NANOMEGA · Novel approach to toxicity testing of nanoparticles mimicing lung exposure. Possible protective effect of omega-3 acids

Novel approach to toxicity testing of nanoparticles mimicing lung exposure. Possible protective effect of omega-3 acids

Despite major efforts to understand the adverse effects of nanoparticles (NP) there is still a serious lack of information concerning the potential hazardous effects of manufactured NP on human health. It has been proposed that ROS play a role in NP genotoxicity but information about the downstream pathways through which NP signal in human cells and induce DNA damage is still very limited. Furthermore, at present there are no data available on the identification of specific ROS responsible for the toxic outcomes of NP. Results published on the toxicity of NP suggest induction of cytotoxicity, oxidative stress, genotoxicity and inflammatory responses and thus possible implication in development of cardiovascular diseases. However, there have been no investigations into possible protective effects against NP injury. In our project we examined the mechanisms of silver nanoparticles (AgNP) and titanium dioxide (TiO2) toxicity in the identification of specific markers of oxidative stress and their role as mediators in the activation of signal pathways associated with DNA damage and repair. Furthermore we addressed for the first time possible influences of nutrition on cardiovascular/cardiopulmonary AgNP toxicity, basing our investigations on the evidence of a beneficial effect of a diet rich in omega 3 fatty acid on cardiovascular disease pathology. We investigated the possible sources of reactive oxygen species (ROS) production and we identified, as a major contributor to oxidative stress induced by AgNP in our experimental systems, the enzymatically produced ROS. In addition, we were able to measure specifically H2O2, using a fluorescence probe called HyPer, insensitive to other ROS. By using the HyPer probe, it has been also possible the investigation of H2O2 localization in different cell compartments through a targeting sequence for mitochondrion and nucleus. Likewise airborne ultrafine particles, ROS and the associated oxidative stress are proposed as a crucial mediator for engineered NP-toxicity. However, how the presence of NP could increase the ROS-formation is not completely understood. The natural property for many NP to bind transition metals is believed to enhance ROS-induced toxicity. Furthermore, the surface chemistry of particles can lead to direct ROS-formation. Additionally NP can directly interfere with the mitochondria or enzymes producing ROS, such as NADPH-oxidases (NOX). Evidence that NOX2 is involved in ROS generation was shown in a mouse pulmonary endothelium model following exposure to ultrafine particles. Similar evidence was suggested by our results where the source of O2•− and H2O2, following O2•−dismutation, appeared to be due to activation of one or more NOX isoforms, as exposure to DPI, a nonspecific but widely used inhibitor of NOX family, reduced the release of AgNP-induced ROS from the cell. As ROS are short-lived species, due to the cellular antioxidant defense, it is unlikely that the O2•− /H2O2 produced by NOX2 or any other cytosolic NOX, could cause damage to DNA. As we found production of H2O2 in the cytoplasm, mitochondria and nucleus, one possibility is that activation of the different NOX isoforms by AgNP depends on its cell localization which in turn causes different cell damage. We also examined the role of MAPK pathways in the toxicity of AgNP. Interestingly we found that the activation of JNK and ERK resulted mediated by a O2•− /H2O2-dependent mechanism and that ERK activation is crucial in the process of DNA repair of AgNP-induced DNA oxidation damage. To investigate a possible protective role of nutrition (omega 3 fatty acid diet) on NP induced toxicity we used a cell model in which the membrane lipid composition was varied: we increased the percentage of DHA on the membrane growing human endothelial cells in DHA enriched media followed by AgNP exposure. Our results showed that DHA was able to significantly inhibit the AgNP induction of DNA oxidation lesions keeping the percentage of oxidized bases at the control level. This was achieved by increasing the ability of the cells to recognize and repair the oxidative DNA damages which in turn depended on the increase in the expression of DNA repair enzymes.

Data: CORDIS, © European Union