Investigation on the effects of TiO2 nanoparticles and bulk counterpart in Vicia faba L.

TiO2 nanoparticles (NPs) are among the top five NPs used in consumer products (toothpastes, sunscreens, cosmetics, food products, medicines and pharmaceuticals, agriculture and environmental cleanup products) for their high stability, anticorrosive properties, redox selectivity, low production costs and wide spectrum of applications (1). NPs have been recently included among the emerging contaminants by USEPA (2) since living organisms may be affected by their exposition to NPs released into aquatic, terrestrial and atmosphere environments. The experimental design of the present work was to study the model system Vicia faba L. var. minor and the potential effects of the same concentration (50 mg/l) of a commercial source of TiO2 NPs <100nm (tetragonal crystals, from Sigma-Aldrich) (S), of a NP laboratory sample <10nm (spherical shape, kindly provided by PlasmaTech, Pisa) (P) (3), and of the corresponding bulk material (B) recently classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (4). The above materials were applied to V. faba seeds considering different endpoints such as germination/root elongation, root meristem mitotic activity, possible anomalies and/or disturbances during cell cycle and root ultrastructure in function of the shape and size of NPs. In addition oxidative stress and antioxidant response were evaluated by biochemical approach and in situ histochemical techniques. Germination percentage did not show significant differences among control and treated materials, but roots from bulk-treated seeds were significantly shorter in comparison to the control and to nanoparticle-treated materials after 72 of imbibition. Concerning cytological analysis, the aberration index only evidenced a significant increase in samples treated with S and B materials. Bulk TiO2 induced an oxidative stress in terms of both hydrogen peroxide and TBA-reactive material, higher than in control and in NPs-treated seedlings. Defense system following this treatment seemed to rely mainly on low molecular weight antioxidants, at the expense of the reduced forms of these molecules. NPs exposure caused a lower oxidative stress with a high reducing power of glutathione in P treated seedlings and by a high POD activity in S material. Histological evaluation associated to in situ detection of oxidative stress was effected by different probes for hydrogen peroxide, reactive nitrogen species, lipid peroxidation and peroxidase activity. In root cross sections differences in staining intensity and localization of the signals were observed mainly in S and B treated roots, to evidence a stressful effect of these materials, while, following the P treatment, the staining pattern was less different from the control. Under electron microscope, the cytoplasm in control root cells was rich in well structured organelles, particularly rough endoplasmic reticulum and dictyosomes. The cells in root treated with B material showed the most disorganized cytoplasm, with swollen cisternae of smooth endoplasmic reticulum, not well organized mitochondria and organelles often not well recognizable. When exposed to nano-scaled TiO2, the ultrastructural appearance of cells was less affected after treatment with P than with S NPs. These results suggest that TiO2 NPs may exert different actions with different levels of toxicity, depending on their size and their shape and that the bulk counterpart, in our experimental condition, seems to provoke the major adverse effects in V. faba root. 1) S.M. Gupta, M. Tripathi (2011) Chin. Sci. Bull., 56, 1639–1657 2) USEPA (2010) http://www.epa.gov/region9/mediacenter/nano-ucla 3) E. Giorgetti, M. Muniz Miranda, S. Caporali, P. Canton, P. Marsilia, C. Vergari, F. Giammanco (2014) J. Alloys Comp. http://dx.doi.org/10.1016/j.jallcom.2014.11.117 4) IARC Monographs (2010) http://monographs.iarc.fr/ENG/Monographs/vol93/mono93-7.pdf