomaterials, ultimately affecting total cell uptake and toxicity. My

project used a combination of advanced techniques, synchrotron-based X-ray

fluorescence (XRF) and X-ray microscopy (XRM), as well as more conventional

imaging tools, such as Scanning Electron Microscope, SEM, and Atomic Force

Microscope, AFM, to reveal unknown features of toxic effects of NMs in biological

systems.

Moreover, the attention has been given to a new important aspect of nanotoxicology,

toxicogenomic. In a 2011 study conducted by researchers at the University of Hawaii

Cancer Center and Fox Chase Cancer Center in Philadelphia, Pennsylvania, it was

discovered that people who carried a mutation in BAP1 were susceptible to developing

asbestos-related disease, such as mesothelioma. When these individuals were exposed

to asbestos or a similar mineral like erionite had a higher risk of developing cancer. To

better understand the relationship between environmental exposure and genetic factors

in development of masothelioma we had the opportunity to analyze twenty-nine

samples of lung tissues from asbestos-exposed patients.

The research also aimed to detect the ability of nanomaterials to exert long-term effects

that could lead to genotoxic damage. For this purpose, Raman spectroscopy was

employed, as it allowed evaluating the toxic mechanisms of NMs on DNA, by revealing

43

the chemical changes that alter the DNA following oxidative stress. In particular, the

advantage of this spectroscopic technique seems to be to quickly identify a “fingerprint”

of the oxidative chemical changes, which in turn can be specifically assigned to the

various nitrogen bases.

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60

CHAPTER 2 – BIOCHEMICAL AND

MICROSCOPIC METHODS IN THE STUDY

OF INTRACELLULAR IRON ALTERATION

AFTER NANOMATERIAL EXPOSURE

In light of the many considerations discussed in the introduction, and the techniques that

will explained in this chapter, I will compare a similar iron-related toxicity in human

mesothelial (MeT5A) and placental (BeWo) cell lines exposed to carbon nanotubes

(raw-SWCNT, purified- and highly purified-SCWCNT) or asbestos (crocidolite fibres).

I’ll present here the two study sets, in which the same material and methods have been

adopted (2.1 section). The results section is divided in two parts: Results I (2.2

paragraph), concerning the pleural cell model and Results II (2.3 paragraph), concerning

the placental cells.

2.1 Material and Methods

2.1.1 Carbon Nanotubes

SWCNTs were produced by the HiPCO process (HiPCO stands for high pressure

carbon monoxide), a Chemical Vapor Deposition (CVD) process using continuously

flowing carbon monoxide as the carbon feed-stock and a small amount of iron

pentacarbonyl as the iron-containing catalyst precursor at high pressure. Two types of

commercial carbon nanotubes and two types of manufactured carbon nanotube were

used in the experiments: raw-SWCNT (r-SWCNT) and purified-SWCNT (p-SWCNT),

which are commercial, purified short-SWCNT (s-SWCNT) and highly purified-

SWCNT (hp-SWCNT) which are manufactured [1-2]. The characteristics of two types

of commercial SWCNTs and highly purified-SWCNT are reported in Table 2. All types

of CNTs were provided by Silvia Giordani (ITT, Genova).

61

Table 2. The characteristics of single-walled carbon nanotubes are reported in detail.

.

2.1.2 Crocidolite

Crocidolite Asbestos UICC Standard fibres (SPI#02704-AB) were purchased from SPI

Supplies Division, Structure Probe, Inc. (West Chester, PA 19381-0656, USA) and

suspended in sterile phosphate buffered saline (PBS) at a concentration of 10 mg/ml [3].

2.1.3 Nanofibre suspensions

Just before use SWCNTs and crocidolite fibres were suspended in serum-free cell

culture medium at final concentration of 5 µg/ml. Nanomaterial suspensions were then

rotated for 30 seconds, sonicated for 15 minutes under temperature-controlled

conditions (+4°C), with 15 seconds interruption every 5 minutes for rotating steps.

2.1.4 Iron Sulphate (FeSO4)

FeSO4 heptahydrate was purchased from Sigma Aldrich (St. Louis, MO, USA). Ferrous

sulphate solution was prepared by dissolving the pure powder in sterile H2O at final

concentration of 80 µM.

2.1.5 Cell culture and treatments

Human mesothelial MeT5A and placental BeWo cells (ATCC) were maintained in

Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum

(FBS) (volume/volume [v/v]), L-glutamine 2mM, 100 U/ml penicillin and 100U/ml

streptomycin at 37° C in a 5% CO2 atmosphere. The cells were cultured in 75 cm2

Falcon flask for 2-3 days, then harvested by exposure to trypsin and transferred onto 24-

62

well plates for viability tests by trypan blue exclusion assay or cultured on silicon

nitride (Si3N4) membranes for SR-XRF analysis. After 24 h semi-confluent cells were

treated for 24 h with 500 µl of medium containing SWCNTs and crocidolite fibres to

obtain the final concentration of 5 µg/ml. Just before use the nanofibres were sonicated

in serum-free cell culture medium.

2.1.6 Evaluation of cytotoxicity

Potential toxicity of crocidolite and SWCNTs (raw, purified and highly purified) was

evaluated on cells by trypan blue exclusion dye test. The cytotoxicity of fibres was

tested for 24 at final concentration of 5 µg/ml. After completion of exposure time, the

cells were rinsed twice with sterile phosphate-buffered saline (0.1mM PBS, pH 7.4),

then detached from the well by 0.25% trypsin (Sigma-Aldrich) containing 0.03%

EDTA, pelleted (1200 rpm for 10 min) and resuspended in serum-free cell culture

medium. Then, 10 μl cellular suspension was mixed gently with 40 μl of trypan blue in

an Eppendorf tube and incubated for 5 min at room temperature. The samples were

loaded in a chamber slide and the number of viable cells was measured using the trypan

blue dye exclusion test.

2.1.7 Ferritin assay

The cells were grown in 24-well plates and exposed for 24 h to 5 µg/ml of SWCNTs

and crocidolite fibres, rinsed twice with sterile phosphate-buffered saline (0.1mM PBS,

pH 7.4), then detached from the well by 0.25% trypsin (Sigma-Aldrich) containing

0.03% EDTA and pelleted. The pellet was washed with PBS (0.1mM PBS, pH 7.4) and

transferred to a microcentrifuge tube. 200 µl of M-PER reagent (Mammalian Protein

Extraction Reagent, Thermo Scientific) was added to each samples and shaken gently

for 5 minute. After incubation, the lysates were collected and centrifugated at 14,000 x

g for 10 minutes at 4°C. The supernatants were transferred to a new tube for analysis.

The ferritin concentrations in the lysates was measured using a Roche Cobas® 6000

instrument.

63

2.1.8 Synchrotron-based X-Ray analysis

To perform in vitro treatment experiments with nanomaterials, cells were seeded at a

concentration of 9×104 cell/ml onto 100 nm thick silicon nitride (Si3N4) windows

(Silson Ltd., Northampton, United Kingdom) contained in 24 multiwell plates. The day

after seeding, the culture medium was replaced with fresh medium containing different

nanomaterials at a concentration of 5 µg/ml, and the cells were incubated at 37°C for 24

h. Another set of cells, not exposed to nanomaterials, were used as control sample.

After incubation, samples were fixed at room temperature with 4% paraformaldehyde

(Sigma Aldrich) in PBS pH 7.4, for 20 minutes. Then, samples were washed two times

with PBS and two times in Milli-Q water before the analysis.

In order to identify both the distribution of light and some heavy elements in the cells,

we performed the experiments using soft X-rays (1.15 KeV) at the TwinMic beamline

of Elettra synchrotron (Trieste, Italy) and harder X-rays (7.2 keV) at the ID21 beamline

of ESRF synchrotron (Grenoble, France). In both cases the microscopes were operated

in vacuum to allow the detection of light elements and reduce air absorption. In

particular the harder X-rays were required to excite the K fluorescent lines of Ca, S, P

and Fe, whereas soft X-ray microscopy provided higher quality absorption and phase

contrast images, together with the ability of mapping carbon. The X-ray absorption and

phase contrast images have outlined the morphological features of the sample at sub-

micrometer length scales, while the simultaneous acquisition of the XRF maps

correlated the elemental distribution to the morphology.

Most of the experiments were performed at the TwinMic beamline [4] of the Elettra

synchrotron facility (Elettra, Trieste, Italy, www.elettra.trieste.it/twinmic) using the

scanning X-ray microscopy (SXM) mode.

In the SXM configuration the sample is raster-scanned with respect to a microprobe

generated by a suitable focusing optics. On TwinMic, a configured CCD detector

system [5-6] collets absorption and phase contrast images, while the XRF emission is

detected by means of 8 Silicon drift detectors (SDDs) [7-8].

The experiments was carried out with a photon energy of 1.15 keV and a spot size of

450 nm, which was a good compromise for getting sufficient fluorescence signal for

light elements (C, N, O, Na) as well as for Fe.

SR-XRF data were also acquired at the ID21 X-ray Microscopy beamline [9] of the

ESRF synchrotron facility (Grenoble, France). The samples were prepared with the

64

protocol used described above. A Ni coated double mirror deflecting in the horizontal

plane ensured the harmonics rejection. A Si(111) double-crystal monochromator was

used to select energy of 7.2 keV, above the Fe K absorption edge. A zone plate (Zone

Plates Ltd, UK) focused the beam down to a micro-probe of 200 nm x 900 nm (V x H)

with a photon flux of 3.5 x 109 photons/s. The sample was raster scanned in the micro-

beam to collect 2D fluorescence maps. An SDD detector (Röntec, Germany) was used

to detect the fluorescence photons emitted by the sample. Analysis of the SR-XRF

images and spectra was carried out by using the multiplatform program (PyMCA)

developed by Sole V. and co-workers [10].

2.1.9 Atomic force microscope analysis

The cells were grown on 100 nm thick silicon nitrite (Si3N4) windows (Silson Ltd.,

Northampton, United Kingdom) at concentration of 9×104 cell/ml. The day after

seeding, the culture medium was replaced with fresh medium containing different

nanomaterials at a concentration of 5 µg/ml, and the cells were incubated at 37°C for 24

h. After incubation, samples were fixed at room temperature with 4% paraformaldehyde

(Sigma Aldrich) in PBS pH 7.4, for 20 minutes. Then, samples were washed two times

with PBS and two times in Milli-Q water before the analysis.

AFM images were taken by using Digital Instruments Dimension 3100 Atomic Force

Microscope (AFM Service, ESRF in Grenoble). A digital camera allowed to select the

scanning area, typically 100 × 100 μm. The measurements were carried out in contact

mode under ambient conditions, adjusting the parameters during scanning to prevent

damaging of the biological structures.

65

2.2 RESULTS I: Iron-related toxicity of single-walled carbon nanotubes

and crocidolite in human mesothelial cells (MeT5A) investigated by

Synchrotron XRF microscopy.a,b

a)Part of this study has been presented to the European conference On X-ray

Spectrometry EXRF (BOLOGNA 15-20 Joune 2014) as poster presentation: Altered

morphology and iron content in MeT5A mesothelial cells following exposure to carbon

nanotubes and crocidolite asbestos”. Cammisuli F., Gianoncelli A., Rizzardi C.,

Giordani S., Melato M. and Pascolo L. Abstract for European Conference on X-Ray

Spectrometry (EXRS), Bologna 15-20 Giugno 2014.

b)Results have been adapted from the manuscript in preparation: “Iron related toxicity

of single-walled carbon nanotubes and crocidolite fibers in human mesothelioma cells

(MeT5A) investigated by Synchrotron XRF microscopy”. Cammisuli F., Giordani S.,

Gianoncelli A., Rizzardi C., Radillo O., Da Ros T., Salome M., Melato M., Pascolo L.

2.2.1 State of the art in in vitro tests of mesothelial cell line

There has been little investigation on the toxicological effects of fibres in pleural

mesothelial cell lines. Mesothelial cells form double layer (mesothelium) lining the

serosal cavities (pleural, pericardial and peritoneal) and the organs contained within

these cavities [11]. The primary function of the mesothelium is to provide a protective

non-adhesive surface but also to transport solutes and cells across serosal cavities,

including antigen presentation, inflammation and tissue repair, coagulation and

fibrinolysis and tumour cell adhesion [11]. Particularly, during inflammation processes

mesothelial cells secrete a whole host of mediators including various cytokines such as

interleukins (IL) 1 and 6 as well as a range of chemokines (e.g. IL-8, MCP-1) [12].

A seminal study performed by Murphy A.F. et al. hypothesized that long fibres cause an

inflammatory response in the pleural cavity through frustrated phagocytosis. The

pleural-activated macrophages trigger an amplified pro-inflammatory cytokine response

from the adjacent pleural mesothelial cells (MeT5A), producing a pro-inflammatory

environment in the pleural space exposed to long CNT [12].

The study conducted by Tabet L. et al. [13] evaluated toxic effects of multi-walled

carbon nanotubes (MWCNTs) on a human epithelial cell line (A549) but also in

mesothelial cells (human MeT5A cell line). MWCNTs are compared to two types of

asbestos fibres (chrysotile and crocidolite) as well as to carbon black (CB)

nanoparticles. From the comparison it has been noted that MWCNTs appear as

66

agglomerates attached to cells, while asbestos fibres penetrate into the cells, and both

cause a decreased metabolic activity but not cell membrane permeability.

More recently a study has been performed to unravel the morphological changes of

mesothelial cells (MeT5A) after interaction with crocidolite fibres, by using a

combination of conventional atomic force microscopy (AFM) and scanning near-field

optical microscopy (SNOM) [3]. The results demonstrated that the crocidolite fibres

penetrate the plasma membrane and may reach also the nuclear compartment.

As already discussed in the introduction, even if the mechanism by which asbestos and

other mineral fibres cause cancer still remains largely unknown, iron associated to fibres

seems to play a crucial role in the pathogenic effects of asbestos. About this, a study

investigated the role of iron in the inactivation of epidermal growth factor receptor

(EGFR) after asbestos treatment of human lung epithelial (A549) cells, human pleural

mesothelial (MeT5A) cells [14]. The results confirmed that there is a dephosphorylation

and subsequent inactivation of epidermal growth factor receptor (EGFR) and iron seems

to be the responsible of this effect on the receptor following internalization fibres. In

fact, the inhibition of fibre endocytosis and iron removal from fibres by using

desferrioxamine B or phytic acid, prevents the decrease in EGFR phosphorylation and

therefore the inactivation of the receptor [14].

The inflammatory stimuli to mesothelial cells include microbes and environmental dust.

These cells are actively phagocytic cells and some reports from Ghio AJ (one of the

most important researcher on asbestos toxicity) indicated that mesothelial cells respond

to inflammatory stimuli with an increase in the ferritin production [15]. This was

demonstrated exposing MeT5A cells to asbestos fibres as well as to talc [15].

Here I reported the iron related response of MeT5A cells to crocidolite and CNTs at

different grades of purification (reducing the iron presence), by both checking ferritin

response and investigating other chemical changes.

2.2.2 Results of viability test

The results were expressed as percentage of dead cells as compared to a control, and are

shown in Figure 11. The results revealed that the cells treated with highly purified-

SWCNTs (hp-SWCNTs) were viable at 90%. While the cell viability was over 80% for

cells exposed to purified SWCNTs (p-SWCNTs). A reduction around 70% was

observed for cells treated with raw-SWCNTs (r-SWCNTs) and to about 65% for

67

treatments with crocidolite fibres and iron sulphate (FeSO4). Moreover, the results

revealed that after 48h of incubation there was an reduction of vitality of an additional

10-15% with respect to 24 h for each nanomaterial.

Based on these results the concentration of 5 µg/m and the incubation at 24 h for all

nanomaterials was selected for X-ray fluorescence and microscopy analyses and ferritin

assay.

Figure 11. Quantification of mesothelial cell viability during the treatments with nanomaterials (5 µg/ml)

for 24 h and 48 h.

2.2.3 Results of elemental mapping with XRF microscopy

After a selection under light microscopy, cells were raster scanned at the ID21 beamline

of the European Synchrotron Radiation Facility (ESRF) using 7.2 keV photon energy,

monitoring the XRF fluorescent signature of P, S, Ca, K and Fe.

The deconvolution of elemental maps was performed by using PyMCA software. The

interpretation of X-ray fluorescence maps was based on temperature colorimetric scale,

ranging from blue for low concentrations in the element of interest to red for high

concentrations.

Panel A (Figure 12) shows the elemental maps of a MeT5A control cell: phosphorus (P)

and sulfur (S) are constitutive elements of cells, and their distribution delineated the cell

shape. In particular the P-rich zone in the central part of the cell could be attributed to

68

the nucleus. The other elements potassium and calcium showed a rather homogeneous

distribution, while the counts for the iron fluorescent signal were low, indicating a very

low presence in healthy cells.

Panel B (Figure 12) shows the elemental maps of cells exposed to iron sulphate

(FeSO4). The iron (Fe) map revealed iron-rich regions, vesicles or aggregates, probably

most of them extracellular. In addition, there was a diffuse signal from intracellular iron

increase derived from the uptake into the cell.

Figure 12. X-ray fluorescence analysis of untreated and treated cells with iron sulphate. (A) Visible

light image of the control cell (a) and the corresponding P, S, Ca, K, Fe XRF maps (32µm x 42,5µm)

showing the distribution of different elements. (B) Visible light image of treated cell with FeSO4 (a) and

the corresponding P, S, Ca, K, Fe XRF maps (52µm x 60,5µm). The XRF maps were acquired at 7.2 keV

incident photon energy.

69

Figure 13 and Figure 14 show the elemental maps of cells exposed to crocidolite. The

iron (Fe) map revealed iron-rich segments that localize the crocidolite fibres, most of

them intracellular as perceived by comparison with the optical image.

Due to the extremely high abundance of iron in the asbestos fibres (about 30 wt%) the

Fe signal was reported on a logarithmic scale in order to highlight also its low

concentration presence in the cells. In fact, the logarithmic scale allowed revealing a

diffuse intracellular iron increase that could derive both from an iron dissolution from

the fibres and an increased iron uptake into the cells. Compared to control cells, some

cells exposed to asbestos showed a reduced K presence, compatible with a cell

sufferance condition.

Figure 13. µXRF and X-ray microscopy of treated cells with crocidolite fibres. Visible light image of

two cells exposed to crocidolite fibers (a) and the corresponding P, S, Ca, K, Fe XRF maps (56µm x

76.5µm) showing the distribution of different elements. Fe map is displayed using a logarithmic scale.

Below are shown the X-ray microscopy absorption (b) and phase contrast (c) images of the corresponding

XRF maps. The absorption and phase contrast images were measured at 0.9 KeV photon energy, whereas

the XRF maps were acquired at 7.2 keV.

70

Figure 13 and Figure 14 show the X-ray absorption (b) and phase contrast (c) images of

MeT5A treated cells with crocidolite fibres. The contrast of the X-ray images (c) clearly

indicated the different density between nucleus and other cell compartments (nucleolus

and cytoplasm). Moreover, X-ray microscopy allowed to precisely reveal the fibres not

only in absorption mode (b) (the fibres are more absorbing than the surrounding cellular

matter), but also in differential phase contrast (c). Indeed, most of the fibres could not

be seen in the optical image since they were internalized. The combination of

synchrotron X-ray fluorescence and soft X-ray microscopy allowed to study the fibres

uptake in mesothelial cells and to distinguish the intracellular fibres from extracellular

ones.

Furthermore, for their needle-like shape the fibres possess an enhanced capacity to

perforate the plasma membrane and only the smaller fibres are able to reach sub-cellular

components of the cells up to nuclear regions. In Figure 13 is clearly visible how

asbestos fibres perforate the plasma membrane as shown black arrow. In red circle was

highlighted a region of the plasma membrane in which was occurred the perforation and

the entry of a very small fibre: the plasma membrane appeared to be folded up like

during the invagination processes (i.e., endocytosis and exocytosis).

In our study, we applied other microscopic techniques, such as scanning electron

microscopy (SEM) and atomic force microscopy (AFM) - the results are shown in

appendix A - to investigate the interaction of these cells with asbestos fibres. It is

important to note that all microscopic techniques used in this study have the advantage

to allow to obtain image with high spatial resolution and without procedures of

labelling. The results reported in Figure A1 of Appendix A explain the promising

combination of X-ray microscopy with AFM. It was possible localize most of the

crocidolite fibres internalized into the cells (Figure A1, lane A and B), and short CNTs

deposited onto cellular surface (Figure A1, lane C). The SEM images showed in Figure

A2 of Appendix A explain the cell membrane penetration by asbestos fibres (E,G,H) or

their wrapping by the cell membrane (F). An aggregate of raw-SWCNTs is visible onto

the cell (L), while localization of short-SWCNTs (purified) was complicated since they

are very similar to cellular materials (O,P,Q,R). Moreover, SEM images allowed to

reveal the presence of plasma membrane blebs in mesothelial cells exposed to all

nanomaterials, probably it was a cellular response linked to sufferance state.

71

In addition, the optical image (Figure 13a) shows the presence of cell adhesion filament

which could be confused with a long asbestos fibre. But by the comparison with the

other microscopic images we had the certainly that it was not asbestos fibre.

In cell sample reported in Figure 14 there was a considerable morphological damage.

The cytoplasm is occupied by numerous membrane vesicles. Soft X-ray microscopy

allowed to investigate this alteration of the morphologic aspects of the surfaces of

mesothelial cells and to reveal in detail the increased numbers of filopodia and vesicles.

After a careful analysis we have noted that the iron-rich regions (shown in Fe map)

were in proximity but did not localize with these vesicles which, however, could be a

cellular response to nanomaterial toxicity.

Figure 14. µXRF and X-ray microscopy of treated cells with crocidolite fibres. Visible light image of

two cells exposed to crocidolite fibers (a) and the corresponding P, S, Ca, K, Fe XRF maps (56µm x

76.5µm) showing the distribution of different elements. Fe map is displayed using a logarithmic scale.

Below are shown the X-ray microscopy absorption (b) and phase contrast (c) images of the corresponding

XRF maps. The absorption and phase contrast images were measured at 0.9 KeV photon energy, whereas

the XRF maps were acquired at 7.2 keV.

72

These techniques do not need staining or metal-coating procedures, in fact MeT5A cells

were only fixed, therefore they can be considered non-invasive analyses, which allowed

to investigate the morphology of cells and to reveal the cellular localization of the

crocidolite fibres, by providing a better spatial resolution compared to the optical

images and therefore much more detailed information.

Figure 15 shows the elemental maps of cells exposed to raw-SWCNTs. Merging with

the cellular shape (P and sulphur S maps) the iron (Fe) map revealed several iron-rich

spots inside or surrounding the cells. Bases on their dimensions and intensities, these

iron-spots could be attributed to iron containing nanotubes or to aggregates of residual

iron particles detached from them [16]. Looking the potassium (K) map, it was

interesting to note that there was an homogeneous increase of its intracellular presence;

K increase can be linked to some alteration in the membrane potential mechanisms (i.e.,

sodium potassium pump alteration), but further investigations are necessary. In the case

of calcium (Ca) signal, there was no notable change compared to control cell.

The X-ray contrast and absorption images (b and c) clearly indicated the increased

density mainly due to the higher thickness in the samples. In this cell, raw carbon

nanotubes were not visible, as expected by the low dimensions and density. In addition,

they often were aggregated in the medium and hardly interacted with plasma membrane.

Moreover, from the absorption images (c) a notable observation was that the presence

of small vesicles near the nucleus, suggesting that the cell was in a serious status of

sufferance. Notably, in this case, although the XRF map did not showed any Fe increase

in clear correspondence with these vesicles, iron aggregates were concentrated in close

proximity.

In general, the toxicity of CNTs is attributed to their physico-chemical characteristics,

such as length, diameter, shape, purity, surface area and surface chemistry. In addition

to these properties, the CNT contamination by catalyst residues which occurs during

their production, has been demonstrated to affect the CNT safety.

In our study, the iron content raw-SWCNTs (non-purified carbon nanotubes) was

approximately 30% (wt % iron). The iron impurities can cause not only the reduction of

cellular viability (as shown in Figure 11), but also a clearly alteration of iron

metabolism, and this study was the first clear-cut evidence for it. Although a lot of the

iron nanoparticles seemed to be extracellular as confirmed by rich iron-spots in Fe map

(Figure 15 panel A and B), it was also possible to recognize a homogenous iron increase

73

inside the cell. The fluorescence signal levels were clearly higher than those of control

cells.

Figure 15. µXRF and X-ray microscopy of treated cells with raw-SWCNTs. (A) Visible light image

of cell exposed to raw carbon nanotubes (r-SWCNT) (a) and P, S, Ca, K, Fe XRF maps (54µm x

70,5µm). The X-ray microscopy phase contrast (b) and absorption (c) images of the corresponding cell

are reported to the side. Panel (B) shows the visible image of another treated cells (d) and the

corresponding P, S, Ca, K, Fe XRF maps (62µm x 50,5µm). Fe map is displayed using a logarithmic

scale. The absorption and phase contrast images were measured at 0.9KeV photon energy, whereas the

XRF maps were acquired at 7.2 keV.

In some case, we had the perception that small raw-SWCNTs were internalized by

mesothelial cells. Probably, most of them were aggregated in cytoplasm, while smaller

74

ones seemed to interact with the plasma membrane depositing along cellular edges (as

indicated by black arrow in Fe map of Figure 15 panel B).

Figure 16 (Panel A) shows the X-ray contrast (a) and absorption (b) images and the

corresponding XRF carbon maps of cells exposed to purified short-SWCNTs (s-

SWCNTs). Their iron content was less than 15% (wt%).

The X-ray contrast (a) and absorption (b) images showed the increased density, mainly

ascribable to the higher thickness in the samples, in particular in the regions of a clear s-

SWCNT internalization (indicated by black arrows). This carbon nanotube

internalization is also confirmed by C map, where there was an increase of carbon level

in correspondence of CNTs intracellular localization.

In this sample we had the certainty that was occurred the internalization of bundles of

purified carbon nanotubes. The results obtained by the different microscopic techniques

allowed to monitor the s-SWCNTs localization and their cellular internalization.

Therefore, it was demonstrated the powerful combination of the advanced microscopic

techniques, based on synchrotron radiation, in obtaining crucial details of cell-nanofibre

interactions and their cellular toxicity.

Figure 16 (Panel B) shows the phosphorus (P) and sulphur (S) maps that allowed to

identify the cellular shape, while the iron (Fe) map revealed several iron-rich spots

inside or surrounding the cells. Bases on dimensions and intensities, these iron-spots

could be attributed to iron containing nanotubes or to residual iron particles detached

from them [16]. The logarithmic scale allowed revealing a diffuse intracellular iron

increase that could derive also from Fe uptake of the cell.

Moreover, the zoom inset of panel B (Figure 16) shows the specific localization of

internalized s-SWCNT. Carbon and iron map confirmed the presence of s-SWCNT:

carbon is the constituent of this nanomaterial, while iron represents the impurities that

derive from their synthesis process. The notable spatial resolution of phase contrast

image (Panel B, a) highlighted the amazing opportunity to reveal the exact shape and

orientation of small carbon nanotubes. Besides, synchrotron X-ray fluorescence, in

association with soft X-ray microscopy, allowed simultaneous mapping of multi-

element distributions with high resolution. In this sample, the optical image (Panel A, c)

obtained in transmission mode, allowed to localize CNTs, even if the most of important

details are surely missing.

In addition, in this cells exposed to purified s-SWCNT, the iron-rich zones showed

colocalization with the highest P signal (Figure 16, panel A), suggesting a possible

75

SWCNT interaction with the nuclear or perinuclear region. Similar evidences have also

been reported by other studies [17-18]. The phosphorus alteration could be linked to

genotoxic effects exerted by carbon nanotubes [19].

Figure 16. µXRF and X-ray microscopy of treated cells with purified s-SWCNTs. (A) The X-ray

microscopy absorption (a) and phase contrast (b) and visible light (c) images of exposed cells to purified

short-SWCNT (s-SWCNT). The C map, collected at the TwinMic beamline with photon energy 1.15

keV, is shown to the side. P, S and Fe maps, obtained at ID21 beamline, are reported below (52µm x

73.5µm). Panel (B) shows phase contrast (a, b) images and corresponding C and Fe maps of zones

indicated with the black and red circle. Fe map is displayed using a logarithmic scale. Moreover, panel B

shows the specific localization of internalized s-SWCNT in a zoomed area of the absorption and phase

contrast images. C and Fe maps confirm intracellular localization of carbon nanotubes in the region with

the highest concentrations of both. The absorption and phase contrast images were measured at 0.9 KeV

photon energy, whereas the XRF maps were acquired at 7.2 keV.

Figure 17 (Panel A) shows the XRF maps of cell exposed to purified carbon nanotubes

(p-SWCNTs). Looking at the iron (Fe) map, it was interesting to note that there was an

76

inhomogeneous increase of its intracellular presence; again the iron-spots could be

linked to iron containing nanotubes (15 wt % iron) or to residual iron particles detached

from them.

Figure 17. µXRF and X-ray microscopy of treated cells with p-SWCNTs and hp-SWCNTs. (A)

Visible light image of cell exposed to purified carbon nanotubes (p-SWCNT) (a) and P, S, Ca, K, Fe XRF

maps (36µm x 46,5µm). The C map, collected at the TwinMic beamline, is shown to the side. P, S and Fe

maps, obtained at ID21 beamline, are reported below. Panel (B) shows visible light and absorption (a, b)

images of cell treated with higly purified carbon nanotubes (hp-SWCNT). The C map was obtained at

1.15 keV, while S, P and Fe maps were collected using a 7.2 keV excitation energy, and are also reported

in the panel. Fe map is displayed using a linear scale.

Figure 17 (Panel B) shows the phosphorus (P) and sulphur (S) maps that identified the

cellular shape, while the iron (Fe) map revealed several iron-rich spots inside the cell. In

these samples treated with highly purified carbon nanotubes (hp-SWCNTs) we

observed a marginal iron alteration, surely due to the higher purity grade of CNTs. The

C map seemed to reveal a thickening of cellular borders allowing to suppose the

77

internalization of carbon nanotubes, in some cases confirmed by iron spot. The

thickening was clearly visible in the absorption image (Panel B, b).

2.2.4 Results of ferritin assay

After X-ray fluorescence analyses we tried to understand if this iron increase was due to

release from nanomaterials or to a biochemical response of cells. Therefore, we

performed an assay in order to evaluate an intracellular ferritin level.

Ferritin is a protein that stores iron and releases it in a controlled amount in the body.

Ferritin has a spherical shape and iron is stored in the Fe(III) oxidation state inside the

sphere. When the body needs iron, it must be changed from the Fe(III) to the Fe(II)

oxidation state. Then, the iron leaves through channels in the spherical structure.

Figure 18. Intracellular concentrations of ferritin after in vitro exposure of mesothelial cells (MeT5A) to

all nanomaterials (5 µg/ml) for 24h and 48h. *Significantly increased is found in samples treated with

raw-SWCNTs, crocidolite and iron sulphate.

The results, displayed in Figure 18, highlighted a ferritin increase in the cells exposed to

crocidolite fibres and iron sulphate (FeSO4), as compared to ferritin concentration found

in control cells. Cells treated with raw-SWCNTs had a similar stimulation although less

evident than crocidolite fibres. The cells treated with purified-SWCNTs had lower

78

stimulation than cells treated with raw-SWCNTs, while cells treated with hp-SWCNTs

had a similar stimulation of control cells (as shown in Figure 18).

The results demonstrated that crocidolite fibres and raw carbon nanotubes gave a severe

stimulation of intracellular ferritin, while highly purified carbon nanotubes gave an

amount of intracellular ferritin comparable to control cells (at 24 h). This means that

raw carbon nanotubes and crocidolite fibres share a similar toxicity mechanism linked

to iron impurities (30% for crocidolite fibres and <30% for raw-SWCNTs). Moreover,

the results revealed that after 48h of incubation there was an increase of ferritin level of

an additional 5-20% with respect to 24 h for each nanomaterial (as shown in Figure 18).

Ghio AJ and co-workers [15] have performed in vitro study to test the disruption in iron

homeostasis after talc exposure of mesothelial cells. They found that there is a

significantly increased iron importation and concentrations of the storage protein

ferritin. Previously, the same author has examined the increased expression of ferritin

after exposure of alveolar macrophages to silica [20]. The findings confirmed that the

expression of ferritin, a storage protein, is controlled by a post-transcriptional

mechanism that is dependent on the concentration of available iron. Iron complexed to

the surface of silica can catalyze the generation of free radicals. The alveolar

macrophage, using superoxide, can mobilize the metal from the surface of a mineral

oxide by reducing it to the ferrous state [21]. When the metal is isolated in this storage

protein has a chemically less reactive state. Therefore, the sequestration of reactive iron

confers a protective effect in the macrophages and limits the iron toxicity.

Similarly, in our study the increased concentration of ferritin after nanomaterial

exposures could be a protective mechanism in order to prevent the oxidative damage.

The mesothelial cells (MeT5A), as well as alveolar macrophages, are able to control the

ferritin expression by iron-dependent manner.

Our study was the first to test this ability in the presence of carbon nanotubes.

79

2.3 RESULTS II: Toxicity effects of single-walled carbon nanotubes and

crocidolite in human placental cells (BeWo) investigated by Synchrotron

XRF microscopy and AFM microscopec

c) Results have been adapted from the manuscript in preparation: “Toxicity effects of

single-walled carbon nanotubes and crocidolite in human placental cells (BeWo)

investigated by Synchrotron XRF microscopy and AFM microscope”. Cammisuli F.,

Gianoncelli A., Altissimo M., Salome M., Rizzardi C., Giordani S., Radillo O., Melato

M. and Pascolo L.

2.3.1 State of the art in in vitro tests of placental cell line

It has been largely confirmed that the BeWo cells are a good in vitro model for

placental functions, also used to study the ability of nanoparticles (NPs) to translocate

across the placental barrier [22].

It has been reported that the uptake and transport of iron oxide and silica nanoparticles

can transfer extensively across the placental barrier model depending on physico-

chemical characteristics such as surface chemistry. The BeWo cell model can be used

efficiently to predict the capacity of NPs to reach the fetus [23].

A recently study investigated the safety of silica NPs by using this choriocarcinoma cell

line and showed a decreased in cell viability only when using concentrations higher than

100 µg/ml. The results showed a low transfer of silica NPs to the fetal compartment,

although the biocompatibility could limit the application of unmodified silica NPs in

biomedical imaging or therapy [24].

Another important study analyzed the transfer of PEGylated gold nanoparticles across

the perfused human placenta [25]. The PEGylated gold nanoparticles of the size 10-

30nm do not cross the perfused human placenta, and cannot be found in detectable

amounts in the fetal circulation within 6h. While during in vitro experiments, the gold

nanoparticles are taken up by BeWo choriocarcinoma cells and retained inside the cells

for an extended period of 48h [25].

These findings indicate that the ability of nanoparticles to cross the placental barrier

depends on their type, size and surface modification, causing complications of the

reproductive system and fetus. However, the mechanism of this process remains

unclear.

80

Moreover, it has been reported that asbestos fibres can pass through the walls of the

digestive tract and travel throughout the body. Another work has also indicated that

asbestos could cross the placenta [26].

An autopsy study was conducted to investigate whether there is transplacental transfer

of asbestos in humans. The study demonstrated the presence of short and thin asbestos

fibres in stillborn infants and their positive association with working mothers [27-28].

Another study showed that repeated administrations of CNTs cause reversible testicular

damage without compromising fertility in male mice [29]. Other researchers have

focused on the effects of CNTs on the female reproductive system. Similarly to

nanoparticles and asbestos, CNTs could cross the placental barrier and to damage both

placenta and fetus.

A further work has studied the mechanism by which oxidised MWCNTs (o-MWCNTs)

may cross the placental barrier and determine the abortion rates in mice with different

pregnancy times. It has also investigated the maternal and fetal toxicity of o-MWCNTs.

The results showed that o-MWCNTs are able to cross the placental barrier and reach the

fetus body, leading to placental dysfunctions and also fetal growth delay with

complications of fetal heart and brain. The abortion rates in pregnant mice depend on

pregnancy times [30].

In addition to direct effects of NMs on placental barrier, other studies have investigated

the indirect effects of NMs on fetus, after the inflammatory and oxidative stress

responses in the exposed mothers [31-32].

An interesting study put forward the hypothesis that pulmonary exposure to NMs during

gestation induces inflammation in the exposed mother and leads to secondary effects in

the fetus [33]. It has been used a toxicogenomics approach to investigate the hypothesis

that maternal exposure to nanosized particles (such as carbon black, CB) during

pregnancy affects the fetal development. In fact, the results have revealed that there is a

change in the expression of several genes and proteins associated with inflammation in

maternal lungs after carbon black (CB) exposure, while the newborns respond with

changes in metabolism-related genes which can also manifest later in life [33].

A more recent study has performed toxicological tests to investigate PEG-SWCNTs

effects in order to evaluate their possible use as biomedical carriers in pregnancy [34].

PEG-SWCNT are intravenously injected in CD1 pregnant mice at different doses and in

single or multiple administrations. The results showed no adverse effects on embryos up

81

to the dose of 10 μg/mouse, while at the dose of 30 μg/mouse, are detected teratogenic

effects, in association with placental damage [34].

From examining the literature, there are few studies about exposure of the human

placenta to asbestos fibres and carbon nanotubes: none, at the best of our knowledge,

comparing the two material classes.

Therefore, our aim was to explore asbestos and carbon nanotube toxicity, as well as iron

related responses, by using in vitro barrier model of the placenta (BeWo cell line).

2.3.2 Results of viability test

The results were expressed as % dead cells when compared with control as shown in

Figure 19. The results have revealed that after 24h of crocidolite (5 µg/ml) and iron

sulphate (80 µm) incubation the viability of cells was to around 65%. The cells exposed

to 5 µg/ml of raw-SWCNTs (r-SWCNTs) showed a decrease of vitality to about 70%,

while in the ones treated with purified-SWCNTs (p-SWCNTs) this was more than 80%.

The highly purified-SWCNTs exposure (hp-SWCNTs) gave a cellular vitality of about

90% (Figure 19).

Similarly to the conditions of MeT5A treatments, the concentration of 5 µg/ml and the

incubation at 24 h were selected for X-ray fluorescence and microscopy analyses and

ferritin assay.

Figure 19. Quantification of placental cells (BeWo) viability during the treatments with each

nanomaterial (5 µg/ml for 24 h).

82

2.3.3 Results of elemental mapping with XRF and AFM microscopy

After a selection under light microscopy, cells were raster scanned at the ID21 beamline

of the European Synchrotron Radiation Facility (ESRF) using 7.2 keV photon energy.

monitoring the characteristic X-ray emissions of P, S, Ca and Fe. Figure 20 shows the

XRF maps of control and treated BeWo cells with iron sulphate (FeSO4).

Figure 20. XRF maps of untreated and treated cells with iron sulphate. (A) Visible light image of the

control cell (a) and the corresponding P, S, Ca and Fe XRF maps (152 µm x 105 µm) showing the

distribution of different elements. (B) Visible light image of treated cell with FeSO4 (a) and the

corresponding P, S, and Fe XRF maps (56 µm x 62,5 µm). The XRF maps were acquired at 7.2 keV

excitation energy.

83

In panel A (Figure 20) the elemental maps of a group of control BeWo cells are

presented: phosphorus (P) and sulphur (S) are constitutive elements of cells, and their

distribution allowed to delineate the cell shape. In particular the maximally P-rich zones

in the central part of the cell could be attributed to the nucleoli. Calcium map (Ca)

showed a rather homogeneous distribution, while iron (Fe) appeared close to the

detection limit, suggesting a very low presence in healthy cells. Looking optical image

(a) and sulphur (S) map, we immediately noted the different morphology of placental

cells compared to mesothelial ones. In fact, BeWo cells are able to fuse and form

multinucleated syncytia.

Panel B (Figure 20) shows the elemental maps of cells exposed to iron sulphate

(FeSO4). The iron (Fe) map revealed iron-rich regions that localize the iron aggregates,

probably most of them probably extracellular. In addition, it seemed there was a diffuse

intracellular iron increase that could derive from Fe uptake of the cell.

Heaton SJ et al. [35], performed in vitro study by using placenta BeWo cells to

demonstrate that they are a valuable cell model for the study of iron transport in the

placenta. Their results suggested that free iron is likely to undergo paracellular diffusion

across the BeWo cell layer readily, while Fe complexed to transferrin protein (Fe-Tf) is

transported across the cell layer predominantly transcellular route.

Figure 21 shows the elemental maps of placental cells exposed to crocidolite. The iron

(Fe) map revealed iron-rich segments that localize the crocidolite fibres: those of small

dimension seemed to be intracellular, as perceived by comparison with phase contrast

(b) absorption (c) images. But they could be also attached onto cell surface. In addition,

the logarithmic scale of the iron map allowed revealing a diffuse intracellular iron

increase that could derive both from iron dissolution of fibres and by an increased Fe

uptake of the cell. Compared to control cells, cells exposed to asbestos showed an

increased Ca presence. This element tends to co-localize on extracellular parts of the

fibres, suggesting that the material attracts ions present in the culture media.

The phase contrast and absorption images (b, c) clearly indicate the different density

between nucleoli and cytoplasm. The technique allowed to precisely reveal all the fibres

which were internalized, in fact most of them were not observable from optical image

(a). In addition, it was possible to investigate the alteration of morphological aspects of

cellular surface revealing in detail the presence of vesicles which were localized in the

cytoplasmic compartment.

84

Figure 21. µXRF and X-ray microscopy of treated cells with crocidolite. Visible light image of a

group of cells exposed to crocidolite fibers (a) and the corresponding P, S, Ca, Fe XRF maps (129 µm x

136 µm) showing the distribution of different elements. Fe map is displayed using a logarithmic scale. On

the right side are showed the X-ray microscopy phase contrast (b) absorption (c) images of the

corresponding XRF maps. The absorption and phase contrast images were measured at 0.9 KeV photon

energy, whereas the XRF maps were acquired at 7.2 keV.

However, in BeWo cells there were some difficulties in interpreting the results due to

their morphology. Frequently, the syncytial form of the trophoblast-derived BeWo cells

did not allowed to easily localize the nanomaterials, especially CNTs. Therefore, we

performed AFM analysis in combination with X-ray fluorescence microscopy to better

monitor cell-nanomaterial interactions and particularly try to discriminate intracellular

from extracellular localizations.

In Figure 22 are reported XRF maps and AFM images (b,c) of treated cells with

crocidolite fibres. In particular, the bidimensional map of P/Fe colocalization was

analyzed in association with AFM images. The combination of X-ray fluorescence with

the AFM microscopy allowed to discriminate the cells that phagocytized the fibres, and

the others which were surely outside the cells, as confirmed from visible image (a).

Looking calcium (Ca) map it was observed an calcium deposition on external fibres,

85

while iron map seemed to confirm that there was an intracellular iron increase as well as

a dissociation of iron from crocidolite fibres. Again, the combination of XRF and AFM

provided a detailed morphological description about the interaction of crocidolite fibres

with the placental cells and allowed to discriminate between intra- and extracellular

asbestos fibres and to identify their interaction of with specific cellular compartments.

Figure 22. µXRF and AFM images of treated cells with crocidolite. Visible light image of a group of

cells exposed to crocidolite fibers (a) and the corresponding P, S, Ca, Fe XRF maps (80 µm x 56,5 µm)

showing the distribution of different elements. Fe map is displayed using a logarithmic scale. Below are

showed P/Fe XRF map and the corresponding AFM images (b,c).

86

Figure 23. µXRF and X-ray microscopy of treated cells with r-SWCNTs. P, S, Ca, Fe XRF maps

(152 µm x 97 µm) image of cell exposed to raw carbon nanotubes (r-SWCNT). Visible light image (a),

the X-ray microscopy absorption (b) and phase contrast (c) images of the corresponding cells are also

reported below. The absorption and phase contrast images were measured at 0.9 KeV photon energy,

whereas the XRF maps were acquired at 7.2 keV.

The elemental maps of cells exposed to raw-SWCNTs are displayed in Figure 23.

Merging with the cellular shape (P and sulphur S maps) the iron (Fe) map revealed

several iron-rich spots inside or surrounding the cells. Bases on their dimensions and

intensities, these iron-spots could be attributed to iron containing nanotubes or to

residual iron particles detached from them [16]. The X-ray absorption and contrast

images (b and c) revealed that raw carbon nanotubes were not easily visible. But from

comparison with iron map (Fe) and visible image (a) it was possible distinguish r-

SWCNTs deposited on the cell surface, which they correspond to more intense spots in

the X-ray absorption image (b). The contrast image (c) showed some spots that were

thicker than cellular material and may correspond to small raw-SWCNTs or iron

particles released from them. We supposed that some of these iron nanoparticles were

internalized from cells.

87

Figure 24. µXRF and AFM images of treated cells with p-SWCNTs. Visible light image of a group of

cells exposed to purified carbon nanotubes (p-SWCNT) (a) and the corresponding P, S, Ca, Fe XRF

maps (96 µm x 131 µm) images are reported, showing the distribution of different elements. Fe map is

displayed using a logarithmic scale. XRF maps were acquired at at 7.2 keV excitation energy. Below are

showed P/Fe XRF map and the corresponding AFM images (b,c).

Figure 24 shows the elemental maps and the corresponding AFM images of cells

exposed to purified carbon nanotubes (p-SWCNTs). The combination of AFM and the

overlapping signals of P and Fe (in green in the P/Fe panel) suggested the presence of

88

small vesicles with CNTs internalized. Further, the P/Fe overlapping signal in green

allowed to localize small SWCNTS or bundle of them. It was clearly difficult to

discriminate CNTs that were outside cells from those inside, also when combining

AFM. Most of CNTs seem to remain outside of the cells, and depositing onto the

surface. It was interesting that it may be perceived that there was a sort of release of

iron traces from nanotubes.

Figure 25. µXRF and X-ray microscopy of treated cells with hp-SWCNTs. P, S, Ca, Fe XRF maps

(128 µm x 112 µm) image of cell exposed to highly purified carbon nanotubes (hp-SWCNTs). Visible

light image (a), the X-ray microscopy absorption (b) and phase contrast (c) images of the corresponding

cells are also reported below. The absorption and phase contrast images were measured at 0.9 KeV

photon energy, whereas the XRF maps were acquired at 7.2 keV.

Figure 25 shows the XRF maps and absorption (b) and phase contrast (c) images of

cells exposed to highly purified carbon nanotubes (hp-SWCNT). Phosphorus (P) and

sulphur (S) maps identified the cellular shape, while the iron (Fe) map revealed a few

iron-rich spots inside the cell. Looking at the iron (Fe) map, it was interesting to note

that there was an inhomogeneous increase of its intracellular presence and iron-rich

spots could overlap with hp-SWCNTs that deposited onto the cell’s surface. The

89

marginal iron alteration observed in this sample is surely due to the higher purity grade

of hp-SWCNTs (5 wt % iron).

2.3.3 Results of ferritin assay

The results demonstrated that the ferritin was increased in the cell exposed to crocidolite

fibres and iron sulphate (FeSO4) compared to control cells (as shown in Figure 26).

Cells treated with r-SWCNTs had a lower ferritin stimulation than crocidolite fibres.

The cells treated with p-SWCNTs had a lower stimulation than cells treated with r-

SWCNTs, while cells treated with hp-SWCNTs showed a similar ferritin content than

control cells (as shown in Figure 26). The results showed that the amount of

intracellular ferritin of BeWo cells treated with different NMs was lower than that of

treated mesothelial cells (MeT5A) (Figure 18).

Figure 26. Intracellular concentrations of ferritin after in vitro exposure of placental cells to different

nanomaterials. *Significantly increased is found in samples treated with crocidolite and iron sulphate, and

also raw- and purified-SWCNTs.

90

2.4 Conclusions and comparison between pleura and placenta cell responses

We have demonstrated that the combination of advanced synchrotron-based X-ray

microscopy and fluorescence techniques (µXRM and XRF) are promising to study the

toxic mechanisms induced by asbestos and carbon nanotubes in the pleural and the

placental cell lines. The results obtained by simultaneous structural and elemental

analysis showed that there was an increase of iron, which was also confirmed by the

stimulation of ferritin, after the exposure to nanomaterial. The presence of iron

impurities in the nanomaterials seemed to be the main cause of toxic effects that result

in an alteration of iron metabolism.

The cells treated with raw-SWCNTs and crocidolite fibres compared to the control

showed a severe alteration of iron metabolism, which was maximal in the pleural cells

(MeT5A) and was clearly related to the presence of iron into the fibre. Highly purified

nanotubes, in fact, did not altered iron metabolism. X-ray absorption and phase contrast

images confirmed that the toxicity of nanomaterials was characterized by membrane

damage with vesicle secretion and filipodia formation.

The placenta barrier showed a difference in the cell response and alteration of iron

metabolism. In fact, while X-ray fluorescence analyses clearly revealed a homogeneous

intracellular iron increase in pleural cells after fibre exposure, the same technique

showed that the iron changes inside placental cells were restricted to small intracellular

regions just in contact with nanofiber or nanotube. This observation was confirmed by

AFM analysis that allowed to clarify the cell-nanofiber interactions as well as the cell

morphology.

In relation to this toxic mechanism quite complex and still unknown, the evaluation of

intracellular ferritin demonstrated that crocidolite and raw carbon nanotubes gave a

severe stimulation of this protein in both cell models, while highly purified carbon

nanotubes gave values comparable to control. The ferritin stimulation was clearly lower

in placental cells, and clearly linked to a different uptake of fibres in these cells,

suggesting that this barrier is less vulnerable than the pleura.

91

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CHAPTER 3 - A “NANOTOXICOGENOMIC”

STUDY: THE ROLE OF BAP1 IN

MESOTHELIOMA DEVELOPMENTa

a) A manuscript is under preparation: “Puzzling results from BAP1 germline mutations

in a small group of asbestos exposed patients in Northeast Italy”. Cammisuli F.,

Athanasakis E., Rizzardi C., Dal Monego S., Costantinides F., Bassan F.,Licastro D.,

Canzonieri V., Melato M., Pascolo L.

A new route to understand the link between genetic variation and susceptibility to

nanomaterials is the so-called “nanotoxicogenomics”. The harmful effects arising from

exposures to nanomaterials can be different for the same dose, depending on the

genotype and genetic predisposition of an individual.

Asbestos is the main risk factor in the etiology of malignant mesothelioma (MM), while

CNTs may represent a risk factor for the future. Typically, asbestos exposure induces a

chronic inflammatory response in response to biopersistent fibres that may lead to

malignant cell transformation after a 15-40 year latency period following initial fibre

exposure [1]. However, only a small proportion of people exposed to asbestos develop

mesothelioma, suggesting that the genetic susceptibility could play an important role in

its development.

Although the commercialization and industrial use of asbestos have been limited since

1990 and it is almost abolished today, the long latency of the pathology will ensure that

the incidence will peak between the years 2010 and 2020. Epidemiological studies have

estimated that about 86-95% of mesothelioma cases are related to demonstrable

asbestos exposure [2-3].

In addition to asbestos inhalation and inflammatory response, the suggestion that

genetic factors are implicated in the etiology of malignant mesothelioma has been put

forward since several years [5-6]. The correlation has been proved by the association of

BAP1 (BRCA1-associated protein 1) germline mutations with the development of

mesothelioma. A study in three villages in Cappadocia (Turkey) showed a clear

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association among certain families, revealing a genetic susceptibility to development of

mesothelioma that occurs probably in an autosomal dominant way [7].

The BAP1 gene is a tumor-suppressor gene that is located on the short arm of

chromosome 3 (3p21) (Figure 27) and belongs to the ubiquitin C-terminal hydrolase

subfamily of deubiquitinating enzymes, which are involved in the removal of ubiquitin

from proteins. In addition, BAP1 is also involved in regulation of gene transcription,

regulation of cell cycle and growth, response to DNA damage and modulation of

chromatin (epigenetic mechanisms) [8].

Figure 27. Schematic representation of BAP1 domains. Human BAP1 is 729 amino acids (90 kDa).

The amino-terminal region contains ubiquitin C-terminal hydrolases (UCH) domain. BAP1 binds to the

RING finger domain of BRCA1 through its carboxyl-terminal region (594-657 amino acids). Domain

comprised by residues 182-365 of BAP1 interacts with the RING finger domain of BARD1. UCH

domain; BARD1 binding domain; HBM binding domain; BRCA1 binding domain; NLS, nuclear

localization signal. BAP1 interacts with HCF-1 (host cell factor 1) through HCF-binding motif (HBM).

The C-terminal region contains a putative nuclear localization signal.

Germline BAP1 mutations are found in families where there is a high incidence of

malignant tumours, which are often developed in an earlier age with respect to the

general population [9]. A “BAP1 cancer syndrome” has been proposed, which includes

mesothelioma, uveal melanoma, cutaneous melanoma and possibly other malignant

tumours [10].

A further study showed a patient with the BAP1 hereditary cancer predisposition

syndrome. The patient, a 72-year-old woman diagnosed with uveal melanoma,

peritoneal mesothelioma and a primary biliary tract adenocarcinoma [11]. She had a

family history of mesothelioma as well as other malignancies including renal cell

carcinoma. This study revealed a germline BAP1 missense mutation (p.Tyr173Cys) that

alters the active site of the ubiquitin hydrolase domain and it is predicted to generate a

non-functional full-length protein [11]. Other studies reported similar germline BAP1

missense mutation in pleural biphasic mesothelioma (p.Tyr173Cys) [11-12] and uveal

melanoma (p.Ser172Arg) [11,13].

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A recent study showed a novel germline BAP1 nonsense mutation (c.1777C>T) which

produces a truncated BAP1 protein product and segregates with cancer [14]. The patient

had multiple cancers and a family history of melanocytic tumors and cutaneous

melanomas in early age and with autosomal dominant inheritance. In addition, the

proband with the same BAP1 germline mutation developed other tumors including

thyroid cancer [14].

Another paper revealed a homozygous substitution of BAP1 gene (c.2054A>T;

p.Glu685Val) in an MPM cell line derived from a mesothelioma patient [15]. These

results demonstrated that aberrant splicing caused by this mutation creates a novel 5'

splice site and activation of cryptic splice sites, contributing to MPM through disruption

of normal splicing [15].

In addition, somatic BAP1 mutations seem also to contribute to the growth and

aggressiveness of malignant mesothelioma [16].

In the present study, we investigated the possible presence of a genetic predisposition to

develop mesothelioma, as BAP1 gene mutation, in a well-characterized court of

asbestos-exposed subjects who developed mesothelioma. The Northeastern of Italy

(around the cities of Trieste and Gorizia) is one of Italy's areas where occupational

exposure to asbestos occurred in the past and caused an excess incidence of malignant

mesothelioma among the people [4].We examined a total of 29 patients from this

asbestos-related disease endemic area, revealing a total of two new Bap1 mutations.

3.1 Material and Methods

3.1.1 Patients and tissue samples

Tissues samples were obtained from twenty-nine autoptic cases randomly selected from

the archives of the Institute of Forensic Medicine of the University of Trieste. All the

cases were subjected to legal medical advice to establish the cause of death and a

possible correlation to a professional exposure to asbestos and/or to the presence of

asbestos-related diseases. All the patients were residents of Trieste, a ship-building town

in North-Eastern Italy with a high incidence of mesothelioma among workers and in

general population.

In Table B1 of Appendix B are reported the clinical features of 29 patients. They are

sex, age of diagnosis and death, survival time, cancer and concurrent diseases,

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malignant mesothelioma histotype, and treatment including surgery, chemotherapy, and

radiation, asbestos exposure circumstances, occupation and asbestos bodies count.

The 21 cases of mesothelioma (20 pleural and 1 peritoneal) were histologically

diagnosed either during their clinical course or at post-mortem examination, according

to standard histological and immunohistochemical criteria, and classified according to

the WHO classification of pleural tumors [17]. Among these, 7 cases were epithelioid, 2

sarcomatoid and 11 biphasic, while the peritoneal mesothelioma was the epithelioid

histological type (Table B1, in Appendix B).

In addition, the causes of death included cell carcinoma (2 cases) and adenocarcinoma

(1 case) of the lung, and squamous cell carcinoma of the oral cavity metastasized to the

lungs in 1 case (Table B1, in Appendix B).

3.1.2 Nucleic Acid Extraction and PCR Amplification

Genomic DNA of 29 patients was extracted from frozen tissues using QIAamp DNA

Mini Kit (Qiagen, Hilden, Germany) according to the manufacture’s protocol. PCR

amplification of 17 BAP1 exons and of all their exon-intron boundaries (NCBI

accession number: NM_004656.3) was performed as previously described by Bortot et

al. [18]. Briefly, primer designs was performed using the Primer3web v4.0.0 on-line

software [19-20] (Table 3). All amplifications was carried out using the KAPA2G Fast

ReadyMix (Kapa Biosystems, Cape Town, South Africa) in a 96-well PCR plate and

sharing a common annealing temperature in a two-cycle step touchdown protocol: first,

an initial denaturation step at 96°C for 3 minutes was followed by a touchdown step

planning to decrease the temperature by 0.5°C/cycle, through 10 cycles: 95°C for 15

seconds, 62°C for 15 seconds, and 72°C for 1 second; the second endpoint-PCR step

through 30 cycles was performed as follows: 95°C for 10 seconds, 59°C for 10 seconds,

and 72°C for 1 second.

Total RNA was isolated from frozen tissues using the EuroGOLD Total RNA Kit

(EuroClone, Milan, Italy). Reverse transcription (RT) reaction was performed using the

GoScript Reverse Transcription System (Promega, Madison, WI, USA) according to the

manufacture’s protocol. RT was performed in a reaction volume of 5 µl containing 5 µg

of total RNA and 15 pM of each sequence-specific primer (Table 3). Primers were

designed using the on-line NCBI Primer-BLAST tool [21] and their specificity was

checked on the Refseq mRNA database. Finally, cDNA PCR amplification was

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conducted according to the above describe touchdown protocol using the same

sequence-specific primers of each RT reaction.

3.1.3 Sanger Sequencing and Mutation Data Handling

PCR products were purified using Illustra ExoStar 1-Step kit (GE Healthcare, UK)

according to the manufacturer’s protocol. Sequencing of the PCR fragments was carried

out using the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies, Foster

City, CA, USA) following the manufacturer’s instructions. Automated electrophoreses

were performed on an ABI 3500Dx Genetic Analyzer and the sequencing results were

analyzed using the SeqScape v2.7 software (Life Technologies, Foster City, CA, USA).

All identified variants were annotated using a custom bioinformatics pipeline basic on

Annovar software [22] and referring to several databases as reported in the Table 3. To

detect alterations in exon-intron boundary regions and splicing motifs due to nucleotide

changes, the bioinformatics tools for splice site prediction HSF [23], NNSplice [24],

NetGene [25] and SPANR [26] were used. All selected variants were reconfirmed either

on genomic DNA or, in case of splicing site mutations, on cDNA by Sanger sequencing.

An in silico mutation analysis was performed for the non-synonymous selected

mutations to predict if the aminoacid substitution could involve a structural

modification of BAP1 protein. Phyre2 v2.0 [27], a web-based resource for template-

based modeling, was used. Wild type aminoacid sequence (NCBI accession number:

NP_004647.1) was submitted through the intensive method in order to obtain a PDB

structure. The outcome wild type protein structure presented a structural homology at

100% of confidence with the ubiquitin carboxyl-terminal hydrolase isozyme l5 (PDB

ID: 3IHR) [28] from the 5 to the 238 amino acids of query sequence. The modeling of

the remain amino acid sequence was predicted. In a second attempt, the mutated

aminoacid sequence was aligned using the same tool on the wild type protein obtained

previously. Analyses of all docked poses were performed using the molecular

visualization software UCSF-Chimera v1.10.2 [29].

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Exon number Forward 5‟ to 3‟ Reverse 5‟ to 3‟

1 gttcgccttcgagcgcatg cacgagcagggtgaagaggc

2 and 3 gaataagggctggctggagctg gccctgttctctgggaccttc

4 cacagcaaggacacctgagtgatg cttcctccatttccacttcccaagc

5 gttgtccagatatgactgacctgctc catgtggtagcattcccagtgg

6 and 7 cgtctgtgttccttccgattcctg gctggtcgggcaatatggtgtag

8 ctacaccatattgcccgaccagc cccatgatctaagcctgatcttgcc

9 tgccaggatatctgcctcaacct gctgaagcccagatctacaagagagt

10 gaatgggtagagccaaggcc agactttccctgtttaggcctccc

11 gcttgctgactcccattgcac accacatgggaaaattgcctgttg

12 gactcagtctggaaaaccatgttggc aggtgctcaacattatctgctgca

13 gtcgggatgtatttaagccattctgggt tgcaggacactttgtggtcacttg

14 gtgatctgggtcctgtcatcagc aggcaaggatgagcagcgagtc

15 and 16 ctcgctgctcatccttgcct caaggtctgctcaagcctcagga

17 tcctgaggcttgagcagaccttg agggcacgatggaaggaatgtg

Exons and cDNA start-

end position

Forward 5‟ to 3‟ Reverse 5‟ to 3‟

Exons 9-11, 715 - 941 atcaagtatgaggccaggctg tctgcaccatctgtgtggttg

Exons 10-12, 921- 1127 cgctggtgctggaagcaaac tcttcttcctcctgcatggg

Exons 13-14, 1628 -

1845

agcctgctgcgtgttgactg catccccgtcttctctctgctgtc

Table 3. List of primers sequences used for Sanger sequencing of DNA (BAP1, NG_031859.1) and

cDNA (BAP1 NM_004656.3, CCDS2853.1) target regions.

3.1.4 Multiplex Ligation-Dependent Probe Amplification

The BAP1 gene were analyzed by MLPA [30] using the SALSA MLPA P471-BAP1 -

LOT1011 (B1) probemix (MRC Holland, Amsterdam, the Netherlands). MLPA

reactions were performed starting from 75 ng of genomic DNA according to the

101

manufacturer’s instructions. The products were separated by capillary electrophoresis

on an ABI 3130XL Genetic Analyzer and controlled using the Gene Mapper v4.0

software (Life Technologies, Foster City, CA, USA). Copy number variations were

predicted in each patient using the Coffalyzer.Net v140721 software (MRC Holland,

Amsterdam, the Netherlands).

3.2 Results

Sanger sequencing of BAP1 gene in 29 patients identified one non-synonymous variant

and two intronic variants. While Sanger sequencing of cDNA revealed no alternative

splicing due to the nucleotide change for each mutations. MLPA analysis revealed no

significant copy number variations at exon level in all patient tumor samples.

Figure 28. Germline BAP1 missense mutation found in patient 9. Electropherogram shows BAP1

missense mutation (c.1028T>C; p.L343P) within exon 11.

In detail, patient 9 carried in heterozygous a missense variant (c.T1028C; p.L343P) at

exon 11 (Figure 28). Mutation pathogenicity prediction web tools reported this variant

as possible benign. Sanger sequencing of cDNA revealed no alternative splicing due to

the nucleotide change. In silico mutation analysis was performed in a predicted protein

structure of BAP1 protein without any significant possible effect of the amino acid

change (Figure 29).

102

Figure 29. In silico mutation analysis. The predicted protein structure of BAP1 protein without any

significant possible effect of the amino acid change. A web-based resource for template-based modeling,

Phyre2 v2.0 is used. Protein structures of wild type aminoacid sequence (NCBI accession number:

NP_004647.1) and of the mutated aminoacid sequence are displayed in figure. The outcome wild type

protein structure present a structural homology at 100% of confidence with the ubiquitin carboxyl-

terminal hydrolase isozyme l5 (PDB ID: 3IHR) from the 5 to the 238 amino acids of query sequence. The

modeling of the remain amino acid sequence is predicted. Analyses of all docked poses are performed

using the molecular visualization software UCSF-Chimera v1.10.2. The yellow star is referred to BAP1

missense mutation, also highlighted in yellow in mutated protein. The in silico analysis was performed by

Simeone Dal Monego (CBM, Trieste).

Patient 4 carried in heterozygous a known intronic variation 8 bases downstream of the

exon 13 (c.1729+8T>C (IVS 13)) (Figure 30). This variant was already described as

SNP (rs150945583) with a minor allele frequency between 0.0030 and 0.0055.

Alternative splicing prediction web tools gave as possible alteration of intronic splicing

site. Sanger sequencing of cDNA revealed no alternative splicing due to the nucleotide

change.

103

Figure 30. Germline BAP1 mutation found in patient 4. Electropherogram shows BAP1 mutation

(c.1729+8T>C) within intron region at 8 bases downstream of the exon 13.

Finally, in patient 15 a heterozygous intronic variation was identified, 8 bases upstream

of the exon 10 (c.784-8G>A (IVS9)) (Figure 31). Alternative splicing prediction web

tools gave contradictory results. Sanger sequencing of cDNA no alternative splicing due

to the nucleotide change.

Figure 31. Germline BAP1 mutation found in patient 15. Electropherogram shows BAP1 mutation

(c.784-8G>A) within intron region at 8 bases upstream of the exon 10.

3.3 Discussion and conclusion

Despite the relatively small number of cases investigated, our study gave puzzling

results on the effects of Bap1 mutation and the concomitant exposure to asbestos.

In total we revealed three mutations for Bap1, two of them in patients with

mesothelioma and one in a patient with only fibrosis.

104

As reported in literature, the tumors with germline BAP1 mutations are much less

aggressive than ones with somatic mutations. It has been reported that the individuals

with germline mutation have a survival time 5 times longer than others. [31].

It was interesting to note that our study reports the case of patient nine, having

mesothelioma, presenting a germline BAP1 missense mutation (c.T1028C; p.L343P)

and with unusual survival time (Table 4). This patient had a story of heavy occupational

exposure and presented the pleural epithelioid mesothelioma in association with diffuse

pleural fibrosis and desmoid-type fibromatosis. The patient underwent a multimodality

therapy and died 3.5 years after the diagnosis from advanced stage mesothelioma with

pulmonary, pericardial, mediastinal, diaphragmatic, peritoneal and osseous metastases.

The survival time of this patient with malignant mesothelioma is clearly unusually long

compared to the median survival time, typically, around 6-12 months.

Table 4. The detailed characteristics of three patients (four, nine and fifteen) with germline BAP1

mutations. In table are displayed gender, age of tumor diagnosis, age of death, cancer and other diseases,

asbestos exposure circumstances, occupation and asbestos bodies count for each patient.

Differently, the survival of patient fifteen, also with pleural mesothelioma, was about 1

year. In this case, the patient had a high content of asbestos bodies (Table 4) and

presented intronic germline BAP1 mutation (c.784-8G>A (IVS9)). It seemed that this

mutation does not affects survival.

A very interesting case was that of patient four, who had pulmonary fibrosis and died

following respiratory failure. His asbestos exposure history was not well documented

and the asbestos bodies count in the lung is quite low, close to environmental exposure.

105

Moreover, the autopsy of this patient with intronic germline BAP1 mutation

(c.1729+8T>C (IVS13)) revealed the presence of renal cell carcinoma.

Renal cell carcinoma was recently associated with BAP1 cancer syndrome. Particularly,

sporadic BAP1 mutations seems to have been identified in a small proportion of renal

cell carcinoma [32], while other renal cell carcinoma can be referred to germline

mutations [33-34].

This means that the mutation, although intronic and considered non dangerous by

computational analyses, clearly was associated to a tumor. Moreover, the patient was

not exposed to high quantities of asbestos, but developed an idiopathic fibrosis, strongly

suggesting a high susceptibility to inflammatory insults in lung.

In conclusion, our findings explained three cases of germline BAP1 missense mutations

in individuals with tumors related to BAP1 cancer syndrome and also exposed to

asbestos. Although the combination of different molecular analyses supported that

BAP1 variants were not a pathogenic mutations, two mesotheliomas and a renal cancer

were associated to mutations. Moreover, in agreement with literature, one patient (nine)

presented significantly increased survival time of three-year and half.

106

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F, Roberts I, Shanahan SJ, Claas A, Dunham A, May AP, Rosenfeld N, Forshew T,

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KDM6A, and JARID1c in clear cell renal cell carcinoma. Genes Chromosomes

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110

CHAPTER 4 - UV RESONANT RAMAN

SPECTROSCOPY FOR INVESTIGATION OF

OXIDATIVE DNA DAMAGE INDUCED BY

CARBON NANOTUBESa

a) The results shown in this chapter are already published: D'Amico F, Cammisuli F,

Addobbati R, Rizzardi C, Gessini A, Masciovecchio C, Rossi B, Pascolo L. Oxidative damage in

DNA bases revealed by UV resonant Raman spectroscopy. Analyst. 2015 Mar 7;140(5):1477-85

In this chapter, I will report the molecular study that allowed testing the feasibility of

UV resonant Raman spectroscopy to reveal oxidative DNA damage after nanomaterial

exposure [1].

Every day DNA molecules are affected by the normal metabolic processes and

environmental factors that can cause damage to its structure [2]. Since an individual cell

can suffer up to one million DNA changes per day, the cells have developed a number

of mechanisms able to detect and repair the different types of damage that can occur in

DNA [3]. But failures of DNA repair mechanisms can lead to accumulation of

mutations and damage.

One of the major mechanisms associated with DNA damage is attributed to generation

of reactive oxygen species (i.e., superoxide, hydrogen peroxide, and hydroxyl radicals)

[4]. ROS are produced in cells during normal metabolic processes involving oxygen and

they have an important catalytic role. The presence of excess ROS may result from an

imbalance between the ROS generation and the inability of a biological system to

readily detoxify all the reactive species. These last can thus attack and damage cellular

components like proteins, lipids and nucleic acids DNA (i.e., oxidation of DNA

nucleobases, de-purination and DNA strand breaks) [5].

The production of ROS may be significantly increased by exposure to different

environmental contaminants derived from industry, agriculture, air pollution, or tobacco

smoke [6].

Oxidative stress induced by nanomaterials is due to their physical and chemical

properties as well as presence of metal impurities in their composition, while cellular

mechanisms such as mitochondrial respiration, nanomaterial-cell interaction, and

111

activation of immune system are responsible for ROS-mediated damage [7]. When

nanomaterials interact with cells trigger oxidative stress events leading to others

harmful effects such as genotoxicity, inflammation, and fibrosis [8].

The transition metals that are present on nanomaterial surface can generate reactive

oxygen species through the Fenton’s reaction [6].

In this study, an oxidative environment was created in vitro by using carbon nanotubes

(raw-SWCNTs), containing metal impurities (30% of iron), and free radicals OH•

(derived from H2O2). In this condition the elements of DNA, in particular the

nucleotides (dATP, dCTP, dGTP and dTTP), result in increased susceptibility to

oxidative damage.

UV Raman spectroscopy is shown to be maximally efficient to reveal changes in the

nitrogenous bases during this oxidative damage occurring on these molecules.

This chapter is divided in two sections. In the first I will describe the experimental

methods used in this study, and in the second I will discuss the results obtained.

4.1 Material and Methods

4.1.1 UV resonant Raman spectroscopy

Raman spectroscopy has been extensively used in the past few years to characterize the

chemical structure of DNA [7-8] and it has recently been applied to reveal changes in

the DNA structures (i.e., chain breaks and oxidative damage) [9-10].

However, the biggest difficulty in interpreting the Raman spectra of biological

macromolecules is the great number of overlapping vibration modes. Furthermore, in

the specific case of DNA study, the analysis of Raman spectra is complicated by the

presence of interfering fluorescence backgrounds due to the use of visible light as

excitation probe.

Nonetheless, the use of ultraviolet (UV) excitation to obtain Raman spectra has several

important advantages: (i) it allows to obtain spectra without any intense fluorescence

background, (ii) it permits to select the vibrations coming from the DNA bases,

matching the absorption band due to the aromatic rings centered at 260 nm. In this way

it is possible to avoid the overlapping of the bands coming from deoxyribose and

phosphate in the same spectrum [11] and focus the attention on how the oxidation

occurs at the level of nitrogenous bases [12].

112

The study was carried out by using the recently developed UV Raman set-up at the

IUVS beamline of Elettra synchrotron (Italy) [13].

It is an optical set-up able to collect UV resonant Raman (UVRR) spectra from liquid

and solid samples. The scheme of UVRR set-up is reported in Figure 32.

Figure 32. Scheme of the developed UVRR scattering set-up. The SR provided by the Elettra

synchrotron is monochromatized by a CT monochromator Czerny-Turner monochromator. Downstream,

the SR light is focused on the sample using either (I) a plano-convex lens, (II) an off-axis parabolic

mirroror (III) a microscope objective lens, depending on the experimental requirements. The radiation

scattered by the sample is collected in a backscattering geometry by the same focusing element and

focused by a lens into the three-stages Czerny-Turner analyzer (cases (I) and (III)), while in the case (II)

the Raman scattering is collected and focused into the analyzer by two off-axis parabolic mirrors.

SOURCES: D’Amico F. et al. in Nuclear Instruments and Methods in Physics Research [13].

Excitation sources at 532 nm and 266 nm were used with the solutions placed in

standard quartz cuvettes. Samples were continuously agitated during the measurements

to avoid local decomposition due to UV exposure. Spectra were collected in a

backscattering configuration employing an f =750 mm Czerny-Turner spectrometer,

equipped with an holographic reflection grating of 3600 grooves mm−1

and a nitrogen-

cooled back-thinned CCD [1].

At the same time, accurate computational simulations were obtained by using a hybrid

density functional theory (Gaussian-03 software), which allowed us to recognize the

contribution of specific oxidized products in the experimental spectra [1].

113

4.1.2 Computational simulations: Gaussian-03 software

Quantum mechanical simulations have been carried out to obtain the vibrational

frequencies and the Raman scattering activities of the molecules considered in the

present work [1].

We used the hybrid DFT model proposed by Becke and co-authors (B3LYP) [14,15],

where the exchange–correlation function used is the three-parameter Lee–Yang–Parr

function [16]. In addition, we used the basis-set of orbital functions developed by Pople

and co-authors that involves the use of spatially diffused p-(hydrogen atoms) and d-

polarized (carbon, nitrogen and oxygen atoms) functions [17,18]. It has been

demonstrated [19,20] that the choice of these parameters gives optimal results in

predicting the vibrational spectra of aromatic organic molecules against the moderate

use of computational resources.

The simulated spectra were obtained from the sum of different Gaussians, one for each

vibrational mode derived from the simulations, centred at the frequencies νi of the

normal modes with area Ai proportional to

where Ri is the Raman scattering activity derived from quantum simulations and ν0 is

the frequency of the excitation source [21,22]. The full width half maximum were

selected to be 20 cm−1

and 45 cm−1

for the C=O stretching peak, to better fit the

experimental lineshape.

4.1.3 Oxidative DNA damage

Nucleotide and DNA aqueous solutions were exposed to hydrogen peroxide (H2O2) and

iron containing carbon nanotubes (CNTs) to produce Fenton’s reaction and induce

oxidative damage [1].

Commercial dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),

dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate) solution

(100 mM) were diluted to 10 mM in MilliQ water. Plasmid circular DNA (pUC19

vector) was amplified in bacterial cells (DH5α), then purified with the QIAGEN

114

Plasmid Midi Kit and diluted to a final concentration of 0.5 μg/mL. Aliquots of single

nucleotide and plasmid DNA samples were incubated in ice for 3 hours with iron

containing 5 μg/mL raw single-walled carbon nanotubes (raw-SWCNTs) and 1% H2O2,

in order to produce oxidation from Fenton’s reaction (Figure 33). The incubation was

stopped by removing the carbon nanotubes through a centrifugation step, carried out at

7500 x g for 10 minutes. The samples were then placed in standard quartz cuvettes and

immediately measured by Raman spectroscopy.

Figure 33. The metallic iron nanoparticle impurities, which remain in carbon nanotubes from their

synthesis, are exposed to hydrogen peroxide and cause the iron-mediated production of hydroxyl free

radicals via the Fenton’s reaction.

4.2 Results and discussion

4.2.1 dNTPs Raman analysis before Fenton‟s reaction

Figure 34, panels (a) and (b) respectively, shows the Raman spectra of nucleotides in

aqueous solutions acquired by using 266 and 532 nm excitation wavelengths. Figure 34

(b) also shows the spectrum of pure water for a qualitative comparison with the

nucleotide spectra.

All the spectra recorded at 532 nm exhibited a common feature at 1120 cm−1

, which

could be assigned to the stretching vibration of the three-phosphate group [23,24] and

the characteristic O–H bending mode of water centred at ≈1630 cm−1

.

115

Both these features were almost absent in the Ultraviolet Resonant Raman spectra

(UVRR) collected at 266 nm. The absence of the phosphate signature in the UVRR

spectra (200–300 nm range) [8,11] allowed better recognition of the vibrational modes

associated with nitrogenous bases.

Figure 34. Panel (a): Resonant Raman spectra of nucleotides (shown on the right side) aqueous solutions

collected at 266 nm incident wavelength. Spectra are vertically shifted for a better visualization. Panel

(b): Raman spectra of the same nucleotide solutions collected at 532 nm. The Raman spectrum of pure

water has been included for comparison. Spectra are vertically shifted for a better visualization. The

dotted line evidences the phosphate stretching band. SOURCE: D’amico F et al. in Analyst, 2015, 140,

1477 [1].

In order to provide a correct assignment of the experimental Raman features, the visible

and UV vibrational spectra of each nucleotide were compared with the theoretical

Raman activities obtained for the structure of the four nitrogenous bases (Figure 35).

116

Figure 35. Raman spectra of dATP (panel a), dGTP (panel b), dCTP (panel c) and dTTP (panel d)

collected at 266 and 532 nm of incident radiation, compared with the simulated spectrum of the

corresponding nitrogenous bases (simul.). SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

The spectra of dATP collected at 532 and 266 nm (panel (a) of Figure 35) presented the

same features, although the observed vibrational peaks exhibited different intensities in

the two spectra. The Raman spectrum obtained at 266 nm was dominated by three

intense vibrational features centred at 1336, 1481 and 1581 cm−1

, respectively. The

comparison of the experimental profile with the simulated spectrum of adenine (panel a,

lower spectrum) allowed us to assign the first 2 features to the overlapping C–C and C–

N stretching occurring within the aromatic rings. The third peak was associated with the

vibrational stretching modes of the aromatic rings and with many CH2 scissoring

vibrations. It was important to note that the signature at 1581 cm−1

was detectable only

117

in the UV spectrum, due to the absence (or marginal presence) of the O–H water band

contribution.

The Raman spectrum of dGTP, collected at 266 nm, was characterized by the

appearance of an intense and sharp peak centred at 1485 cm−1

that could be assigned to

the C–C and C–N stretching modes involving the aromatic rings, similar to what

observed in the dATP spectrum. The 266 nm-excited spectra matched with good

approximation to the simulated spectra. Another intense feature centred at 1575 cm−1

could be associated with the combination of C–C/C–N bending and CH2 scissoring

vibrations. The feature at 1678 cm−1

could be assigned to the C=O stretching mode. The

broadening of this component was larger than the others, and its frequency was

significantly lower than the simulated one. Both effects were well known and could be

addressed to the hydrophobic interactions of the carbonyl group with the surrounding

water molecules [13,25].

The 266 nm-excited spectrum of dCTP (Figure 35, panel c) exhibited two intense peaks

at 1250 and 1292 cm−1

which were assigned to the 5C–H and 6C–H bending modes,

while the intense peaks found at 1472 and 1528 cm−1

were associated with the aromatic

ring stretching modes. Finally, the feature centred at 1638 was the result of the

overlapping of C=O stretching and NH2 scissoring vibrations.

At wavenumbers below 1300 cm−1

, the dTTP (Figure 35, panel d) spectrum showed an

unexpected discrepancy between the experimental and simulated spectrum. Both visible

and UV Raman spectra exhibited multiple peaks that were clearly underestimated in the

intensity by the simulations. This may be due to the vibrations of the methyl group

added to the base in the simulation that affects the spectra in this wavenumber region. In

the region above 1300 cm−1

the simulations matched more closely the experimental

data.

In this spectral range, we identified the peak at 1374 cm−1

which was related to the

aromatic ring stretching modes, while the intense peak at 1653 cm−1

could be assigned

to the C=O stretching mode of both the carbonyls present in the structure and to the

aromatic ring stretching vibrations.

118

4.2.2 dNTPs Raman analysis after Fenton‟s reaction

Figure 36 displays the changes observed in the dATP solutions after oxidative damage.

Figure 36, panel (a) shows the spectra of the aqueous solution of dATP before and after

the Fenton’s oxidative reaction, while panel (b) displays the simulated spectra of

oxidized products: 2-hydroxyadenine, 8-hydroxyadenine, 8-oxoadenine and 4,6-

diamino-5-fomamidopyrimidine.

Figure 36. Panel (a): Raman spectra of dATP solution before (dATP) and after (dATP ox) the Fenton’s

oxidation process. Panel (b): Simulated spectra of 2-hydroxyadenine, 8-hydroxyadenine, 8-oxoadenine

and 4,6- diamino-5-formamidopyrimidine. The colored bars highlight the main vibrational features of the

spectra. SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

119

The comparison between the experimental spectrum of oxidized dATP and the

simulated spectrum of 2-hydroxyadenine demonstrated that they are very similar

spectra. A similar shape for the structures centred at 1326 cm−1

(highlighted in red in the

figure) was in fact found in both the spectra. These vibrational features were associated

with the C–C/C–N stretching modes of the aromatic rings. The small peak at 1417 cm−1

(shown in green) was attributed to a combination of stretching modes occurring in the

aromatic rings and it was found both in the control (although shifted by 3 cm−1

) and

oxidized ATP solution. But the main fingerprint came from the two structures

(highlighted in cyan) between 1472 and 1506 cm−1

, both thought to be caused by the 6-

NH2 scissoring vibrations combined with multiple internal stretching of the aromatic

rings. Finally, there was a good correspondence for the peaks at 1560 and 1610 cm−1

(highlighted in yellow), involved in the 6-NH2 scissoring modes. The last vibration was

downshifted by 29 cm−1

for the oxidized solution with respect to the same feature from

the non-oxidized one (1581 cm−1

). Although the 2-hydroxyadenine appeared to be the

dominant product, the presence of additional oxidation derivatives could be excluded.

Figure 37 displays the spectra of the solution of dGTP before and after the oxidative

stress (panel a), as well as the simulated spectra of 8-hydroxyguanine, 8-oxoguanine

and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (panel b).

The results demonstrated that there was a slight oxidative damage for guanine

molecules. In fact, it was to be noted the persistence of the peak at 1485 cm−1

in the

spectrum of the oxidized dGTP (highlighted in cyan). The most interesting feature of

the oxidized spectrum was the presence of an intense peak at 1607 cm−1

(highlighted in

red), which could be assigned to the overlapping of two types of vibrations involving

respectively C–C/C–H stretching within the aromatic ring and scissoring of the 2-NH2

atoms. This feature was clearly distinguishable in the simulated spectra for 8-

hydroxyguanine, in which these vibrational modes were more intense. Since there were

several bands that overlap above 1650 cm−1

and below 1400 cm−1

, it was very difficult

to assign the features coming from the different oxidized species. The smooth band

above 1600 cm−1

was probably due to 8-OH group bending modes of the 8-

hydroxyguanine 6C=O stretching vibration. Nevertheless, it was not possible to exclude

contributions in the spectra arising from 8-oxoguanine and 2,6-diamino-4-hydroxy-5-

formamidopyrimidine C=O stretching vibrations. The superposition of many vibrational

modes coming from the whole oxidized forms of guanine may also explain the broad

shape of the Raman spectrum below 1400 cm−1

.

120

Figure 37. Panel (a): Raman spectra of dGTP solution before (dGTP) and after (dGTP ox) the Fenton’s

oxidation process. Panel (b): Simulated spectra of 8-hydroxyguanine, 8-oxoguanine and 2,6-diamino-4-

hydroxy-5-formamidopyrimidine. The colored bars highlight the main vibrational features of the spectra.

SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

The oxidized product of dCTP generated with Fenton’s reaction was identified as the 5-

hydroxycytosine. This was confirmed by comparison between the simulated spectrum

(Figure 38, panel (b)) with the experimental spectrum of the damaged dCTP solution

(panel (a)). Specifically, the intense peak at 1257 cm−1

(highlighted in red) could be

assigned to the 5-OH group bending on 5-hydroxycytosine. This peak appeared to be

shifted towards lower wavenumbers of about 35 cm−1

with respect to the same

vibrational structure observed in the spectrum of pristine cytosine. The two low

intensity features at 1385 and 1444 cm−1

(highlighted in green) could be attributed to a

combination of 5-OH bending, 6C–H bending, and C–C/C–N stretching modes

121

occurring within the aromatic ring. Finally, the peak at 1547 cm−1

(highlighted in cyan)

could be assigned to 4-NH2 scissoring vibrations while the large band above 1600 cm−1

could be attributed to the overlapping of vibrational modes involving 4-NH2 scissoring,

6C–H bending and C–C/ C–N stretching modes of the aromatic ring.

Figure 38. Panel (a): Raman spectra of dCTP solution before (dCTP) and after (dCTP ox) the Fenton’s

oxidation process. Panel (b): Simulated spectra of 5-hydroxycytosine. The colored bars highlight the

main vibrational features of the spectra. SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

Despite a general oxidative damage in the other nucleotides, no changes were detected

in the dTTP’s Raman spectra before and after oxidation (Figure 39). This result

suggested that our experimental conditions did not produce chemical changes in this

nitrogen base when part of a triphosphate acid.

122

Figure 39. Raman spectra of dTTP solution before (dTTP) and after (dTTP ox) the Fenton’s oxidation

process. SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

4.2.3 Plasmid DNA Raman analysis

Figure 40, panel (a) shows the spectra of original (pDNA) and oxidized (pDNA ox)

plasmid DNA compared with the water spectrum. In contrast to what was seen with

nucleotides, the intensity of oxidized pDNA spectrum was at least three times lower

than that of control pDNA (see panel b of Figure 40). Moreover, the oxidation observed

in the plasmid DNA differed significantly from the one observed in nucleotide

solutions.

The comparison between the pDNA ox spectrum and the curve dNTP ox suggested an

almost total disappearance of the peak band centred at 1326 cm−1

, characteristic of

adenine and found also in the oxidized dATP solution spectra. Similarly, the vibrational

bands at 1479 and 1506 cm−1

, characteristics of the oxidation of adenine and guanine,

were damped in the spectra of oxidized plasmid DNA. These changes demonstrated that

the oxidation of adenine in the DNA was much more drastic than in the dATP solution,

causing the break of the aromatic ring at the 8C position with a probable consequent

production of derivatives like 4,6-diamino-5-fomamidopyrimidine. A comparable

reasoning could be applied to guanosine: although there was a clear fingerprinting for

the presence of 8-hydroxyguanosine (1610 cm−1

), the intensity of the peak was quite

low. This seemed to indicate that for this base additional degradation products were

formed such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine.

123

Figure 40. Panel (a): Raman spectra of pristine (pDNA) and damaged (pDNA ox) plasmid DNA

solutions. The spectrum of water is still reported for comparison. Panel (b): Raman spectra of damaged

plasmid DNA without the water contribution (pDNAox – H2O). The curve dNTP ox is still reported for

comparison. SOURCE: D’amico F et al. in Analyst, 2015, 140, 1477 [1].

The smaller Raman activity observed for the vibrational modes in 4,6-diamino-5-

fomamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine with respect

to the corresponding closed-ring oxidized forms (see Figure 36 and 37) supported this

interpretation. Furthermore, it was impossible to detect markers of the oxidation of

cytosine and thymine because of the relatively low intensity of the fingerprint band in

the total spectrum of both nucleotides mix and DNA.

124

4.3 Conclusions

Our study demonstrated the feasibility of using UV-Raman spectroscopy to reveal

oxidation changes in DNA aqueous solutions after Fenton’s reaction. In this conditions,

the main specie responsible for DNA damage was the hydroxyl radical (OH˙) that acted

on the DNA bases, generating a number of DNA base derivatives. It was interesting that

some bases were more susceptible than the others to ROS. Initially we analyzed the

nucleotides (dATP, dGTP, dCTP, dTTP), which are the subunits of DNA. Then, the

Raman spectra obtained for nucleotides, were used as a reference to better understand

the oxidative plasmid DNA damage, recognizing the contribute of single nucleotides.

The results showed that the incident radiation at 266 nm seemed to be more efficient to

detect the chemical modifications on the nitrogenous bases.

Our analysis when combined with computational simulations revealed that the oxidation

on three-phosphate nucleotides in aqueous solutions generated mainly 2-

hydroxyadenine, 8-hydroxyguanine and 5-hydroxycytosine, while 8-hydroxyguanine

was clearly also a product of the DNA oxidation. However, in this case there was more

severe damage of the nitrogenous bases, most probably leading to the opening of the

adenine and guanine aromatic rings.

Under these experimental conditions, the elements of DNA, particularly the dATP,

dCTP, dGTP and dTTP nucleotides, resulted to be more susceptible to oxidative

damage.

The results demonstrated that UV-Raman spectroscopy is useful to reveal the chemical

changes that affect the nitrogenous bases after nanomaterials exposure, providing a

“fingerprint” of the oxidative DNA damage.

125

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128

CHAPTER 5 - GENERAL CONCLUSIONS AND

OUTLOOK

This PhD work was a multidisciplinary study to reveal something new on the toxic

effect of nanomaterials in biological systems, with particular interest to investigate

similarities with an old killer: asbestos. As we have already discussed, the number of

subjects exposed to nanomaterials is increasing, in contexts where the exposure’s

impact on occupational health and safety is difficult to predict. Fetuses, newborns and

children will need protection.

Carbon nanotubes are promising products in industry and medicine, but there are

several concerns for human pulmonary exposure since their fibrous structure might

cause asbestos-like pathology. Our findings demonstrated that the combination of

advanced synchrotron-based X-ray microscopy and fluorescence techniques (µXRM

and XRF) are promising to study the toxic mechanisms induced by asbestos and carbon

nanotubes in the pleural (Met5A) and the placental (BeWo) cell lines.

The morphological and elemental X-ray analysis showed that in both cell models there

was an increase of intracellular iron, which was also confirmed by the stimulation of

ferritin, after the exposure to nanomaterials. It was interesting to highlight that the

presence of iron impurities in the nanomaterial itself seemed to be the main cause of

toxic effects that resulted in an alteration of iron metabolism. The pleural cells treated

with raw-SWCNT and crocidolite fibres compared to the control showed a severe

alteration of iron metabolism, while the same techniques showed that the iron changes

inside placental cells were restricted to small intracellular regions, thought to be just in

contact with the exogenous fibre. This study also found that highly purified nanotubes

did not alter iron metabolism, at least after 24 hrs of treatment. X-ray microscopy

images (absorption and phase contrast imaging), in combination with AFM and SEM

analysis, confirmed that the toxicity of nanomaterials was characterized by membrane

damage with vesicle secretion and filipodia formation. Moreover, the ferritin assay

resulted to be a valid test for toxicity, confirming that the concentration of this iron

transporter was clearly lower in placental cells than in pleural ones. This seems linked

to a different/lower uptake of fibres, suggesting that this barrier is less vulnerable than

the pleura.

129

We were also interested to evaluate the genetic factors involved in susceptibility to

nanomaterial exposure by using conventional molecular techniques (i.e., PCR and

Sanger sequencing).

We investigated the relation between genetic predisposition to develop mesothelioma

and asbestos exposure by looking for BAP1 gene mutations in 29 cases of

mesothelioma or related diseases. All the patients were asbestos-exposed workers.

Sanger sequencing of BAP1 gene in the 29 patients allowed to identify one non-

synonymous variant and two intronic variants: patient 9 carried in heterozygous a

missense variant (c.T1028C; p.L343P) at exon 11; patient 4 carried in heterozygous a

known intronic variation 8 bases downstream of the exon 13 (c.1729+8T>C); in patient

15 carried a heterozygous intronic variation 8 bases upstream of the exon 10 (c.784-

8G>A). Sanger sequencing of cDNA revealed no alternative splicing due to the

nucleotide change for each mutations. In silico mutation analysis demonstrated that

there were no significant possible effect of the amino acid change about exonic

mutations of patient 9 in a predicted protein structure of BAP1 protein. MLPA

(Multiplex ligation-dependent probe amplification) analysis revealed no significant

copy number variations at exonic level in all samples. Although the combination of

different molecular analysis fails to predict that BAP1 variants were pathogenic

mutations, two mesotheliomas and a renal cancer were associated to mutations.

Moreover, in agreement with literature, one patient (nine) presented significantly

increased survival time of three and half years with respect to the average life

expectancy.

Overall the results are puzzling and there was no clear correlation with type and extent

of exposure. However they are the first analyses of BAP1 genotyping in patients of

Friuli Venezia Giulia (Italy) region, which is a known endemic area for asbestos related

diseases.

Based on these results, we intend to expand our casuistic and we will evaluate the

expression levels of BAP1 in both mesothelial (MeT5A) and placental (BeWo) cells

treated with asbestos and carbon nanotubes in order to demonstrate the possible

interference of nanomaterials on genetic (epigenetic) mechanisms.

We are also strongly interested to test the feasibility of UV-Raman (IUVS beamline,

Elettra Synchrotron of Trieste) spectroscopy to reveal oxidative DNA damage after

nanomaterial exposure. The transition metals that are present on nanomaterial surface

can generate reactive oxygen species through the Fenton’s reaction.

130

The oxidative environment created in vitro by using carbon nanotubes (raw-SWCNT),

which contain some impurity in metal traces (iron), and free radicals OH• (derived from

H2O2) caused the oxidative damage of the nucleotides (dATP, dCTP, dGTP and dTTP).

The results showed that the incident radiation at 266 nm seems to be more efficient to

detect the chemical modifications on the nitrogenous bases.

The combination of the experimental analysis with computational simulations allowed

to detect the oxidation on three-phosphate nucleotides in aqueous solutions that

generated mainly 2-hydroxyadenine, 8-hydroxyguanine and 5-hydroxycytosine, while

8-hydroxyguanine was clearly also a product of the DNA oxidation. When we analyzed

the DNA oxidative damage, we found a more severe damage of the nitrogenous bases,

most probably leading to the opening of the adenine and guanine aromatic rings. These

results demonstrated that UV-Raman spectroscopy is useful to reveal the chemical

changes that affect the nitrogenous bases after nanomaterials exposure, providing a

“fingerprint” of the oxidative DNA damage.

After this success, we intend to continue with UV-Raman investigation to reveal

epigenetic changes (DNA methylation) in placental DNA of asbestos-exposed women,

since we believe that the placental methylation alterations may have harmful effects on

fetal development. This new spectroscopic approach may help to unravel the link

between nanomaterial exposure and epigenetic changes.

In conclusion, the power of this multidisciplinary study which encompasses the

biochemistry - study of the role of iron in in vitro cell models after exposure to

nanomaterials - and the molecular biology - the study of genetic predisposition and

molecular damage after exposure to nanomaterials - was the combination of different

microscopic and molecular techniques that allowed to detect the toxic effects and the

cellular interaction of asbestos and carbon nanotubes in biological systems.

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Appendix A

A1. AFM and µXRM imaging studies on MeT5A cells

Figure A1. Combination of optical, µXRM (absorption and phase contrast, respectively) and

AFM images of MeT5A cells exposed to crocidolite fibres (lane A and B) and to purified s-

SWCNTs (lane C). The mesothelial cells are seeded on Si3N4 windows and the day after are

exposed for 24h to 5μg/mL of each nanomaterial. The absorption and phase contrast images are

measured at 0.9KeV photon energy at TwinMic Beamline (Elettra, Trieste). AFM images are

obtained at Laboratorio TASC-INFM (Elettra, Trieste) with collaboration of PhD Damiano

Cassese.

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A2. SEM imaging studies on MeT5A cells

Figure A2. SEM images of control MeT5A (A,B,C,D), crocidolite-treated cells (E,F,G,H) and

cells exposed to raw SWCNTs (I,L,M,N) and purified s-SWCNTs (O,P,Q,R). The zoom images

of some detail are shown in right side of the panel for each nanomaterial (D,H,N,R). MeT5A

cells are seeded on ˪-poly-lysine coated coverslipes and day after are exposed for 24 h to

5µg/mL of each nanomaterial. Then the cells are washed in PBS and fixed with 2.5%

glutaraldheyde at room temperature for 20 min, rinsed in PBS and in the end dehydrated in

ascending ethanol concentrations (30%, 50%, 70%, 90%, 100%). At this point, the samples are

transferred to a critical point dryer (Bal-Tec; EM Technology and Application, Furstentum,

Liechtenstein) in 100% ethanol and dried through CO2. Coverslips are mounted on aluminium

sample stubs and gold coated by sputtering (Edwards S150A apparatus, Edwards High Vacuum,

Crawley, West Sussex, UK). SEM measurements are performed by using Leica Stereoscan 430i

scanning electron microscope (Leica Cambridge Ltd., Cambridge, UK), with collaboration of

Dr Francesca Vita at University of Trieste.

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Appendix B

B1. The clinical features of 29 patients

Case

n.

Gender

Age

of MM

diagnosis

(y)

Age

of

death

(y)

MM

survival

(m)

Cancer

MM

histotype

Other

diseases

Treatment

Asbestos

exposure

circumstances

Occupation

Asbestos

bodies

count

1 M 77.9 77.9 0.5

Pleural

mesothelioma;

prostatic

carcinoma

Epithelioid / Pallliative Occupational Dockworker 13

2 M 87.3 87.6 2.6 Pleural

mesothelioma Epithelioid / Palliative Occupational

Ship

mechanic 34300

3 M NA 72.2 NA

Laryngeal

carcinoma;

prostatic

carcinoma; lung

carcinoma; oral

carcinoma with

lung metastases

/ /

Laringectomy;

pulmonary lobectomy;

emiglossopelvectomy;

partial

pharingoglossectomy;

bilateral neck

dissection; radiotherapy

Occupational Sailor 8400

4 M NA 70.5 NA Renal cell

carcinoma /

Pulmonary

fibrosis / Occupational

Chemical

factory

worker

104

5 F 84.9 85.0 0.5 Pleural

mesothelioma Biphasic / None NA 94

6 M 71.5 72.9 16.8

Pleural

mesothelioma

(circumscribed

non evolutive);

Epithelioid / None Occupational Dockworker 5100

134

oral carcinoma

7 M 82.1 82.6 5.23 Pleural

mesothelioma Epithelioid / Palliative Occupational

Engine

factory

worker

20000

8 M 57.9 68.7 126.6

Pleural

mesothelioma

(healed);

bladder

carcinoma;

contralateral lung

carcinoma

NA / Pneumectomy NA NA 55

9 M 68.3 71.8 41.1 Pleural

mesothelioma Epithelioid

Diffuse

pleural

fibrosis;

desmoid-

type

fibromatosis

Pleurectomy and

decortication, multiple

pulmonary resections;

chemo- and

radiotherapy

Occupational

Ship engine

room worker,

shipyard

worker

229

10 F 68.4 69.9 17.6 Pleural

mesothelioma Epithelioid NA

Pleuropneumectomy;

chemo- and

radiotherapy

Household Housewife 121

11 M 81.5 81.5 0.6 Pleural

mesothelioma Biphasic / None NA

Steel mill

worker 231

12 M 82.1 83.6 17.5 Pleural

mesothelioma Epithelioid /

Chemo- and

radiotherapy NA Electrician 513

13 M NA 78.6 NA / / Asbestosis None Occupational Dockworker 5025

14 M NA 77.1 NA / / Asbestosis None Occupational Ship cook 6580

135

15 M 62.6 64.1 18.1 Pleural

mesothelioma Epithelioid / Chemotherapy Occupational Welder 31330

16 M 66.5 69.4 34.2 Pleural

mesothelioma Epithelioid / Pleurectomy Occupational Dockworker 19840

17 M NA 69.9 NA / / / / Occupational

Engine

factory

worker

6690

18 M 57.3 60.1 32.9 Peritoneal

mesothelioma Epithelioid /

Intestinal resection;

chemotherapy NA NA 25

19 M 82.7 82.9 2.1 Pleural

mesothelioma Epithelioid / Chemotherapy Occupational

Shipyard

worker 119

20 M 64.3 65.5 14.5 Lung carcinoma / / Chemotherapy Occupational Glass factory

worker 136

21 M 64.4 70.5 72.1 Pleural

mesothelioma Epithelioid /

Pleurectomy and

decortication;

chemotherapy

Occupational Elevator

technician 485

22 M 74.3 74.4 1.4 Pleural

mesothelioma NA / None NA NA 75

23 M 55.5 58.0 29.4 Pleural

mesothelioma Epithelioid /

Pleurectomy and

decortication;

chemotherapy

Occupational

Occupational

health and

safety

technician

18

24 M 86.8 87.0 3.0 Pleural

mesothelioma Epithelioid / Palliative Occupational Navy officer 1660

25 M 86.8 87.3 5.7 Pleural

mesothelioma Biphasic / Palliative Occupational Sailor 1320

136

26 M 65.1 65.8 8.2 Lung

adenocarcinoma / /

Chemo- and

radiotherapy Occupational Dockworker 11150

27 M 85.7 88.4 31.1 Pleural

mesothelioma Biphasic / None NA NA 1270

28 F 78.9 79.9 12.3

Pleural

mesothelioma

(regressed)

Epithelioid / Chemo- and

radiotherapy NA NA 0

29 M NA 91.1 NA

Lung carcinoma;

colorectal

carcinoma

/ Asbestosis NA NA NA 1670

Table B1. The table summarizes the clinical features of 29 patients. They are sex, age of diagnosis and death, survival time, cancer and concurrent diseases,

malignant mesothelioma histotype, and treatment including surgery, chemotherapy, and radiation, asbestos exposure circumstances, occupation and asbestos

bodies count. The table was provided by Dr Clara Rizzardi (University of Trieste).

137

ABBREVIATION LIST

NMs: nanomaterials

BSI: British Standard Institute

DNA: deoxyribonucleic acid

SWCNTs: single-walled carbon nanotubes

CNT: carbon nanotube

MWCNTs: multi-walled carbon nanotubes

BBB: blood–brain barrier

BTB: the blood–testis barrier

ROS: reactive oxygen species

COPD: chronic obstructive pulmonary diseases

UFPs: ambient ultrafine particles

AM: alveolar macrophage

TEM: transmission electron microscope

FPP: fibre pathogenicity paradigm

RNS: reactive nitrogen species

IARC: International Agency for Research on Cancer

MAPK: mitogen-activated protein kinase

NF-κB: nuclear factor-kappa B

XRF: X-Ray Fluorescence

XANES: X-ray Absorption Near Edge Spectroscopy

ZP: Zone Plate

OSA: Order Sorting Aperture

PAHs: polycyclic aromatic hydrocarbons

MM: malignant mesothelioma

BAP1: BRCA1-associated protein 1

MBAITs: melanocytic BAP1-mutated atypical intradermal tumours

HiPCO: high pressure carbon monoxide

CVD: Chemical Vapor Deposition

r-SWCNTs: raw-SWCNTs

p-SWCNTs: purified-SWCNTs

hp-SWCNTs: highly purified-SWCNTs

s-SWCNTs: short-SWCNTs

138

PBS: phosphate buffered saline

DMEM: dulbecco’s modified Eagle’s medium

FBS: fetal bovine serum

M-PER: mammalian protein extraction reagent

XRF: X-ray fluorescence

SXM: scanning X-ray microscopy

SDDs: silicon drift detectors

SR-XRF: synchrotron radiation X-ray fluorescence

CB: carbon black

AFM: atomic force microscope

SNOM: scanning near-field optical microscopy

EGFR: epidermal growth factor receptor

ESRF: European Synchrotron Radiation Facility

SEM: scanning electron microscopy

NPs: nanoparticles

o-MWCNTs: oxidised MWCNTs

WHO: World Health Organization Classification

PCR: Polymerase Chain Reaction

NCBI: National Center for Biotechnology Information

RT: Reverse transcription

HSF: Human Splicing Finder

NNSplice: Neural Network Splice

SPANR: Splicing-based Analysis of Variants

PDB: Protein Data Bank

MLPA: Multiplex ligation-dependent probe amplification

dATP: deoxyadenosine triphosphate

dCTP: deoxycytidine triphosphate

dGTP: deoxyguanosine triphosphate

dTTP: deoxythymidine triphosphate

UVRR: ultraviolet resonant Raman spectra

pDNA: plasmid DNA

pDNA ox: oxidized plasmid DNA

139