Effects of nanomaterials on biological barriers, fetal and post-natal, and evaluation of epigenetic toxicity

Beyond the opportunities offered by nanotechnology research, there is a great need of studies aimed at understanding the harmful effects of general exposure to nanomaterials. My Ph.D. project aimed to be part of this evaluation, focusing on the interaction and the induction of possible toxic effects of two fibrous nanomaterials (asbestos and carbon nanotubes) at two critical internal biological barriers: the pleura and the placenta. The work is carried out by using advanced microscopy (\ub5XRM and XRF) based on synchrotron radiation and other microscopes (SEM and AFM), and also conventional molecular analysis (PCR and Sanger sequencing) and advanced spectroscopic measurements (UV-Raman). We conducted biochemical studies by using the advanced X-Ray microscopy and fluorescence (\ub5XRM and XRF) techniques in order to reveal mechanisms of toxicity in human mesothelial (MeT5A) and placental cell lines (BeWo) exposed to carbon nanotubes (raw-SWCNT, purified- and highly purified-SCWCNT) or asbestos (crocidolite fibres). Other microscopes (AFM, atomic force microscopy and SEM, Scanning Electron Microscope) are added in some experiments, to better investigate the morphology and the cell-nanofiber interactions. The results obtained with the combination of microscopic techniques allowed to reveal similar as well as different toxic mechanisms in the two internal barriers. The cells treated with raw-SWCNT and crocidolite fibres compared to the control showed an severe alteration of iron metabolism, which is maximal in the pleural cells and is clearly related to the presence of iron into the fibre. X-ray microscopy images (absorption and phase contrast imaging) confirm that the toxicity of nanomaterials is characterized by membrane damage with vesicle secretion and filipodia formation. In relation to this toxic mechanism quite complex and still unknown we evaluated the presence of intracellular ferritin in treated cells. The results demonstrated that crocidolite and \u201craw\u201d carbon nanotubes increase the amount of intracellular ferritin in both cell models, while purified and highly purified carbon nanotubes give values comparable to control. The stimulation is clearly lower in placental cells, clearly linked to a different or lower uptake of fibres in these cells, suggesting that this barrier is less vulnerable than the pleura. We also investigated the genetic effects and genetic predisposition to toxicity of nanomaterials (nanotoxicogenomic). Since we have to learn from asbestos, one study investigates the possible genetic predisposition to develop mesothelioma after asbestos exposure by looking for BAP1 gene mutations in 30 cases of mesothdelioma. Sanger sequencing of BAP1 gene in the 30 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. 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 has been created in vitro by using carbon nanotubes (raw-SWCNT), which contain some impurity in metal traces (iron), and free radicals OH\u2022 (derived from H2O2). In this condition the nucleotides (dATP, dCTP, dGTP and dTTP) result 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 \u201cfingerprint\u201d of the oxidative DNA damage