AUTHOR=Martin Sebastin , de Haan Laura , Miro Estruch Ignacio , Eder Kai Moritz , Marzi Anne , Schnekenburger Jürgen , Blosi Magda , Costa Anna , Antonello Giulia , Bergamaschi Enrico , Riganti Chiara , Beal David , Carrière Marie , Taché Olivier , Hutchison Gary , Malone Eva , Young Lesley , Campagnolo Luisa , La Civita Fabio , Pietroiusti Antonio , Devineau Stéphanie , Baeza Armelle , Boland Sonja , Zong Cai , Ichihara Gaku , Fadeel Bengt , Bouwmeester Hans TITLE=Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials JOURNAL=Frontiers in Toxicology VOLUME=4 YEAR=2022 URL=https://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2022.974429 DOI=10.3389/ftox.2022.974429 ISSN=2673-3080 ABSTRACT=

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA (“biomaterial risk management”) an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 “benchmark” nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.