Effects of silver nanoparticles on oxidative stress and Nrf2 pathway in an in vitro model of intestinal epithelium cells

Abstract Considering the very interesting physicochemical properties of nanomaterials, it is not surprising that during the last few years, nanomaterial applications expended exponentially. Among nanoparticles, silver nanoparticles (AgNPs) are the most commonly used in consumer products, mainly because of their antimicrobial properties. AgNPs are already incorporated in many products of the food sector such as food packaging materials, antimicrobial sprays and even dietary supplements. These applications could lead to abnormally high oral exposure for the consumer. Many authors have demonstrated AgNPs toxicity towards various biological systems. Furthermore, there is still a huge lack of knowledge concerning the mechanisms of toxicity related to AgNPs and the contribution of Ag+ involved in AgNPs toxicity. Therefore, this work was devoted to study AgNPs and Ag+ effects on oxidative stress and the related cellular responses. In order to do so, monocultures of Caco-2 cells were used. They are indeed a widely used in vitro model of the human intestinal epithelium. First of all, the ability of AgNPs and Ag+ to generate ROS and induce oxidative stress was evaluated by performing an NBT assay. A significant increase in ROS production was observed for both AgNPs and Ag+ with a nano-specific effect. The second objective of this thesis was to determine the impact of AgNPs on enzymatic activity of several oxidative stress-related enzymes (CAT, GR, GP and GST). Unfortunately, we were not able to detect any significant changes in these enzymatic activities after 3h incubation with 15 µg/ml AgNPs. Thirdly, we studied the impact of AgNPs/Ag+-induced ROS generation on Nrf2 signalling pathway by performing an ELISA quantification of HO-1, which is an Nrf2 specific gene target. Nrf2 is a major pathway involved in protecting cells from oxidative stress, through the induction of antioxidant-responsive genes. Surprisingly, we did not measure any significant effect on HO-1 production. Finally, the effect of AgNPs on inflammation was evaluated, along with the potential implication of the Nrf2 signalling pathway. Indeed, preliminary work at the lab indicated that the pro-inflammatory response related to AgNPs was not due to activation of the NF-κB pathway. The alternative Nrf2 pathway was therefore studied to explain the production of the inflammatory marker IL-8 measured by ELISA. According to our results, Nrf2 does not seem to be involved in IL-8 production following AgNPs treatments. In conclusion, although AgNPs were not cytotoxic at the studied concentrations, we have observed an increased ROS production. Numerous studies confirm our results regarding ROS production, but in contrast to our observations, suggest the implication of the Nrf2/HO-1 signalling pathway to explain the cellular responses related to the AgNPs-induced ROS production.